Genomic Exploitation of Genetic Variation for Crop Improvement

John Z. Yu

USDA-ARS

Southern Plains Agricultural Research Center

College Station, Texas, USA

An International Conference that Examines the Issues of Yesterday, the Challenges of Today, and the Opportunities for Tomorrow

Plant Molecular Biology and Biotechnology (PMBB)

An International Conference that Examines the Issues of Yesterday, the Challenges of Today, and the Opportunities for Tomorrow

An International Conference that Examines the Issues of Yesterday, the Challenges of Today, and the Opportunities for Tomorrow

An International Conference that Examines the Issues of Yesterday, the Challenges of Today, and the Opportunities for Tomorrow

Plant Molecular Biology and Biotechnology (PMBB)

From Green Revolution to Gene Revolution Norman E. Borlaug (1914-2009) Nobel Laureate Professor of Plant Breeding Texas A&M University

• Genotype to Phenotype • Phenotype to Genotype • Genotype and Phenotype

• Genomics and Phenomics

COTTON

Source: FAO

Variety panel Exotic panel

Origin and evolution of the AD-genome polyploid cottons

Other Gossypium diploid sp.

G. thurberi G. trilobum G. lobatum G. aridum G. laxum G. schwendimanii G. raimondii G. gossypioides G. armourianum

G. turneri G. harknessii

G. klotzschianum G. davidsonii

G. arboreum G. herbaceum

D-genome diploids 13 species, New World:

A-genome diploids 2 species, Africa, Asia

AD

-genome allopolyploids

5 species, New

World

G. tomentosum

G. hirsutum

G. barbadense

G. darwinii

G. mustelinum 5 – 10 MYA

1 – 2 MYA

Seelanan et al. 1997; Small et al. 1998; Wendel et al., 2000

Yu et al., 2012

Xu et al., 2008a

An integrated map of G. hirsutum chromosome 12

Wilkins et al., 2005

Fiber initiation

-3 ~ +3 DPA

Fiber Elongation

3 ~ 20 DPA

Second cell wall deposition

20 ~ 45 DPA

Fiber maturation

45 ~ 60 DPA

Four Stages of Fiber Development

Xu et al., 2008b

Four chromosomes that predominantly control fiber development.

Gene ontology (GO) categorization of fiber genes mapped to the four chromosomes.

Expression profiles of fiber genes associated with specific chromosomes and developmental stages in upland cotton.

List of the anchored fiber development genes/transcription factors assembled with STS markers and their functions and locations in the tetraploid cotton genome.

Fiber gene name

Gene/ Factors

STS name

Function annotated

Location in Genome

Overlapped base number

Gene-ctg10 Gene-GhEF1A2

COAU0001M07 Fiber elongation

Chr.01-[97.3] 559

Gene-ctg22 Gene-P2B08 Gate4DB11 Fiber elongation

Chr.26-[114.1] 431

Gene-ctg45 f-DT544876 CBV028F22_R Heat stress transcription factor

Chr.26-[92.1] 549

Xu et al., 2010

Distribution patterns of fiber development genes and transcription factors in AD tetraploid cottons.

Subgenome/genome origination No. of contigs from 103 transcription factors

No. of contigs from 173 SSR- containing fiber ESTs

No. of contigs from 259 fiber development genes

No. of contigs from total 535 collections

At (Expected distribution under H0:no difference in genome distribution between At and Dt)

3(9.04)** 104(94.02) 46(50.02)) 135

Dt 12(5.96) 52(61.98) 37 (32.98) 89

15 156 83 224

X2 test* <0.01 <0.01 <0.01

Shared AtDt 29 111 88 157

DD 1 15 5 16

Unallocated 10 0 92 102

Total 55 182 268 499

*: X2/df 5 is 60.92; **: values in the parentheses are the expected values.

• More fiber development genes were from At subgenome than Dt;

• More transcription factors were from Dt subgenome than At;

• Interaction of two subgenomes in one nucleus enhances the fiber development and production.

Cultivated Tetraploid Cottons

Gossypium hirsutum L. (AD1)

Gossypium barbadense L. (AD2)

n=26 (13 At and 13 Dt)

C=2,425 Mb (ca. 3,500 cM)

Estimated 50,000 genes

Probable Subgenome Progenitors A2 genome: Gossypium arboreum, n=13, C=1,746 Mb, Cultivated Old World D5 genome: Gossypium raimondii, n=13, C=885 Mb, Wild New World

Wang et al., 2012

Li et al., 2014

Diagrammatic representation of BAC-pools and their homology across the pools and respective BAC clone ABI assemblies in Ctg-3301 and Ctg-465.

Buyyarapu et al., 2013

Comparative genomic analysis of Ctg-3301 (Chromosome A12) and Ctg-465 (Chromosome D12) of Upland cotton with Arabidopsis genome.

Gossypium barbadense Field Trials

Photoperiod Dependent Photoperiod Independent

P1: photoperiod-dependent parent (D)

Selection for Photoperiod-Independence

X

P2: photoperiod-independent parent (I)

F1 BC1 BC2 BC3

BC4:F2

3D : 1I segregation

X X X X

Segregating F2

Genotype-By-Sequencing • GBS method (HinP1I or BsrGI) is used to

sequence specific genomic regions (marker loci) across different samples utilizing restriction enzymes.

• Narrow down regions showing genetic polymorphisms in a characterized mapping population.

• Independent method to narrow down genome regions containing the gene(s) influencing photoperiod independence.

Candidate Experiment Results Barcode ID Barcode Floral Queue MID Parsed Sequences

AF1 ACGAGTGCGT Gossypium raimondii D5 Diploid Photoperiodic 22693AF2 ACGCTCGACA Gossypium herbaceum A1 Diploid Photoperiodic 11825AF3 AGACGCACTC Gossypium barbadense 379 Allotetraploid Photoperiod Independent 23166AF4 AGCACTGTAG Gossypium hirsutum TM1 Allotetraploid Photoperiod Independent 19141AF5 ATCAGACACG Gossypium hirsutum TX231 Allotetraploid Photoperiodic 8152AF6 ATATCGCGAG Gossypium barbadense K46 Allotetraploid Photoperiodic 7010AF7 CGTGTCTCTA Gossypium barbadense PS6 Allotetraploid Photoperiod Independent 2920AF8 CTCGCGTGTC Gossypium incanum E4 Diploid Photoperiodic 4792

Ungrouped 4531

Mid Identifiers

Targeted GBS • Similar to GBS • Can be utilized in segregating populations • Key differences

– Specific Barcoded Primer containing BsrGI site and particular bases

– Targets only certain loci within the BsrGI digestion sites, while avoiding others

P5

P7

Forward (in-line) Barcode

Read1

Index Barcode

Index Read

Forward Specific Primer

GBS Conclusions • Narrow down to nine regions implicated in

photoperiodic flowering; • Allow to focus on most important SNPs and

discard regions with lowest probability; • Reference genome sequence allows for

integration of GBS and candidate gene data; • Ongoing validation of GBS SNPs associated

with photoperiodic flowering.

Acknowledgments

USDA-ARS Russell Kohel

Richard Percy

Lori Hinze

James Frelichowski

China Cotton Inst Shuxun Yu

Fuguang Li

Kunbo Wang

Peking University Yuxian Zhu

BGI Shenzhen Jun Wang

Texas A&M Alan Pepper

Carla Young

Alabama A&M Govind Sharma

Dow Agrosci Ramesh Buyyarapu

Uzbek Academy of Sciences Ibrokhim Abdurakhmonov

THANKS