2014 12-09-oulu

Genomic architecture underlying a major transition in

social evolution

[email protected] http://wurmlab.github.io2014-12-09-Oulu

mailto:[email protected]

http://yannick.poulet.org

© National Geographic

Atta leaf-cutter ants

Oecophylla Weaver ants

© ameisenforum.de

© ameisenforum.de

Fourmis tisserandes

© ameisenforum.de

Oecophylla Weaver ants

Tofilski et al 2008

Forelius pusillus

Tofilski et al 2008

Forelius pusillus hides the nest entrance at night

Avant

Workers staying outside die« preventive self-sacrifice »

Tofilski et al 2008

Forelius pusillus hides the nest entrance at night

Dorylus driver ants: ants with no home

© BBC

Animal biomass (Brazilian rainforest)

from Fittkau & Klinge 1973

Other insects AmphibiansReptiles

Birds

Mammals

Earthworms

Spiders

Soil fauna excluding earthworms,

ants & termites

Ants & termites

Well-studied:• behavior

• morphology

• evolutionary context

• ecology

Genetic basis?

www.sciencemag.org SCIENCE VOL 331 25 FEBRUARY 2011 1067

REPORTS

on

Mar

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2, 2

013

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w.s

cien

cem

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oade

d fro

m

Solenopsis invicta fire ants are a big problem!very well studied!

Ascunce et al 2011

Solenopsis invicta fire ant: two social forms

•1 large queen•Independent founding•Highly territorial•Many sizes of workers

•2-100 smaller queens•Dependent founding•No inter-colony aggression•All workers similar size

Single-queen form: Multiple-queen form:

Fire ants+

Population genetics: Allozyme screen

Ken Ross

“starch gel”+

1 2 3

L. Keller

Allozyme screen Social form associated to Gp-9 locus

Frequency of the most

common allele

Locus!

0.3!0.4!0.5!0.6!0.7!0.8!0.9!1.0!

Single queen!Multiple queen!

Est-6!Est-4!G3pdh-1!Ca-4!Pgm-4!Ddh-1!Pro-5!

Pgm-3!

Acoh-5!

acoh-1!

Acy-1!

Pgm-1!

Aat-2!

Gp-9!

Ken Ross and colleaguesLaurent Keller and colleagues

Single queen form Multiple queen form


Social form completely associated to Gp-9 locus

bbbbBB BB Bb bb




(>15% ) (< 5% )

bbBB BB Bb

x

Gp-9 bb females rareKen Ross and colleagues

Laurent Keller and colleagues



(>15% ) (< 5% )

BB BB Bb




(>15% ) (< 5% )

BB BB Bb

xKen Ross and colleagues




(>15% ) (< 5% )

BB BB Bb

x xKen Ross and colleagues



Single queen form Multiple queen form(>15% ) (< 5% )

BB BB Bb

x x xKen Ross and colleagues





• Is this gene the single überregulator?

• Is this gene the single überregulator?


maybe 1/14th of the genome?

•Only 14 allozyme markers were used

Locus!

0.3!0.4!0.5!0.6!0.7!0.8!0.9!1.0!

Single queen!Multiple queen!

Est-6!Est-4!G3pdh-1!Ca-4!Pgm-4!Ddh-1!Pro-5!

Pgm-3!

Acoh-5!

acoh-1!

Acy-1!

Pgm-1!

Aat-2!

Gp-9!

This changes everything.454

IlluminaSolid...

Any lab can sequence anything!

Are other genes linked to Gp-9?Sequenced:

•Genome

Single ♂:

His brothers:

11×

4×

(330bp-insert paired reads) (normal single-end reads)45× +

Sequencing from haploid males (for easier assembly):

(8,000 & 20,000bp-insert paired reads)

B 20x

Genome of a Gp-9 B ♂

★ Expansion of lipid-processing gene families (for Cuticular Hydrocarbons?)

★ 420 putative olfactory receptors (more than any other insect!)

0.05

SiOR

04648+10

SiOR01968+4

SiOR0

0899+7

SiOR02814+3

SiOR04171+6

SiO

R04609+4

SiOR00330+28

SiO

R02

694+25

SiO

R04609+20

SiOR05285+6

SiOR00330+25

SiOR04510+15

SiOR00330+18

SiOR04609+23

SiOR01

968+23

SiO

R03

952+4

SiOR0

4648+16

SiO

R05

901+2

SiO

R02

944+4

SiOR01968+5

SiOR04171+19 SiOR

04648+5

SiOR10535+3

SiOR0

1224+3

SiOR06723+2

SiOR01968+9

SiOR02883+1

SiOR0

0899+3

SiOR01629+1

SiOR04171+1

SiOR01629+11

SiOR04171+10

SiOR04171+13

SiOR02694+3

SiOR04171+20

SiO

R02694+35

SiOR04171+15

SiOR04609+7

SiOR05118+2

SiOR07

837+2

SiO

R02

694+27

SiOR01968+10

SiOR04648+17

SiOR0196

8+19

SiO

R02

694+17

SiOR04648+13

SiOR01968+6

SiOR

00899+1

2

SiOR0

5901+1

SiOR00330+20

SiO

R02

648+2

SiO

R02

659+2

SiOR01968+1

6

SiOR0

0899+11

SiO

R02974

SiOR04171+2

SiOR03952+2

SiOR06792+2

SiOR04510+4

SiOR04171+28

SiOR05285+5

SiOR05285+9

SiOR

00899+1

5

SiOR

04648+3

SiO

R02694+36

SiOR10535+1

SiO

R02

694+19

SiO

R02

694+23

SiOR02694+1

SiO

R04609+14

SiOR01122

SiOR

04648+9

SiO

R02694+34

SiOR01629+8

SiOR

04648+8

SiOR04510+8

SiOR06573

SiO

R02

944+1

SiOR00330+26

SiO

R00330+1S

iOR02

694+15

SiO

R03038

SiOR05285+7

SiOR0

0899+5

SiOR04609+10

SiO

R04609+3

SiOR04339

SiOR0

8068

SiOR

04648+6

SiOR04510+2

SiOR05285+8

SiOR01

573+4

SiOR0366

3

SiOR04171+8

SiOR01858+2

SiOR01968+2SiOR01968+1

SiOR02694+5

SiOR01968+3

SiOR06723+3

SiOR01968+15

SiOR05285+1

SiOR0

0899+4

SiOR04609+22

SiOR04171+9

SiOR02694+9

SiO

R02

648+1

SiOR06792+3

SiOR0

1573+2

SiO

R02

694+20

SiOR1

0542

SiO

R04609+15

SiOR02694+8

SiOR00330+16

SiOR0

0899+2

SiOR02694+10

SiOR04510+9

SiOR05285+3

SiOR04171+24

SiO

R04609+2

SiOR05285+11

SiOR02

694+14

SiOR01

573+1

SiOR05285+2

SiOR00613

SiOR0196

8+22

SiOR

00899+9

SiO

R06

843+2

SiO

R02694+37

SiOR0

0899+1

SiOR04609+9

SiOR05431+2

SiOR10535+2

SiOR00330+15

SiO

R02

694+18

SiOR0

1224+2

SiOR04510+11

SiOR00330+23

SiO

R02

694+29

SiOR05416

SiOR05285+10

SiOR02694+2

SiOR01629+9

SiOR05431+1

SiOR02883+2

SiOR04171+14

SiO

R08

341

SiO

R02

694+22

SiOR01

224+1

SiOR01968+12

SiOR05431+3

SiOR04510+6

SiOR02694+7

SiOR04609+5

SiO

R02

944+2

SiOR03

952+3

SiOR01968+8

SiOR04609+24

SiO

R02

694+30

SiOR01629+10

SiOR04510+14

SiOR00565

SiOR05118+3

SiO

R01321

SiO

R04609+19

SiOR00330+14

SiO

R02694+38

SiOR04609+8

SiOR04171+16

SiOR10455

SiO

R04609+16

SiO

R04609+21

SiO

R02

694+28

SiO

R02

659+1

SiOR04171+5

SiOR00330+29

SiOR01968+14

SiOR03983

SiOR00330+27

SiOR05285+4

SiOR04510+1

SiOR

00899+8

SiOR

04648+7

SiO

R04609+17

SiO

R00330+5

SiO

R02

694+21

SiOR02814+4

SiOR00330+7

SiOR01

629+3

SiOR01

968+26

SiO

R02694+31

SiOR0

4648+2

SiO

R02694+39

SiOR04171+25

SiOR06577

SiOR01

968+25

SiOR04171+21

SiOR06792+6

SiO

R04609+11

SiOR02694+11

SiOR06792+1

SiOR04171+4

SiOR01629+5

SiOR00330+21

SiOR0

4648+15

SiO

R00330+6

SiO

R02

694+16

SiOR

04648+11

SiOR

04648+4

SiO

R00330+3

SiO

R06

535

SiOR04171+7

SiOR10493

SiO

R02694+32

SiOR06792+4

SiOR04510+3

SiO

R06

890

SiOR0196

8+20

SiO

R04609+12

SiOR04171+3

SiOR01968

+18

SiOR01968+11

SiO

R04609+13

SiOR01629+12

SiOR00330+22

SiO

R02694+33

SiOR00330+13

SiOR01

573+3

SiOR04510+16

SiOR04171+12

SiOR04510+13

SiOR

05118+1

SiO

R02

944+3

SiOR04171+26

SiOR04171+17

SiOR

00899+14

SiOR04171+29

SiOR

00899+1

3

SiOR02694+13

SiOR00330+24

SiOR00330+19

SiOR04171+27

SiO

R02

694+24

SiOR04510+5

SiOR07090

SiOR03952+1

SiOR04510+10

SiOR00330+17

SiO

R02

694+26

SiO

R04609+1

SiOR0

0899+6

SiOR04648+12

SiOR02814+2

SiOR00330+11

SiOR04171+18

SiOR01968+1

7

SiOR00330+10

SiOR00330+9

SiOR0162

9+2

SiOR04171+11

SiOR04510+12

SiOR0196

8+21

SiOR00330+8

SiOR01858+1

SiOR04510+7

SiOR05431+4

SiOR02694+6

SiOR01968+13

SiO

R00330+4

SiO

R04609+18

SiOR0

0899+10

SiOR00330+12

SiOR00330+31

SiO

R06

843+1

SiOR07

837+1

SiO

R00330+2

SiOR01629+4

SiOR0

4648+1

SiOR01

968+24

SiOR04171+23

SiOR0162

9+7

SiOR0

4648+14

SiO

R06

843+3

SiOR06792+5

SiOR01968+7

SiOR01629

+6

SiOR02883+3

SiOR02694+12

SiOR

05118+4

SiOR04171+22

SiO

R01080

SiOR04609+6

SiOR02814+1

SiOR00330+30

SiOR05285+12

SiOR06723+1

SiOR02694+4

Wurm et al 2011

> 400Nasonia wasp 225honey bee 165

6510

fire antGenome of a Gp-9 B ♂

★ Expansion of lipid-processing gene families (for Cuticular Hydrocarbons)


★ Functional DNA-methylation system

Wurm et al 2011

0.05

SiOR

04648+10

SiOR01968+4

SiOR0

0899+7

SiOR02814+3

SiOR04171+6

SiO

R04609+4

SiOR00330+28

SiO

R02

694+25

SiO

R04609+20

SiOR05285+6

SiOR00330+25

SiOR04510+15

SiOR00330+18

SiOR04609+23

SiOR01

968+23

SiO

R03

952+4

SiOR0

4648+16

SiO

R05

901+2

SiO

R02

944+4

SiOR01968+5

SiOR04171+19 SiOR

04648+5

SiOR10535+3

SiOR0

1224+3

SiOR06723+2

SiOR01968+9

SiOR02883+1

SiOR0

0899+3

SiOR01629+1

SiOR04171+1

SiOR01629+11

SiOR04171+10

SiOR04171+13

SiOR02694+3

SiOR04171+20

SiO

R02694+35

SiOR04171+15

SiOR04609+7

SiOR05118+2

SiOR07

837+2

SiO

R02

694+27

SiOR01968+10

SiOR04648+17

SiOR0196

8+19

SiO

R02

694+17

SiOR04648+13

SiOR01968+6

SiOR

00899+1

2

SiOR0

5901+1

SiOR00330+20

SiO

R02

648+2

SiO

R02

659+2

SiOR01968+1

6

SiOR0

0899+11

SiO

R02974

SiOR04171+2

SiOR03

952+2

SiOR06792+2

SiOR04510+4

SiOR04171+28

SiOR05285+5

SiOR05285+9

SiOR

00899+1

5

SiOR

04648+3

SiO

R02694+36

SiOR10535+1

SiO

R02

694+19

SiO

R02

694+23

SiOR02694+1

SiO

R04609+14

SiOR01122

SiOR

04648+9

SiO

R02694+34

SiOR01629+8

SiOR

04648+8

SiOR04510+8

SiOR06573

SiO

R02

944+1

SiOR00330+26

SiO

R00330+1S

iOR02

694+15

SiO

R03038

SiOR05285+7

SiOR0

0899+5

SiOR04609+10

SiO

R04609+3

SiOR04339

SiOR0

8068

SiOR

04648+6

SiOR04510+2

SiOR05285+8

SiOR01

573+4

SiOR0366

3

SiOR04171+8

SiOR01858+2

SiOR01968+2SiOR01968+1

SiOR02694+5

SiOR01968+3

SiOR06723+3

SiOR01968+1

5

SiOR05285+1

SiOR0

0899+4

SiOR04609+22

SiOR04171+9

SiOR02694+9

SiO

R02

648+1

SiOR06792+3

SiOR0

1573+2

SiO

R02

694+20

SiOR1

0542

SiO

R04609+15

SiOR02694+8

SiOR00330+16

SiOR0

0899+2

SiOR02694+10

SiOR04510+9

SiOR05285+3

SiOR04171+24

SiO

R04609+2

SiOR05285+11

SiOR02

694+14

SiOR01

573+1

SiOR05285+2

SiOR00613

SiOR0196

8+22

SiOR

00899+9

SiO

R06

843+2

SiO

R02694+37

SiOR0

0899+1

SiOR04609+9

SiOR05431+2

SiOR10535+2

SiOR00330+15

SiO

R02

694+18

SiOR0

1224+2

SiOR04510+11

SiOR00330+23

SiO

R02

694+29

SiOR05416

SiOR05285+10

SiOR02694+2

SiOR01629+9

SiOR05431+1

SiOR02883+2

SiOR04171+14

SiO

R08

341

SiO

R02

694+22

SiOR01

224+1

SiOR01968+12

SiOR05431+3

SiOR04510+6

SiOR02694+7

SiOR04609+5

SiO

R02

944+2

SiOR03

952+3

SiOR01968+8

SiOR04609+24

SiO

R02

694+30

SiOR01629+10

SiOR04510+14

SiOR00565

SiOR05118+3

SiO

R01321

SiO

R04609+19

SiOR00330+14

SiO

R02694+38

SiOR04609+8

SiOR04171+16

SiOR10455

SiO

R04609+16

SiO

R04609+21

SiO

R02

694+28

SiO

R02

659+1

SiOR04171+5

SiOR00330+29

SiOR01968+14

SiOR03983

SiOR00330+27

SiOR05285+4

SiOR04510+1

SiOR

00899+8

SiOR

04648+7

SiO

R04609+17

SiO

R00330+5

SiO

R02

694+21

SiOR02814+4

SiOR00330+7

SiOR01

629+3

SiOR01

968+26

SiO

R02694+31

SiOR0

4648+2

SiO

R02694+39

SiOR04171+25

SiOR06577

SiOR01

968+25

SiOR04171+21

SiOR06792+6

SiO

R04609+11

SiOR02694+11

SiOR06792+1

SiOR04171+4

SiOR01629+5

SiOR00330+21

SiOR0

4648+15

SiO

R00330+6

SiO

R02

694+16

SiOR04648+11

SiOR

04648+4

SiO

R00330+3

SiO

R06

535

SiOR04171+7

SiOR10493

SiO

R02694+32

SiOR06792+4

SiOR04510+3

SiO

R06

890

SiOR0196

8+20

SiO

R04609+12

SiOR04171+3

SiOR01968

+18

SiOR01968+11

SiO

R04609+13

SiOR01629+12

SiOR00330+22

SiO

R02694+33

SiOR00330+13

SiOR01

573+3

SiOR04510+16

SiOR04171+12

SiOR04510+13

SiOR

05118+1

SiO

R02

944+3

SiOR04171+26

SiOR04171+17

SiOR

00899+14

SiOR04171+29

SiOR

00899+1

3

SiOR02694+13

SiOR00330+24

SiOR00330+19

SiOR04171+27

SiO

R02

694+24

SiOR04510+5

SiOR07090

SiOR03952+1

SiOR04510+10

SiOR00330+17

SiO

R02

694+26

SiO

R04609+1

SiOR0

0899+6

SiOR04648+12

SiOR02814+2

SiOR00330+11

SiOR04171+18

SiOR01968+1

7

SiOR00330+10

SiOR00330+9

SiOR0162

9+2

SiOR04171+11

SiOR04510+12

SiOR0196

8+21

SiOR00330+8

SiOR01858+1

SiOR04510+7

SiOR05431+4

SiOR02694+6

SiOR01968+13

SiO

R00330+4

SiO

R04609+18

SiOR0

0899+10

SiOR00330+12

SiOR00330+31

SiO

R06

843+1

SiOR07

837+1

SiO

R00330+2

SiOR01629+4

SiOR0

4648+1

SiOR01

968+24

SiOR04171+23

SiOR0162

9+7

SiOR0

4648+14

SiO

R06

843+3

SiOR06792+5

SiOR01968+7

SiOR01629

+6

SiOR02883+3

SiOR02694+12

SiOR

05118+4

SiOR04171+22

SiO

R01080

SiOR04609+6

SiOR02814+1

SiOR00330+30

SiOR05285+12

SiOR06723+1

SiOR02694+4


★ Expansion of lipid-processing gene families (for Cuticular Hydrocarbons)


★ Functional DNA-methylation system ★Ant-specific duplication and subfunctionalization

of vitellogenin (in bees: involved in reproduction & division of labor)

Wurm et al 2011

significance of these duplication events in vitellogenins, odorperception genes, and a family of lipid-processing genes. We alsodiscuss additional features of interest in the fire ant genome rel-evant to the complex social biology of this species, including sexdetermination genes, DNA methylation genes, telomerase, andthe insulin and juvenile hormone pathways.

