Bioinformatics and MAS

8/13/2019 Bioinformatics and MAS

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Bioinformatics and MAS

- an Indian Experience

N. K. Singh and T. R. Sharma

NRC Plant BiotechnologyIARI, New Delhi


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Outline1. Informatics in MAS2. Rice genome sequence information for mapping,

tagging and map-based cloning of genes3. Mining novel alleles of cloned genes in the

germplasm

4. Synteny and colinearity- transferring rice genome

information to wheat

5. Databases and web-based tools to assist

breeders

6. High throughput genotyping to save time andcost

7. Proposed activities for the Indo-Aus wheat MAS

network


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1. Informatics in MAS


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Applications incrop improvement

(MAS/ Transgenics)

Identify DNA

markers linkedwith traits(Simple/ QTL)

Basic Resources :Germplasm, mutant lines, knock outs, mapping populations,

GSTs, ESTs, BAC libraries, BAC-end sequences, Bioinformatics

Functional

genomics

Genotype

Transcriptome

Proteome

Metabolome

Phenome

Pilot genome

sequencing

High density

molecular genetic map

Large scale

genome

sequencing

Gene based markers

Map based cloning

High throughput gene/ marker discovery

Arrow

ofTime

Phase 2

Phase 3

Phase1

Phase 4

Different Phases of Plant Genomics Research

Rice

Tomato

SoybeanSorghum

Medicago

Brassica

ChickpeaPigeonpea

Mango

Banana

Maize

Wheat

Cotton

Sugarcane


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Status of genome maps

5 (Medicago,Lotus, Soybean,

Sorghum, Tomato)

3 (Arabidopsis,Rice, Poplar)

Plants

108 (Human, Chimp,

Mouse, Rat, Fruitfly, Mosquito, Fogu

fish)

Animals

543268Microbes

In progressCompletedGroup

Source: NCBI


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Enormous size

of crop genomes1pg = 1 bill ion base pairs (1000 Mbp)

Arabidopsis

125 Mb

Rice

390 Mb

Sorghum1000 Mb

Human3000 Mb

Maize

2500 Mb

Barley

6000 Mb

Wheat16000 Mb

Microbes

5 Mb


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What is needed?

1. De novo tools- Better maps, ESTs, BACs

(Sequencing, genotyping facility)

2. Comparative genomics- leverage

information from model species

(Genome-informatics facility, human resource)

3. Create novel genetic variation- wide

crosses, transgenics

(Green houses, tissue culture facility, gene constructs, IPR)

(for Molecular Breeding in Orphan Crops)


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NRCPBGenoinformatics

Centre


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2. Rice genome sequence

information for mapping,tagging and map-based

cloning of genes


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Ref: IRGSP,Nature 11 August 2005

Maps of the 12 Sequenced Rice Chromosomes

Size = 388.8 Mb


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Mapping of QTLs/Genes for

important traits in rice

Basmati quality traits

Grain number

Salt tolerance

Blast resistance


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Gene Discovery: Efforts at NRCPB, IARI and CSSRI

Effect of 100 mM NaCl on salt susceptible (MI 48) and

salt tolerant (CSR 27) varieties of rice

Fine mapping

of QTLs, and

expression

profiling

(micro array/proteomics) of

genes for

complex

agronomictraits


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P1 P2 RILs

0

5

10

15

20

25

30

5.51

-5.71

5.91

-6.11

6.31

-6.51

6.71

-6.91

7.11

-7.31

7.51

-7.71

7.91

-8.11

8.31

-8.51

8.71

-8.91

9.11

-9.31

Grain length (in mm)

Frequency

P2

P1

Distribution of Grain length in RILs

Grain length


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Overview of Quality QTLs in Pusa 1121


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Effect of 100 mM NaCl on salt susceptible (MI 48)

and salt tolerant (CSR 27) varieties of rice

Salt tolerance


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Graphic display of detected QTLs for 17 salt tolerance parameters


