Development Of Gossypium Anomalum-derived Micro Satellite Markers And Their Use For Construction Of G.anomalum Introgression Library

With the development of social production,higher and higher demands are proposed for cotton breeding work.The breeding of new variety with high yield,good quality and resistant to pests and diseases become more and more urgent.Wild cotton which has a lot of untapped and excellent genes is the important genetic resources for cotton variety improvement.However,there are many problems between upland cottons and diploid wild species due to differences in chromosome ploidy level,such as hybrid incompatibility,F1 hybrid sterility,crazy separation of offspring.A large number of excellent genes in wild have yet to be developed.Gossypium anomalum?2n = 2x = 26,B1?is a wild species belonging to the B1 genome.As a member of the subsection of Anomala Todaro,G.anomalum possesses several desirable characters such as extremely fine fibre with high maturity,good strength,low fiber weight,resistance to insect pests,and tolerance to water deficit due to its endemic to relatively dry areas.This study takes the G.hirsutum 86-1 as the female Parent,G.anomalum as the male parent.We developed a set of Gossypium anomalum-derived microsatellite markersby RNA-Seq Technology.By continuous backcrossing and marker-assisted selectin,we constructed a set of G.anomalum introgression lines.It broaded the genetic basis of upland cotton and provided important basic materials for the study of gene structures and functional genes analysis.1.Development of Gossypium anomalum-derived microsatellite markersA total of 13963 SSR were found throught MISA software,and 6656 pairs of SSR primers were designed,682 of which were redundant with primers in CMD.These primers amplified clear and polymorphic bands between G.hirsutum and G.anomalum.Combed G.anomalum-derived SSRs with the publicly available whole-genome marker map,Thenumber of G.anomalum-derived SSR markers on the chromosomes of the combined marker map ranged from 272 to 737 per chromosome,whih covered approximately 98.5%of the physical length of the whole cotton D genome.The marker density along each chromosome ranged from 5.8 to 10.3 markers per Mb,with an average of 7.4 markers per Mb.Provided a set of effective SSR markers for the detection of G.anomalumgenome.2.Genome-wide screening of a set of informative G.anomalum-specific SSR loci based onBC2F1 segregation dataThis study takes the G.hirsutum 86-1 as the female parent,G.anonmalum as the male parent.Through continuous backcross obtained BC2F1 populations?384 individuals?.Based on the combined marker map and genetic map of guo et al,we selected 1484 SSR markers?including 642 G.anomalum-derived SSR primer?were selected to analyze the threeparents and four hexaploid hybrid plants.248 evenlydistributed,well-amplified,informative microsatellite markers?including 133 G.anomalum-derivedSSR primers?were selected to genotype the entire BC2F1 population.Segregation data from the above 248 SSR loci wereused to conduct linkage analysis via MapMaker/EXP 3.0b,revealing 243 SSR loci in 13 linkage groups.230 marker loci with positionalinformation on chromosomes were included on the linkage map.The number of G.anomalum-specific marker loci per chromosome varied from 12 to 27.The mean distancebetween the adjacent marker loci varied from 0-35.8 cM,with an average of 10.5 cM.The coverage of G.anomalum-specific SSR marker loci on every chromosome varied from86.1%?chromosome 10?to 99.49%?chromosome 1?,with an average of 95.72%.There were two gaps of more than 30 cM were still present on chromosomes 5 and 9.3.Identifying recombination events in the BC2F1 populationA total of 323 recombination events?50 recombination types?were identified among the 384 BC2F1 individuals.The frequency of recombination events varied greatly between individual chromosomes,ranging from one?chromosome 3 and chromosome 4?to 171?chromosome 11?.A high frequency of recombination events was observed in the marker regions JAAS1148-NAU5100 on chromosome 1 and JAAS0426-NAU998 on chromosome 2.The estimated donor segment length ranged from 15.4 to 235.05 cM.The donor genomewas represented on between 43.31%?chromosome 4?and 99.37%?chromosome 12?of each chromosome.The total length of the introgressed G.anomalum genome was 1882.7 cM,covering 81.48%of the donor genome.4.Identifying recombination events in the BC₃F₁ populationA total of 772 recombination events?40 recombination types?were identified among the 4349 BC3FI individuals.The frequency of recombination events varied greatly between individual chromosomes,ranging from 0?chromosome 7,chromosome 8,chromosome 13?to 282?chromosome 11?.The estimated donor segment length ranged from 13.65-tol75.35 cM.The donor genome was represented on between 0?chromosome 7,chromosome 8,chromosome 13?and 99.66%?chromosome 4?of each chromosome.The total length of the introgressed G.anomalum genome was 1346.05cM,covering 58.25%of the donor genome.There are 12 new recombinations,such as Chr1:NAU2083-NAU4045;Chr2:NAU1190-JAAS0426;Chr4:NAU2363-JAAS5943,JAAS2662-JAAS4808,NAU3508-JAAS5943,JAAS2022-JAAS5943;Chr5:HAU1248-JAAS0657,NAU5486-NAU911;Chr6:NAU4969-JAAS2480;Chr10:JAAS3294-NAU5359,NAU4881-JAAS0321.5.Identifying recombination events in the BC4FI populationA total of 521 recombination events?56 recombination types?were identified among the 8345 BC4F1 individuals.The frequency of recombination events varied greatly between individual chromosomes,ranging from 1?chromosome 7?to 143?chromosome 11?.The estimated donor segment length ranged from 8.05 to 175.35 cM.The donor genomewas represented on between 33.39?chromosome 7?and 99.66%?chromosome 4?of each chromosome.The total length of the introgressed G.anomalum genome was 1848.22cM,covering 80.0%of the donor genome.There are 16 new recombinations,such as Chrl:NAU3615-NAU2182,NAU3615-NAU2083,NAU5100-NAU3714,NAU3714-JAAS2596,NAU2083-JAAS2569;Chr2:NAU2929?JAAS4258;Chr3:NAU1072-JAAS2579,NAU895-JAAS4015;Chr4:NAU2363?JAAS2977;Chr6:NAU2714-NAU905;Chr8:JAAS1049-JA AS0199,NAU2914-JAAS4933,NAU1262-JAAS0199;Chr10:JAAS3294-JAAS5359,NAU4881-JAAS0321;Chrl3:NAU2871-NAU3398.We identified 9 recombinants through using the addition lines backcross with su8269,such as Chr7:NAU3678-JAAS5041;Chr8:JAAS1049-JAAS0199,NAU2914-JAAS4933,NAU1262-JAAS0199;Chr12:JAAS6317-JAAS6372;Chrl3:JAAS4570-JAAS6264,JAAS0784-JAAS0049,JAAS3946-JAAS0049,NAU2871-NAU3398.