Development And Evaluation Of Genome-Wide Molecular Markers In Two Cultivated Allotetraploid Cottons,Gossypium Hirsutum And Gossypium Barbadense

Cotton is one of the important cash crops in the world,cotton fiber is the most important natural material for textile industry and cottonseeds can be used for the production of edible oil.Four cotton species include two American tetraploids(G.hirsutum and G.barbadense)and two African-Asian diploids(G.arboreum and G.herbaceum).Upland cotton has high yield,strong adaptability,and the most extensive planting area.Sea-island cotton,characterized by its extra-long staple(ELS)and strong and fine fibers,and disease resistance,provides important natural fiber for textile industry,also key donor for improving agronomic traits of upland cotton.The use of molecular marker technology for cotton variety identification and genetic diversity analysis has provided important scientific and production significance for cotton breeding practice and variety improvement.Based on resequencing technology and chip technology,this experiment developed a batch of SSR,InDel,and SNP markers.These markers were used to study genetic diversity of upland cotton and island cotton materials for variety identification.This study provides a theoretical basis for cotton germplasm identification and diversity of germplasm resources.The results of this study are as follows:1.Based on the TM-1 genomic information released by Wang(Wang et al,2018),the MISA software was used to analyze the TM-1 sequencing data,and a total of 695,235 SSR sites were obtained,with an average of 3.38 Kb.Tetranucleotide are the most repeated.Based on the resequencing data of upland cotton materials from 10 different sources,2,361,956 InDel markers were detected using TM-1 as the reference genome,with an average of 90,844 on each chromosome.Among the 2,361,956 InDel markers,A subgroups had an average of 101 markers per 100 kb,D subgroups had an average of 113 markers per 100 kb.Combined with the annotation information of the TM-1 genome,2,171,266 InDel(92%)were located in the intergenic region.2.A total of 324.691 InDel markers with polymorphism among 10 upland cotton materials from different sources were selected to compare the SSR position in the genome.Of these 10,325 InDel sites containing SSR sites were defined as polymorphic SSR sites(PSSR).The remaining 314,366 InDel sites were further defined as PInDel.These polymorphic sites are mainly distributed at the starts and ends of the chromosome,and the maximum variation range between 2,400,001 and 2,500,000 bp on the D01 chromosome.Through mass design of primers,7,935 PSSR and 287,646 PInDel site detection primers were developed.Through e-PCR correction,3,253 pairs of PSSR primers and 110,585 pairs of PInDel primers with unique amplification sites on the genome were obtained.106 pairs of PInDel and 101 pairs of PSSR primers were selected respectively.Based on polyacrylamide gel electrophoresis,the upland cotton materials from 32 different sources were used to detect the polymorphism of the markers.Polymorphism of PlnDel was 86%and polymorphism of PSSR was 78%.3.To obtain the SNP core loci for fingerprinting construction of sea-island cotton accessions,SNP genotyping was performed within 282 accessions using the CottonSNP80K array.Following the selective criteria:call frequency for each locus>0.95;loci with polymorphism;minor allele frequency(MAF)>0.01;heterozygosity rate<0.05;and removal of the same genotype,2594 high-quality core SNP loci were obtained.Further,the number of optimization for the core loci was screened by gradients analysis.As the number of loci increasing,the recognition rate of the island cotton accessions increased gradually.When loci was 200,the recognition rate was 89%;however,the number of loci increased to 1500,the recognition rate was 99%.No significant increased recognition rate was detected,when loci were further improved.Based on the combination of 1500 core loci,the average MAF value was 0.14,the average heterozygosity rate was 0.007,and the average polymorphism information content was 0.21.The core SNP loci were also verified by polyacrylamide gel electrophoresis with the consistency between SNP-PCR and chip typing as high as 98.3%.This study provides a set of core SNP loci suitable for the construction of fingerprinting of sea-island cotton accessions,which can be used for genetic diversity analysis and fingerprinting identification of Sea island cotton.