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sizeBAC Acc.No

MARKER cM

148Kb

40.5 AC 121327149Kb

150Kb

AC118340134 kb

AC 145349

171 Kb

RM224 136 Kb

AC 122143

4.5

1.3

9.5

AC 109832

AC125780

AC125782

AC10484610.7

3.6

Pi kh0.5

0.7TRS 26

TRS 33

130 Kb

AC 145349

TRS 2635,016-35059

142 Kb

CG

TRS 3316,903-16 972

171Kb

AC104846

170502-171522

136 Kb

size BAC Acc.No

R

gene

GENETIC & PHYSICAL MAP of Pi-kh locus - Comparative Genomics Approaches

Genetic map ofPi kh locus,Chr 11

Physical map ofPi-kh locus

in Nippon bare

Candidate gene identified

28 MB ~1 MB 142kb 1.5kb

RM206 0

Search for NewSSR markers inNippon bare

RM2190

RM6965

CAPS100

RM202

RM536

(7400 genes) (18 genes)


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Cloning of Disease Resistance Gene in Rice

R-gene like sequences (Nipponbare)

Design PCR primers flanking to the R-gene Expected PCR product


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Tetep

ATT Poly A

990AAcAAA

990 bp ORF

P2TACP1

-343 -269 -101 -64 -47 T1WUN-1

MotiffMeJA resp

Element

CAAT

BOX

TATA

BOX

GT1

S

- 221

Isolation & Structure of the Candidate Gene

Comparison of gene structure between NB & cloned gene

PCR amplification

ofPi-kh gene from

Tetep and HP2216

Gene structure

HP2216

ATT Poly A

990AAcAAA

P2TACP1

-343 -269 -101 -64 -47 G1WUN-1

MotiffMeJA resp

Element

CAAT

BOX

TATA

BOX

GT1

S

- 221

Sharma et al. Mol Gen Genomics: 274:569-578


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Konishi et al. (2006)qSH 1 (BEL1-type homeobox)Seed

shattering

10

Ma et. (2006)Lsi 1 (Silicon transporter)Lodging

tolerance

9

Xu et al. (2006)Sub1 (Ethylene response factor-

like)

Submergence

tolerance

8

Sharma et al. (2005)Pikh (NBS-LRR type protein)Blast

resistance

7

Bradburry et al. (2005)BAD2 (betaine aldehyde

dehydrogenase 2)

Grain aroma6

Ren et al. (2005)SKC1 (a HKT type transpor ter)Salt tolerance5Ashikari et al. (2005)OsCKX2 (cytokinin oxydase)Grain number4

Liu et al. (2004)Sbe 3 (starch branching enzymes)Amylose

content

3

Sasaki et al. (2002)Sd 1 (gibberellin-20-oxidase)Plant height2

Song et al. (1995)Xa 21 (NBS-LRR type receptor

kinase)

Bacterial leaf

blightresistance

1

ReferenceGeneTraitS.

no.

Table: Growing number of genes for important agronomic traits cloned

recently making use of the rice genome sequence information


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3. Mining novel alleles

of cloned genes in the

germplasm


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Phenotypic analysis of O .s a t i v a

lines and species with M . g r i s e a

Resistant: O. punctata, O. latifolia,

O. officinalis, O. rhizomatis,

Coloro, Jatto, K-60,

Fukunishiki and Tetap

Susceptible: O. rufipogon, O. nivara,O. minuta, O. gradiglumis,

HP2216, Co-39, Bhrigudhan

Lesions


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1 2 3 4 5 6 7 8 9 10 11 12 13 14M M

10001500

750

250

PCR amplification of Pi-Kh allele from rice

lines and wild Oryza species

bp

Quantification (A) and Restriction analysis (B) ofplasmid to check presence of insert

2027

bp

23224361

pUC1 2 3 4 5 6M UC 1 2 3 4 5 6 7 8 UC M

A B

M

PCR amplification of Pi-kh gene from Different Rice Lines

F- Primer R- Primer


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Consensus sequence derived from

individual reads


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Tetep

HP2216

Coloro

K-60

Jatto

Co-39

Bhrigudhan

Fukunishiki

Nipponbare

O. nivara

O .rufipogon

O. latifolia

O.officinalis

O. rhizomatis

O. punctata

O. minuta

O.gradiglumis

1bp 1765 bp

ORFs and number of exons predicted in P i - k hgene isolated fromdifferent O . sa t iv alines and wild species

Mining blast resistance genes from wild species of Rice


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0

20

40

60

80

100

Transition

Transversion

Indels

Num

berofSNPs

Lines/Species

HP2216

Nipp

onba

re

O.niva

ra

O.rufip

ogon

O.min

uta

O.rhizo

matis

O.latis

folia

O.officin

alis

O.gran

diglu

mis

O.punc

tata

Co-39

Colo

ro

Bhrigu

dhan

Jatto

K-60

Fuk

unishk

i

Number of transitions, transversions and

indels in P i - khallele

Indica Japonica Wild species


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Phylogenetic relationship among P i - khgenes amplified from

different wild species of rice.

