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