Development Of EST-SSR Markers And Genetic Diversity Analysis In Nanyang Yellow Cattle

Nanyang Yellow Cattle is one of the five best cattle breeds in China.In recent years,this indigenous cattle breed encounters problems such as breed depression and the decrease in genetic diversity.The investigation and analysis of the current status of genetic diversity of Nanyang Yellow Cattle is a key prerequisite for the sustainable development and reasonable utilization of this important local germplasm resources.DNA polymorphisms,such as microsatellites(simple sequence repeat,SSR),play an important role in assessing genetic diversity in various populations.In this study,we searched for SSR loci from known bovine expressed sequence tags(EST),and selected SSR-containing sequences related to important economic traits for polymerase chain reaction(PCR)primer design.Genomic DNA was then collected from different bovine breeds to screen for polymorphic SSR markers that could be used to detect the genetic diversity of Nanyang Yellow Cattle.The results of this study are as follows:1.A total of 1387262 cattle(Bos taurus)EST sequences were retrieved from the GenBank database,and 45364 non-redundant UniGene sequences were obtained after splicing.A total of 4453 SSR loci containing 2-6 base repeats were obtained and distributed over 3660 sequences.An average of 12.71 Kb in the bovine transcript sequence will result in an SSR locus with a detection rate of 8.07%.The obtained bovine EST-SSR was mainly double base(49.02%)and three base(47.43%)repeats,of which the core sequence AT/TA(16.45%)was the most common.2.Sequence alignment and structural annotation of 3660 sequences containing SSR loci were performed by blastx and interproscan,respectively.It was found that 2346 bovine transcript sequences containing SSR loci could find homologous protein products.Based on the gene ontology database,the functional annotation and cluster analysis of these sequences were performed,and sequences related to bovine growth and development,meat quality and reproduction were selected.The pairs of PCR primers were designed to amplify the SSR locus.3.Blood samples were taken from multiple samples of various cattle breeds,including Nanyang Yellow cattle,Simmental cattle,Charolais Cattle,Piedmontese cattle and Xinjiang Cattle.Genomic DNA was then isolated and amplified by PCR using above-mentioned SSR primers.The products were separated by polyacrylamide gel and stained by silver staining and fluorescence staining,respectively.The results showed that:(1).The silver-stained method and the fluorescent staining method revealed similar DNA band patterns.The procedure of fluorescent staining is more convenient than that of silver staining,and could replace the traditional silver staining method for SSR electrophoresis observation.(2).The ten pairs of SSR primers can produce effective amplification in all samples.Among them,primers for SSR4,which is located in insulin-like growth factor-2can produce differential amplified fragments between different varieties of Nanyang yellow cattle and the other cattle breeds,indicating this SSR can be used as effective markers for the study of genetic diversity of Nanyang yellow cattle.