Vitellogenins. In contrast to other insects that mainly have only oneor two vitellogenins, the fire ant genome harbors four adjacent

copies of vitellogenin (Vg1-4) (Fig. 3A), all of which are at leastpartially supported by EST reads. A phylogenetic analysis revealsthat an ancestral vitellogenin gene first underwent duplication,followed by possibly independent duplications of each of thedaughter vitellogenins, thus giving rise to Vg1 and Vg4 and to Vg2andVg3. All of these duplications occurred after the ancestor offireants split from wasps and bees (Fig. 3B). The single vitellogeninfound inA.mellifera is amultifunctional protein (26) involved in theregulation of life span (27, 28) and division of labor (29). Quanti-tative RT-PCR shows thatVg1 andVg4 are preferentially expressedin workers and Vg2 and Vg3 in queens (Fig. 3C, SI Materials andMethods, and Table S1G). Vitellogenin expression in S. invictaworkers is surprising because they lack ovaries. Given the super-organism properties of ant societies, the expression patterns sug-gest that vitellogenins underwent neo- or subfunctionalizationafter duplication to acquire caste-specific functions.

Odor Perception. Consistent with studies in other insects, we finda single S. invicta ortholog to DmOr83b, a broadly expressed ol-factory receptor (OR) required to interact with other ORs forDrosophila and Tribolium castaneum olfaction (30–32). BeyondOR83b, OR number varies greatly between insect species. Blastsearches and GeneWise searches using an HMM profile con-structed with aligned ORs from N. vitripennis (33) and Pogono-myrmex barbatus identified more than 400 loci in the S. invictagenome with significant sequence similarity to ORs. Preliminarywork on gene model reconstruction identified 297 intact full-length proteins. Many S. invicta ORs are in tandem arrays (Fig.S2A) and derive from recent expansions. S. invicta may thus har-bor the largest identified insect OR repertoire because there are10 ORs in Pediculus humanus (34), 60 in Drosophila, 165 inA. mellifera, 225 in N. vitripennis (33), and 259 in T. castaneum(32). The large numbers of N. vitripennis and T. castaneum ORsare thought to be due to current or past difficulties in host andfood finding. As has been suggested for A. mellifera (35), the largenumber of S. invicta ORs may result from the importance ofchemical communication in ants. The odorant-binding proteins(OBPs) are another family of genes also known to play roles inchemosensation in Drosophila (36). Intriguingly, the social orga-nization of S. invicta colonies is completely associated with se-

Eum

etaz

oa

No hits 3424

Not assigned 274

Cnidaria 100

Art

hro

po

da

Ne

op

tera

Coe

lom

ata

Bila

teri

a

Nematoda 25

Deuterostomia 173

Arachnida 50

Paraneoptera 577

End

opte

rygo

ta

Hym

en

op

tera

Apoidea 7398

Chalcidoidea 3271

Ichneumonoidea 94

Diptera 404

Lepidoptera 29

Coleoptera 713

Fig. 2. Taxonomic distribution of best blastp hits of S. invicta proteins to thenonredundant (nr) protein database (E < 10−5). Results were first plottedusing MEGAN software (22) and then branches with fewer than 20 hits wereremoved, branch lengths were reduced for compactness, and tree topologywas adjusted to reflect consensus phylogenies (23, 24).

Vg1Vg4 Vg3 Vg2

2,330,000 bp 2,360,000 bpA

B CSolenopsis Vg1Solenopsis Vg4Solenopsis Vg2Solenopsis Vg3

Apis VgBombus Vg

Nasonia Vg1

Nasonia Vg2Pteromalus Vg

Encarsia VgPimpla Vg

Athalia Vg

Apocrita

Tenthedinoidea

Vespoidea

Apoidea

Aculeata

Chalcidoidea

Ichneumonoidea 0

5000

10000

15000

20000

25000 Vg3Vg2

142 389 17820 1.4 9269 0.61 40WQ WQW Q WQ

*** ***Vg1 Vg4* ***

0

100

200

300

400

500

600

Fig. 3. S. invicta vitellogenins. (A) Four vitellogenins are located within a single 40,000-bp region of the S. invicta genome. (B) Parsimony tree of known hy-menopteran vitellogenin protein sequences suggests that two rounds of vitellogenin duplication occurred after the split between ants and other hyme-nopterans including bees and wasps. (C) Quantitative RT-PCR of the four putative S. invicta vitellogenins on whole bodies of major workers (W) and matedqueens (Q) (n = 10). The y axis indicates mRNA concentrations for the different vitellogenins. Values depicted by each bar are shown below the x-axis labels.Error bars represent SEs. Expression differences between queens and workers were significant (Bonferroni-corrected two-tailed t tests: *P < 0.05, ***P < 10−10).

Wurm et al. PNAS Early Edition | 3 of 6

EVOLU

TION

0.05

SiOR

04648+10

SiOR01968+4

SiOR0

0899+7

SiOR02814+3

SiOR04171+6

SiO

R04609+4

SiOR00330+28

SiO

R02

694+25

SiO

R04609+20

SiOR05285+6

SiOR00330+25

SiOR04510+15

SiOR00330+18

SiOR04609+23

SiOR01

968+23

SiO

R03

952+4

SiOR0

4648+16

SiO

R05

901+2

SiO

R02

944+4

SiOR01968+5

SiOR04171+19 SiOR

04648+5

SiOR10535+3

SiOR0

1224+3

SiOR06723+2

SiOR01968+9

SiOR02883+1

SiOR0

0899+3

SiOR01629+1

SiOR04171+1

SiOR01629+11

SiOR04171+10

SiOR04171+13

SiOR02694+3

SiOR04171+20

SiO

R02694+35

SiOR04171+15

SiOR04609+7

SiOR05118+2

SiOR07

837+2

SiO

R02

694+27

SiOR01968+10

SiOR04648+17

SiOR0196

8+19

SiO

R02

694+17

SiOR04648+13

SiOR01968+6

SiOR

00899+1

2

SiOR0

5901+1

SiOR00330+20

SiO

R02

648+2

SiO

R02

659+2

SiOR01968+1

6

SiOR0

0899+11

SiO

R02974

SiOR04171+2

SiOR03

952+2

SiOR06792+2

SiOR04510+4

SiOR04171+28

SiOR05285+5

SiOR05285+9

SiOR

00899+1

5

SiOR

04648+3

SiO

R02694+36

SiOR10535+1

SiO

R02

694+19

SiO

R02

694+23

SiOR02694+1

SiO

R04609+14

SiOR01122

SiOR

04648+9

SiO

R02694+34

SiOR01629+8

SiOR

04648+8

SiOR04510+8

SiOR06573

SiO

R02

944+1

SiOR00330+26

SiO

R00330+1S

iOR02

694+15

SiO

R03038

SiOR05285+7

SiOR0

0899+5

SiOR04609+10

SiO

R04609+3

SiOR04339

SiOR0

8068

SiOR

04648+6

SiOR04510+2

SiOR05285+8

SiOR01

573+4

SiOR0366

3

SiOR04171+8

SiOR01858+2

SiOR01968+2SiOR01968+1

SiOR02694+5

SiOR01968+3

SiOR06723+3

SiOR01968+1

5

SiOR05285+1

SiOR0

0899+4

SiOR04609+22

SiOR04171+9

SiOR02694+9

SiO

R02

648+1

SiOR06792+3

SiOR0

1573+2

SiO

R02

694+20

SiOR1

0542

SiO

R04609+15

SiOR02694+8

SiOR00330+16

SiOR0

0899+2

SiOR02694+10

SiOR04510+9

SiOR05285+3

SiOR04171+24

SiO

R04609+2

SiOR05285+11

SiOR02

694+14

SiOR01

573+1

SiOR05285+2

SiOR00613

SiOR0196

8+22

SiOR

00899+9

SiO

R06

843+2

SiO

R02694+37

SiOR0

0899+1

SiOR04609+9

SiOR05431+2

SiOR10535+2

SiOR00330+15

SiO

R02

694+18

SiOR0

1224+2

SiOR04510+11

SiOR00330+23

SiO

R02

694+29

SiOR05416

SiOR05285+10

SiOR02694+2

SiOR01629+9

SiOR05431+1

SiOR02883+2

SiOR04171+14

SiO

R08

341

SiO

R02

694+22

SiOR01

224+1

SiOR01968+12

SiOR05431+3

SiOR04510+6

SiOR02694+7

SiOR04609+5

SiO

R02

944+2

SiOR03

952+3

SiOR01968+8

SiOR04609+24

SiO

R02

694+30

SiOR01629+10

SiOR04510+14

SiOR00565

SiOR05118+3

SiO

R01321

SiO

R04609+19

SiOR00330+14

SiO

R02694+38

SiOR04609+8

SiOR04171+16

SiOR10455

SiO

R04609+16

SiO

R04609+21

SiO

R02

694+28

SiO

R02

659+1

SiOR04171+5

SiOR00330+29

SiOR01968+14

SiOR03983

SiOR00330+27

SiOR05285+4

SiOR04510+1

SiOR

00899+8

SiOR

04648+7

SiO

R04609+17

SiO

R00330+5

SiO

R02

694+21

SiOR02814+4

SiOR00330+7

SiOR01

629+3

SiOR01

968+26

SiO

R02694+31

SiOR0

4648+2

SiO

R02694+39

SiOR04171+25

SiOR06577

SiOR01

968+25

SiOR04171+21

SiOR06792+6

SiO

R04609+11

SiOR02694+11

SiOR06792+1

SiOR04171+4

SiOR01629+5

SiOR00330+21

SiOR0

4648+15

SiO

R00330+6

SiO

R02

694+16

SiOR04648+11

SiOR

04648+4

SiO

R00330+3

SiO

R06

535

SiOR04171+7

SiOR10493

SiO

R02694+32

SiOR06792+4

SiOR04510+3

SiO

R06

890

SiOR0196

8+20

SiO

R04609+12

SiOR04171+3

SiOR01968

+18

SiOR01968+11

SiO

R04609+13

SiOR01629+12

SiOR00330+22

SiO

R02694+33

SiOR00330+13

SiOR01

573+3

SiOR04510+16

SiOR04171+12

SiOR04510+13

SiOR

05118+1

SiO

R02

944+3

SiOR04171+26

SiOR04171+17

SiOR

00899+14

SiOR04171+29

SiOR

00899+1

3

SiOR02694+13

SiOR00330+24

SiOR00330+19

SiOR04171+27

SiO

R02

694+24

SiOR04510+5

SiOR07090

SiOR03952+1

SiOR04510+10

SiOR00330+17

SiO

R02

694+26

SiO

R04609+1

SiOR0

0899+6

SiOR04648+12

SiOR02814+2

SiOR00330+11

SiOR04171+18

SiOR01968+1

7

SiOR00330+10

SiOR00330+9

SiOR0162

9+2

SiOR04171+11

SiOR04510+12

SiOR0196

8+21

SiOR00330+8

SiOR01858+1

SiOR04510+7

SiOR05431+4

SiOR02694+6

SiOR01968+13

SiO

R00330+4

SiO

R04609+18

SiOR0

0899+10

SiOR00330+12

SiOR00330+31

SiO

R06

843+1

SiOR07

837+1

SiO

R00330+2

SiOR01629+4

SiOR0

4648+1

SiOR01

968+24

SiOR04171+23

SiOR0162

9+7

SiOR0

4648+14

SiO

R06

843+3

SiOR06792+5

SiOR01968+7

SiOR01629

+6

SiOR02883+3

SiOR02694+12

SiOR

05118+4

SiOR04171+22

SiO

R01080

SiOR04609+6

SiOR02814+1

SiOR00330+30

SiOR05285+12

SiOR06723+1

SiOR02694+4


Wurm et al 2011


BB BB Bb

x x x


Are other genes linked to Gp-9?

Sequenced:•Genome of a Gp-9 B ♂•Genome of a Gp-9 b ♂

“Next Generation Genotyping.”RAD sequencing

Are other genes linked to Gp-9?

“Next Generation Genotyping.”

Bb

unfertilised eggs

haploid ♂

Gp-9 B Gp-9 b Gp-9 B Gp-9 b Gp-9 b Gp-9 B

RAD sequencing

38 B♂ & 38 b♂

EcoR1 EcoR1 EcoR1

Gp-9 B

RAD sequencing of haploid ♂ for SNP discovery & genotyping

EcoR1 EcoR1 EcoR1

AACTG

AACTG

AACTG

AACTG

Gp-9 B


Gp-9 B

AACTGGp-9 B

Gp-9 B

GGCCT

Gp-9 B

Gp-9 B

AAGGTGp-9 B

Gp-9 B

CCAGTGp-9 b

Gp-9 b

TAAATGp-9 b

Gp-9 b

GGAATGp-9 b

Gp-9 b

38 Gp-9 B males

38 Gp-9 b males


Identify polymorphismindividual x locus

genotype table

RADseq: sequencing the same 0.01% of the genome in many individuals

A B C D E FL1 A C A A C CL2 G G T - T GL3 - A G A - GL4 C - - G G CL5 T T C T C -L6 G A A - - G

2419

loci

38 B♂ & 38 b♂

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20+

Amount of variance explained per principal component

Principal Component

% V

aria

nce

Exp

lain

ed

05

1015

2025

30

12.7%

6.1% 5.4% 4.8% 4.7% 3.9% 3.5% 3.2% 3.1% 2.9% 2.8% 2.6% 2.4% 2.3% 2.2% 2.0% 1.9% 1.7% 1.6%

