RICE PHYLOGENY RICE PHYLOGENY REVISITEDREVISITED

N. K H I R I P E TN. K H I R I P E T ANDAND V. POTLURIV. POTLURI

Some BackgroundSome Background

Rice is staple for more than half of the Rice is staple for more than half of the world’s populationworld’s population

Large collection of germplasm [ >10000 Large collection of germplasm [ >10000 accessions] held at IRRIaccessions] held at IRRI

Plant breeders/genecists use these to Plant breeders/genecists use these to develop news cultivarsdevelop news cultivars

For this purpose, evolutionary For this purpose, evolutionary history/phylogeny is importanthistory/phylogeny is important

Rice geneticsRice genetics

Rice is usually a diploid, but tetraploids are Rice is usually a diploid, but tetraploids are not uncommonnot uncommon

The diploid genome is classified asThe diploid genome is classified asAA,BB,CC,DD,EE,FF,GG,HH.KKAA,BB,CC,DD,EE,FF,GG,HH.KK

The tetraploids are mostly alloThe tetraploids are mostly alloBBCC,CCDD,HHJJ,HHKKBBCC,CCDD,HHJJ,HHKK

Taxa in the Genus Taxa in the Genus OryzaOryza: the species and : the species and genome groupsgenome groups

Table 1-1.  Taxa in the genus Table 1-1.  Taxa in the genus OryzaOryza:  the species and genome groups. [From IRRI]:  the species and genome groups. [From IRRI]  Species2nGenomeDistributionSpecies2nGenomeDistribution

Sect. OryzaSect. Oryza        Ser. Ser. Sativae Sativae       O. barthiiO. barthii  24 AA Sub-Saharan Africa  24 AA Sub-Saharan AfricaO. glaberrima O. glaberrima 24 AA West Africa24 AA West AfricaO. glumaepatulaO. glumaepatula  24 AA South, Central America  24 AA South, Central AmericaO. longistaminataO. longistaminata  24 AA Sub-Saharan Africa  24 AA Sub-Saharan AfricaO. meridionalisO. meridionalis  24 AA Tropical Australia  24 AA Tropical AustraliaO. nivaraO. nivara 24 AA Tropical, Subtropical Asia 24 AA Tropical, Subtropical AsiaO. rufipogonO. rufipogon  24 AA Tropical, Subtropical Asia, Tropical Australia  24 AA Tropical, Subtropical Asia, Tropical Australia

O. sativa O. sativa 24 AA Worldwide24 AA WorldwideSer. Ser. Latifoliae Latifoliae O. eichingeri O. eichingeri 24 CC South Asia, East Africa 24 CC South Asia, East Africa      O. altaO. alta  48 CCDD South, Central America  48 CCDD South, Central America O. grandiglumisO. grandiglumis 48 CCDD South, Central America 48 CCDD South, Central America O. O. latifolia latifolia 48 CCDD South, Central America48 CCDD South, Central America O. minutaO. minuta 48 BBCC Philippines, Papua New Guinea 48 BBCC Philippines, Papua New Guinea O. punctataO. punctata 24, 48 BB, BBCC Sub-Saharan Africa 24, 48 BB, BBCC Sub-Saharan Africa O. rhizomatisO. rhizomatis 24 CC Sri Lanka 24 CC Sri Lanka O. officinalisO. officinalis 24,48 CC, BBCC Tropical, Subtropical Asia 24,48 CC, BBCC Tropical, Subtropical AsiaSer. Ser. AustraliensesAustralienses O. australiensisO. australiensis 24 EE ropical Australia 24 EE ropical AustraliaSect. BrachyanthaSect. Brachyantha      Ser. Ser. BrachyanthaeBrachyanthae      O. brachyanthaO. brachyantha 24 FF Sub-Saharan Africa 24 FF Sub-Saharan AfricaSect. PadiaSect. Padia      Ser.Ser. Meyerianae Meyerianae       O. granulataO. granulata  24 GG South, Southeast Asia  24 GG South, Southeast Asia O. meyerianaO. meyeriana 24 GG Southeast Asia 24 GG Southeast Asia O. neocaledonica O. neocaledonica 24 ?? New Caledonia 24 ?? New CaledoniaSer. Ser. RidleyanaeRidleyanae        O. longiglumis O. longiglumis 48 HHJJ Indonesia (Irian Jaya), Papua New Guinea48 HHJJ Indonesia (Irian Jaya), Papua New Guinea O. ridleyi O. ridleyi 48HHJJSoutheast Asia48HHJJSoutheast AsiaSer. Ser. Schlechterianae Schlechterianae      O. schlechteriO. schlechteri 48 HHKK Indonesia (Irian Jaya), Papua New Guinea 48 HHKK Indonesia (Irian Jaya), Papua New Guinea

So, What is the Problem?So, What is the Problem?

