| Cotton (Malvaceae Gossypium) is an important cash crop and the uppermost source of textile fiber. Along with the society’s advance, cultivation of varieties with high yield and super fiber quality is becoming increasingly important. However, it is difficult for conventional breeding methods to synchronously improve several characters in a short time. With rapid development of molecular biology and biotechnology, combination of molecular breeding technology and conventional breeding technology may be an efficient approach to cotton breeding.Laying molecular marker technology at the core, this study launched three parts of work aiming at providing germplasm resource and theoretical direction to cotton breeding:(1) construction of introgression lines in cotton;(2) development and evaluation of new markers in cotton;(3) construction of high-density genetic linkage map in cotton.1. Construction of introgression lines in cottonIn this study, introgression lines were constructed using molecular marker-assisted selection, with substitution segments of plants checked by515markers evenly selected from the linkage map. Up to2012,82.79%of the total337plants harbored only three or less substitution segments. Totally speaking, substitution segments from the donor parent (3-79) comparatively well covered the cotton genome, with coverage of77.67%. However, coverage in different chromosomes varied from57.14%(Chr05) to100.00%(Chr23). In addition, numbers of substitution segments in different plants also varied greatly, ranging from1to10. It is expected that ILs constructed in this study will greatly accelerate research progress of quantitative traits, and will provide new valuable resource for cotton breeding.2. Development and evaluation of new markers in cotton(1) Development of functional markers related to cotton fiber developmentA total of331gene primers and164protein primers were designed from functional sequences related to cotton fiber development. After genotyping using SSCP method,52primers (10.51%) showed polymorphism, and produced58polymorphic loci. After linkage analysis,53loci representing48genes or proteins were distributed randomly throughout the cotton genome, indicating that cotton fiber development was regulated by the whole genome. Besides, GO analysis of functional sequences also illustrated the complexity of fiber development. Both RT-PCR analysis and qRT-PCR analysis of polymorphic primers obtained similar expression tendencies with previous reports, and they also support field experimental results obtained in our laboratory. These expression patterns may provide some guidance to improve fiber quality of G. hirsutum, which could be accomplished by transforming those genes preferentially expressed in G. barbadense and controlling fiber quality obviously.(2) Development of SNP and IDP markers in cottonA total of1349SNP/IDP markers were developed using in-silico analysis in this study, including455Ghi-prefixed primers,356HAU-SNP-prefixed primers,415GhlDP-prefixed primers, and123PCtIDP-prefixed primers. After genotyping using SSCP analysis,137primers (10.16%) showed polymorphism, and produced142polymorphic loci. Genetic mapping showed that133polymorphic loci randomly distributed throughout the cotton genome, which is in accordance with the fact that SNPs/IDPs always pervade the whole genome, and indicates that SNP and IDP markers are valuable resource to increase density of linkage map. In addition, mapped SNPs and IDPs also could supplement previous research of segregation distortion. Statistic analysis of base substitutions showed that bias of base transitions exists in cotton genome, with the fact that base transitions occupied55.78%of the total SNPs. Functional accumulation analysis showed that numbers of SNPs in different functional categories varied obviously, which may provide some guidance to research of genes belonging to different functional categories. Generally speaking, in-silico analysis is a double-faced method to develop SNP markers in cotton revealed by sequence analysis, as the practical SNPs were not always in accordance with the predicted ones.(3) Development of subgenome-specific markers in cottonA total of260At-subgenome-specific primers and545Dt-subgenome-specific primers were developed in this study. SSCP analysis made25(9.62%) At-subgenome-specific primers and25(4.59%) Dt-subgenome-specific primers produce50polymorphic loci, indicating that difference between G. hirsutum and G. barbadense may mainly originate from the At-subgenome. After linkage analysis,15loci of the24mapped Ga-Gh-prefixed loci were located on the At-subgenome, and other9loci were located on the Dt-subgenome. As far as the21Gr-Gh-prefixed mapped loci,15of them were located on the At-subgenome, while only6loci were located on the Dt-subgenome. Reasons why experimental results deviated from anticipation tremendously are as follows. On the one hand, sequence shortage of cotton resulted in the fact that designed primers were not really specific to subgenome. On the other hand, unfavorable results may largely originate from complexity of the cotton genome-high homology/collinearity between homologous chromosomes of the At-subgenome and Dt-subgenome.3. Construction of high density genetic linkage map in cottonA total of13507primers, including new published SSRs in CMD (http://www.cottonmarker.org/), new kinds of markers reported by previous researchers, and three parts of primers designed in this study, were used to detect polymorphism between the two mapping parents using two genotyping methods. As a result,2498(18.49%) primers showed polymorphism, and produced2751polymorphic loci. Gathering the original2528polymorphic loci obtained by Yu (2011) and other353polymorphic loci related to mic-RNAs, targets, transcription factors, anti-disease genes, cell development and so on, a total of5632polymorphic loci were used for linkage analysis using Joinmap3.0. Finally, a high density interspecific linkage map including5152loci was constructed, with4696.03cM in total length and0.91cM in average distance between adjacent markers.Colinearity analysis among cotton chromosomes showed that it harbored the highest value between homologous chromosomes. However, comparatively high colinearity appeared between non-homologous chromosomes in14chromosomes which are homologous chromosomes in pairs. It indicates that this study is useful to analyze complex genome of cotton, and is helpful to research evolution history of cotton.Comparative analysis between allotetraploid cotton and diplontic cotton (G.raimondii) showed that most of the allotetraploid cotton chromosomes (except Chr03and Chr04) had the highest autoploidy with homologous chromosomes of the diplontic cotton. Comparative analysis between allotetraploid cotton and T. cocoa showed that percentage of successfully blasted markers was46.55%, and Tc01of T.cocoa was the most frequent chromosome which had the highest autoploidy with chromosomes of cotton. Comparative analysis between allotetraploid cotton and Arabidopsis showed that percentage of successfully blasted markers was13.74%, and Atl of Arabidopsis was the most frequent chromosome which had the highest autoploidy with chromosomes of cotton. Totally speaking, all above provides revelation and guidance to analysis of complex genome of allotetraploid cotton, through exploiting genome sequences of G.raimondii, T.cocoa and Arabidopsis. |
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