30.2%

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20+

Amount of variance explained per principal component

Principal Component

% V

aria

nce

Exp

lain

ed

05

1015

2025

30

PCA: Principal Component Analysis

pc: 2 % variance: 6.073

pc: 3

%

var

ianc

e: 5

.441

-0.2

-0.1

0.0

0.1

0.2

-0.2 -0.1 0.0 0.1 0.2pc: 2 % variance: 6.073

pc: 3

%

var

ianc

e: 5

.441

-0.2

-0.1

0.0

0.1

0.2

-0.2 -0.1 0.0 0.1 0.2

Principal Components: PC2 vs PC3

Gp-9 B ♂Gp-9 b ♂


pc: 2

%

var

ianc

e: 6

.073

-0.2

-0.1

0.0

0.1

0.2

-0.10 -0.05 0.00 0.05 0.10 0.15

Principal Components: PC1 vs PC2


pc: 2

%

var

ianc

e: 6

.073

-0.2

-0.1

0.0

0.1

0.2

-0.10 -0.05 0.00 0.05 0.10 0.15

Gp-9 B ♂Gp-9 b ♂

Confirmed in 8 additional families (sons of Bb queens) with RAD (3 families) and PCR (5 families). BBqueens: normal recombination throughout (RAD 3 families) ⟹ Gp-9 is not a “recombination cold-spot”

brc_m013_0001..brc_m013_0005brc_m013_0006..brc_m013_0014brc_m013_0015..brc_m013_0017brc_m013_0018brc_m013_0019..brc_m013_0020brc_m013_0021..brc_m013_0029brc_m013_0030..brc_m013_0031brc_m013_0032..brc_m013_0034brc_m013_0035..brc_m013_0036brc_m013_0037..brc_m013_0038brc_m013_0039..brc_m013_0043brc_m013_0044brc_m013_0045brc_m013_0046..brc_m013_0048brc_m013_0049brc_m013_0050..brc_m013_0051brc_m013_0052..brc_m013_0056brc_m013_0057..brc_m013_0061

brc_m013_0062..brc_m013_0075brc_m013_0076..brc_m013_0078brc_m013_0079..brc_m013_0081

brc_m013_0082..brc_m013_0088

brc_m013_0089..brc_m013_0092

brc_m013_0093..brc_m013_0096brc_m013_0097brc_m013_0098..brc_m013_0113brc_m013_0114..brc_m013_0119brc_m013_0120..brc_m013_0130brc_m013_0131brc_m013_0132..brc_m013_0134brc_m013_0135..brc_m013_0136brc_m013_0137..brc_m013_0139brc_m013_0140..brc_m013_0142brc_m013_0143brc_m013_0144..brc_m013_0146brc_m013_0147..brc_m013_0154brc_m013_0155

brc_m013_0156brc_m013_0157..brc_m013_0180brc_m013_0181brc_m013_0182brc_m013_0183..brc_m013_0188brc_m013_0189..brc_m013_0208brc_m013_0209brc_m013_0210..brc_m013_0211brc_m013_0212..brc_m013_0215brc_m013_0216brc_m013_0217brc_m013_0218..brc_m013_0224brc_m013_0225..brc_m013_0228brc_m013_0229..brc_m013_0237brc_m013_0238..brc_m013_0245

brc_m013_0246..brc_m013_0270

brc_m013_0271..brc_m013_0274brc_m013_0275brc_m013_0276..brc_m013_0278

brc_m013_0279

brc_m013_0280

brc_m013_0281..brc_m013_0294

brc_m013_0295..brc_m013_0303brc_m013_0304brc_m013_0305..brc_m013_0307brc_m013_0308brc_m013_0309..brc_m013_0314brc_m013_0315..brc_m013_0317brc_m013_0318..brc_m013_0320brc_m013_0321..brc_m013_0326brc_m013_0327brc_m013_0328brc_m013_0329..brc_m013_0330brc_m013_0331..brc_m013_0333brc_m013_0334..brc_m013_0339brc_m013_0340brc_m013_0341..brc_m013_0343brc_m013_0344brc_m013_0345..brc_m013_0349brc_m013_0350

brc_m013_0351..brc_m013_0354brc_m013_0355brc_m013_0356brc_m013_0357..brc_m013_0361brc_m013_0362..brc_m013_0376brc_m013_0377..brc_m013_0390brc_m013_0391..brc_m013_0393brc_m013_0394..brc_m013_0400brc_m013_0401..brc_m013_0439brc_m013_0440..brc_m013_0478brc_m013_0479..brc_m013_0480

0

20

40

60

80

100

120

140

160

180

LG1brc_m013_0481brc_m013_0482..brc_m013_0484brc_m013_0485..brc_m013_0488brc_m013_0489..brc_m013_0502brc_m013_0503brc_m013_0504..brc_m013_0519brc_m013_0520..brc_m013_0532brc_m013_0533..brc_m013_0534brc_m013_0535..brc_m013_0537brc_m013_0538..brc_m013_0540brc_m013_0541..brc_m013_0543brc_m013_0544..brc_m013_0545brc_m013_0546..brc_m013_0549

brc_m013_0550..brc_m013_0554

brc_m013_0555..brc_m013_0560

brc_m013_0561..brc_m013_0562brc_m013_0563..brc_m013_0565

brc_m013_0566brc_m013_0567brc_m013_0568..brc_m013_0570brc_m013_0571..brc_m013_0573

brc_m013_0574..brc_m013_0575brc_m013_0576..brc_m013_0578brc_m013_0579..brc_m013_0580brc_m013_0581brc_m013_0582..brc_m013_0584brc_m013_0585brc_m013_0586brc_m013_0587brc_m013_0588..brc_m013_0591brc_m013_0592brc_m013_0593brc_m013_0594..brc_m013_0612brc_m013_0613..brc_m013_0614brc_m013_0615..brc_m013_0632brc_m013_0633brc_m013_0634..brc_m013_0648brc_m013_0649..brc_m013_0655brc_m013_0656brc_m013_0657..brc_m013_0694brc_m013_0695..brc_m013_0703brc_m013_0704brc_m013_0705brc_m013_0706..brc_m013_0707brc_m013_0708brc_m013_0709..brc_m013_0711brc_m013_0712..brc_m013_0715brc_m013_0716..brc_m013_0721brc_m013_0722brc_m013_0723brc_m013_0724..brc_m013_0728brc_m013_0729brc_m013_0730brc_m013_0731brc_m013_0732..brc_m013_0735brc_m013_0736..brc_m013_0769brc_m013_0770..brc_m013_0771brc_m013_0772..brc_m013_0773brc_m013_0774..brc_m013_0775brc_m013_0776..brc_m013_0782brc_m013_0783brc_m013_0784..brc_m013_0795brc_m013_0796..brc_m013_0798brc_m013_0799..brc_m013_0801brc_m013_0802..brc_m013_0805brc_m013_0806..brc_m013_0809brc_m013_0810..brc_m013_0811brc_m013_0812..brc_m013_0824brc_m013_0825..brc_m013_0826brc_m013_0827..brc_m013_0829brc_m013_0830..brc_m013_0831brc_m013_0832..brc_m013_0842brc_m013_0843..brc_m013_0854brc_m013_0855..brc_m013_0861brc_m013_0862brc_m013_0863..brc_m013_0864brc_m013_0865..brc_m013_0867brc_m013_0868..brc_m013_0883brc_m013_0884..brc_m013_0893brc_m013_0894brc_m013_0895..brc_m013_0897brc_m013_0898..brc_m013_0906brc_m013_0907..brc_m013_0910brc_m013_0911..brc_m013_0925brc_m013_0926..brc_m013_0928brc_m013_0929..brc_m013_0931

0

20

40

60

80

100

120

140

LG2brc_m013_0932..brc_m013_0941brc_m013_0942..brc_m013_0943brc_m013_0944..brc_m013_0945brc_m013_0946..brc_m013_0949brc_m013_0950..brc_m013_0952

brc_m013_0953..brc_m013_0975

brc_m013_0976..brc_m013_1019brc_m013_1020brc_m013_1021brc_m013_1022..brc_m013_1061brc_m013_1062brc_m013_1063

brc_m013_1064..brc_m013_1065

brc_m013_1066..brc_m013_1068brc_m013_1069brc_m013_1070

brc_m013_1071..brc_m013_1074

brc_m013_1075

brc_m013_1076..brc_m013_1081

brc_m013_1082..brc_m013_1086brc_m013_1087..brc_m013_1088brc_m013_1089..brc_m013_1098brc_m013_1099..brc_m013_1106brc_m013_1107..brc_m013_1116brc_m013_1117brc_m013_1118..brc_m013_1121brc_m013_1122..brc_m013_1127brc_m013_1128brc_m013_1129..brc_m013_1136brc_m013_1137..brc_m013_1138brc_m013_1139..brc_m013_1141brc_m013_1142..brc_m013_1144brc_m013_1145..brc_m013_1156brc_m013_1157brc_m013_1158..brc_m013_1170brc_m013_1171..brc_m013_1181brc_m013_1182..brc_m013_1185brc_m013_1186brc_m013_1187..brc_m013_1205brc_m013_1206..brc_m013_1218brc_m013_1219..brc_m013_1220brc_m013_1221..brc_m013_1224brc_m013_1225..brc_m013_1228brc_m013_1229brc_m013_1230..brc_m013_1236brc_m013_1237brc_m013_1238..brc_m013_1247brc_m013_1248..brc_m013_1251

brc_m013_1252brc_m013_1253..brc_m013_1268brc_m013_1269..brc_m013_1270brc_m013_1271..brc_m013_1273brc_m013_1274brc_m013_1275..brc_m013_1280brc_m013_1281

brc_m013_1282..brc_m013_1286brc_m013_1287..brc_m013_1298brc_m013_1299..brc_m013_1307brc_m013_1308brc_m013_1309..brc_m013_1313brc_m013_1314..brc_m013_1317brc_m013_1318..brc_m013_1319brc_m013_1320..brc_m013_1326brc_m013_1327..brc_m013_1340brc_m013_1341..brc_m013_1362brc_m013_1363..brc_m013_1385

0

20

40

60

80

100

120

140

LG3brc_m013_1386..brc_m013_1388brc_m013_1389..brc_m013_1398brc_m013_1399..brc_m013_1406brc_m013_1407..brc_m013_1411brc_m013_1412..brc_m013_1413brc_m013_1414..brc_m013_1416

brc_m013_1417brc_m013_1418..brc_m013_1420brc_m013_1421..brc_m013_1424brc_m013_1425..brc_m013_1432brc_m013_1433..brc_m013_1442brc_m013_1443brc_m013_1444..brc_m013_1450brc_m013_1451brc_m013_1452brc_m013_1453..brc_m013_1455brc_m013_1456..brc_m013_1467brc_m013_1468..brc_m013_1469brc_m013_1470brc_m013_1471..brc_m013_1474brc_m013_1475brc_m013_1476brc_m013_1477brc_m013_1478..brc_m013_1482brc_m013_1483

brc_m013_1484brc_m013_1485..brc_m013_1487brc_m013_1488..brc_m013_1490


brc_m013_1497..brc_m013_1500brc_m013_1501brc_m013_1502..brc_m013_1513brc_m013_1514..brc_m013_1562brc_m013_1563..brc_m013_1565brc_m013_1566..brc_m013_1567

brc_m013_1568..brc_m013_1580brc_m013_1581..brc_m013_1587brc_m013_1588..brc_m013_1591brc_m013_1592..brc_m013_1593brc_m013_1594..brc_m013_1604brc_m013_1605..brc_m013_1607brc_m013_1608..brc_m013_1609brc_m013_1610..brc_m013_1611brc_m013_1612..brc_m013_1616brc_m013_1617..brc_m013_1618brc_m013_1619..brc_m013_1620brc_m013_1621..brc_m013_1629brc_m013_1630..brc_m013_1633brc_m013_1634..brc_m013_1638brc_m013_1639..brc_m013_1647brc_m013_1648..brc_m013_1649brc_m013_1650..brc_m013_1656brc_m013_1657..brc_m013_1665brc_m013_1666..brc_m013_1672brc_m013_1673..brc_m013_1674brc_m013_1675..brc_m013_1678brc_m013_1679..brc_m013_1682brc_m013_1683brc_m013_1684brc_m013_1685..brc_m013_1686brc_m013_1687..brc_m013_1700brc_m013_1701..brc_m013_1702brc_m013_1703brc_m013_1704..brc_m013_1707brc_m013_1708..brc_m013_1709brc_m013_1710..brc_m013_1714brc_m013_1715..brc_m013_1728brc_m013_1729..brc_m013_1742

0

20

40

60

80

100

120

140

LG4brc_m013_1743..brc_m013_1750


brc_m013_1769..brc_m013_1772

brc_m013_1773..brc_m013_1779brc_m013_1780brc_m013_1781brc_m013_1782..brc_m013_1783brc_m013_1784brc_m013_1785..brc_m013_1786

brc_m013_1787

brc_m013_1788brc_m013_1789..brc_m013_1790brc_m013_1791..brc_m013_1793brc_m013_1794..brc_m013_1797brc_m013_1798..brc_m013_1800brc_m013_1801..brc_m013_1804brc_m013_1805brc_m013_1806..brc_m013_1808brc_m013_1809brc_m013_1810..brc_m013_1813brc_m013_1814..brc_m013_1818brc_m013_1819..brc_m013_1820brc_m013_1821..brc_m013_1822brc_m013_1823..brc_m013_1824brc_m013_1825brc_m013_1826..brc_m013_1840brc_m013_1841..brc_m013_1842brc_m013_1843brc_m013_1844..brc_m013_1848brc_m013_1849brc_m013_1850brc_m013_1851..brc_m013_1853brc_m013_1854..brc_m013_1858brc_m013_1859..brc_m013_1866brc_m013_1867..brc_m013_1868brc_m013_1869brc_m013_1870..brc_m013_1874brc_m013_1875..brc_m013_1876brc_m013_1877..brc_m013_1878brc_m013_1879..brc_m013_1883brc_m013_1884brc_m013_1885brc_m013_1886..brc_m013_1888brc_m013_1889..brc_m013_1895brc_m013_1896..brc_m013_1899brc_m013_1900..brc_m013_1913brc_m013_1914brc_m013_1915..brc_m013_1922brc_m013_1923brc_m013_1924..brc_m013_1928brc_m013_1929..brc_m013_1942brc_m013_1943..brc_m013_1946brc_m013_1947..brc_m013_1948brc_m013_1949brc_m013_1950..brc_m013_1953brc_m013_1954..brc_m013_1955brc_m013_1956brc_m013_1957..brc_m013_1963brc_m013_1964..brc_m013_1966brc_m013_1967..brc_m013_1968brc_m013_1969..brc_m013_1970brc_m013_1971brc_m013_1972brc_m013_1973..brc_m013_1980brc_m013_1981..brc_m013_1983brc_m013_1984..brc_m013_1990brc_m013_1991..brc_m013_1993brc_m013_1994..brc_m013_1996brc_m013_1997..brc_m013_2009

0

20

40

60

80

100

120

LG5brc_m013_2010..brc_m013_2028

brc_m013_2029..brc_m013_2038brc_m013_2039

brc_m013_2040..brc_m013_2041brc_m013_2042..brc_m013_2047brc_m013_2048..brc_m013_2050brc_m013_2051..brc_m013_2053brc_m013_2054..brc_m013_2062brc_m013_2063brc_m013_2064..brc_m013_2065brc_m013_2066..brc_m013_2067brc_m013_2068brc_m013_2069..brc_m013_2071brc_m013_2072..brc_m013_2081brc_m013_2082

brc_m013_2083brc_m013_2084brc_m013_2085..brc_m013_2099brc_m013_2100brc_m013_2101..brc_m013_2102brc_m013_2103..brc_m013_2107brc_m013_2108brc_m013_2109..brc_m013_2112

brc_m013_2113..brc_m013_2114brc_m013_2115..brc_m013_2123brc_m013_2124..brc_m013_2131brc_m013_2132brc_m013_2133brc_m013_2134..brc_m013_2136

brc_m013_2137

brc_m013_2138..brc_m013_2139

brc_m013_2140..brc_m013_2142brc_m013_2143..brc_m013_2150brc_m013_2151..brc_m013_2152brc_m013_2153..brc_m013_2161brc_m013_2162..brc_m013_2163

brc_m013_2164..brc_m013_2165brc_m013_2166..brc_m013_2170brc_m013_2171..brc_m013_2172brc_m013_2173brc_m013_2174..brc_m013_2182brc_m013_2183..brc_m013_2186brc_m013_2187..brc_m013_2190brc_m013_2191..brc_m013_2193brc_m013_2194brc_m013_2195..brc_m013_2201brc_m013_2202..brc_m013_2203brc_m013_2204..brc_m013_2220brc_m013_2221..brc_m013_2232brc_m013_2233..brc_m013_2239brc_m013_2240..brc_m013_2261brc_m013_2262..brc_m013_2267brc_m013_2268..brc_m013_2269brc_m013_2270..brc_m013_2271brc_m013_2272..brc_m013_2282brc_m013_2283..brc_m013_2284brc_m013_2285..brc_m013_2299brc_m013_2300..brc_m013_2301brc_m013_2302..brc_m013_2305brc_m013_2306..brc_m013_2307brc_m013_2308..brc_m013_2330brc_m013_2331..brc_m013_2337brc_m013_2338..brc_m013_2352