No problem really, butNo problem really, but

We would like to have a better tool to We would like to have a better tool to understand the interrelationships of understand the interrelationships of various cultivars various cultivars

Which in turn should help breeders and Which in turn should help breeders and genecistsgenecists

Well then, what is the plan?Well then, what is the plan?

We chose four important enzymes, vizWe chose four important enzymes, viz

MaturaseMaturase

IntegraseIntegrase

Alcohol dehydrogenase I andAlcohol dehydrogenase I and

Alcohol dehydrogenase IIAlcohol dehydrogenase II

Then,Then,

We got the protein sequence data [from We got the protein sequence data [from NCBI]for these enzymes for 8 cultivarsNCBI]for these enzymes for 8 cultivars

O. sativa O. sativa 24 AA Worldwide24 AA WorldwideSer. Ser. Latifoliae Latifoliae O. eichingeri O. eichingeri 24 CC South Asia, East 24 CC South Asia, East AfricaAfrica      O. altaO. alta  48 CCDD South, Central   48 CCDD South, Central AmericaAmerica O. grandiglumisO. grandiglumis 48 CCDD South, C. America 48 CCDD South, C. America O. O. latifolia latifolia 48 CCDD South, C.America48 CCDD South, C.America O. minutaO. minuta 48 BBCC Philippines, PN G 48 BBCC Philippines, PN G O. punctataO. punctata 24, 48 BB, BBCC Sub-Saharan Africa 24, 48 BB, BBCC Sub-Saharan Africa O. rhizomatisO. rhizomatis 24 CC Sri Lanka 24 CC Sri Lanka

Hold OnHold On

Change of SpeakerChange of Speaker

Methods [contd.]Methods [contd.]

We constructed a phylogenetic tree for We constructed a phylogenetic tree for each enzyme using Workbench using each enzyme using Workbench using ClustalwClustalw

We calculated the distance matrix from We calculated the distance matrix from this tree using ClustalDistthis tree using ClustalDist

[Methods-contd.][Methods-contd.]

We converted the distance matrix to We converted the distance matrix to digital form to be used in JBenzer and digital form to be used in JBenzer and GDEGDE using PERL scripts.using PERL scripts.

ThresholdThreshold

We used a median point between the We used a median point between the distance values as a threshold. We will distance values as a threshold. We will discuss the implications of changing the discuss the implications of changing the threshold threshold after presenting the resultsafter presenting the results

Finally [methods contd.]Finally [methods contd.]

We combined all the distance matrices We combined all the distance matrices and combined in to one matrix andand combined in to one matrix and

Digitized the matrix for use in JBenzer and Digitized the matrix for use in JBenzer and GDE to obtain a single Benzer plotGDE to obtain a single Benzer plot

clustalw

clustdist clustdist2benzer.pl

benzer2gde.pl

Biology Workbench

sequences

Clustdist2benzer.plClustdist2benzer.plOL (1) 0.000 0.002 0.002 0.008 0.006 0.013 0.015 0.015OG (2) 0.002 0.000 0.000 0.006 0.004 0.012 0.013 0.013OA (3) 0.002 0.000 0.000 0.006 0.004 0.012 0.013 0.013OE (4) 0.008 0.006 0.006 0.000 0.002 0.013 0.015 0.015OR (5) 0.006 0.004 0.004 0.002 0.000 0.012 0.013 0.013OS (6) 0.013 0.012 0.012 0.013 0.012 0.000 0.015 0.015OP (7) 0.015 0.013 0.013 0.015 0.013 0.015 0.000 0.000OM (8) 0.015 0.013 0.013 0.015 0.013 0.015 0.000 0.000

