Genetic structure,gene flow pattern,and association analysis of superior germplasm resources in domesticated upland cotton(Gossypium hirsutum L.)

Gene flow patterns and the genetic structure of domesticated crops like cotton are not well understood.Furthermore,marker-assisted breeding of cotton has lagged far behind that of other major crops because the loci associated with cotton traits such as fiber yield and quality have scarcely been identified.In this study,we used 19 microsatellites to first determine the population genetic structure and patterns of gene flow of superior germplasm resources in upland cotton.We then used association analysis to identify which markers were associated with 15 agronomic traits(including ten yield and five fiber quality traits).The results showed that the upland cotton accessions have low levels of genetic diversity(polymorphism information content=0.427),although extensive gene flow occurred among different ecological and geographic regions.Bayesian clustering analysis indicated that the cotton resources used in this study did not belong to obvious geographic populations,which may be the consequence of a single source of domestication followed by frequent genetic introgression mediated by human transference.A total of 82 maker-trait associations were examined in association analysis and the related ratios for phenotypic variations ranged from 3.04% to 47.14%.Interestingly,nine SSR markers were detected in more than one environmental condition.In addition,14 SSR markers were co-associated with two or more different traits.It was noteworthy that NAU4860 and NAU5077 markers detected at least in two environments were simultaneously associated with three fiber quality traits(uniformity index,specific breaking strength and micronaire value).In conclusion,these findings provide new insights into the population structure and genetic exchange pattern of cultivated cotton accessions.The quantitative trait loci of domesticated cotton identified will also be very useful for improvement of yield and fiber quality of cotton in molecular breeding programs.

Generation of mouse anti-human urate anion exchanger antibody by genetic immunization and its identification

Background Human urate anion exchanger (hURAT1) as a major urate transporter expressed on renal tubular epithelial cells regulates blood urate level by reabsorbing uric acid. Antibody is an important tool to study hURAT1. This study aimed, by genetic immunization, to produce mouse anti-hURAT1 polyclonal antibody with high throughput and high specificity and to detect the location of hURAT1 in human kidney.Methods Human renal total RNA was isolated and the entire cDNA of hURAT1 was amplified by RT-PCR. The sequence of intracellular high antigenicity fragment (A280 to R349) was chosen by prediction software of protein antigenicity, and its cDNA was amplified from cDNA of hURAT1, and then cloned into pBQAP-TT vector to construct recombinant plasmid pBQAP-TT-hURAT1-210 for genetic immunization. Mice were inoculated with this recombinant plasmid and two other adjuvant plasmids, pCMVi-GMCSF and pCMVi-Flt3L, which helped to enhance the antibody’s generation. After four weeks, the mice were sacrificed to obtain the anti-hURAT1 antibody from serum. The antibody was identified by western blot analysis and immunohistochemistry. At the same time, rabbit anti-hURAT1 antibody was produced by protein immunization. The specificity and efficiency between the rabbit and mouse anti-hURAT1 antibody were compared by western blot analysis and immunohistochemistry. Results The entire cDNA of hURAT1 and cDNA of its intracellular high immunogenic fragment were amplified successfully. Recombinant plasmid pBQAP-TT-hURAT1-210 for genetic immunization was confirmed by restriction digestion and sequencing. Both!the mouse anti-hURAT1 antibody and rabbit anti-hURAT1 antibody recognized 58kD hURAT1 and 64kD glycosylated hURAT1 protein bands in western blot. Immunohistochemically, hURAT1 was located at the brush border membrane of renal proximal tubular cells. In addition, the throughput and specificity of the mouse anti-hURAT1 antibody were higher than those of the rabbit anti-hURAT1 antibody.Conclusion Genetic immunization can generate anti-hURAT1 polyclonal antibody of high throughput and specificity.