Microsatellite markers in Bermudagrass: linkage and QTL analysis

Sameer Khanal

PGML

docstoc.com

Introduction

• Warm-season perennial grasses

• Resilient nature suitable for turf purposes

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Biology

• Family: Poaceae

• Subfamily: Chloridoideae (PACC clade)

Biology

• Family: Poaceae

• Subfamily: Chloridoideae (PACC clade)

• Genus: Cynodon

• Eight extant species and 10 varieties

(Harlan 1970)

• Major cultivars are triploid hybrids

Germplasm

• Common bermudagrass (Cynodon dactylon)

2n=4x=36

• African bermudagrass (Cynodon transvaalensis)

2n=2x=18

• Hybrids: triploids 2n=3x=27

Framework linkage map

113 F1 mapping population

Marker system: RFLP

Mapping strategy: Two-way pseudo-testcross

African

Bermudagrass (T574)

Common

Bermudagrass (T89)

RFLP markers

• Bermudagrass derived or heterologous RFLP probes

• Restriction digestion gels were blotted with labeled probes

RFLP markers

• Polymorphic fragments were scored

• Parental origin of polymorphic fragments were ascertained

Two-way pseudo-testcross

• Open pollinated/heterozygous parents were crossed

African

Bermudagrass (T574)

Common

Bermudagrass (T89)

Two-way pseudo-testcross

• Fragments originating from one parent and absent in the other parent, but still segregating

T574

Two-way pseudo-testcross

• Fragments originating from one parent and absent in the other parent, but still segregating

T89

Two-way pseudo-testcross mapping

Marker segregation patterns in autotetraploids

Single dose marker: showing 1:1 segregationFor example: Parent 1: oo x Aooo :Parent 2

F1: Aoo:ooo (1:1)

Marker segregation patterns in autotetraploids

Double dose marker: showing 5:1 segregationFor example: Parent 1: oo x AAoo :Parent 2

F1: AAo /Aoo :ooo (5:1)

Framework linkage map

T574 (Diploid): 77 single dose restriction fragments on 18 linkage groups

Seven homologous groups

T89 (Tetraploid): 155 single dose + 17 double dose markers on 35 linkage groups

Four homologous and 4 partial homologous groups identified

Further development

Twenty microsatellite loci + 4 resistant gene analogs were added to this framework

Yet,

Marker coverage is less than 70%

Homologous groups not completely linked

My research

Addition of simple sequence repeat markers to the framework linkage map of bermudagrass

QTL analysis of morphological and turf quality traits

SSR resource

Primers from sugarcane EST templates available (total no. of EST-SSRs: 1,240)

Sugarcane markers have been mapped to several grass species

Further, EST-SSRs are shown to be highly transferable among different grass species

Phase I: parental screening

• Two genotypes: T574 and T89

Phase II: screening a subset

• Parents and 10 F1 progenies were genotyped

T89

T574 F1 progenies

Phase III: population screening

• A total of 96 available primer pairs were screened against two parents and 92 F1s

T89

T574F1 progenies

Marker scoring

• Fragments originating from one parent and absent in the other parent, but segregating in the progeny were scored

T89

Marker scoring

• Fragments originating from one parent and absent in the other parent, but segregating in the progeny were scored

T574

Marker scoring

• Fragments originating from both the parents and segregating (biparental markers) were also scored

Biparental

Marker scoring

• A total of 120 SSR marker loci were scored

Markers # SSR loci # RFLP loci

T89 73 270 SDRF + 64 DDRF

T574 36 103

Biparental 11 0

Mapping

• Software tools used:

• TetraploidMap

• OneMap (R environment)

TetraploidMap

• Handles two-way pseudo testcross data

• Dominant and codominant markers

• Single/double dose, and biparental markers

• Four steps: 1. Segregation analysis

2. Cluster analysis

3. Linkage analysis

4. Linkage phase and maps

Segregation analysis

Cluster analysis

Linkage analysis

Phase determination

Drawing maps

TetraploidMap: T574

• A total of 116 (30 SSRs) markers were mapped

- Framework linkage map had 77 markers

• Map is composed of 20 linkage groups, 17 of them organized into 8 homologous groups

- Framework linkage map had 18 linkage groups, 15 of them organized into 7 homologous group

TetraploidMap: T574

Lack of repulsion phase linkages in one of the groups

TetraploidMap: T574• Map covers a total of 932 cM with 116 markers

separated by an average of 9.6 cM

- Framework map: 973.4 cM with 77 markers separated by an average of 16.5 cM

• Linkage groups are defined by about 6 markers and cover ~50 cM

- Framework map: 4 markers and cover ~54 cM

TetraploidMap: T89

• A total of 282 (62 SSRs, 24 DDRFs) markers were mapped

- Framework linkage map had 155 SDRFs and 17 DDRFs

TetraploidMap: T89

• Map is composed of 39 linkage groups, 34 of them organized into 15 partial homologous groups (of 2-3 linkage groups each)

- Framework map: 35 linkage groups, 16 organized into four complete homologous groups and eight into partial homologous groups

Inferring complete homologous groups Multilocus probe linked in repulsion

Repulsion phase linkage

Multilocus probe

TetraploidMap: T89

• Map covers a total of 1,165 cM with 282 markers separated by an average of 9 cM

- Framework map: 1837.3 cM with 155 markers separated by an average of 15.3 cM

• Linkage groups are defined by about 9 markers and cover ~70 cM

- Framework map: 4 markers and cover ~52.5 cM

Inferring ancestral chromosomes

T574Consensus 1

T89Consensus 4

Inferring ancestral chromosomes

T574Consensus 7

T89Consensus 8

Inferring ancestral chromosomes

T574LG 9

T89Consensus 4

OneMap

• R language based program

• Suitable for diploid outcrossing species

• Does not handle double dose markers

• Routinely used in sugarcane mapping studies

OneMap: T574

# mapped markers

# linkagegroups Total cM

OneMap 115 13 1,364

TetraploidMap 116 20 932

Framework Map

77 18 973

OneMap: T89

# mapped markers

# linkage groups Total cM

OneMap 260 32 3,355

TetraploidMap 282 39 1,165

Framework Map

155 35 1,837

Future Works

• Updating the maps (rescored SSRs, changing input formats)

• Defining homologous groups

• QTL studies of morphological and turf quality traits