Stability Analysis,Genetic Map Construction And QTL Mapping Of A Synthesized Allohexanloid Brassica

A doubled haploid(DH)mapping population was obtained via microspore culture from a hybrid derived from a cross between two allohexaploid Brassica lines.Stability and 6 important phenotypic traits including seed number,seed yield,pod length,plant height,1000-seed weight and pollen viability were investigated at field station.SSR markers were used to construct the first framework linkage map of this allohexaploid Brassica.SNPs derived by RAD-seq were used to construct a high-density linkage map of this allohexaploid Brassica.QTL mapping was performed on 6 phenotypic traits with the aid of the linkage map.The main results are as follows:1.Flow cytometry testing results showed that 85.96%(202 out of 235)DH lines were hexaploid while the rest 14.06%were chimera consisting of hexaploid and triploid cells,indicating that most DH lines were relatively stable hexaploid.Chromosome number observation at root tip further verified this result.Ten DH lines showed high fertility(>90%)while 6 DH lines were almost sterile(<1%).The average pollen viability of 235 DH lines was 52.35%.The 1000-seed weight were significantly higher compared with diploid and tetraploid parents in some DH lines due to the nutritional advantage of polyploid crops.However,yield,seed number,plant height and pollen viability were relatively low in most DH lines due to meiosis disorder of this allohexaploid Brassica.2.The polymorphism of 578 SSR primer pairs were assessed among allohexaploid parents and 189 DH lines and 408(70.6%)were polymorphic.SSR primer pairs specific to A genome(107),B genome(44)and C genome(109)were used to construct a genetic linkage map of the population.Twenty-seven linkage groups were resolved from 274 polymorphic loci on the A genome(109),B genome(49)and C genome(116)covering a total genetic distance of 3178.8 cM with an average distance between markers of 11.60 cM.This is the first genetic framework map for the artificially synthesized Brassica allohexaploid.The linkage groups represent the expected complement of chromosomes in the A,B and C genomes from the original diploid and tetraploid parents.3.RAD libraries of allohexaploid parents and 146 DH progenies derived from H16-1 were constructed by restriction enzymes.SNPs derived from RAD-seq and SSRs that had been integrated into the framework linkage map were used to construct a high density genetic linkage map of this allohexaploid Brassica.A total of 2.87 G illumina reads with an average sequencing depth of 2.59 × in the allohexaploid parents and 1.41 × in the DH progeny were obtained,among which 2.49G were high quality reads.SNPs were obtained from clustering 290,422 RAD reads without reference genome.After a rigorous filtering process,7,950 high quality SNP markers that segregated normally(1:1)in the DH population left.A linkage map was successfully constructed with 7,511 SNPs and 151 SSRs,containing 27 chromosomes(A1-A10,B1-B8,C1-C9)with a total genetic distance of 5,725.19 cM and an average distance of 0.75 cM between adjacent markers.4.Twenty-five QTLs were detected controlling 6 phenotypic traits including seed yield,1000-seed weight,seed number,plant height,pod length and pollen viability,with 2 to 8 QTLs for each phenotypic trait using ICIM(Inclusive composite interval mapping)method of QTL IciMapping 4.1.In addition,44 intra-genomic and 18 inter-genomic epistatic QTL pairs were detected for 4 phenotypic traits(seed yield,1000-seed weight,seed number and pollen viability).78 and 42 candidate gene were also discovered for two QTL hotspots.These additive QTLs and epistasis QTLs can be used for gene mapping,map based cloning and molecular marker assisted breeding thus largely facilitate the breeding speed of allopolyploid Brassica.