Development And Characterization Of Microsatellite Markers For Phylogenetic Analysis Of Musa

In the study, genomic-SSRs and EST-SSRs were developed by using selectively amplified microsatellite (SAM) analysis and searching the ESTdb of NCBI. Meanwhile, those SSR markers were evaluated in a collection of 11 related species/subspecies of Musaceae about 82 banana accessions and 7 M. itinerans populations. The main results and conclusions were as follows:(1) Analysis of 2284 ESTs identified 110 sequences containing 122 SSRs. Among them, there were 37 motifs. The overall average length of SSRs was 20 bp. The trinucelotide repeats appeared to be the most abundant SSRs (47.6%), followed by dinucelotide repeats (33.1%), whereas the tetrnucelotide repeats were less abundant. The rich repeats AG and AGG were predominant, accounting for 75.7% and 36% in dri-and tri-nucelotide repeats respectively. A total of 63 primer pairs were designed and tested against genomic DNA of brazil. Forty-one primers could be validated as usable markers. Nineteen of these primers revealed moderate to high polymorphism information content (PIC) across 49 accessions. The polymorphic bands were ranged from 4 to 12, averagely 7.58. The mean PIC value was 0.7324, ranging from 0.3572 to 0.8744. Forty-nine banana varieties were separated into two major clusters closely corresponding with the genome composition by UPGMA analysis. The results showed that the EST-SSR markers were of great value in evaluation of banana germplasms.(2) Eighty-three and thirty-eight microsatellite sequences were cloned and sequenced from a commercial cultivar’Gongjiao’ (Musa acuminate, AA froup) and’Yejiao’(M. balbisiana, BB group) respectively, using selectively amplified microsatellite (SAM) analysis. A total of 62 microsatellite primers were designed (38 from AA group and 24 from BB group). These primers were pre-screened on’Gongjiao’and’Yejiao’. From those,26 from AA group and 19 from BB group produced clear repeatable amplification patterns and were used to all the samples.(3) A set of 45 Genomic-SSR and 24 EST-SSR markers was identified in a collection of 22 cultivated bananas and 11 related species/subspecies. The results showed that EST-SSR is more polymorphic than Genomic-SSR. But both of them can provide accurate evaluation of genetic relationship among accessions. The ratio of the cross-species/genera amplification detected by Genomic-SSR and EST-SSR were 80% and 70.83% among 11 related wild species representing 10 different species, representative of three important genus of Musaceae family. To further demonstrate the transferability of the developed SSR loci, Sequence comparison of cross-species/genus amplicons generated by four SSR loci for 9-11 related wild species was done. In general, the amplified regions were found to be homologous to the original sequences (from which the SSRs were developed) and their comparisons across species. Sequencing of PCR products confirmed the presence of microsatellite repeats at all loci. Sequencing alleles at four loci showed that, apart from microsatellite length variation, point mutations and insertion/deletions were quite abundant in the flanking region of the alleles sequenced.(4) To well evaluate and use the cultivars, it is necessary to identify them accurately by DNA molecular markers. A total of 56 banana accessions were investigated in this study. Five pre-selected SSR primers showed ample polymorphism and could distinguish all the materials studied. The number of polymorphic bands varied between 11 and 16 among the 56 accessions with an average of 13.8. The mean of polymorphism information content (PIC) values was 0.80, ranging from 0.45 to 0.91. Due to the complex SSR patterns in banana, it is difficult to score each band with number 1 and 0. Here, we scored each SSR pattern with an alphanumeric code instead of with pairs of 1 and 0. By arranging all of pattern numbers, we can build the fingerprint code for every banana accession.(5) On the population level, Na=4.0, Ne=1.5404, Ho=0.3038, He=0.2775. The genetic diversity of Jiujialing population was most (Na=3.40, Ne=1.7054, Ho=0.3043, He=0.3633) and Atuoling population was the least (Na=2.20, Ne=1.4050, Ho=0.2296, He=0.2633). A low level of genetic differentiation among populations was detected based on Nei’s genetic diversity analysis (Fst=0.0677) and main variations was within population (93.23%). UPGMA cluster analysis based on Nei’s genetic distance divided the 7 population into two main groups.