JBENZER81,1,1,0,0,0,0,0,OL1,1,1,0,1,0,0,0,OG1,1,1,0,1,0,0,0,OA0,0,0,1,1,0,0,0,OE0,1,1,1,1,0,0,0,OR0,0,0,0,0,1,0,0,OS0,0,0,0,0,0,1,1,OP0,0,0,0,0,0,1,1,OM

Benzer2gde.plBenzer2gde.pl

JBENZER81,1,1,0,0,0,0,0,OL1,1,1,0,1,0,0,0,OG1,1,1,0,1,0,0,0,OA0,0,0,1,1,0,0,0,OE0,1,1,1,1,0,0,0,OR0,0,0,0,0,1,0,0,OS0,0,0,0,0,0,1,1,OP0,0,0,0,0,0,1,1,OM

"OL","OG""OL","OA""OG","OL""OG","OA""OG","OR""OA","OL""OA","OG""OA","OR""OE","OR""OR","OG""OR","OA""OR","OE""OS","OS""OP","OM""OM","OP"

MaturaseMaturaseOL

OG

OA

OE

OR

OS

OP

OM

IntegraseIntegraseOA

OGOL

OS

OP

OEOR

OM

Alcohol dehydrogenase IAlcohol dehydrogenase IOP

OA

OG

OS

OE

OR

OLOM

Alcohol dehydrogenase IIAlcohol dehydrogenase IIOL

OGOA

OM

OP

OSOE

OR

Combined MatrixCombined Matrix0 0.159 0.146 0.138 0.103 0.131 0.141 0.185 0.128 OP

0.159 0 0.055 0.137 0.118 0.16 0.179 0.264 0.157 OE0.146 0.055 0 0.135 0.092 0.138 0.158 0.253 0.135 OR0.138 0.137 0.135 0 0.124 0.066 0.047 0.223 0.059 OL0.103 0.118 0.092 0.124 0 0.127 0.147 0.225 0.124 OM0.131 0.16 0.138 0.066 0.127 0 0.04 0.228 0.007 OA0.141 0.179 0.158 0.047 0.147 0.04 0 0.238 0.047 OG0.185 0.264 0.253 0.223 0.225 0.228 0.238 0 0.234 OS

Back to the Back to the BeginningBeginning

ConclusionsConclusions

The phylogenetic trees and JBenzer differ The phylogenetic trees and JBenzer differ in some respectsin some respects

JBenjer plots show O.sativa [2n=24,AA]to JBenjer plots show O.sativa [2n=24,AA]to be uniquebe unique but in phylogenic trees,it is but in phylogenic trees,it is clustered with O.punctata [4x,48,BBCC] or clustered with O.punctata [4x,48,BBCC] or O.grandiglumis and O.alta[4x,48,CCDD]O.grandiglumis and O.alta[4x,48,CCDD]

BUTBUT

Remember the threshold values?Remember the threshold values?

We used a median value from a matrix We used a median value from a matrix table and it does give relationships which table and it does give relationships which can be related to their genomes.can be related to their genomes.

IF we change the threshold value, the IF we change the threshold value, the Benjer plot will change which brings the Benjer plot will change which brings the question whether relationships based on question whether relationships based on distance matrices need re-visitationdistance matrices need re-visitation

We realize that phylogenetic trees based We realize that phylogenetic trees based on distance matrices have been used for on distance matrices have been used for so longso longHowever, different scientists calculate and However, different scientists calculate and interpret the distance matrix results interpret the distance matrix results differentlydifferentlyDoes it help if the input data is obtained Does it help if the input data is obtained differently from the beginning in binary differently from the beginning in binary formatformat

We used a very limited data to compare We used a very limited data to compare but it does indicate that we may have to but it does indicate that we may have to look at the phylogenetic relationships look at the phylogenetic relationships using improved methods to minimize using improved methods to minimize and /or to understand better varied and /or to understand better varied interpretationsinterpretations

FinallyFinally

A Gigabyte of Thanks toA Gigabyte of Thanks to

ALL THE BIOQUEST STAFF AND THE ALL THE BIOQUEST STAFF AND THE PARTICIPANTS OF THIS WORKSHOP PARTICIPANTS OF THIS WORKSHOP WHO MADE OUR PARTICIPATION IN WHO MADE OUR PARTICIPATION IN THE WORKSHOP A MEMORABLE AND THE WORKSHOP A MEMORABLE AND REWARDING EXPERIENCEREWARDING EXPERIENCE