A comparison of candidate gene-based and genotyping-by-sequencing (GBS) approaches to trait mapping in Gossypium barbadense L.
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1 A comparison of candidate gene-based and genotyping-by-sequencing (GBS) approaches to trait mapping in Gossypium barbadense L. Authors Carla Jo Logan-Young. Texas A&M University. College Station, TX. United States. (presenting author) John Z. Yu. USDA-ARS. College Station, TX. United States. (corresponding author) Richard G. Percy. USDA-ARS. College Station, TX. United States. (corresponding author) Alan E. Pepper. Texas A&M University. College Station, TX. United States. (corresponding author) Ibrohkim Abdurakhmonov. Center of Genomic Technologies, Academy of Sciences of Uzbekistan. Yuqori Yuz, Qibray region Tashkent, Uzbekistan. (corresponding author) October 11 th, 2012
2 Richard Percy Gossypium barbadense Field Trials Photoperiod Dependent Photoperiod Independent
3 Segregating F 2 P1: photoperiod-dependent parent (D) F1 BC1 BC2 BC3 X X X X X BC4:F2 3D : 1I segregation P2: photoperiod-independent parent (I) Selection for Photoperiod-Independence
4 G. barbadense Phenotypic Results Photoperiod Dependent : Photoperiod Independent Ratio Significance
5 Hypothesis A single gene influences photoperiod independence within the floral network in cotton.
6 Arabidopsis Flowering Pathway A Companion to Plant Physiology, Fourth Edition by Lincoln Taiz and Eduardo Zeiger Essay 25.2 FT Protein, not mrna, is the Phloem-Mobile Signal for Flowering Jan A. D. Zeevaart, MSU-DOE Plant Research Laboratory, Michigan State University, East Lansing, MI Figure 1 Multiple developmental pathways for flowering in Arabidopsis
7 Pathways controlling flowering-time in Arabidopsis. Henderson I R, Dean C (2004) Development 131:
8 Candidate Genes Circadian Clock Genes ELF3 Clock Transcription Factor GI Clock Transcription Factor LHY/CCA1 Clock Transcription Factor TOC1 Clock Transcription Factor ADO3_FKF1 Signaling Molecule ZTL Signaling Molecule Light Dependent Genes CRY1 Photoreceptor CRY2 Photoreceptor CRY3 Photoreceptor HY2 Photoreceptor HY6 Photoreceptor PHYA Photoreceptor PHYB Photoreceptor PHYC Photoreceptor PHYE Photoreceptor Floral MiRNA Activators MiRNA172c MiRNA MiRNA172a MiRNA MiRNA172d MiRNA Floral Integrator Genes COP1 Signaling Molecule DET1 Signaling Molecule LDW1 Signaling Molecule LDW2 Signaling Molecule PIE1 Signaling Molecule PRR5 Signaling Molecule PRR7 Signaling Molecule TFL1 Signaling Molecule TFL2 Signaling Molecule ATGRP7 Transcription Factor CO Transcription Factor COL3 Transcription Factor COL5 Transcription Factor FD Transcription Factor Floral Homeotic Genes FT Signaling Molecule AP1_AGL7 Transcription Factor LFY Transcription Factor SOC1 Transcription Factor Floral Development Genes AGL1 Transcription Factor AGL16 Transcription Factor AGL2 Transcription Factor AGL24 Transcription Factor AGL3_SEP4 Transcription Factor AGL30 Transcription Factor AGL32 Transcription Factor AGL6 Transcription Factor AGL62 Transcription Factor AGL65 Transcription Factor AGL8 Transcription Factor AGL9_SEP3 Transcription Factor AP3 Transcription Factor ARP6 Transcription Factor Pistillata Transcription Factor SPA3 Transcription Factor SPA4 Transcription Factor
9 454 Sequencing Product
10 Introgressed F 5 P1: photoperiod-dependent parent (D) F1 BC1 BC2 BC3 BC4 BC4:F5 X X X X X X X Selection for Photoperiod-Independence P2: photoperiod-independent parent (I)
11 Candidate Gene Exon-Intron Spanning Regions 1
12 Candidate Experiment Results Barcode ID Barcode Mid Identifiers Floral Queue MID Parsed Sequences AF1 ACGAGTGCGT Gossypium raimondii D5 Diploid Photoperiodic AF2 ACGCTCGACA Gossypium herbaceum A1 Diploid Photoperiodic AF3 AGACGCACTC Gossypium barbadense 379 Allotetraploid Photoperiod Independent AF4 AGCACTGTAG Gossypium hirsutum TM1 Allotetraploid Photoperiod Independent AF5 ATCAGACACG Gossypium hirsutum TX231 Allotetraploid Photoperiodic 8152 AF6 ATATCGCGAG Gossypium barbadense K46 Allotetraploid Photoperiodic 7010 AF7 CGTGTCTCTA Gossypium barbadense PS6 Allotetraploid Photoperiod Independent 2920 AF8 CTCGCGTGTC Gossypium incanum E4 Diploid Photoperiodic 4792 Ungrouped 4531