0

20

40

60

80

100

120

LG6brc_m013_2353..brc_m013_2365brc_m013_2366..brc_m013_2369

brc_m013_2370..brc_m013_2372brc_m013_2373..brc_m013_2378brc_m013_2379..brc_m013_2386brc_m013_2387brc_m013_2388..brc_m013_2394brc_m013_2395..brc_m013_2397brc_m013_2398brc_m013_2399brc_m013_2400brc_m013_2401brc_m013_2402..brc_m013_2407brc_m013_2408..brc_m013_2411brc_m013_2412..brc_m013_2416brc_m013_2417brc_m013_2418brc_m013_2419..brc_m013_2436brc_m013_2437..brc_m013_2441brc_m013_2442brc_m013_2443..brc_m013_2444brc_m013_2445brc_m013_2446..brc_m013_2453brc_m013_2454brc_m013_2455..brc_m013_2460brc_m013_2461brc_m013_2462..brc_m013_2470brc_m013_2471..brc_m013_2474brc_m013_2475..brc_m013_2482brc_m013_2483brc_m013_2484..brc_m013_2487brc_m013_2488brc_m013_2489..brc_m013_2492brc_m013_2493..brc_m013_2496brc_m013_2497brc_m013_2498..brc_m013_2504brc_m013_2505brc_m013_2506..brc_m013_2510brc_m013_2511..brc_m013_2523brc_m013_2524..brc_m013_2531brc_m013_2532..brc_m013_2536brc_m013_2537..brc_m013_2555brc_m013_2556..brc_m013_2571brc_m013_2572..brc_m013_2573brc_m013_2574..brc_m013_2579brc_m013_2580..brc_m013_2581brc_m013_2582..brc_m013_2587brc_m013_2588brc_m013_2589..brc_m013_2594

brc_m013_2595

brc_m013_2596..brc_m013_2597brc_m013_2598..brc_m013_2604brc_m013_2605brc_m013_2606..brc_m013_2616brc_m013_2617..brc_m013_2619brc_m013_2620..brc_m013_2623


brc_m013_2629..brc_m013_2630

0

20

40

60

80

100

LG7brc_m013_2631..brc_m013_2632brc_m013_2633brc_m013_2634..brc_m013_2635brc_m013_2636..brc_m013_2642brc_m013_2643..brc_m013_2657brc_m013_2658..brc_m013_2659brc_m013_2660..brc_m013_2661brc_m013_2662

brc_m013_2663

brc_m013_2664brc_m013_2665..brc_m013_2666

brc_m013_2667..brc_m013_2668brc_m013_2669..brc_m013_2670brc_m013_2671..brc_m013_2680brc_m013_2681..brc_m013_2682brc_m013_2683brc_m013_2684..brc_m013_2685brc_m013_2686..brc_m013_2694


brc_m013_2714..brc_m013_2725brc_m013_2726..brc_m013_2727brc_m013_2728..brc_m013_2731brc_m013_2732brc_m013_2733..brc_m013_2753brc_m013_2754..brc_m013_2758brc_m013_2759..brc_m013_2763brc_m013_2764..brc_m013_2779brc_m013_2780brc_m013_2781brc_m013_2782..brc_m013_2784brc_m013_2785..brc_m013_2787brc_m013_2788..brc_m013_2791brc_m013_2792..brc_m013_2797brc_m013_2798..brc_m013_2799brc_m013_2800brc_m013_2801..brc_m013_2804brc_m013_2805..brc_m013_2809

brc_m013_2810..brc_m013_2811

brc_m013_2812..brc_m013_2813brc_m013_2814..brc_m013_2817brc_m013_2818..brc_m013_2827brc_m013_2828brc_m013_2829..brc_m013_2832brc_m013_2833brc_m013_2834..brc_m013_2840brc_m013_2841..brc_m013_2846brc_m013_2847brc_m013_2848..brc_m013_2852brc_m013_2853brc_m013_2854..brc_m013_2856brc_m013_2857..brc_m013_2862brc_m013_2863..brc_m013_2868brc_m013_2869..brc_m013_2874brc_m013_2875..brc_m013_2896

0

20

40

60

80

100

LG8

brc_m013_2897..brc_m013_2920brc_m013_2921..brc_m013_2928brc_m013_2929..brc_m013_2931brc_m013_2932

brc_m013_2933

brc_m013_2934..brc_m013_2935brc_m013_2936brc_m013_2937..brc_m013_2943brc_m013_2944brc_m013_2945..brc_m013_2946brc_m013_2947brc_m013_2948brc_m013_2949..brc_m013_2950brc_m013_2951..brc_m013_2957brc_m013_2958..brc_m013_2961brc_m013_2962..brc_m013_2970brc_m013_2971..brc_m013_2980brc_m013_2981..brc_m013_2992brc_m013_2993..brc_m013_2996brc_m013_2997..brc_m013_2998brc_m013_2999..brc_m013_3000brc_m013_3001brc_m013_3002..brc_m013_3003brc_m013_3004brc_m013_3005brc_m013_3006..brc_m013_3010brc_m013_3011..brc_m013_3014brc_m013_3015brc_m013_3016..brc_m013_3019brc_m013_3020brc_m013_3021..brc_m013_3030brc_m013_3031..brc_m013_3032brc_m013_3033..brc_m013_3034brc_m013_3035..brc_m013_3036brc_m013_3037..brc_m013_3045brc_m013_3046..brc_m013_3052brc_m013_3053brc_m013_3054..brc_m013_3061brc_m013_3062..brc_m013_3066brc_m013_3067..brc_m013_3068brc_m013_3069..brc_m013_3076brc_m013_3077..brc_m013_3084brc_m013_3085..brc_m013_3087brc_m013_3088..brc_m013_3089brc_m013_3090..brc_m013_3096brc_m013_3097..brc_m013_3100brc_m013_3101..brc_m013_3104brc_m013_3105brc_m013_3106..brc_m013_3112brc_m013_3113..brc_m013_3122brc_m013_3123..brc_m013_3124brc_m013_3125..brc_m013_3127brc_m013_3128..brc_m013_3145brc_m013_3146..brc_m013_3159brc_m013_3160..brc_m013_3172

0

20

40

60

80

100

LG9brc_m013_3173..brc_m013_3175brc_m013_3176..brc_m013_3180brc_m013_3181..brc_m013_3189brc_m013_3190..brc_m013_3198brc_m013_3199brc_m013_3200..brc_m013_3201brc_m013_3202..brc_m013_3203brc_m013_3204brc_m013_3205..brc_m013_3206brc_m013_3207..brc_m013_3211brc_m013_3212..brc_m013_3214brc_m013_3215..brc_m013_3227brc_m013_3228..brc_m013_3230brc_m013_3231..brc_m013_3235brc_m013_3236..brc_m013_3238brc_m013_3239..brc_m013_3242brc_m013_3243..brc_m013_3244brc_m013_3245brc_m013_3246..brc_m013_3247brc_m013_3248..brc_m013_3249brc_m013_3250..brc_m013_3252brc_m013_3253..brc_m013_3257brc_m013_3258brc_m013_3259brc_m013_3260..brc_m013_3261brc_m013_3262..brc_m013_3263brc_m013_3264brc_m013_3265..brc_m013_3269brc_m013_3270..brc_m013_3274brc_m013_3275..brc_m013_3276brc_m013_3277..brc_m013_3281brc_m013_3282..brc_m013_3284brc_m013_3285brc_m013_3286..brc_m013_3289brc_m013_3290..brc_m013_3296brc_m013_3297brc_m013_3298..brc_m013_3300brc_m013_3301..brc_m013_3302brc_m013_3303..brc_m013_3305brc_m013_3306..brc_m013_3308brc_m013_3309..brc_m013_3314brc_m013_3315..brc_m013_3317brc_m013_3318..brc_m013_3329brc_m013_3330..brc_m013_3331brc_m013_3332..brc_m013_3338brc_m013_3339..brc_m013_3340brc_m013_3341..brc_m013_3344brc_m013_3345..brc_m013_3349brc_m013_3350..brc_m013_3357brc_m013_3358..brc_m013_3359brc_m013_3360brc_m013_3361..brc_m013_3368brc_m013_3369..brc_m013_3372brc_m013_3373..brc_m013_3376brc_m013_3377brc_m013_3378..brc_m013_3386brc_m013_3387..brc_m013_3388brc_m013_3389..brc_m013_3395brc_m013_3396..brc_m013_3399

0

20

40

60

80

LG10brc_m013_3400..brc_m013_3411brc_m013_3412brc_m013_3413..brc_m013_3424brc_m013_3425brc_m013_3426brc_m013_3427..brc_m013_3429

brc_m013_3430

brc_m013_3431..brc_m013_3432brc_m013_3433..brc_m013_3435brc_m013_3436brc_m013_3437..brc_m013_3439

brc_m013_3440..brc_m013_3441brc_m013_3442

brc_m013_3443..brc_m013_3445brc_m013_3446..brc_m013_3447brc_m013_3448..brc_m013_3449brc_m013_3450..brc_m013_3454brc_m013_3455brc_m013_3456..brc_m013_3462brc_m013_3463..brc_m013_3464brc_m013_3465brc_m013_3466..brc_m013_3467brc_m013_3468..brc_m013_3472brc_m013_3473brc_m013_3474..brc_m013_3476brc_m013_3477..brc_m013_3487brc_m013_3488brc_m013_3489..brc_m013_3491brc_m013_3492..brc_m013_3500brc_m013_3501..brc_m013_3512brc_m013_3513..brc_m013_3514brc_m013_3515..brc_m013_3524brc_m013_3525..brc_m013_3527brc_m013_3528..brc_m013_3531brc_m013_3532..brc_m013_3547brc_m013_3548..brc_m013_3557brc_m013_3558..brc_m013_3566brc_m013_3567..brc_m013_3568brc_m013_3569..brc_m013_3570brc_m013_3571..brc_m013_3574brc_m013_3575..brc_m013_3582brc_m013_3583..brc_m013_3592brc_m013_3593..brc_m013_3605brc_m013_3606..brc_m013_3616brc_m013_3617..brc_m013_3618brc_m013_3619..brc_m013_3622brc_m013_3623..brc_m013_3624brc_m013_3625..brc_m013_3628brc_m013_3629..brc_m013_3635

0

20

40

60

80

LG11



brc_m013_3672


brc_m013_3683..brc_m013_3685

brc_m013_3686..brc_m013_3688

brc_m013_3689..brc_m013_3693

brc_m013_3694..brc_m013_3698brc_m013_3699

brc_m013_3700brc_m013_3701..brc_m013_3702brc_m013_3703..brc_m013_3704brc_m013_3705..brc_m013_3712brc_m013_3713brc_m013_3714..brc_m013_3716brc_m013_3717..brc_m013_3724brc_m013_3725..brc_m013_3730brc_m013_3731..brc_m013_3752brc_m013_3753..brc_m013_3758brc_m013_3759..brc_m013_3789brc_m013_3790..brc_m013_3801brc_m013_3802..brc_m013_3814brc_m013_3815..brc_m013_3818brc_m013_3819..brc_m013_3822brc_m013_3823..brc_m013_3826brc_m013_3827..brc_m013_3832brc_m013_3833..brc_m013_3837brc_m013_3838brc_m013_3839..brc_m013_3841brc_m013_3842..brc_m013_3847

brc_m013_3848..brc_m013_3853brc_m013_3854..brc_m013_3858brc_m013_3859..brc_m013_3868brc_m013_3869..brc_m013_3871brc_m013_3872..brc_m013_3901brc_m013_3902brc_m013_3903..brc_m013_3909brc_m013_3910brc_m013_3911..brc_m013_3926brc_m013_3927..brc_m013_3931brc_m013_3932..brc_m013_3948

0

20

40

60

80

LG12


brc_m013_3972..brc_m013_3975brc_m013_3976

brc_m013_3977..brc_m013_3985

brc_m013_3986brc_m013_3987..brc_m013_3994brc_m013_3995..brc_m013_3997brc_m013_3998..brc_m013_4004

brc_m013_4005..brc_m013_4006brc_m013_4007..brc_m013_4008brc_m013_4009..brc_m013_4010brc_m013_4011..brc_m013_4013brc_m013_4014brc_m013_4015brc_m013_4016..brc_m013_4019brc_m013_4020..brc_m013_4021brc_m013_4022..brc_m013_4025brc_m013_4026..brc_m013_4032brc_m013_4033..brc_m013_4036brc_m013_4037..brc_m013_4041brc_m013_4042..brc_m013_4043

brc_m013_4044..brc_m013_4046brc_m013_4047..brc_m013_4056brc_m013_4057brc_m013_4058..brc_m013_4063brc_m013_4064..brc_m013_4071brc_m013_4072..brc_m013_4075brc_m013_4076brc_m013_4077brc_m013_4078..brc_m013_4085brc_m013_4086..brc_m013_4089brc_m013_4090..brc_m013_4091brc_m013_4092..brc_m013_4093brc_m013_4094..brc_m013_4095brc_m013_4096..brc_m013_4114brc_m013_4115..brc_m013_4117brc_m013_4118..brc_m013_4131brc_m013_4132..brc_m013_4133brc_m013_4134..brc_m013_4146

0

20

40

60

80

LG13


brc_m013_4168brc_m013_4169

brc_m013_4170brc_m013_4171..brc_m013_4172brc_m013_4173..brc_m013_4175brc_m013_4176brc_m013_4177..brc_m013_4178brc_m013_4179..brc_m013_4183brc_m013_4184..brc_m013_4185brc_m013_4186..brc_m013_4187brc_m013_4188..brc_m013_4191brc_m013_4192brc_m013_4193..brc_m013_4194brc_m013_4195..brc_m013_4206brc_m013_4207..brc_m013_4210brc_m013_4211..brc_m013_4213brc_m013_4214brc_m013_4215..brc_m013_4217brc_m013_4218..brc_m013_4221brc_m013_4222..brc_m013_4223brc_m013_4224..brc_m013_4231brc_m013_4232..brc_m013_4234brc_m013_4235..brc_m013_4239brc_m013_4240..brc_m013_4246brc_m013_4247..brc_m013_4248brc_m013_4249..brc_m013_4258brc_m013_4259..brc_m013_4260brc_m013_4261..brc_m013_4269brc_m013_4270..brc_m013_4271brc_m013_4272..brc_m013_4278brc_m013_4279..brc_m013_4280brc_m013_4281..brc_m013_4284brc_m013_4285brc_m013_4286..brc_m013_4288brc_m013_4289..brc_m013_4294brc_m013_4295..brc_m013_4296brc_m013_4297..brc_m013_4301brc_m013_4302..brc_m013_4313brc_m013_4314..brc_m013_4320brc_m013_4321..brc_m013_4322brc_m013_4323..brc_m013_4345brc_m013_4346..brc_m013_4351

0

20

40

60

LG14brc_m013_4352..brc_m013_4366brc_m013_4367brc_m013_4368brc_m013_4369..brc_m013_4373brc_m013_4374..brc_m013_4381brc_m013_4382..brc_m013_4383brc_m013_4384..brc_m013_4385brc_m013_4386brc_m013_4387..brc_m013_4388brc_m013_4389brc_m013_4390..brc_m013_4404brc_m013_4405brc_m013_4406..brc_m013_4409brc_m013_4410..brc_m013_4411brc_m013_4412..brc_m013_4418brc_m013_4419..brc_m013_4434brc_m013_4435..brc_m013_4442brc_m013_4443..brc_m013_4448brc_m013_4449..brc_m013_4451brc_m013_4452..brc_m013_4461brc_m013_4462..brc_m013_4471brc_m013_4472..brc_m013_4475brc_m013_4476..brc_m013_4477brc_m013_4478brc_m013_4479brc_m013_4480..brc_m013_4485brc_m013_4486brc_m013_4487..brc_m013_4491brc_m013_4492brc_m013_4493brc_m013_4494..brc_m013_4495brc_m013_4496..brc_m013_4501brc_m013_4502..brc_m013_4510brc_m013_4511..brc_m013_4531brc_m013_4532brc_m013_4533..brc_m013_4534brc_m013_4535..brc_m013_4541brc_m013_4542..brc_m013_4543brc_m013_4544..brc_m013_4545brc_m013_4546..brc_m013_4548brc_m013_4549..brc_m013_4551brc_m013_4552..brc_m013_4555brc_m013_4556..brc_m013_4561

