Analysis of SSRs Information in Capsicum spp.from EST Database

SSR markers are useful in pepper linkage mapping and gene location.446 SSR markers have been reported,but they are insufficient.It is costly to develop SSR markers from DNA library,whereas it seems much easy to find in EST sequences in the GenBank of pepper through internet.In this study,attempts have been made to develop SSR markers in the EST sequences by using bioinformatics.EST sequences were trimmed by ‘est-trimmer.pl' software,while 116 915 EST sequences were obtained without poly ‘A' or poly ‘T',ranged between 100 and 700 bp.Using ‘e-PCR' and ‘del.pl' softwares,SSR sequences were identified.2 508 microsatellite loci (larger than 20 repeats) were established and 755 SSR primers were designed using SSR finder software and Primer 3 software.There were 498 (0.43％) mono-,1 026 (0.89％) di-,5 1 8 (0.45％) tri-,245 (0.21％) tetra-,114 (0.10％) penta-,and 107 (0.09％) hexa-nucleotide SSRs.The estimated frequency of SSRs was approximately 1/25.12 kb.According to the distribution of SSRs in pepper,the mean length of pepper SSRs was 22.68 bp and the adenine rich repeats such as A/T,AG,AT,AAG,AAAT,and AAAC were predominant in each type of SSRs (mono-,di-,tri-,tetra-,penta-,and hexa-),whereas the C/G,CG,CCG repeats were less abundant.210 primers were tested in 8 pepper cultivars and the PCR result revealed the existence of polymorphism among 127 (60.48％) SSR primers within 8 pepper cultivars.It is confirmed that pepper EST database could be efficiently exploited for availability of SSR markers.     

高粱非编码区SSR引物设计以及e-PCR的验证

本研究通过在NCBI公共数据库的高粱全基因序列中进行简单重复序列SSR搜索,结果显示共有87950个SSR位点,共有1134种重复,其中二元碱基和三元碱基重复所占比例最大,分别占43.36%,32.38%。根据SSR搜索结果设计了高粱SSR引物,通过两次e-PCR验证和筛选引物,随机合成了50对引物。以先锋和乐食两个高丹草品种为材料进行引物的筛选和评价。结果表明,50对引物中有46对引物出带,但只有8对引物具有多态性。这说明在高粱非编码区设计的引物多态性并不高于EST-SSR引物,但对高粱的SSR引物的开发可以起到补充和促进作用。     

基于e-PCR的小麦、山羊草属SSR电子指纹图谱的开发

电子指纹图谱的绘制可对电泳结果进行规范化、可视化操作，进而提高小麦分子标记的流通性，加速小麦育种进程。本研究用MicroSAtellite (MISA)软件对小麦属、山羊草属共61个叶绿体和13个线粒体的全基因组SSR标记进行搜索，发现SSR位点的出现频率与基因组类型有关，线粒体、叶绿体基因组均以2次重复的五核苷酸和六核苷酸重复基序为主，同一种类型的基序重复次数与SSR数目成反比；基于电子PCR技术 (e-PCR)并结合MapChart软件共筛选出30对引物，其中来源于二倍体和四倍体山羊草属叶绿体全基因组的引物分别有16和2对，来源于四倍体和六倍体小麦属叶绿体全基因组的引物分别有5和2对，来源于六倍体小麦属线粒体全基因组的引物有5对，最后用MapChart软件绘制电子指纹图谱。本研究采用了一种新的SSR引物设计和筛选方法，完成了小麦属、山羊草属叶绿体和线粒体共74个全基因组序列SSR分子标记的初步开发和电子指纹图谱的绘制，为引物开发提供了新思路，有利于提高分子标记的流通性以及小麦种质资源亲缘关系的鉴定。