Construction Of Molecular Linkage Map And QTL Mapping Of Cultivated Diploid Cotton (Gossypium Arboreum L.)

Molecular linkage maps of cotton based upon DNA markers provide essential tools for genome research,investigating genomic structure and evolution of cotton,also facilitating QTL mapping,marker-assisted selection and map-based cloning of important gene.Asiatic cotton(Gossypium arboreum L.) has been domesticated and cultivated for almost 2000 years in China since it was first introduced from India.Through growth in many natural ecological environments and through natural and artificial selection,many landraces with characteristic Chinese forms have been produced.These landraces are still used as germplasm resources in the present cotton breeding program due to their early maturity, strong tolerance to stress,resistance to disease and insects,high fiber strength and excellent plasticity.The low rate of polymorphism among the A-genome Gossypium species,in comparison with the D-genome species,has been a major constraint in the development of a high-density A-genome map.With the recent development of EST-SSRs,a larger number of SSRs are now available to counter the constraint of modest level of polymorphism in the A genome.The SSRs and EST-SSRs and comparison of linkage map and QTL between tetraploid and diploid provide structural and functional genomic information that will be useful for dig of potential gene in Asian cotton,understanding the origin and evolution of allpolyploid,studying the genome structure of important agronomic trait and the relationship of gene structure and function,elucidating the influence to the trait by the interaction between genes,which is important in crop improvement.In this study,198 F₂ and 176 F_2:3 population were developed by single seed descended from the cross of two Asia cotton cultivars(Gossypium arboreum L.)(Jianglingzhongmian and Zhejiangxiaoshanliishu).With the two parents,F₁and F₂,the multiple-PCR techniques were studied;A-genome diploid cotton map was constructed with SSR markers,the function of some genomic SSRs and EST-SSRs were analyzed;the major gene plus polygene mixed inheritance model was used to analyze the genetics of 6 main fiber qualities and 5 main yield traits by using multiple generations of P₁,P₂,F₁,F₂ and F_2:3 of the crosses;furthermore,QTL for important traits were tagged using multiple-QTLs model based the data of the F₂ and F_2:3 populations.The results were listed as the following:1.SSR multiple-PCRMultiple-SSR PCR is an efficient and economical method of PCR because several markers can be detected exactly in one production as single-pair primer PCR but with less consuming of reagent and time.Two Asia cotton cultivars(Gossypium arboreum L.),F₁ and F₂ population were analyzed by SSR markers multiple-PCR and single-pair SSR marker PCR techniques based on silver staining.Six pairs of SSR primers selected were used in PCR amplification under three kinds of two-plexe multiple PCR and five different reaction systems.Different types of two-plexe post-PCR detection were also analyzed.As the result,the products of two-plexe multiple PCR were same under the five reaction systems,and the polymorphic loci in two-plexe multiple-PCR products can be detected as same as single-pair primer PCR and two-plexe post-PCR detection.The data can be used for further employment of multiple-SSR PCR in mapping for cotton efficiently.2.Construction of Asiatic cotton intraspeciflc molecular mapA genetic linkage map of A-genome diploid cotton was constructed with a newly developed molecular marker-SSR(simple sequence repeat) using a population consisting of 189 F₂ individuals derived from the intraspecific cross of two Asiatic cotton cultivars (Gossypium arboreum) Jianglingzhongmian(JLZM) and Zhejiangxiaoshanlushu (ZJXSLS).A total of 6092 SSR primer pairs were used to detect polymorphisms between the two parents and 268 primer pairs with better polymorphisms were picked out to analyze the F₂ population.320 polymorphic bands were generated in total,and used to construct linkage map with JoinMap3.0 and 267 loci including three phenotypic traits were mapped at a LOD=3.5 on 13 linkage groups.The total length of the map is 2508.71 cM,and the average distance between adjacent markers is 9.40 cM.All the markers are distributed evenly among the linkage groups without clustering of loci.Chromosome assignments were according to the association of linkages with our backbone tetraploid specific map using the 89 similar SSR loci.Comparisons among the 13 suites of orthologous linkage groups revealed that the A-genome chromosomes are largely collinear with the At and Dt sub-genome chromosomes accompanied with some inversions suggesting that allopolyploidy accompanied by homologous chromosomal rearrangement,and the interchromosome duplicated loci supply a molecular evidence that the A-genome diploid Asiatic cotton is paleopolyploid and support the result of other researchers.3.Major gene and polygene genetic analysis of Asiatic cotton fiber and yield qualityThe method of major gent plus polygene mixed inheritance model was used to analyze the genetic of 6 main fiber qualities and 5 main yield traits in the crosses Jianglingzhongmian×Zhejiangxiaolushu was made among two Asiatic cotton parents, which are landraces of Gossypium arboretum L..One parent with long fiber length,high fiber strength,and the other parent with short fiber length and low fiber strength.The results of analysis of multiple generations of P₁,P₂,F₁,F₂ and F_2:3 of the crosses showed the optimum inheritance model in six major fiber qualities and five yield traits characters: fiber length,fiber strength,Micronaire,fiber uniformity,short fiber index,fiber elongation, bolls/plant,boll weight,lint percentage,seed index,cotton wcight/plant.The optimum inheritance model in fiber length,fiber strength,Micronaire,fiber uniformity,short fiber index,seed index are all polygenes models without major gene,where the optimum inheritance model in fiber elongation,bolls/plant,boll weight,lint index,cotton weight/plant are two major genes plus polygenes mixed inheritance models.4.QTL mapping of fiber qualities,yield traits and yield-components in Asiatic cottonBased on the Asiatic cotton intraspecific map,QTL for all traits were analyzed by multiple QTLs model(MQM) method using MapQTL5.0.Twelve significant and 21 putative QTLs for yield traits and yield components were detected in F₂ population,and 12 significant and 29 putative QTLs for yield traits and yield components were detected in F_2:3 families,2 QTLs for cotton-seed weight/plant,2QTLs lint index and 3 QTLs for seed index were consistent in the two generation.Fourteen significant and 20 putative QTLs for fiber qualities were detected in F_2:3 families,among them,52%of them were located both of the corresponding At-subchromosomes and Dt-subchrosomes of tetraploid cotton,5%of them were located only on the corresponding At-subchromosomes,and 20%of them were located only on the corresponding Dt-subchrosomes of tetraploid cotton.This suggested that conservation of evolution of the QTLs for fiber qualities,the genetic base for fiber quality of diploid may be same with tetraploid.It was assumed recombination event occurred randomly in At or Dt subgenome homologous region during the process of the tetraploid cotton.Most QTLs for fiber qualities and yield traits were located at the same interval or neighbor interval,especially at A2.A2 carried 3 QTLs for fiber length,2 QTLs for cotton weight/plant,1 QTL for strength,fiber uniformity,short fiber index and fiber elongation, respectively.This reveal that the negative correlation between fiber qualities and yield traits may be the result either as pleiotropic effect of one gene or correlated effects of multiple closely-linked genes.Probably the greatest value of MAS is in the reduction of linkage drag during introgression.