0

20

40

60

LG15

brc_m013_4562..brc_m013_4577brc_m013_4578..brc_m013_4594brc_m013_4595..brc_m013_4599brc_m013_4600..brc_m013_4625brc_m013_4626..brc_m013_4638brc_m013_4639..brc_m013_4642brc_m013_4643..brc_m013_4644brc_m013_4645..brc_m013_4650brc_m013_4651..brc_m013_4663brc_m013_4664..brc_m013_4668brc_m013_4669..brc_m013_4670brc_m013_4671..brc_m013_4674brc_m013_4675..brc_m013_4679brc_m013_4680..brc_m013_4681brc_m013_4682brc_m013_4683..brc_m013_4688brc_m013_4689..brc_m013_4692brc_m013_4693..brc_m013_4695brc_m013_4696..brc_m013_4701brc_m013_4702brc_m013_4703..brc_m013_4712brc_m013_4713..brc_m013_4717brc_m013_4718..brc_m013_4720brc_m013_4721brc_m013_4722..brc_m013_4726brc_m013_4727..brc_m013_4728brc_m013_4729..brc_m013_4742brc_m013_4743brc_m013_4744..brc_m013_4746brc_m013_4747brc_m013_4748..brc_m013_4749brc_m013_4750..brc_m013_4752

brc_m013_4753..brc_m013_4756brc_m013_4757..brc_m013_4759brc_m013_4760..brc_m013_4762brc_m013_4763brc_m013_4764..brc_m013_4766brc_m013_4767..brc_m013_4769brc_m013_4770brc_m013_4771..brc_m013_4774brc_m013_4775..brc_m013_4776brc_m013_4777..brc_m013_4778brc_m013_4779..brc_m013_4780brc_m013_4781..brc_m013_4793brc_m013_4794brc_m013_4795..brc_m013_4798brc_m013_4799..brc_m013_4802brc_m013_4803..brc_m013_4806brc_m013_4807brc_m013_4808..brc_m013_4814brc_m013_4815..brc_m013_4819brc_m013_4820brc_m013_4821brc_m013_4822..brc_m013_4823brc_m013_4824brc_m013_4825..brc_m013_4855brc_m013_4856..brc_m013_4858brc_m013_4859..brc_m013_4863brc_m013_4864..brc_m013_4865brc_m013_4866..brc_m013_4875brc_m013_4876..brc_m013_4881brc_m013_4882..brc_m013_4891brc_m013_4892..brc_m013_4895brc_m013_4896..brc_m013_4911brc_m013_4912..brc_m013_4938brc_m013_4939brc_m013_4940..brc_m013_4957brc_m013_4958..brc_m013_4959brc_m013_4960..brc_m013_4972brc_m013_4973..brc_m013_4981brc_m013_4982..brc_m013_4983

0

20

40

60

80

100

120

LGSB

Gp-9

Figure 1a b

Si_gnF.scaffold00779_nt2778431.7

Si_gnF.scaffold00779_nt1255229Si_gnF.scaffold02684_nt10884.2


Si_gnF.scaffold00779_nt3746833Si_gnF.scaffold00779_nt374687927.3Si_gnF.scaffold00779_nt382158728.5




Si_gnF.scaffold05266_nt63430678.4Si_gnF.scaffold05266_nt65952779.7





Si_gnF.scaffold03404_nt128925Si_gnF.scaffold03404_nt228606Si_gnF.scaffold03404_nt241461

104.4


Si_gnF.scaffold00413_nt1035856109.6Si_gnF.scaffold01573_nt108462110.8Si_gnF.scaffold01573_nt447618112.1Si_gnF.scaffold00899_nt377419Si_gnF.scaffold00899_nt686574114.4Si_gnF.scaffold00899_nt236146Si_gnF.scaffold00899_nt332715Si_gnF.scaffold00899_nt335756

115.8

Si_gnF.scaffold00469_nt794118.0Si_gnF.scaffold00690_nt229012Si_gnF.scaffold00690_nt415290119.3



LGS Bfrom M013




22.2



Si_gnF.scaffold01573_nt10846280.7Si_gnF.scaffold00255_nt314067Si_gnF.scaffold00255_nt407778Si_gnF.scaffold00413_nt1035856Si_gnF.scaffold00413_nt676115Si_gnF.scaffold00469_nt794Si_gnF.scaffold00690_nt229012Si_gnF.scaffold00690_nt415290Si_gnF.scaffold00899_nt236146Si_gnF.scaffold00899_nt332715Si_gnF.scaffold00899_nt335756Si_gnF.scaffold00899_nt377419Si_gnF.scaffold00899_nt686574Si_gnF.scaffold01573_nt447618Si_gnF.scaffold01957_nt412242Si_gnF.scaffold03404_nt128925Si_gnF.scaffold03404_nt228606Si_gnF.scaffold03404_nt241461Si_gnF.scaffold05266_nt634306Si_gnF.scaffold05266_nt659527Si_gnF.scaffold05266_nt733643Si_gnF.scaffold05266_nt753644Si_gnF.scaffold06914_nt297673Si_gnF.scaffold07090_nt1051771Si_gnF.scaffold07090_nt710010Si_gnF.scaffold09758_nt222732

81.8


LGS B/bfrom P034

Gp-9

Total285 non-recombiningmarkersbrc_m013_0001..brc_m013_0005

brc_m013_0006..brc_m013_0014brc_m013_0015..brc_m013_0017brc_m013_0018brc_m013_0019..brc_m013_0020brc_m013_0021..brc_m013_0029brc_m013_0030..brc_m013_0031brc_m013_0032..brc_m013_0034brc_m013_0035..brc_m013_0036brc_m013_0037..brc_m013_0038brc_m013_0039..brc_m013_0043brc_m013_0044brc_m013_0045brc_m013_0046..brc_m013_0048brc_m013_0049brc_m013_0050..brc_m013_0051brc_m013_0052..brc_m013_0056brc_m013_0057..brc_m013_0061

brc_m013_0062..brc_m013_0075brc_m013_0076..brc_m013_0078brc_m013_0079..brc_m013_0081

brc_m013_0082..brc_m013_0088

brc_m013_0089..brc_m013_0092

brc_m013_0093..brc_m013_0096brc_m013_0097brc_m013_0098..brc_m013_0113brc_m013_0114..brc_m013_0119brc_m013_0120..brc_m013_0130brc_m013_0131brc_m013_0132..brc_m013_0134brc_m013_0135..brc_m013_0136brc_m013_0137..brc_m013_0139brc_m013_0140..brc_m013_0142brc_m013_0143brc_m013_0144..brc_m013_0146brc_m013_0147..brc_m013_0154brc_m013_0155

brc_m013_0156brc_m013_0157..brc_m013_0180brc_m013_0181brc_m013_0182brc_m013_0183..brc_m013_0188brc_m013_0189..brc_m013_0208brc_m013_0209brc_m013_0210..brc_m013_0211brc_m013_0212..brc_m013_0215brc_m013_0216brc_m013_0217brc_m013_0218..brc_m013_0224brc_m013_0225..brc_m013_0228brc_m013_0229..brc_m013_0237brc_m013_0238..brc_m013_0245

brc_m013_0246..brc_m013_0270


brc_m013_0279

brc_m013_0280

brc_m013_0281..brc_m013_0294

brc_m013_0295..brc_m013_0303brc_m013_0304brc_m013_0305..brc_m013_0307brc_m013_0308brc_m013_0309..brc_m013_0314brc_m013_0315..brc_m013_0317brc_m013_0318..brc_m013_0320brc_m013_0321..brc_m013_0326brc_m013_0327brc_m013_0328brc_m013_0329..brc_m013_0330brc_m013_0331..brc_m013_0333brc_m013_0334..brc_m013_0339brc_m013_0340brc_m013_0341..brc_m013_0343brc_m013_0344brc_m013_0345..brc_m013_0349brc_m013_0350

brc_m013_0351..brc_m013_0354brc_m013_0355brc_m013_0356brc_m013_0357..brc_m013_0361brc_m013_0362..brc_m013_0376brc_m013_0377..brc_m013_0390brc_m013_0391..brc_m013_0393brc_m013_0394..brc_m013_0400brc_m013_0401..brc_m013_0439brc_m013_0440..brc_m013_0478brc_m013_0479..brc_m013_0480

0

20

40

60

80

100

120

140

160

180

LG1brc_m013_0481brc_m013_0482..brc_m013_0484brc_m013_0485..brc_m013_0488brc_m013_0489..brc_m013_0502brc_m013_0503brc_m013_0504..brc_m013_0519brc_m013_0520..brc_m013_0532brc_m013_0533..brc_m013_0534brc_m013_0535..brc_m013_0537brc_m013_0538..brc_m013_0540brc_m013_0541..brc_m013_0543brc_m013_0544..brc_m013_0545brc_m013_0546..brc_m013_0549

brc_m013_0550..brc_m013_0554

brc_m013_0555..brc_m013_0560


brc_m013_0566brc_m013_0567brc_m013_0568..brc_m013_0570brc_m013_0571..brc_m013_0573

brc_m013_0574..brc_m013_0575brc_m013_0576..brc_m013_0578brc_m013_0579..brc_m013_0580brc_m013_0581brc_m013_0582..brc_m013_0584brc_m013_0585brc_m013_0586brc_m013_0587brc_m013_0588..brc_m013_0591brc_m013_0592brc_m013_0593brc_m013_0594..brc_m013_0612brc_m013_0613..brc_m013_0614brc_m013_0615..brc_m013_0632brc_m013_0633brc_m013_0634..brc_m013_0648brc_m013_0649..brc_m013_0655brc_m013_0656brc_m013_0657..brc_m013_0694brc_m013_0695..brc_m013_0703brc_m013_0704brc_m013_0705brc_m013_0706..brc_m013_0707brc_m013_0708brc_m013_0709..brc_m013_0711brc_m013_0712..brc_m013_0715brc_m013_0716..brc_m013_0721brc_m013_0722brc_m013_0723brc_m013_0724..brc_m013_0728brc_m013_0729brc_m013_0730brc_m013_0731brc_m013_0732..brc_m013_0735brc_m013_0736..brc_m013_0769brc_m013_0770..brc_m013_0771brc_m013_0772..brc_m013_0773brc_m013_0774..brc_m013_0775brc_m013_0776..brc_m013_0782brc_m013_0783brc_m013_0784..brc_m013_0795brc_m013_0796..brc_m013_0798brc_m013_0799..brc_m013_0801brc_m013_0802..brc_m013_0805brc_m013_0806..brc_m013_0809brc_m013_0810..brc_m013_0811brc_m013_0812..brc_m013_0824brc_m013_0825..brc_m013_0826brc_m013_0827..brc_m013_0829brc_m013_0830..brc_m013_0831brc_m013_0832..brc_m013_0842brc_m013_0843..brc_m013_0854brc_m013_0855..brc_m013_0861brc_m013_0862brc_m013_0863..brc_m013_0864brc_m013_0865..brc_m013_0867brc_m013_0868..brc_m013_0883brc_m013_0884..brc_m013_0893brc_m013_0894brc_m013_0895..brc_m013_0897brc_m013_0898..brc_m013_0906brc_m013_0907..brc_m013_0910brc_m013_0911..brc_m013_0925brc_m013_0926..brc_m013_0928brc_m013_0929..brc_m013_0931

0

20

40

60

80

100

120

140

LG2brc_m013_0932..brc_m013_0941brc_m013_0942..brc_m013_0943brc_m013_0944..brc_m013_0945brc_m013_0946..brc_m013_0949brc_m013_0950..brc_m013_0952

brc_m013_0953..brc_m013_0975

brc_m013_0976..brc_m013_1019brc_m013_1020brc_m013_1021brc_m013_1022..brc_m013_1061brc_m013_1062brc_m013_1063

brc_m013_1064..brc_m013_1065


brc_m013_1071..brc_m013_1074

brc_m013_1075

brc_m013_1076..brc_m013_1081

brc_m013_1082..brc_m013_1086brc_m013_1087..brc_m013_1088brc_m013_1089..brc_m013_1098brc_m013_1099..brc_m013_1106brc_m013_1107..brc_m013_1116brc_m013_1117brc_m013_1118..brc_m013_1121brc_m013_1122..brc_m013_1127brc_m013_1128brc_m013_1129..brc_m013_1136brc_m013_1137..brc_m013_1138brc_m013_1139..brc_m013_1141brc_m013_1142..brc_m013_1144brc_m013_1145..brc_m013_1156brc_m013_1157brc_m013_1158..brc_m013_1170brc_m013_1171..brc_m013_1181brc_m013_1182..brc_m013_1185brc_m013_1186brc_m013_1187..brc_m013_1205brc_m013_1206..brc_m013_1218brc_m013_1219..brc_m013_1220brc_m013_1221..brc_m013_1224brc_m013_1225..brc_m013_1228brc_m013_1229brc_m013_1230..brc_m013_1236brc_m013_1237brc_m013_1238..brc_m013_1247brc_m013_1248..brc_m013_1251

brc_m013_1252brc_m013_1253..brc_m013_1268brc_m013_1269..brc_m013_1270brc_m013_1271..brc_m013_1273brc_m013_1274brc_m013_1275..brc_m013_1280brc_m013_1281

brc_m013_1282..brc_m013_1286brc_m013_1287..brc_m013_1298brc_m013_1299..brc_m013_1307brc_m013_1308brc_m013_1309..brc_m013_1313brc_m013_1314..brc_m013_1317brc_m013_1318..brc_m013_1319brc_m013_1320..brc_m013_1326brc_m013_1327..brc_m013_1340brc_m013_1341..brc_m013_1362brc_m013_1363..brc_m013_1385

0

20

40

60

80

100

120

140

LG3brc_m013_1386..brc_m013_1388brc_m013_1389..brc_m013_1398brc_m013_1399..brc_m013_1406brc_m013_1407..brc_m013_1411brc_m013_1412..brc_m013_1413brc_m013_1414..brc_m013_1416

brc_m013_1417brc_m013_1418..brc_m013_1420brc_m013_1421..brc_m013_1424brc_m013_1425..brc_m013_1432brc_m013_1433..brc_m013_1442brc_m013_1443brc_m013_1444..brc_m013_1450brc_m013_1451brc_m013_1452brc_m013_1453..brc_m013_1455brc_m013_1456..brc_m013_1467brc_m013_1468..brc_m013_1469brc_m013_1470brc_m013_1471..brc_m013_1474brc_m013_1475brc_m013_1476brc_m013_1477brc_m013_1478..brc_m013_1482brc_m013_1483



brc_m013_1497..brc_m013_1500brc_m013_1501brc_m013_1502..brc_m013_1513brc_m013_1514..brc_m013_1562brc_m013_1563..brc_m013_1565brc_m013_1566..brc_m013_1567

brc_m013_1568..brc_m013_1580brc_m013_1581..brc_m013_1587brc_m013_1588..brc_m013_1591brc_m013_1592..brc_m013_1593brc_m013_1594..brc_m013_1604brc_m013_1605..brc_m013_1607brc_m013_1608..brc_m013_1609brc_m013_1610..brc_m013_1611brc_m013_1612..brc_m013_1616brc_m013_1617..brc_m013_1618brc_m013_1619..brc_m013_1620brc_m013_1621..brc_m013_1629brc_m013_1630..brc_m013_1633brc_m013_1634..brc_m013_1638brc_m013_1639..brc_m013_1647brc_m013_1648..brc_m013_1649brc_m013_1650..brc_m013_1656brc_m013_1657..brc_m013_1665brc_m013_1666..brc_m013_1672brc_m013_1673..brc_m013_1674brc_m013_1675..brc_m013_1678brc_m013_1679..brc_m013_1682brc_m013_1683brc_m013_1684brc_m013_1685..brc_m013_1686brc_m013_1687..brc_m013_1700brc_m013_1701..brc_m013_1702brc_m013_1703brc_m013_1704..brc_m013_1707brc_m013_1708..brc_m013_1709brc_m013_1710..brc_m013_1714brc_m013_1715..brc_m013_1728brc_m013_1729..brc_m013_1742

