Mapping of Li1 Gene Based on the Combination of PCR-SSCP Technique and Comparative Genomics in Cotton (Gossypium spp.)
Polymerase chain reaction-single strand conformation ploymorphism (PCR-SSCP),as an important technique in detecting minor differences between genomic DNA bases,has the characteristics of simplicity,rapidity and sensitivity.So far,a large number of genome sequences provide lots of DNA sequences for the development of novel genetic markers through comparative genomics and the mapping of target genes.Hence,the possibility was investigated on the application of PCR-SSCP technique combined with comparative genomics to develop novel molecular markers in cotton (Gossypium spp.) genetic mapping in the study,taking Ligon lintless-1 (Li1) mutant gene mapping as an example and the published sequences of Arabidopsis and cotton genome D as references.The optimization of SSCP conditions for samples of large fragment PCR products (＞400 bp) were carried out in the experiment.The results showed that the optimal experiment conditions were as follows:voltage of 150 V,gel concentration of 10％,electrophoresis temperature of 4 ℃,and 5 ∶ 1 of loading dye to PCR product.According to the linear segments of Arabidopsis and cotton,we selected 102 cotton EST sequences to develop the SSCP primers.The optimized PCR-SSCP system was utilized to construct a genetic map consisting of 28 markers and spanning 149.6 cM.This genetic map contained 4 genes with functions closely related to Li1 loci.Additionally,using genome D sequences,185 SSCP primers were developed and there were 86 primers showing polymorphism between the two parents (Li1 and H7124).In this experiment,Li1 gene was firstly mapped in a genetic map consisting of 17 SSCP markers and the Li1 loci,which spanned 40.3 cM.The Li1 gene was flanked by W058 and P095,which were the nearest markers,and the distance from Li1 loci was 0.6 and 0.3 cM respectively.Using the published Li1 genetic map to analyze the relationship between D genome sequences and two genetic maps constructed in this experiment,the study suggested that there were good homologous between these genetic maps.The results presented here could offer a useful foundation for future research work on fine mapping and cloning of Li1 gene.In addition,with the development of the third generation genome sequencing technology,constantly enrichment of the GenBank and the publishment of cDNA sequences,the optimization of SSCP technology plays an important role in cotton genetic linkage map construction,map-based cloning,molecular markerassisted breeding and other aspects.
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维普