13 SNP Estimates for 454 Data A/D SNP Ratio Exon: 1:68 bp Intron: 1:195 bp Intraspecific (cult. vs. wild) Exon: 1:769 bp Intron: 1:73 bp Interspecfic (G. hirsutum vs. G. barbadense) Exon: 1:622 bp Intron: 1:197 bp
14 D 5 Chromosome Alignment of Candidate Genes AGL65 GASA/GAST/Snakin family protein Chromosome Mbp 40 Mbp 55,868,233 PRMT5 VRN2 AGL16 Like1 PIF3 like 2 ELF3 AGL8_FUL Like 1 GASA6 AGL3 Chromosome Mbp 40 Mbp 62,769,430 AGL24 Like 1 AGL62 PI Contig 2 BLH4 Like Chromosome Mbp 40 Mbp 45,769,430 FD ATGRP3 AP3 TOC1 AGL8_FUL Like 2 CO EMF2 LSN(PNY) Chromosome Mbp 40 Mbp 62,178,258 ZTL Like 2 VIN3 Like 1 COL5 Contig A LD COL5 Contig B AGL20 SOC1 Like 1 FT Like 1 TSF
15 Chromosome Mbp 40 Mbp 64,140,413 FPA Like 1 GI Contig A PI Contig 1 LDW1 Contig B PFT1 AGL9_SEP3 AGL24 Like 2 CRY2 Contig B MADS Box Transcription Factor Chromosome Mbp 40 Mbp 51,074,515 GASA4 HY2 BLH4 Like 1 PRR7 Contig 1 CRY2 Contig A AGL15 Like Chromosome Mbp 40 Mbp 60,982,465 AGL2 AGL21 AGL6 PHYC COP1 CLF PIF5 Like PIF3 Like SPA4 SPA3 TFL2 PRR7 Contig 2 LHY Contig 1 Chromosome Mbp 40 Mbp 57,128,820 COL2 CCA1 LHY Contig 2 FKF1 VIP4 PHOT2 AGL20 SOC1 Like 1 Chromosome Mbp 40 Mbp 60 Mbp 70,713,020 ZTL VIN3 Like 2 FVE BLH7 LDL1 CRY1 Contig A CRY1 Contig B COL4 FT Like 2 AGL16 Like 2 GI Contig B FLD Like 2 BLH6 PIE1 ATC, Centroradialis TFL1
16 Chromosome Mbp 40 Mbp 62,175,169 ARP6 CRY3 FLD Like 1 COL3 Chromosome Mbp 40 Mbp 62,681,010 VIP5 PHYE CKA2 FPA Like 2 LDW1 Contig A LDW2 PHYB FCA FY Chromosome Mbp 35,429,946 PRR5 AG DET1 Chromosome Mbp 40 Mbp 58,321,163 ATGRP7 AGL32 AGL30 AP1 FIE LDL2 HY6 PHYA Contig 2 PHYA Contig 1
17 Candidate Gene Conclusions 48 candidate genes sequenced SNPs characterized Mapped to D-genome reference sequence Ongoing Experiments Verification of SNPs linked to photoperiodic flowering Characterize pattern of sequence evolution of flowering genes
18 Genotype By Sequencing Independent method to narrow down genome regions containing the gene(s) influencing photoperiod independence. GBS method is used to sequence specific genomic regions (marker loci) across different samples utilizing restriction enzymes.
19 Introgressed F 5 P1: photoperiod-dependent parent (D) F1 BC1 BC2 BC3 BC4 BC4:F5 X X X X X X X Selection for Photoperiod-Independence P2: photoperiod-independent parent (I)
20 Progeny-Dependent SNPs 1 to 3 SNPs 1 to 3 SNPs HinP1I Total Loci Loci 2 parents Loci 2 parents w/ progeny PS-5 Progeny Undomesticated Progeny 379/TM n/a n/a n/a 379/TX n/a n/a n/a PS5/K PS5/PI TM1/TX n/a n/a n/a HinP1I Cross 1: Intraspecific PS-5 x K % HinP1I Cross 2: Intraspecific PS-5x PI % 1 to 3 SNPs 1 to 3 SNPs BsrGI Total Loci Loci 2 parents Loci 2 parents w/ progeny PS-5 Progeny Undomesticated Progeny 379/TM n/a n/a n/a 379/TX n/a n/a n/a PS5/K PS5/PI TM1/TX n/a n/a n/a A1/D n/a n/a n/a BsrGI Cross 1: Intraspecific PS-5 x K % BsrGI Cross 2: Intraspecific PS-5x PI %
21 GBS Data for Heat Map BsrGI HEAT MAP Highest High Low Lowest SNPs with Progeny A/D
22 SNPs on D 5 Chromosomes 2, 3, and 5 At Chromosome 2 bb bb bb aa 1 20 Mbp 40 Mbp 62,769,430 Dt Genes At Chromosome 3 Dt 1 20 Mbp 40 Mbp 45,769,430 Genes At Chromosome 5 Dt Genes bb bb bb 1 20 Mbp 40 Mbp 64,140,413 aa
23 SNPs on D 5 Chromosomes 9 and 11 At bb 1 Chromosome 9 20 Mbp 40 Mbp 60 Mbp 70,713,020 aa bb Dt Genes aa At bb bb bb aa Chromosome Mbp 40 Mbp 62,681,010 Dt aa Genes
24 GBS Conclusions Narrow down to 9 regions implicated in photoperiodic flowering Allow focus on most important SNPs and discard regions with lowest probablity D 5 Reference Genome allows for integration of GBS and candidate gene data Ongoing validation of GBS SNPs associated with photoperiodic flowering
25 Segregating F 2 P1: photoperiod-dependent parent (D) F1 BC1 BC2 BC3 X X X X X BC4:F2 3D : 1I segregation P2: photoperiod-independent parent (I) Selection for Photoperiod-Independence
26 Acknowledgements Lab Members Angela Hawkins Millie Burrell Dan Grum Sponsors Undergraduate Research Assistants Chris Lyle Natalie Ware Ryan McCormick Contributing Lab Affliation Patricia Klein Andy Paterson
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