0

20

40

60

80

100

120

140

LG4brc_m013_1743..brc_m013_1750


brc_m013_1769..brc_m013_1772

brc_m013_1773..brc_m013_1779brc_m013_1780brc_m013_1781brc_m013_1782..brc_m013_1783brc_m013_1784brc_m013_1785..brc_m013_1786

brc_m013_1787

brc_m013_1788brc_m013_1789..brc_m013_1790brc_m013_1791..brc_m013_1793brc_m013_1794..brc_m013_1797brc_m013_1798..brc_m013_1800brc_m013_1801..brc_m013_1804brc_m013_1805brc_m013_1806..brc_m013_1808brc_m013_1809brc_m013_1810..brc_m013_1813brc_m013_1814..brc_m013_1818brc_m013_1819..brc_m013_1820brc_m013_1821..brc_m013_1822brc_m013_1823..brc_m013_1824brc_m013_1825brc_m013_1826..brc_m013_1840brc_m013_1841..brc_m013_1842brc_m013_1843brc_m013_1844..brc_m013_1848brc_m013_1849brc_m013_1850brc_m013_1851..brc_m013_1853brc_m013_1854..brc_m013_1858brc_m013_1859..brc_m013_1866brc_m013_1867..brc_m013_1868brc_m013_1869brc_m013_1870..brc_m013_1874brc_m013_1875..brc_m013_1876brc_m013_1877..brc_m013_1878brc_m013_1879..brc_m013_1883brc_m013_1884brc_m013_1885brc_m013_1886..brc_m013_1888brc_m013_1889..brc_m013_1895brc_m013_1896..brc_m013_1899brc_m013_1900..brc_m013_1913brc_m013_1914brc_m013_1915..brc_m013_1922brc_m013_1923brc_m013_1924..brc_m013_1928brc_m013_1929..brc_m013_1942brc_m013_1943..brc_m013_1946brc_m013_1947..brc_m013_1948brc_m013_1949brc_m013_1950..brc_m013_1953brc_m013_1954..brc_m013_1955brc_m013_1956brc_m013_1957..brc_m013_1963brc_m013_1964..brc_m013_1966brc_m013_1967..brc_m013_1968brc_m013_1969..brc_m013_1970brc_m013_1971brc_m013_1972brc_m013_1973..brc_m013_1980brc_m013_1981..brc_m013_1983brc_m013_1984..brc_m013_1990brc_m013_1991..brc_m013_1993brc_m013_1994..brc_m013_1996brc_m013_1997..brc_m013_2009

0

20

40

60

80

100

120

LG5brc_m013_2010..brc_m013_2028

brc_m013_2029..brc_m013_2038brc_m013_2039

brc_m013_2040..brc_m013_2041brc_m013_2042..brc_m013_2047brc_m013_2048..brc_m013_2050brc_m013_2051..brc_m013_2053brc_m013_2054..brc_m013_2062brc_m013_2063brc_m013_2064..brc_m013_2065brc_m013_2066..brc_m013_2067brc_m013_2068brc_m013_2069..brc_m013_2071brc_m013_2072..brc_m013_2081brc_m013_2082

brc_m013_2083brc_m013_2084brc_m013_2085..brc_m013_2099brc_m013_2100brc_m013_2101..brc_m013_2102brc_m013_2103..brc_m013_2107brc_m013_2108brc_m013_2109..brc_m013_2112

brc_m013_2113..brc_m013_2114brc_m013_2115..brc_m013_2123brc_m013_2124..brc_m013_2131brc_m013_2132brc_m013_2133brc_m013_2134..brc_m013_2136

brc_m013_2137

brc_m013_2138..brc_m013_2139

brc_m013_2140..brc_m013_2142brc_m013_2143..brc_m013_2150brc_m013_2151..brc_m013_2152brc_m013_2153..brc_m013_2161brc_m013_2162..brc_m013_2163

brc_m013_2164..brc_m013_2165brc_m013_2166..brc_m013_2170brc_m013_2171..brc_m013_2172brc_m013_2173brc_m013_2174..brc_m013_2182brc_m013_2183..brc_m013_2186brc_m013_2187..brc_m013_2190brc_m013_2191..brc_m013_2193brc_m013_2194brc_m013_2195..brc_m013_2201brc_m013_2202..brc_m013_2203brc_m013_2204..brc_m013_2220brc_m013_2221..brc_m013_2232brc_m013_2233..brc_m013_2239brc_m013_2240..brc_m013_2261brc_m013_2262..brc_m013_2267brc_m013_2268..brc_m013_2269brc_m013_2270..brc_m013_2271brc_m013_2272..brc_m013_2282brc_m013_2283..brc_m013_2284brc_m013_2285..brc_m013_2299brc_m013_2300..brc_m013_2301brc_m013_2302..brc_m013_2305brc_m013_2306..brc_m013_2307brc_m013_2308..brc_m013_2330brc_m013_2331..brc_m013_2337brc_m013_2338..brc_m013_2352

0

20

40

60

80

100

120

LG6brc_m013_2353..brc_m013_2365brc_m013_2366..brc_m013_2369

brc_m013_2370..brc_m013_2372brc_m013_2373..brc_m013_2378brc_m013_2379..brc_m013_2386brc_m013_2387brc_m013_2388..brc_m013_2394brc_m013_2395..brc_m013_2397brc_m013_2398brc_m013_2399brc_m013_2400brc_m013_2401brc_m013_2402..brc_m013_2407brc_m013_2408..brc_m013_2411brc_m013_2412..brc_m013_2416brc_m013_2417brc_m013_2418brc_m013_2419..brc_m013_2436brc_m013_2437..brc_m013_2441brc_m013_2442brc_m013_2443..brc_m013_2444brc_m013_2445brc_m013_2446..brc_m013_2453brc_m013_2454brc_m013_2455..brc_m013_2460brc_m013_2461brc_m013_2462..brc_m013_2470brc_m013_2471..brc_m013_2474brc_m013_2475..brc_m013_2482brc_m013_2483brc_m013_2484..brc_m013_2487brc_m013_2488brc_m013_2489..brc_m013_2492brc_m013_2493..brc_m013_2496brc_m013_2497brc_m013_2498..brc_m013_2504brc_m013_2505brc_m013_2506..brc_m013_2510brc_m013_2511..brc_m013_2523brc_m013_2524..brc_m013_2531brc_m013_2532..brc_m013_2536brc_m013_2537..brc_m013_2555brc_m013_2556..brc_m013_2571brc_m013_2572..brc_m013_2573brc_m013_2574..brc_m013_2579brc_m013_2580..brc_m013_2581brc_m013_2582..brc_m013_2587brc_m013_2588brc_m013_2589..brc_m013_2594

brc_m013_2595

brc_m013_2596..brc_m013_2597brc_m013_2598..brc_m013_2604brc_m013_2605brc_m013_2606..brc_m013_2616brc_m013_2617..brc_m013_2619brc_m013_2620..brc_m013_2623


brc_m013_2629..brc_m013_2630

0

20

40

60

80

100

LG7brc_m013_2631..brc_m013_2632brc_m013_2633brc_m013_2634..brc_m013_2635brc_m013_2636..brc_m013_2642brc_m013_2643..brc_m013_2657brc_m013_2658..brc_m013_2659brc_m013_2660..brc_m013_2661brc_m013_2662

brc_m013_2663

brc_m013_2664brc_m013_2665..brc_m013_2666

brc_m013_2667..brc_m013_2668brc_m013_2669..brc_m013_2670brc_m013_2671..brc_m013_2680brc_m013_2681..brc_m013_2682brc_m013_2683brc_m013_2684..brc_m013_2685brc_m013_2686..brc_m013_2694


brc_m013_2714..brc_m013_2725brc_m013_2726..brc_m013_2727brc_m013_2728..brc_m013_2731brc_m013_2732brc_m013_2733..brc_m013_2753brc_m013_2754..brc_m013_2758brc_m013_2759..brc_m013_2763brc_m013_2764..brc_m013_2779brc_m013_2780brc_m013_2781brc_m013_2782..brc_m013_2784brc_m013_2785..brc_m013_2787brc_m013_2788..brc_m013_2791brc_m013_2792..brc_m013_2797brc_m013_2798..brc_m013_2799brc_m013_2800brc_m013_2801..brc_m013_2804brc_m013_2805..brc_m013_2809

brc_m013_2810..brc_m013_2811

brc_m013_2812..brc_m013_2813brc_m013_2814..brc_m013_2817brc_m013_2818..brc_m013_2827brc_m013_2828brc_m013_2829..brc_m013_2832brc_m013_2833brc_m013_2834..brc_m013_2840brc_m013_2841..brc_m013_2846brc_m013_2847brc_m013_2848..brc_m013_2852brc_m013_2853brc_m013_2854..brc_m013_2856brc_m013_2857..brc_m013_2862brc_m013_2863..brc_m013_2868brc_m013_2869..brc_m013_2874brc_m013_2875..brc_m013_2896

0

20

40

60

80

100

LG8


brc_m013_2933

brc_m013_2934..brc_m013_2935brc_m013_2936brc_m013_2937..brc_m013_2943brc_m013_2944brc_m013_2945..brc_m013_2946brc_m013_2947brc_m013_2948brc_m013_2949..brc_m013_2950brc_m013_2951..brc_m013_2957brc_m013_2958..brc_m013_2961brc_m013_2962..brc_m013_2970brc_m013_2971..brc_m013_2980brc_m013_2981..brc_m013_2992brc_m013_2993..brc_m013_2996brc_m013_2997..brc_m013_2998brc_m013_2999..brc_m013_3000brc_m013_3001brc_m013_3002..brc_m013_3003brc_m013_3004brc_m013_3005brc_m013_3006..brc_m013_3010brc_m013_3011..brc_m013_3014brc_m013_3015brc_m013_3016..brc_m013_3019brc_m013_3020brc_m013_3021..brc_m013_3030brc_m013_3031..brc_m013_3032brc_m013_3033..brc_m013_3034brc_m013_3035..brc_m013_3036brc_m013_3037..brc_m013_3045brc_m013_3046..brc_m013_3052brc_m013_3053brc_m013_3054..brc_m013_3061brc_m013_3062..brc_m013_3066brc_m013_3067..brc_m013_3068brc_m013_3069..brc_m013_3076brc_m013_3077..brc_m013_3084brc_m013_3085..brc_m013_3087brc_m013_3088..brc_m013_3089brc_m013_3090..brc_m013_3096brc_m013_3097..brc_m013_3100brc_m013_3101..brc_m013_3104brc_m013_3105brc_m013_3106..brc_m013_3112brc_m013_3113..brc_m013_3122brc_m013_3123..brc_m013_3124brc_m013_3125..brc_m013_3127brc_m013_3128..brc_m013_3145brc_m013_3146..brc_m013_3159brc_m013_3160..brc_m013_3172

0

20

40

60

80

100

LG9brc_m013_3173..brc_m013_3175brc_m013_3176..brc_m013_3180brc_m013_3181..brc_m013_3189brc_m013_3190..brc_m013_3198brc_m013_3199brc_m013_3200..brc_m013_3201brc_m013_3202..brc_m013_3203brc_m013_3204brc_m013_3205..brc_m013_3206brc_m013_3207..brc_m013_3211brc_m013_3212..brc_m013_3214brc_m013_3215..brc_m013_3227brc_m013_3228..brc_m013_3230brc_m013_3231..brc_m013_3235brc_m013_3236..brc_m013_3238brc_m013_3239..brc_m013_3242brc_m013_3243..brc_m013_3244brc_m013_3245brc_m013_3246..brc_m013_3247brc_m013_3248..brc_m013_3249brc_m013_3250..brc_m013_3252brc_m013_3253..brc_m013_3257brc_m013_3258brc_m013_3259brc_m013_3260..brc_m013_3261brc_m013_3262..brc_m013_3263brc_m013_3264brc_m013_3265..brc_m013_3269brc_m013_3270..brc_m013_3274brc_m013_3275..brc_m013_3276brc_m013_3277..brc_m013_3281brc_m013_3282..brc_m013_3284brc_m013_3285brc_m013_3286..brc_m013_3289brc_m013_3290..brc_m013_3296brc_m013_3297brc_m013_3298..brc_m013_3300brc_m013_3301..brc_m013_3302brc_m013_3303..brc_m013_3305brc_m013_3306..brc_m013_3308brc_m013_3309..brc_m013_3314brc_m013_3315..brc_m013_3317brc_m013_3318..brc_m013_3329brc_m013_3330..brc_m013_3331brc_m013_3332..brc_m013_3338brc_m013_3339..brc_m013_3340brc_m013_3341..brc_m013_3344brc_m013_3345..brc_m013_3349brc_m013_3350..brc_m013_3357brc_m013_3358..brc_m013_3359brc_m013_3360brc_m013_3361..brc_m013_3368brc_m013_3369..brc_m013_3372brc_m013_3373..brc_m013_3376brc_m013_3377brc_m013_3378..brc_m013_3386brc_m013_3387..brc_m013_3388brc_m013_3389..brc_m013_3395brc_m013_3396..brc_m013_3399

0

20

40

60

80

LG10brc_m013_3400..brc_m013_3411brc_m013_3412brc_m013_3413..brc_m013_3424brc_m013_3425brc_m013_3426brc_m013_3427..brc_m013_3429

brc_m013_3430

brc_m013_3431..brc_m013_3432brc_m013_3433..brc_m013_3435brc_m013_3436brc_m013_3437..brc_m013_3439

brc_m013_3440..brc_m013_3441brc_m013_3442

brc_m013_3443..brc_m013_3445brc_m013_3446..brc_m013_3447brc_m013_3448..brc_m013_3449brc_m013_3450..brc_m013_3454brc_m013_3455brc_m013_3456..brc_m013_3462brc_m013_3463..brc_m013_3464brc_m013_3465brc_m013_3466..brc_m013_3467brc_m013_3468..brc_m013_3472brc_m013_3473brc_m013_3474..brc_m013_3476brc_m013_3477..brc_m013_3487brc_m013_3488brc_m013_3489..brc_m013_3491brc_m013_3492..brc_m013_3500brc_m013_3501..brc_m013_3512brc_m013_3513..brc_m013_3514brc_m013_3515..brc_m013_3524brc_m013_3525..brc_m013_3527brc_m013_3528..brc_m013_3531brc_m013_3532..brc_m013_3547brc_m013_3548..brc_m013_3557brc_m013_3558..brc_m013_3566brc_m013_3567..brc_m013_3568brc_m013_3569..brc_m013_3570brc_m013_3571..brc_m013_3574brc_m013_3575..brc_m013_3582brc_m013_3583..brc_m013_3592brc_m013_3593..brc_m013_3605brc_m013_3606..brc_m013_3616brc_m013_3617..brc_m013_3618brc_m013_3619..brc_m013_3622brc_m013_3623..brc_m013_3624brc_m013_3625..brc_m013_3628brc_m013_3629..brc_m013_3635

0

20

40

60

80

LG11



brc_m013_3672


brc_m013_3683..brc_m013_3685

brc_m013_3686..brc_m013_3688

brc_m013_3689..brc_m013_3693

brc_m013_3694..brc_m013_3698brc_m013_3699

brc_m013_3700brc_m013_3701..brc_m013_3702brc_m013_3703..brc_m013_3704brc_m013_3705..brc_m013_3712brc_m013_3713brc_m013_3714..brc_m013_3716brc_m013_3717..brc_m013_3724brc_m013_3725..brc_m013_3730brc_m013_3731..brc_m013_3752brc_m013_3753..brc_m013_3758brc_m013_3759..brc_m013_3789brc_m013_3790..brc_m013_3801brc_m013_3802..brc_m013_3814brc_m013_3815..brc_m013_3818brc_m013_3819..brc_m013_3822brc_m013_3823..brc_m013_3826brc_m013_3827..brc_m013_3832brc_m013_3833..brc_m013_3837brc_m013_3838brc_m013_3839..brc_m013_3841brc_m013_3842..brc_m013_3847

brc_m013_3848..brc_m013_3853brc_m013_3854..brc_m013_3858brc_m013_3859..brc_m013_3868brc_m013_3869..brc_m013_3871brc_m013_3872..brc_m013_3901brc_m013_3902brc_m013_3903..brc_m013_3909brc_m013_3910brc_m013_3911..brc_m013_3926brc_m013_3927..brc_m013_3931brc_m013_3932..brc_m013_3948

0

20

40

60

80

LG12


brc_m013_3972..brc_m013_3975brc_m013_3976

brc_m013_3977..brc_m013_3985

brc_m013_3986brc_m013_3987..brc_m013_3994brc_m013_3995..brc_m013_3997brc_m013_3998..brc_m013_4004

brc_m013_4005..brc_m013_4006brc_m013_4007..brc_m013_4008brc_m013_4009..brc_m013_4010brc_m013_4011..brc_m013_4013brc_m013_4014brc_m013_4015brc_m013_4016..brc_m013_4019brc_m013_4020..brc_m013_4021brc_m013_4022..brc_m013_4025brc_m013_4026..brc_m013_4032brc_m013_4033..brc_m013_4036brc_m013_4037..brc_m013_4041brc_m013_4042..brc_m013_4043

brc_m013_4044..brc_m013_4046brc_m013_4047..brc_m013_4056brc_m013_4057brc_m013_4058..brc_m013_4063brc_m013_4064..brc_m013_4071brc_m013_4072..brc_m013_4075brc_m013_4076brc_m013_4077brc_m013_4078..brc_m013_4085brc_m013_4086..brc_m013_4089brc_m013_4090..brc_m013_4091brc_m013_4092..brc_m013_4093brc_m013_4094..brc_m013_4095brc_m013_4096..brc_m013_4114brc_m013_4115..brc_m013_4117brc_m013_4118..brc_m013_4131brc_m013_4132..brc_m013_4133brc_m013_4134..brc_m013_4146

