An improved 2b-RAD approach (I2b-RAD) offering genotyping tested by a rice (Oryza sativa L.) F2 population

BMC Genomics

Hongliang Zhang³  Xun Xu¹  Zichao Li³  Jianping Yu³  Lu Wu¹  Yong Liu¹  Yan Liu²  Lianbo Song³  Dongming Fang¹  Hui Yuan¹  Yu Guo¹ 

[1] Beijing Genome Institute-Shenzhen, Beishan Industrial Zone, Yantian District, Shenzhen 518083, China;National Institute of Biological Sciences, Beijing, Zhongguancun Life Science Park, Changping District, Beijing 100026, China;Beijing Key Laboratory of Crop Genetic Improvement, College of Agronomy and Biotechnology, China Agricultural University, Beijing 100193, China

关键词: Genetic linkage map;    Genotyping;    2b-RAD;   

Others  :  1127734
DOI  :  10.1186/1471-2164-15-956

 received in 2013-11-06, accepted in 2014-10-24,  发布年份 2014

【 摘 要 】

Background

2b-RAD (type IIB endonucleases restriction-site associated DNA) approach was invented by Wang in 2012 and proven as a simple and flexible method for genome-wide genotyping. However, there is still plenty of room for improvement for the existent 2b-RAD approach. Firstly, it doesn’t include the samples pooling in library preparation as other reduced representation libraries. Secondly, the information of 2b-RAD tags, such as tags numbers and distributions, in most of species are unknown. The purposes of the research are to improve a new 2b-RAD approach which possesses samples pooling, moreover to figure out the characteristic and application potentiality of 2b-RAD tags by bioinformatics analysis.

Results

Twelve adapter1 and an adapter2 were designed. A library approach comprising digestion, ligation, pooling, PCR and size selection were established. For saving costs, we used non-phosphorylated adapters and indexed PCR primers. A F2 population of rice (Oryza sativa .L) was genotyped to validate the new approach. On average, 2000332 high quality reads of each sample were obtained with high evenness. Totally 3598 markers containing 3804 SNPs were discovered and the missing rate was 18.9%. A genetic linkage map of 1385 markers was constructed and 92% of the markers’ orders in the genetic map were in accordance with the orders in chromosomes. Meanwhile, the bioinformatics simulation in 20 species showed that the BsaXI had the most widespread recognition sites, indicating that 2b-RAD tags had a powerful application potentiality for high density genetic map. Using modified adapters with a fix base in 3′end, 2b-RAD was also fit for QTL studies with low costs.

Conclusions

An improved 2b-RAD genotyping approach was established in this research and named as I2b-RAD. The method was a simple, fast, cost-effective and multiplex sequencing library approach. It could be adjusted by selecting different enzymes and adapters to fit for alternative uses including chromosomes assembly, QTL fine mapping and even natural population analysis.

【 授权许可】

   
2014 Guo et al.; licensee BioMed Central Ltd.

【 图 表 】

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