MOLECULAR MAPS AND MARKERS FOR DIPLOID ROSES
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1 MOLECULAR MAPS AND MARKERS FOR DIPLOID ROSES Patricia E Klein, Mandy Yan, Ellen Young, Jeekin Lau, Stella Kang, Natalie Patterson, Natalie Anderson and David Byrne Department of Horticultural Sciences, Texas A&M University, College Station, TX 77843
2 A HIGH-DENSITY SNP-BASED CONSENSUS MAP FOR DIPLOID ROSES JoinMap 4.1
3 A HIGH-DENSITY SNP-BASED CONSENSUS MAP FOR DIPLOID ROSES Fluorescence anchor SSRs (Spiller et al.)
4 Optimization of the GBS protocol and alignment results Enzyme DNA extraction method % reads mapped to strawberry genome (more complete and the chromosome order is known) Fsel CTAB+Zymo PCR inhibitor 35-36% Fastprep 36-37% NgoMIV CTAB+Zymo PCR inhibitor 54-57% Fastprep 59-60% Nhel Fastprep+Zymo clean&con % More reads from NgoMIV were mapped compared with the other two enzymes. NgoMIV (G^CCGGC) is a methylation-sensitive enzyme and there are 16,326 predicted NgoMIV sites in the F. vesca genome.
5 Mapping using JoinMap 4.1 CP: cross pollination population LOD 8 Seven linkage groups Maximum likelihood mapping algorithm
6 Use of Graphical Genotyping to Aid in Marker Order
7 Initial Rose Populations Developed Female Male Population size J14-3 (R) LC (S) 69 J14-3 (R) RF (S) 124 J14-3 (R) SC (S) 58 J14-3 (R) VS (S) 83 J3-3 (R) RF (S) 19 J4-6 (R) RF (S) 62 M4-4 (R) SC (S) 10 M4-4 (R) VS (S) 5 OB (MS) J3-6 (R) 93 OB (MS) M4-4 (R) 10 OB (MS) RF (S) 82 SC (S) J14-3 (R) 24 SC (S) J4-6 (R) 10 SC (S) M4-4 (R) 53 VS (S) J14-3 (R) Colors White Grey Black Indicate Founder or intermediate parents with no DNA information nor phenotypic data Direct parents of the mapping populations with DNA information Mapping families used for map construction and QTL discovery
8 LG1 comparison for J14-3 x LC, J14-3 x VS, OB x RF and ICD (consensus map).
9 A HIGH-DENSITY SNP-BASED CONSENSUS MAP FOR DIPLOID ROSES Statistical Summary of Linkage Group Data Marker Density along the 7 Rose LGs Linkage groups Population Map LG1 LG2 LG3 LG4 LG5 LG6 LG7 Overall J14-3 x LC J14-3 x VS OB x RF ICD Marker number Map length (cm) Marker number Map length (cm) Marker number Map length (cm) Marker number Map length (cm)
10 INSIGHTS INTO THE HIGH SYNTENY BETWEEN FRAGARIA VESCA AND ROSA Diploid Rosa Fragaria vesca LGs Populations J14-3xLC J14-3xVS OBxRF ICD J14-3xLC J14-3xVS OBxRF ICD J14-3xLC J14-3xVS OBxRF ICD J14-3xLC J14-3xVS OBxRF ICD J14-3xLC J14-3xVS OBxRF ICD J14-3xLC J14-3xVS OBxRF ICD J14-3xLC J14-3xVS OBxRF ICD
11 INSIGHTS INTO THE HIGH SYNTENY BETWEEN FRAGARIA VESCA AND ROSA a. b. Yan et al. (2018) Horticulture Research. DOI /s