0

20

40

60

80

LG13


brc_m013_4168brc_m013_4169

brc_m013_4170brc_m013_4171..brc_m013_4172brc_m013_4173..brc_m013_4175brc_m013_4176brc_m013_4177..brc_m013_4178brc_m013_4179..brc_m013_4183brc_m013_4184..brc_m013_4185brc_m013_4186..brc_m013_4187brc_m013_4188..brc_m013_4191brc_m013_4192brc_m013_4193..brc_m013_4194brc_m013_4195..brc_m013_4206brc_m013_4207..brc_m013_4210brc_m013_4211..brc_m013_4213brc_m013_4214brc_m013_4215..brc_m013_4217brc_m013_4218..brc_m013_4221brc_m013_4222..brc_m013_4223brc_m013_4224..brc_m013_4231brc_m013_4232..brc_m013_4234brc_m013_4235..brc_m013_4239brc_m013_4240..brc_m013_4246brc_m013_4247..brc_m013_4248brc_m013_4249..brc_m013_4258brc_m013_4259..brc_m013_4260brc_m013_4261..brc_m013_4269brc_m013_4270..brc_m013_4271brc_m013_4272..brc_m013_4278brc_m013_4279..brc_m013_4280brc_m013_4281..brc_m013_4284brc_m013_4285brc_m013_4286..brc_m013_4288brc_m013_4289..brc_m013_4294brc_m013_4295..brc_m013_4296brc_m013_4297..brc_m013_4301brc_m013_4302..brc_m013_4313brc_m013_4314..brc_m013_4320brc_m013_4321..brc_m013_4322brc_m013_4323..brc_m013_4345brc_m013_4346..brc_m013_4351

0

20

40

60

LG14brc_m013_4352..brc_m013_4366brc_m013_4367brc_m013_4368brc_m013_4369..brc_m013_4373brc_m013_4374..brc_m013_4381brc_m013_4382..brc_m013_4383brc_m013_4384..brc_m013_4385brc_m013_4386brc_m013_4387..brc_m013_4388brc_m013_4389brc_m013_4390..brc_m013_4404brc_m013_4405brc_m013_4406..brc_m013_4409brc_m013_4410..brc_m013_4411brc_m013_4412..brc_m013_4418brc_m013_4419..brc_m013_4434brc_m013_4435..brc_m013_4442brc_m013_4443..brc_m013_4448brc_m013_4449..brc_m013_4451brc_m013_4452..brc_m013_4461brc_m013_4462..brc_m013_4471brc_m013_4472..brc_m013_4475brc_m013_4476..brc_m013_4477brc_m013_4478brc_m013_4479brc_m013_4480..brc_m013_4485brc_m013_4486brc_m013_4487..brc_m013_4491brc_m013_4492brc_m013_4493brc_m013_4494..brc_m013_4495brc_m013_4496..brc_m013_4501brc_m013_4502..brc_m013_4510brc_m013_4511..brc_m013_4531brc_m013_4532brc_m013_4533..brc_m013_4534brc_m013_4535..brc_m013_4541brc_m013_4542..brc_m013_4543brc_m013_4544..brc_m013_4545brc_m013_4546..brc_m013_4548brc_m013_4549..brc_m013_4551brc_m013_4552..brc_m013_4555brc_m013_4556..brc_m013_4561

0

20

40

60

LG15

brc_m013_4562..brc_m013_4577brc_m013_4578..brc_m013_4594brc_m013_4595..brc_m013_4599brc_m013_4600..brc_m013_4625brc_m013_4626..brc_m013_4638brc_m013_4639..brc_m013_4642brc_m013_4643..brc_m013_4644brc_m013_4645..brc_m013_4650brc_m013_4651..brc_m013_4663brc_m013_4664..brc_m013_4668brc_m013_4669..brc_m013_4670brc_m013_4671..brc_m013_4674brc_m013_4675..brc_m013_4679brc_m013_4680..brc_m013_4681brc_m013_4682brc_m013_4683..brc_m013_4688brc_m013_4689..brc_m013_4692brc_m013_4693..brc_m013_4695brc_m013_4696..brc_m013_4701brc_m013_4702brc_m013_4703..brc_m013_4712brc_m013_4713..brc_m013_4717brc_m013_4718..brc_m013_4720brc_m013_4721brc_m013_4722..brc_m013_4726brc_m013_4727..brc_m013_4728brc_m013_4729..brc_m013_4742brc_m013_4743brc_m013_4744..brc_m013_4746brc_m013_4747brc_m013_4748..brc_m013_4749brc_m013_4750..brc_m013_4752

brc_m013_4753..brc_m013_4756brc_m013_4757..brc_m013_4759brc_m013_4760..brc_m013_4762brc_m013_4763brc_m013_4764..brc_m013_4766brc_m013_4767..brc_m013_4769brc_m013_4770brc_m013_4771..brc_m013_4774brc_m013_4775..brc_m013_4776brc_m013_4777..brc_m013_4778brc_m013_4779..brc_m013_4780brc_m013_4781..brc_m013_4793brc_m013_4794brc_m013_4795..brc_m013_4798brc_m013_4799..brc_m013_4802brc_m013_4803..brc_m013_4806brc_m013_4807brc_m013_4808..brc_m013_4814brc_m013_4815..brc_m013_4819brc_m013_4820brc_m013_4821brc_m013_4822..brc_m013_4823brc_m013_4824brc_m013_4825..brc_m013_4855brc_m013_4856..brc_m013_4858brc_m013_4859..brc_m013_4863brc_m013_4864..brc_m013_4865brc_m013_4866..brc_m013_4875brc_m013_4876..brc_m013_4881brc_m013_4882..brc_m013_4891brc_m013_4892..brc_m013_4895brc_m013_4896..brc_m013_4911brc_m013_4912..brc_m013_4938brc_m013_4939brc_m013_4940..brc_m013_4957brc_m013_4958..brc_m013_4959brc_m013_4960..brc_m013_4972brc_m013_4973..brc_m013_4981brc_m013_4982..brc_m013_4983

0

20

40

60

80

100

120

LGSB

Gp-9

Figure 1a b




Si_gnF.scaffold00779_nt3746833Si_gnF.scaffold00779_nt374687927.3Si_gnF.scaffold00779_nt382158728.5




Si_gnF.scaffold05266_nt63430678.4Si_gnF.scaffold05266_nt65952779.7






104.4


Si_gnF.scaffold00413_nt1035856109.6Si_gnF.scaffold01573_nt108462110.8Si_gnF.scaffold01573_nt447618112.1Si_gnF.scaffold00899_nt377419Si_gnF.scaffold00899_nt686574114.4Si_gnF.scaffold00899_nt236146Si_gnF.scaffold00899_nt332715Si_gnF.scaffold00899_nt335756

115.8




LGS Bfrom M013




22.2



Si_gnF.scaffold01573_nt10846280.7Si_gnF.scaffold00255_nt314067Si_gnF.scaffold00255_nt407778Si_gnF.scaffold00413_nt1035856Si_gnF.scaffold00413_nt676115Si_gnF.scaffold00469_nt794Si_gnF.scaffold00690_nt229012Si_gnF.scaffold00690_nt415290Si_gnF.scaffold00899_nt236146Si_gnF.scaffold00899_nt332715Si_gnF.scaffold00899_nt335756Si_gnF.scaffold00899_nt377419Si_gnF.scaffold00899_nt686574Si_gnF.scaffold01573_nt447618Si_gnF.scaffold01957_nt412242Si_gnF.scaffold03404_nt128925Si_gnF.scaffold03404_nt228606Si_gnF.scaffold03404_nt241461Si_gnF.scaffold05266_nt634306Si_gnF.scaffold05266_nt659527Si_gnF.scaffold05266_nt733643Si_gnF.scaffold05266_nt753644Si_gnF.scaffold06914_nt297673Si_gnF.scaffold07090_nt1051771Si_gnF.scaffold07090_nt710010Si_gnF.scaffold09758_nt222732

81.8


LGS B/bfrom P034

Gp-9

Total285 non-recombiningmarkers

No recombination between B and b over ⅔ of a chromosme!

4% of genome linked to Gp-9

Sex chromosomes

X Y

Gp-9 B

Gp-9 b

SB Sb

“Social chromosomes”

?

Why non-recombining?

Small portion (200,000bp) of social chromosome

Structural differences between B and blikely inhibit recombination

SB

Sb

Sb SB Gp-9B

genetic map

E3

E17

A22

Gp-9 B male Gp-9 b male Figure 2 a

b Diploid Gp-9 Bb

Sb SB Gp-9B

genetic map

E3

E17

A22


b Diploid Gp-9 Bb

Sb SB Gp-9B

genetic map

E3

E17

A22


b Diploid Gp-9 Bb

Sb SB Gp-9B

genetic map

E3

E17

A22


b Diploid Gp-9 Bb

Flourescence in situ Hybridization

John Wang @ Taipei

Structural differences between B and blikely inhibit recombination

Gp-9 B ♂ Gp-9 b ♂

Gp-9 B

Gp-9 b

SB Sb

“Social chromosomes”

>10 rearrangments

X

♀

X X Y

♂

>10 rearrangements

•Prediction:

SBSB SB Sb

Single queen colony Multiple queen colony

Differences between SB and Sb?•genes?

Region contains 800 genes! only small differences⟹ Prediction: directional (antagonistic?) selection?

Sb is degenerating?

SB

Sb

• More, larger repetitive DNA in Sb compared to SB• larger introns in Sb• larger intergenic regions in Sb• assembly worse (smaller scaffolds) in Sb

• increased dN/dS

[b] vs. [c]: p < 10-4

[a] vs. [c]: p < 10-7

Gp-9B male Gp-9b male

Region:

Genome assembly:

Normally recombiningregions from all 16

linkage groups

Normally recombiningregions from all 16

linkage groups

Sb region without recombination

in Gp-9 Bb queens

SB region without recombination

in Gp-9 Bb queens

Sca

ffold

leng

th (b

p)

0

1,000,000

2,000,000

3,000,000

4,000,000

5,000,000

6,000,000

[a] [a], [b] [a] [c]

SB Sb

Sb is degenerating?

X

♀

X X Y

♂

Likely several rearrangements

•Prediction:

SBSB SB Sb

Single queen colony Multiple queen colony

Differences between SB and Sb?•genes?

Region contains 800 genes! only small differences⟹ Prediction: directional (antagonistic?) selection?

Sb is degenerating?•Probably ♂ haploidy: strong purifying selection ⟹ slower degeneration

Formica selysi

Purcell et al 2014Convergent social chromosome architecture

J Meunier


Alpine silver antCommon ancestor with fire ant: 130 MYA

≠ social chromosomes

Purcell et al 2014

Solenopsis invicta social chromosome

Formica selysi social

chromosome

Social chromosomes also for other major social transitions?

Summary

Fire ant Solenopsis invicta queen number determined by Gp-9 genotypes: •only BB workers ➔ single BB queen•with Bb workers ➔ multiple Bb queens

Genome sequencing + RAD Genotyping >500 individuals•Gp-9 marks ~4% of genome ➔ social chromosomes:

SB is like X; Sb is like YStructural differences between SB and Sb ➔ no recombination

Ants are cool.

Formica selysi: Convergent evolution of social chromosomes

genome evolution social evolutionGenerally

Major transitions:

Social parasitism

Strengths of selection in social evolution

concepts & mechanisms

Candidate gene studiesVitellogeninSex determination genes

Tools for genomics work on emerging model organisms

Caste differentiation (Medically relevant)

Single- vs. Multiple queennessEusociality

Pollinator health

Four tools

suck less. Four tools that

Four tools

suck less. (hopefully)

Four tools that

1. SequenceServer

“Can you BLAST this for me?”

“Can you BLAST this for me?”

Antgenomes.org SequenceServer BLAST made easy

(well, we’re trying...)

@yeban Anurag Priyam

http://antgenomes.org/blast

http://www.sequenceserver.com/

(requires a BLAST+ install)

Do you have BLAST-formatted databases? If not: sequenceserver format-databases /path/to/fastas

1. Installinggem install sequenceserver

# ~/.sequenceserver.conf bin: ~/ncbi-blast-2.2.25+/bin/ database: /Users/me/blast_databases/

2. Configure.

sequenceserver ### Launched SequenceServer at: http://0.0.0.0:4567

3. Launch.

@yebanAnurag Priyam

http://www.sequenceserver.com/

2. Bionode

http://bionode.io

@bmpvieira

http://bionode.io

# Get descriptions for papers related to SRA search bionode ncbi search sra Solenopsis invicta | tool-‐stream extractProperty uid | bionode ncbi link sra pubmed | tool-‐stream extractProperty destUID | bionode ncbi search pubmed

# Get URL of Solenopsis invicta genome bionode-‐ncbi urls assembly Solenopsis invicta | grep genomic.fna http://ftp.ncbi.nlm.nih.gov/genomes/all/GCA_000188075.1_Si_gnG/GCA_000188075.1_Si_gnG_genomic.fna.gz

http://bionode.io in the terminal

# Get all FASTQ of Arthropod short reads bionode-‐ncbi download sra arthropoda | bionode-‐sra

# Get all GFF of bacterial genome annotations bionode-‐ncbi download gff bacteria

@bmpvieira

http://ftp.ncbi.nlm.nih.gov/genomes/all/GCA_000188075.1_Si_gnG/GCA_000188075.1_Si_gnG_genomic.fna.gz

http://bionode.io

Difficulty writing scalable, reproducible andcomplex bioinformatic pipelines.Solution: Node.js everywhereStreams var ncbi = require('bionode-ncbi') var tool = require('tool-stream') var through = require('through2') var fork1 = through.obj() var fork2 = through.obj()

ncbi .search('sra', 'Solenopsis invicta') .pipe(fork1) .pipe(dat.reads)

fork1 .pipe(tool.extractProperty('expxml.Biosample.id')) .pipe(ncbi.search('biosample')) .pipe(dat.samples)

fork1 .pipe(tool.extractProperty('uid')) .pipe(ncbi.link('sra', 'pubmed'))

Complex pipelines using Node.js

@bmpvieira

Working with Gene Predictions

Gene prediction/identificationDozens of software algorithms: dozens of predictions

20% failure rate: •missing pieces•extra pieces•incorrect merging•incorrect splitting

Visual inspection... and manual fixing required.

1 gene = 5 minutes to 3 days

Yand

ell &

Enc

e 20

13 N

RG

GTCTACAATGCGATTGTAAAATAGCACGAgAGGTGCATATGATGAACGACTATGTTCCACAACCACAGCTCATATATAACATGATTTtGTTTGCCGAATTCATACACGCATTACAACACACATTGAATTCAATAATAATATCAAATTCACATTCAAAGCTTTCAAGTTAGACAAAAGTTTTAATGCCGTTTTtACCTGTTTTtGAAAAGGTAATTTTCTTTAGATATATTATGTTGAATaTTAGGGTTTTTATAAAGAATGTGTATATTGUTTACAATATAAAAGACACAATTGCAAACTAGCATGATTGTAAACAATTGCTAAACGGATCAATATAAATTAAAATTGTAATATTAAGTATCAAACCGATAATTTTTATTTATTGTTCATTGTTTGTTCTTTATTTTGTTATTTGTAAATAATGAAA

Evidence

Consensus:

3. GeneValidator

Monica Dragan

Ismail Moghul

https://github.com/monicadragan/GeneValidator


GeneValidator

Run on:

★ whole geneset: identify most problematic predictions

★ alternative models for a gene (choose best)

★ individual genes (while manually curating)

4. Afra: Crowdsourcing gene model curation

Dozens of software algorithms: dozens of predictions

20% failure rate: •missing pieces•extra pieces•incorrect merging•incorrect splitting

Visual inspection... and manual fixing required. 1 gene = 20 minutes to 3 days15,000 genes * 20 species = impossible. Ya

ndell

& E

nce

2013

NRG

GTCTACAATGCGATTGTAAAATAGCACGAgAGGTGCATATGATGAACGACTATGTTCCACAACCACAGCTCATATATAACATGATTTtGTTTGCCGAATTCATACACGCATTACAACACACATTGAATTCAATAATAATATCAAATTCACATTCAAAGCTTTCAAGTTAGACAAAAGTTTTAATGCCGTTTTtACCTGTTTTtGAAAAGGTAATTTTCTTTAGATATATACAGTTTGTAATaTTAGGTATTTTATAAACAGTGTGTATATTTCTTACAATATAAAAGACACAATTGCAAACTAGCATGATTGTAAACAATTGCTAAACGGATCAATATAAATTAAAATTGTAATATTAAGTATCAAACCGATAATTTTTATTTATTGTTCATTGTTTGTTCTTTATTTTGTTATTTGTAAATAATGAAA

Evidence

Evidence

Consensus:

Gene prediction/identification

Afra: Crowdsourcing gene model curation

Challenges• Recruiting & retaining contributors

• Ensuring qualityBegin

Needs curation

Create initial tasks

Being curated

Curate

Being curated

Curate

Being curated

Curate

Submit Submit Submit

Auto-check

Done

Inconsistent: create

“review” task

Consistent: create next required task

@yebanAnurag Priyam

@yebanAnurag Priyam

Timelines• Rolled out to:

• 8 MSc students

• 20 3rd year students

• Need to improve tutorials/guidance/documentation

• Roll out to 200 first years (few months)

• Expand

@yebanAnurag Priyam

Summary

Fire ant Solenopsis invicta queen number determined by Gp-9 genotypes: •only BB workers ➔ single BB queen•with Bb workers ➔ multiple Bb queens

Genome sequencing + RAD Genotyping >500 individuals•Gp-9 marks ~4% of genome ➔ social chromosomes:

SB is like X; Sb is like YStructural differences between SB and Sb ➔ no recombination

Ants are cool.