Allele mining for disease resistance genes


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An overview of the sequence contigs of the badh1 gene of 16 rice

varieties based on sequence reads obtained using 16 pair ofprimers, assembled using Phred/Phrap/Consed software)


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Screen shot of Consed window showing location of

one of the 20 SNPs discovered by sequencing of the

badh1 gene fragments from 16 rice varieties.


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Location of PCR primers (reverse primer underlined) and 20 SNPs (highlighted)

in the badh1 gene of rice. The gene has 15 exons (in bold) and 14 introns


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Summary of the SNP alleles in 16 rice varieties and the reference

variety Nipponbare at 20 different positions in the badh1 gene,

starting from the ATG codon of Nipponbare..

SNP: BADH1- S1 S2 S3 S4 S5 S6 S7 S8 S9 S10 S11 S12 S13 S14 S15 S16 S17 S18 S19 S20


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4. Synteny and colinearity

-transferring riceinformation to wheat


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Rice chromosome 11 long arm:

comparison with wheat

0

5

10

15

20

25

30

35

40

45

No.

ofGenes

1 2 3 4 5 6 7

Wheat Chromosome Group

Genome wide analysis of homology between 56298 predicted


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Genome wide analysis of homology between 56298 predicted

rice gene CDS (from the IRGSP sequence) and 39,813 wheat EST

contigs (from wheat SNP consortium, build 3), plus 3792 bin-mapped wheat EST contigs (USDA-NSF wheat genome project,

version Aug. 03)

Homology plot of 5840 rice genes mapped on 21 wheat chromosomes


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Ancient duplications in the Rice GenomeAncient duplications in the Rice Genome

Rice-Sorghum gene colinearity


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Rice-Sorghum gene colinearity

sorghum

rice

Rice Maize gene colinearity


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Rice-Maize gene colinearity

rice

maize


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Typical Patterns of Synteny between rice and wheat

Distribution of Copy Number among the


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Distribution of Copy Number among the

4659 Rice Gene Homologs of Wheat


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Conserved Synteny of

Single Copy Rice Genes with Wheat


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Conserved Synteny

of single copy ricegenes with wheat

A. Rice chr 1/ wheat chr 3

B. Rice chr 2/ wheat chr 6

C. Rice chr 6/ wheat chr 8

Transposition ofgenes concentrated

near wheat

centromeres

Wheat ChromosomesRice-Wheat Colinearity


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1 2 3 4 5 6 7 8 9 10 11 12

Rice Chromosomes

y

based on 1063 single

copy rice genes

Conclusions:1. Seven wheat chromosomes

seem to have evolved fromthe 12 ancestral rice

chromosomes by 3 centric

fusion and 6 translocation

events

1. W1 = R5 + R10

W2 = R7 + R4

W3 = R1

W4 = R3 + R11

W5 = R12 + R9 +R3

W6 = R2

W7 = R6 + R8

Predicting wheat bin location of 6178 unmapped single copy rice gene homologs


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Predicting wheat bin location of 6178 unmapped rice gene homologs


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V lid ti f th di t d h t bi l ti


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1. Chinese Spring ; 2. CS del 6AL8; 3. CS del 6BL5; 4. CS del 6DL10

Validation of the predicted wheat bin location

of unmapped single copy rice genes

Experimental Validation of the Predicted


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35% of the 213

single copyrice gene

homologs,

representingall 12 rice and

all 7 wheat

chromosomes,

mapped totheir predicted

bin location

Map Location of genes in Wheat


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5. Databases and

web-based tools

to assist breeders


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Containing Info on 56298 genes


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Distribution of R-like Genes and Defense


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Response Genes in Rice Genome

1 7378 115 1.56 28 0.38

2 5770 84 1.46 10 0.17

3 6544 46 0.7 28 0.434 5831 38 0.65 4 0.07

5 4977 54 1.08 20 0.4

6 5071 63 1.24 16 0.32

7 4858 62 1.28 11 0.23

8 4536 62 1.37 13 0.29

9 3561 44 1.24 8 0.22

10 3933 39 0.99 8 0.2

11 4436 115 2.59 11 0.25

12 4355 53 1.22 10 0.23

Total 61250 775 15.38 167 3.19

Def.