12 Addition of Two Maps to Improve the ICD for FlexQTL J4-6 x RF (62 progeny) LG Length (cm) No. of markers No. of distorted markers (P<0.05) No. of bin markers Biggest gap Overall OB x J3-6 (93 progeny)
13 BLACK SPOT QTL DISCOVERY IN FIFTEEN RELATED DIPLOID ROSE POPULATIONS chr7_ chr7_ chr7_ chr4_ chr7_ chr7_ chr7_ chr7_ chr7_ chr7_ chr7_ chr7_ chr7_ chr7_ chr7_ chr7_ chr7_ chr7_ chr7_ chr7_ chr7_ chr7_ chr7_ chr7_ chr7_ chr7_ chr7_ chr7_ chr7_ chr7_ chr7_ chr7_ chr7_ chr7_ chr7_ chr7_ chr7_ chr7_ chr7_ chr7_ chr7_ chr7_ chr7_ chr7_ chr5_ chr7_ chr7_ chr7_ chr7_ chr7_ chr7_ chr7_ chr7_ chr7_ chr7_ chr7_ chr7_ chr7_ chr7_ chr7_ chr7_ chr7_ chr5_ chr7_ chr7_ chr7_ chr7_ chr7_ chr7_ chr7_ chr7_ chr7_ chr7_ chr7_ chr7_ chr7_ chr7_ chr7_ chr1_ chr1_ chr1_ chr1_ chr1_ chr1_ chr1_ chr1_ chr1_ chr1_ chr1_ chr1_ chr1_ chr1_ chr1_ chr1_ chr1_ chr1_ chr1_ chr1_ chr1_ chr1_ chr1_ chr1_ chr1_ chr1_ chr1_ chr1_ chr1_ chr1_ chr1_ chr1_ chr1_ chr1_ chr1_ chr1_ chr1_ chr1_ chr1_ chr1_ chr1_ chr1_ chr1_ chr1_ chr1_ chr1_ chr1_ chr1_ chr1_ chr1_ chr1_ chr1_ chr1_ chr1_ chr1_ chr1_ chr1_ chr1_ chr1_ chr1_ chr1_ chr1_ chr1_ chr1_ chr1_ chr1_ chr1_ chr1_ chr1_ chr1_ chr1_ chr1_ chr1_ chr1_ chr1_ chr1_ chr1_ chr1_ chr1_ chr1_ chr1_ chr1_ chr1_ chr1_ chr1_ chr1_ chr1_ chr1_ chr1_ chr1_ chr1_ chr1_ chr6_ chr6_ chr6_ chr6_ chr6_ chr1_ chr1_ chr6_ chr6_ chr6_ chr6_ chr6_ chr6_ chr3_ chr6_ chr6_ chr6_ chr6_ chr6_ chr6_ chr6_ chr6_ chr6_ chr6_ chr6_ chr6_ chr6_ chr6_ chr6_ chr6_ chr6_ chr6_ chr6_ chr6_ chr6_ chr6_ chr6_ chr6_ chr6_ chr6_ chr6_ chr6_ chr6_ chr6_ chr6_ chr6_ chr6_ chr6_ chr6_ chr6_ chr6_ chr6_ chr6_ chr6_ chr6_ chr6_ chr6_ chr6_ chr6_ chr6_ chr6_ chr6_ chr6_ chr6_ chr6_ chr6_ chr6_ chr6_ chr6_ chr6_ chr6_ chr6_ chr6_ chr6_ chr6_ chr6_ chr6_ chr6_ chr6_ chr6_ chr6_ chr6_ chr6_ chr6_ chr6_ chr6_ chr6_ chr6_ chr6_ chr6_ chr6_ chr6_ chr6_ chr6_ chr6_ chr6_ chr6_ chr6_ chr6_ chr6_ chr6_ chr6_ chr6_ chr6_ chr6_ chr6_ chr6_ chr6_ chr6_ chr6_ chr6_ chr6_ chr6_ chr6_ chr6_ chr6_ chr6_ chr6_ chr6_ chr6_ chr6_ chr6_ chr6_ chr6_ chr6_ chr6_ chr6_ chr6_ chr6_ chr6_ chr6_ chr6_ chr6_ chr6_ chr6_ chr6_ chr6_ chr6_ chr6_ chr6_ chr6_ chr6_ chr6_ chr6_ chr6_ chr6_ chr6_ chr6_ chr6_ chr6_ chr6_ chr6_ chr6_ chr6_ chr6_ chr6_ chr6_ chr6_ chr4_ chr6_ chr4_ chr1_ chr1_ chr3_ chr3_ chr4_ chr4_ chr4_ chr1_ chr4_ chr4_ chr4_ chr4_ chr4_ chr4_ chr4_ chr4_ chr4_ chr4_ chr4_ chr1_ chr1_ chr1_ chr4_ chr4_ chr4_ chr4_ chr4_ chr4_ chr4_ chr4_ chr4_ chr4_ chr4_ chr4_ chr4_ chr4_ chr4_ chr4_ chr4_ chr4_ chr4_ chr4_ chr4_ chr4_ chr4_ chr4_ chr4_ chr4_ chr4_ chr4_ chr4_ chr4_ chr4_ chr4_ chr4_ chr4_ chr4_ chr4_ chr4_ chr3_ chr4_ chr3_ chr4_ chr4_ chr4_ chr4_ chr4_ chr4_ chr4_ chr4_ chr4_ chr4_ chr4_ chr4_ chr4_ chr4_ chr4_ chr4_ chr4_ chr4_ chr4_ chr4_ chr4_ chr4_ chr4_ chr4_ chr4_ chr4_ chr4_ chr4_ chr4_ chr4_ chr4_ chr4_ chr4_ chr4_ chr4_ chr4_ chr4_ chr4_ chr4_ chr4_ chr4_ chr4_ chr4_ chr4_ chr4_ chr4_ chr4_ chr4_ chr4_ chr4_ chr4_ chr4_ chr4_ chr3_ chr3_ chr3_ chr3_ chr3_ chr3_ chr3_ chr3_ chr3_ chr3_ chr3_ chr3_ chr3_ chr3_ chr3_ chr3_ chr3_ chr3_ chr3_ chr4_ chr2_ chr3_ chr6_ chr3_ chr3_ chr3_ chr3_ chr3_ chr3_ chr3_ chr3_ chr3_ chr3_ chr3_ chr3_ chr3_ chr3_ chr3_ chr3_ chr3_ chr2_ chr2_ chr3_ chr3_ chr3_ chr3_ chr3_ chr3_ chr3_ chr3_ chr3_ chr3_ chr3_ chr3_ chr3_ chr3_ chr5_ chr3_ chr3_ chr3_ chr3_ chr5_ chr5_ chr3_ chr3_ chr3_ chr3_ chr3_ chr3_ chr3_ chr3_ chr3_ chr3_ chr1_ chr3_ chr3_ chr3_ chr3_ chr3_ chr3_ chr3_ chr3_ chr3_ chr3_ chr3_ chr3_ chr3_ chr3_ chr3_ chr3_ chr3_ chr5_ chr3_ chr3_ chr3_ chr3_ chr3_ chr3_ chr3_ chr3_ chr3_ chr3_ chr3_ chr3_ chr3_ chr3_ chr3_ chr3_ chr3_ chr3_ chr3_ chr3_ chr3_ chr3_ chr2_ chr2_ chr2_ chr2_ chr2_ chr2_ chr2_ chr2_ chr2_ chr2_ chr2_ chr2_ chr2_ chr2_ chr2_ chr2_ chr2_ chr2_ chr2_ chr2_ chr2_ chr2_ chr5_ chr2_ chr2_ chr2_ chr2_ chr2_ chr2_ chr2_ chr2_ chr2_ chr2_ chr2_ chr2_ chr2_ chr2_ chr2_ chr2_ chr2_ chr2_ chr2_ chr2_ chr2_ chr2_ chr2_ chr2_ chr2_ chr2_ chr2_ chr2_ chr2_ chr2_ chr2_ chr2_ chr2_ chr2_ chr2_ chr2_ chr2_ chr2_ chr2_ chr2_ chr2_ chr2_ chr2_ chr2_ chr2_ chr2_ chr2_ chr2_ chr2_ chr2_ chr2_ chr2_ chr2_ chr2_ chr2_ chr2_ chr2_ chr2_ chr2_ chr2_ chr2_ chr2_ chr2_ chr2_ chr2_ chr2_ chr2_ chr2_ chr2_ chr2_ chr2_ chr2_ chr2_ chr2_ chr2_ chr2_ chr2_ chr2_ chr2_ chr3_ chr3_ chr3_ chr2_ chr2_ chr2_ chr2_ chr2_ chr2_ chr2_ chr2_ chr2_ chr2_ chr2_ chr2_ chr2_ chr2_ chr2_ chr2_ chr2_ chr2_ chr2_ chr2_ chr2_ chr3_ chr2_ chr2_ chr2_ chr2_ chr2_ chr5_ chr5_ chr5_ chr5_ chr5_ chr5_ chr5_ chr5_ chr5_ chr5_ chr5_ chr5_ chr5_ chr5_ chr5_ chr5_ chr5_ chr5_ chr5_ chr5_ chr5_ chr1_ chr5_ chr5_ chr5_ chr5_ chr5_ chr5_ chr5_ chr5_ chr5_ chr5_ chr5_ chr5_ chr5_ chr5_ chr5_ chr5_ chr5_ chr5_ chr5_ chr5_ chr5_ chr5_ chr5_ chr5_ chr5_ chr5_ chr5_ chr5_ chr5_ chr5_ chr5_ chr5_ chr5_ chr5_40345 chr5_ chr5_98049 chr5_ chr5_ chr5_ chr5_ chr5_ chr5_ chr5_ chr5_ chr5_98223 chr5_ chr5_ chr5_ chr5_ chr5_40337 chr5_ chr3_ chr5_ chr5_ chr1_ chr5_ chr5_ chr5_ chr5_ chr5_ chr5_ chr5_ chr5_ chr5_ chr5_ chr2_ chr5_ chr5_ chr5_ chr1_ chr1_ chr6_ chr6_ chr5_ chr5_ chr5_ chr5_ LG Length SNP no Max gap Total Updated consensus map utilizing 5 populations. Map length, SNP number and maximum gaps between SNPs for each LG from the final consensus map for FlexQTL analysis.
