日本沼虾ITS1序列分析及SNPs位点的筛选

SEQUENCING OF ITS1 AND IDENTIFICATION OF SNPS LOCI IN MACROBRACHIUM NIPPONENSE

研究采用直接测序法,分析日本沼虾(Macrobrachium nipponense)rDNA基因内转录间隔区ITS1的DNA序列,以筛选日本沼虾SNPs位点。共分析了32个太湖水域野生日本沼虾样本,结果表明,日本沼虾ITS1序列平均长度为1749.8bp,是迄今已报道的最长的ITS1序列,A、G、T和C的平均含量分别为29.9%、28.3%、27.7%、14.0%,G+C的含量平均为42.3%。通过序列比对,共筛选出22个SNPs位点,SNPs位点出现频率为0.0126,其中9个为C/T转换(占40.91%),4个为A/G转换(占18.18%),2个为A/T颠换(占9.09%),5个为T/G颠换(占22.73%),1个为A/C颠换(占4.55%),1个A/T或C颠换(占4.55%)。日本沼虾ITS1序列的22个SNP位点中,21个位点为2个等位基因,1个位点出现了3个等位基因,为复等位基因位点。日本沼虾ITS1序列中还发现3个具有多态性的微卫星位点、1个高度变异区以及大量的缺失、插入。研究首次对日本沼虾ITS1序列进行了分析,并发现了大量的SNP位点,为日本沼虾遗传育种研究提供了新的分子标记。

Macrobrachium nipponense is one of the most important freshwater prawns for aquaculture in China, especially in the southern regions of the country. But in recent years, 34. nipponense farming has seriously declined due to diseases and small body size. Investigation of genetic background and genetic selection may help this situation for M. nipponense, and development of genetic marker is the basis. Single nucleotide polymorphisms （SNPs） is a type of genetic marker based on single DNA base differences between individuals at defined positions. The possible alleles for a SNP locus are four bases of A, T, C and G. SNPs is a new molecular marker with high value because of its abundance, genetic stability and easy to check. In agricultural economical animals, SNPs were developed in livestock, domestic birds as well as aquatic animals, including fish, shrimp, crab and shellfish. The reported applications of SNPs included determination of relativeness, survey of genetic polymorphisms, QTL localization, marker-assisted selection, etc. So far, no work was reported on development and application of SNPs in M. nipponense. Nuclear rDNA internal transcribed spacer （ITS） were composed of ITS1 fragment between 18S and 5.8S rRNA and ITS2 fragment between 5.8S and 28S rRNA. In this paper, ITS1 sequences were analyzed and SNPs in ITS1 were screened in M. nipponense. Totally 32 samples was sequenced, and the average length of ITS1 sequence was 1749.8bp, which was the longest among reported ITS1 sequences. The average contents of A, G, T and C was 29.9%, 28.3%, 27.7% and 14.0% respectively, and that of G＋C was 42.3%. By comparison of sequences from different individuals, 22 SNP loci were found, including 9 C/T transitions （40.91%）, 4 A/G transitions （18.18%）, 2 A/T transversions （9.09%）, 5 T/G transversions （22.73%）, 1 A/C transversions （4.55%）, 1 A/T or C transversion （4.55%）. Among 22 SNP Loci, 21 loci were two alleles, and 1 was three alleles In addition, 3 SSR loci with polymorphisms, 1 highly variable segment （243-384 bp）, and a great deal of deletions and insertions were found in the ITS1 sequences of M. nipponense. Total 130 deletions and 124 insertions were found, and deletion length was between 1-13 bp, insertions length between 1-80 bp. This paper firstly analyzed the ITS1 sequence of M. nipponense, and filled the gap of its ITS 1 characterization, and it also filled the gap of SNP loci development in M. nipponense, and provided new available molecular markers for research of genetics and genetic breeding in M. nipponense.