Response

Def.Response

(%)

Chromoso

me No.

Total

genes

R-

genes

R-genes

(%)

R- gene like seq.= NBS-LRR, LZ-NBS-LRR, LRR-TM, Misc. (putative, known genes)

Defense response genes = chitinases, glucanases, thaumatin like proteins

Different types of R-Like Genes Distributed on


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Rice Chromosomes

0

10

20

30

40

50

60

70

80

1 2 3 4 5 6 7 8 9 10 11 12

Rice Chrom osom e

No.

of

R-genes

NBS-LRR LZ-NBS-LRR LRR-TM

Misc Def.Resp

Distribution of Defense Response Genes on


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p

Rice Chromosomes

0

5

10

15

20

25

D

ef.

R

esp

o

n

se

G

ene

s

N

o

.

1 2 3 4 5 6 7 8 9 10 11 12

Rice Chromosome

Thaumatin Glucanases Chitinases

Mapping of R-like Genes on Rice Chromosome 1


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Chr 1 Chr 2

Chr 3 Chr 4

Chr 5 Chr 6



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A total o f 176 R- and DR-genes clusters ident if ied

Chr 7 Chr 8

Chr 9 Chr 10

Chr 12Chr11



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To sum up


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VansanuDhan-A Rice Genome Database created at NRCPB contains

562898 genes info. It is being used in functional and comparative

genome analysis in rice.

We found 942 R-gene and Defense Response gene like sequences in

the rice genome. The physical location and orientation of each genedelineated.

Comparative analysis of indica- japonica sequences helped us in

mapping and cloning of a new Rice blast gene Pi-kh.

Extensive sequence variation was observed between the Pi-kh alleles

amplified from wild species and land races of rice. These alleles are

being used in functional validation experiments.

Analysis of Pi-kh locus in indica- japonica provided an insight in the

presence of SSR elements in this region which may play an important

role in shuff ling of genes in the genome.


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6. High throughput

genotyping to save

time and cost

Multipex SNP assays using Sequenom MassARRAY system


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Sequence of flanking pre-amplification primers(PCRP) and single nucleotide extension primers(UEP) for genotyping of 20 SNPs of badh1 gene

Assaying SNPs by MALDI-ToF MS


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Once a SNP is identified, 3 primers are required to enable genotyping including:-

- Two PCR primers to amplify the region around the SNP

5 3

- One Extension primer which anneals directly adjacent to the SNP.

5 3

MALDI-TOF Mass Spectrometry


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DNA samples (384 no.) are mixed with amatrix, spotted onto a MALDI plate and

loaded into the Mass Spectrometer.

Each spot is subjected to pulses of

nitrogen laser (337nm) in vacuum, which

vaporises and ionises sample. Matrixabsorbs most of the laser energy,

preventing degradation of the sample,

and allows ionisation of some of the DNA

substrate.

Application of an electric field causes

DNA ions to enter flight tube and are

accelerated towards Mass detector.

All ions gain same kinetic energy, so

larger ions take longer to reach detector.

The variation in Time of Flight allows

separation based on size.

Assaying SNPs by MALDI-ToF MS


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0

20

40

60

80

100

120

4300 4400 4500 4600 4700 4800 4900 5000

C = 273 Da

T = 288 Da

A = 297 DaG = 313 Da

Difference in peaksize reveals the SNPallele for that rice

variety

UnextendedSNP primer

Extended SNPprimer

GenotypingGenotyping


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Genotyping spectra for one well (11Genotyping spectra for one well (11 SNPsSNPs))


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Genotyping of badh1_S5 SNP Using Sequenom MassARRAY

badh1_S5 badh1_S5Another Another


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Pusa

NPT11

Pusa

1342

badh1_S5

Unextended

primer

Primer

extended

with C

Primer

extendedwith T

Primer

of theMultiplex

Primer

of theMultiplex


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7. Proposed activities for

the Indo-Australianwheat MAS project

Proposed Activities


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p

Database of markers and cloned genes

ESTs, GSS, HTGS, SSRs,, SNPs, Traits

Allele mining for specified genesGBSS-1, Glu-1, Amylase, Lr, Sr and Yr genes etc.

High throughput genotypingSequenom Mass Array

Capillary sequencer fragment analysis


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Thank You

Very Much

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