14 BLACK SPOT QTL DISCOVERY IN FIFTEEN RELATED DIPLOID ROSE POPULATIONS
15 BLACK SPOT QTL DISCOVERY IN FIFTEEN RELATED DIPLOID ROSE POPULATIONS QTL position Genotype probabilities (positive evidence) LG3 QTL is segregating in parents VS, J4-6 and J14-3 with positive support (76% probability). Genotype probabilities (strong evidence) LG3 QTL is segregating in parents J4-6 and J14-3 with strong support (95% probability).
16 BLACK SPOT QTL DISCOVERY IN FIFTEEN RELATED DIPLOID ROSE POPULATIONS Eleven haplotypes (H1-7 parental, 8-11 recombinant progeny) identified among individuals composed of seven SNPs on LG3 in QTL region cm covering the QTL peak at 41 cm. Comparison between two haplotypes in J14-3 and J4-6 within resulting families J14-3xRF and J4-6xRF on black spot overall rating in 2016 in the QTL region. Comparisons of means were conducted by student s t- test at α = Dots represent each individual s phenotype.1 H3 and H1 are the resistant haplotypes from J14-3 and J4-6, respectively. The resistant H3 haplotype from J14-3 decreased the overall disease rating by 0.9, while the resistant H1 haplotype from J4-6 decreased disease rating by 1.1. z Levels not connected by the same letter are significantly different (α=0.05) y QTL genotype predicated at LG3 41cM x Number of progeny in each QTL genotype class
17 BLACK SPOT QTL DISCOVERY USING PEDIGREE BASED ANALYSIS IN FIFTEEN RELATED DIPLOID ROSE POPULATIONS Documented pedigree of nine directed parents of mapping populations generated by Pedimap 1.2. Traced 7 SNP haplotypes across the LG 3 QTL through the pedigrees to identify inheritance patterns. No definitive conclusion on the origin of the two resistance haplotypes yet. Can conclude the H3 resistant haplotype in J14-3 does not come from DD but from an unknown paternal pollen source (PP-J14-3) and the H1 resistant haplotype might have come from either OB or Rosa wichurana Basye s Thornless (R.wichTless).
18 Next Steps Genome sequence of Old Blush doubled haploid was sequenced at INRA Reanalyzed GBS data from the 15 inter-related diploid rose populations using the rose genome sequence Will allow for marker development for mapped traits which can be used in the breeding program and will aid in the confirmation of the origin of disease resistance Have completed GBS on several of the progenitors of these populations Will improve tracing the origin of the two black spot resistance haplotypes GBS analysis on 435 progeny from 8 additional diploid rose populations for map construction and FlexQTL analysis GBS analysis on 112 rose cultivars and species for GWAS Incorporated the KGD method to estimate relatedness of the progeny from each population based on the SNPs and read depths Tetraploid populations will be genotyped using the Axiom 68k SNP array, followed by map construction and FlexQTL analysis
19 Acknowledgments David Byrne Robert E. Basye Endowment in Rose Genetics Mandy Yan Ellen Young Jeekin Lau Stella Kang Natalie Patterson Natalie Anderson Jizhou Yang Qianni Dong This work was partially funded by the USDA s National Institute of Food and Agriculture (NIFA) Specialty Crop Research Initiative project, Combating Rose Rosette Disease: Short Term and Long Term Approaches ( /SCRI) and RosBREEDII: Combining disease resistance with horticultural quality in new rosaceous cultivars
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