Formica selysi: Convergent evolution of social chromosomesA few tools for genomics: • http://sequenceserver.com (easy custom BLAST server)• http://bionode.io (Agile data & analysis workflows)• http://afra.sbcs.qmul.ac.uk (Crowdsourcing gene feature curation)• http://genevalidator.sbcs.qmul.ac.uk Identifying gene prediction

problems

http://sequenceserver.com

http://bionode.io

http://afra.sbcs.qmul.ac.uk

http://genevalidator.sbcs.qmul.ac.uk

Ecology & Evolution & Vital-IT @ LausanneOrganismal Biology & Psych @ Queen Mary

QMUL lab (R Pracana, B Vieira, E Stolle, A Priyam, I Moghul et al) Laurent Keller lab (J WANG, D SHOEMAKER,O RIBA-GROGNUZ, M Nipitwattanaphon)

http://wurmlab.github.io

M Corona, S Nygaard, BG Hunt, KK Ingram, L Falquet, M Nipitwattanaphon, D Gotzek, MB Dijkstra, J Oettler, F Comtesse, CJ Shih, WJ Wu, CC Yang, J Thomas, E Beaudoing, S Pradervand, V Flegel, ED Cook, R Fabbretti, H Stockinger, L Long, WG Farmerie, J Oakey, JJ Boomsma, P Pamilo, SV Yi, J Heinze, MAD Goodisman, L Farinelli, K Harshman, N

Hulo, L Cerutti, Ioannis Xenarios

Michel Chapuisat, Jess Purcell, Alan Breslford, Nicolas Perrin

http://www.unil.ch/dee/page32298_en.html

Age of the region based on dS

0

50

100

150

200

250

0.00 0.05 0.10 0.15 0.20 0.25leafcutterDndsSubset$dS

coun

tleafcutterdS

0

50

100

150

0.00 0.05 0.10 0.15 0.20 0.25subset(dndsdata, gp9linked == TRUE)$dS

coun

t

gp9linkedSolenopsisdS

## Min. 1st Qu. Median Mean 3rd Qu. Max.

## 0.00000 0.08160 0.10300 0.10559 0.12600 0.32190

Richard thinks the mega-dS values are likely not orthologs. WARNING: We still have some leafcutterD-ndsSubset dS > 1 here!!!!

1.3.4 Calculating age using mean based on dS

10000000 * median(subset(dndsdata, gp9linked == TRUE)$dS)/median(leafcutterDndsSubset$dS)

## [1] 330097.09


## [1] 264077.67

Richard Nichols says I can use Maximum Likelihood to compare such distributions. See section below.

1.3.5 Calculating age based on dN

What do my dN distributions look like?

multiplot(qplot(x = subset(leafcutterDndsSubset, dN < 0.1)$dN, binwidth = 0.002, main = "leafcutterdNs"), qplot(x = subset(dndsdata,

gp9linked == TRUE & dN < 0.1)$dN, binwidth = 0.0005, main = "gp9linkedSolenopsisdNs"), cols = 1)

3

0

50

100

150

200

250

0.00 0.05 0.10 0.15 0.20 0.25leafcutterDndsSubset$dS

coun

tleafcutterdS

0

50

100

150

0.00 0.05 0.10 0.15 0.20 0.25subset(dndsdata, gp9linked == TRUE)$dS

coun

t

gp9linkedSolenopsisdS

## Min. 1st Qu. Median Mean 3rd Qu. Max.

## 0.00000 0.08160 0.10300 0.10559 0.12600 0.32190

Richard thinks the mega-dS values are likely not orthologs. WARNING: We still have some leafcutterD-ndsSubset dS > 1 here!!!!

1.3.4 Calculating age using mean based on dS


## [1] 330097.09


## [1] 264077.67

Richard Nichols says I can use Maximum Likelihood to compare such distributions. See section below.

1.3.5 Calculating age based on dN

What do my dN distributions look like?

multiplot(qplot(x = subset(leafcutterDndsSubset, dN < 0.1)$dN, binwidth = 0.002, main = "leafcutterdNs"), qplot(x = subset(dndsdata,

gp9linked == TRUE & dN < 0.1)$dN, binwidth = 0.0005, main = "gp9linkedSolenopsisdNs"), cols = 1)

3

Leafcutter common ancestor: 8,000,000-10,000,000 years ago

Maximum Likelihood Estimation of SB/Sb age:280,000-425,000⟹ little time for degeneration

Module countsNode = “NPM”

Most BB vs Bb gene expression differences map to S

Non-recombing region of S contains 800 genes

polygyne colonies (Table 2 and Table S2; all P,0.05). The 11genes encoding mitochondrial and prefoldin complex (molecularchaperone) proteins were all up-regulated in polygyne comparedto monogyne workers. Increased mitochondrial gene expressionmay reflect increased oxidative metabolism, while increasedprefoldin expression may indicate higher protein synthesis rates,possibly in relation to the relatively smaller size and highermetabolic rates of polygyne workers [42].

The pattern of expression of the six genes in the viral genecategory is consistent with the expectation that differences in socialorganization affect susceptibility to pathogens and parasites. In themonogyne form, there is intense selection against susceptibleinfected individuals during independent colony founding, a stagethat colonies of the polygyne form never display [26]. Accordingly,we found that workers in the polygyne form express moresequences corresponding to viral genes than their counterparts inthe monogyne form, presumably because of relaxed selection andgenerally greater susceptibility in the former (see also [43]). Basedon sequence similarity and correlated expression across ourexperiments, we identified six gene products that likely representthree different viruses, a ssRNA negative-strand (2) virus and twossRNA positive-strand (+) viruses, one of which is the SINV-2 virus[44]. All 20 polygyne study colonies showed evidence of infectionwith at least one virus (mean number of viral types per colony,2.560.67), whereas only three of the 20 monogyne coloniesshowed evidence of infection, in all cases by a single viral type.Finally, the pattern of expression of another socially-regulated

gene, this one encoding a defensin (a class of small proteinantibiotics active against viruses, bacteria, and fungi [45]), also isconsistent with greater selection for resistance in the monogyneform, as this gene was more highly expressed in monogyne thanpolygyne workers.

The numbers of genes differentially expressed in the genotype (39)and social form (91) comparisons are relatively low compared to thenumbers expected based on other published microarray experi-ments. There are several possible explanations for this. First, the useof whole worker bodies as the source of RNA may decrease theprobability of detecting genes whose level of expression varies amongcells or tissues. Second, our comparisons were performed on groupsof workers originating from different colonies, thus adding a colony-level effect to, and thus increasing the total variance in, geneexpression. Finally, workers of alternate genotype or social formapparently exhibit fewer phenotypic differences than queens[20,21,46,47], possibly reflecting the involvement of fewer differen-tially expressed genes in the former caste.

Remarkably, there was almost no overlap between genes whoselevel of expression was influenced by the focal workers’ Gp-9genotypes and genes whose expression was influenced by the socialenvironment, with only one of the 129 differentially expressedgenes appearing in both categories. This demonstrates an almostcomplete decoupling of the direct effects of the genomic regionmarked by Gp-9 and the indirect effects mediated by socialinteractions within colonies. Moreover, there is little indication ofan interaction between these direct and indirect effects; genes

Figure 1. Expression profiles between S. invicta adult workers with the BB and Bb genotypes of Gp-9. Expression profiles for 39differentially expressed genes are depicted (ANOVA, 10% false discovery rate [FDR]). Each row represents data for one gene, and each columnrepresents data for a pool of 7–10 nestmates with the same Gp-9 genotype sampled from each of twenty colonies of each social form. Colonies werecollected from Georgia (2004, checkered peach bar; 2006, solid peach bar) and Louisiana (2006, solid red bar), USA (data from each polygyne colonyare presented in the identical order for the alternate genotype groupings). Expression levels for each gene are depicted relative to the average levelacross all experimental samples (blue, low levels; yellow, high levels). Genes are arranged by hierarchical clustering. See Text S1 and Table S1 forconfirmation of selected gene expression results with quantitative RT-PCR (qRT-PCR).doi:10.1371/journal.pgen.1000127.g001

Gene Expression Patterns for a Social Trait

PLoS Genetics | www.plosgenetics.org 3 July 2008 | Volume 4 | Issue 7 | e1000127

Gene expression: Gp-9 Bb vs BB workers in multiple queen colonies

29 significant genes are in the SB/Sb region

(p<10-10)

20 of

Similar for BB vs Bb queens; & for B vs b males. Wang et al 2008

Algorithm discovery by protein folding game playersFiras Khatiba, Seth Cooperb, Michael D. Tykaa, Kefan Xub, Ilya Makedonb, Zoran Popovićb,David Bakera,c,1, and Foldit PlayersaDepartment of Biochemistry; bDepartment of Computer Science and Engineering; and cHoward Hughes Medical Institute, University of Washington,Box 357370, Seattle, WA 98195

Contributed by David Baker, October 5, 2011 (sent for review June 29, 2011)

Foldit is a multiplayer online game in which players collaborateand compete to create accurate protein structure models. For spe-cific hard problems, Foldit player solutions can in some cases out-perform state-of-the-art computational methods. However, verylittle is known about how collaborative gameplay produces theseresults and whether Foldit player strategies can be formalized andstructured so that they can be used by computers. To determinewhether high performing player strategies could be collectivelycodified, we augmented the Foldit gameplay mechanics with toolsfor players to encode their folding strategies as “recipes” and toshare their recipes with other players, who are able to further mod-ify and redistribute them. Here we describe the rapid social evolu-tion of player-developed folding algorithms that took place in theyear following the introduction of these tools. Players developedover 5,400 different recipes, both by creating new algorithms andby modifying and recombining successful recipes developed byother players. The most successful recipes rapidly spread throughthe Foldit player population, and two of the recipes became parti-cularly dominant. Examination of the algorithms encoded in thesetwo recipes revealed a striking similarity to an unpublished algo-rithm developed by scientists over the same period. Benchmarkcalculations show that the new algorithm independently discov-ered by scientists and by Foldit players outperforms previouslypublished methods. Thus, online scientific game frameworks havethe potential not only to solve hard scientific problems, but also todiscover and formalize effective new strategies and algorithms.

citizen science ∣ crowd-sourcing ∣ optimization ∣ structure prediction ∣strategy

Citizen science is an approach to leveraging natural humanabilities for scientific purposes. Most such efforts involve

visual tasks such as tagging images or locating image features(1–3). In contrast, Foldit is a multiplayer online scientific discoverygame, in which players become highly skilled at creating accurateprotein structure models through extended game play (4, 5). Folditrecruits online gamers to optimize the computed Rosetta energyusing human spatial problem-solving skills. Players manipulateprotein structures with a palette of interactive tools and manipula-tions. Through their interactive exploration Foldit players also uti-lize user-friendly versions of algorithms from the Rosetta structureprediction methodology (6) such as wiggle (gradient-based energyminimization) and shake (combinatorial side chain rotamer pack-ing). The potential of gamers to solve more complex scientific pro-blems was recently highlighted by the solution of a long-standingprotein structure determination problem by Foldit players (7).

One of the key strengths of game-based human problem ex-ploration is the human ability to search over the space of possiblestrategies and adapt those strategies to the type of problem andstage of problem solving (5). The variability of tactics andstrategies stems from the individuality of each player as well asmultiple methods of sharing and evolution within the game(group play, game chat), and outside of the game [wiki pages (8)].One way to arrive at algorithmic methods underlying successfulhuman Foldit play would be to apply machine learning techniquesto the detailed logs of expert Foldit players (9). We chose insteadto rely on a superior learning machine: Foldit players themselves.

As the players themselves understand their strategies better thananyone, we decided to allow them to codify their algorithmsdirectly, rather than attempting to automatically learn approxi-mations. We augmented standard Foldit play with the ability tocreate, edit, share, and rate gameplay macros, referred to as“recipes” within the Foldit game (10). In the game each playerhas their own “cookbook” of such recipes, from which they caninvoke a variety of interactive automated strategies. Players canshare recipes they write with the rest of the Foldit community orthey can choose to keep their creations to themselves.

In this paper we describe the quite unexpected evolution ofrecipes in the year after they were released, and the striking con-vergence of this very short evolution on an algorithm very similarto an unpublished algorithm recently developed independentlyby scientific experts that improves over previous methods.

ResultsIn the social development environment provided by Foldit,players evolved a wide variety of recipes to codify their diversestrategies to problem solving. During the three and a half monthstudy period (see Materials and Methods), 721 Foldit players ran5,488 unique recipes 158,682 times and 568 players wrote 5,202recipes. We studied these algorithms and found that they fellinto four main categories: (i) perturb and minimize, (ii) aggressiverebuilding, (iii) local optimize, and (iv) set constraints. The firstcategory goes beyond the deterministic minimize functionprovided to Foldit players, which has the disadvantage of readilybeing trapped in local minima, by adding in perturbations to leadthe minimizer in different directions (11). The second categoryuses the rebuild tool, which performs fragment insertion withloop closure, to search different areas of conformation space;these recipes are often run for long periods of time as they aredesigned to rebuild entire regions of a protein rather than justrefining them (Fig. S1). The third category of recipes performslocal minimizations along the protein backbone in order to im-prove the Rosetta energy for every segment of a protein. The finalcategory of recipes assigns constraints between beta strands orpairs of residues (rubber bands), or changes the secondary struc-ture assignment to guide subsequent optimization.

Different algorithms were used with very different frequenciesduring the experiment. Some are designated by the authors aspublic and are available for use by all Foldit players, whereasothers are private and available only to their creator or theirFoldit team. The distribution of recipe usage among differentplayers is shown in Fig. 1 for the 26 recipes that were run over1,000 times. Some recipes, such as the one represented by theleftmost bar, were used many times by many different players,while others, such as the one represented by the pink bar in the

Author contributions: F.K., S.C., Z.P., and D.B. designed research; F.K., S.C., M.D.T., andF.P. performed research; F.K., S.C., M.D.T., K.X., and I.M. analyzed data; and F.K., S.C., Z.P.,and D.B. wrote the paper.

The authors declare no conflict of interest.

Freely available online through the PNAS open access option.1To whom correspondence should be addressed. E-mail: [email protected].

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1115898108/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1115898108 PNAS ∣ November 22, 2011 ∣ vol. 108 ∣ no. 47 ∣ 18949–18953

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Research themesSocial evolution

Pollinator health• Biomedical approaches • International population genomics surveys • Monitoring via sequencing • Responses to environmental challenges

Modern Bioinformatics for Genomics• Reproducibility • Accuracy • Sustainability

• Versioning • Open source

• Agile & efficient data handling

• Major social transitions » social chromosomes » convergence » eusociality, queen number, parasitism...

• 100-fold intra-specific variation in lifespan • Strengths of selection • Candidate genes/pathway

Hypothesis: influences queen odor & how workers « smell » queens

Gp-9 is an odorant binding protein

Krieger & Ross

dN/dS = infinityKrieger & Ross

2014 12-09-oulu

Science

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