Identification And Development Of Polymorphic EST-SSR Markers In Pepper

The amount of EST derived form pepper in public available sequence database grows dramatically, which offers abundant sequence resource for the development of SSR markers. While, the utilization fo pepper EST sequence information was very limited. In the study, a set of polymorphic EST-SSR loci markers was identified and subsequently validated using about120000pepper EST sequences in NCBI.Firstly, the bioinformatic tools and data mining methods were employed to analyze the occurring and distribution characteristics of EST-SSR in pepper. A total of30759Unigenes covering23.12Mbp were obtained after process and assembly of118060pepper ESTs retrieved from NCBI.3758EST-SSR loci were identified on the Unigenes with the occurring frequency of1/6.15kb. The amount of EST-SSR loci with dinucleotide motif and trinucleotide motif took account of70.7%and26.8%of the total respectively. There were83.5%EST-SSR loci with the repeat number no more than10times. AG/TC, taking account of43.8%of the total, was the dominant motif type. AAG/TTC was the most frequent occurring trinucleotide motif, accounting for6.8%of the total. The distribution density of EST-SSR with dinucleotide motif was20/10kb in5’UTR, which was higher than that of8/20kb in CDS. However, the EST-SSR with trinucleotide motif, the distribution density was4/10kb, which was comparable with the5/10kb in CDS. The results offered valuable information for SSR marker development in pepper.Secondly, the characters of redundancy and heterozygosity of EST sequences in public sequence database were used to identify a set of polymorphic EST-SSR loci by sequence alignment and a set of polymorphic EST-SSR markers were subsequently validated. The contigs containing the redundant EST sequences were screened and a total of68polymorphic SSR loci were identified,65of which had suitable sequence length for primer design. The polymorphisms of the putative polymorphic SSR loci were validated by31pepper genotypes, resulting in33polymorphic EST-SSR loci. The repeating number of the motif for the polymorphic loci ranged from2to10times. There were18loci with the motif repeating number less than5times, accounting for55%of the total. There are27EST-SSR loci having significant homology with the genes with known functions, which covered the physiological proceedings of seed maturity, stress response and so on. There were7,18and15EST-SSR that could transfer to eggplant, tomato and potato respectively. A total of14EST-SSR markers had the transferability on all the corps of eggplant, tomato and potato. Meanwhile, there were6EST-SSR markers that could not transfer to either of eggplant, tomato and potato. The results not only offered a set of SSR markers locating in the transcript regions for pepper genetic breeding researches, but also provided experimental evidences for further mining the sequence information in public database and developing the SSR markers with the low repeating number.Finally, the molecular genetic diversity of31pepper varieties was analyzed by using33polymorphic EST-SSR markers.91alleles were amplified by the33polymorphic markers. The maximum of6and average of2.76alleles per locus were detected respectively. The average observed and expected heterozygosities were0.28and0.39respectively. The polymorphic information content (PIC) ranged from0.03to0.74with the means of0.38. The cluster analysis showed that those varieties could be divided into2major groups according to0.48Jaccard’s similarity. The first group had5varieties,most of which were Capsicum Chinense Jacquin, and the second group had26varieties which could be divided into6subgroup. The results of principal coordinates analysis were basically consistent with the UPGMA cluster. Furthermore, the cluster analysis based on EST-SSR markers revealed that the genetic similarity was not correlation with the morphologic traits of the fruit, such as fruit type, maturity, hot taste, etc.