基于芭蕉属EST序列的地涌金莲SSR引物开发

Development of SSR Markers of Musella lasiocarpa by Data Mining in Musa EST Sequences

对31 308条来自NCBI的芭蕉属(Musa)EST序列进行拼接得到全长为13.51 Mb的21 129条无冗余EST序列(含3 818条contigs及17 311条singletons),其中,4 944条(23.40%)EST序列含有5 416条SSRs,SSR的出现频率为25.63%,平均分布距离2.49 Kb,有234条(1.11%)含有1个以上的SSR。在所检测的SSR中,二、三、四核苷酸重复是主导重复类型,分别占EST-SSR总数的21.80%(1 181条)、52.55%(2 846条)和14.55%(788条)。AG/CT、AAG/CTT与AGG/CCT和AAAG/CTTT与AAAT/ATTT分别是二、三、四核苷酸的优势重复基元。随机设计了238对EST-SSR引物在24个地涌金莲个体中进行筛选,116对有扩增产物,其中,78对EST-SSR引物扩增出清晰稳定的目的片段,49对引物表现出多态性。本研究检测的15对引物扩增等位基因数范围是2～7个,平均3.067个;表观杂合度(Ho)范围是0.042 0.750,平均0.250;期望杂合度(He)范围是0.232～0.823,平均0.522。

21 129 non-redundant clusters,containing 3 818 contigs and 17 311 singletons,were identified from a total of 31 308 publicly available Musa EST sequences.4 944（23.40%） of them contained 5 416（25.63%） SSR motifs,and the di-（21.80%）,tri-（52.55%） and tetra-nucleotide（14.55%） are the main motifs of all SSRs obtained.AG/CT repeats were dominant in di-nucleotide motif,AAG/CTT and AGG/CCT repeats were dominant in tri-nucleotide motif,AAAG/CTTT and AAAT/ATTT repeats were dominant in tetra-nucleotide motif.238 EST sequences were randomly selected on molecular markers development,for PCR amplification and polymorphism analysis in Musella lasiocarpa.Of which,116 pairs of SSR primers successfully amplified PCR product and 78 pairs gave clear bands.49 of them were found to be polymorphic.A set of 15 polymorphic SSR markers from above 49 SSR markers selected were analyzed using 24 individuals from 4 wild M.lasiocarpa populations.The average allele number of 3.067 per locus was detected with a range from 2 to 7.The observed heterozygosities per marker were ranged from 0.042 to 0.750（mean 0.250） and expected heterozygosities were 0.232 to 0.823（mean 0.522）.