Mapping of Quantitative Trait Loci Underlying Cold Tolerance in Rice Seedlings via High-Throughput Sequencing of Pooled Extremes

Zemao Yang,Adrian J Cutler,Yan Zheng,Weiqi Tang,Weiren Wu,Kangjing Liang,Daiqing Huang,Rongling Wu

doi:10.1371/journal.pone.0068433

Abstract

Low temperature is a major limiting factor in rice growth and development. Mapping of quantitative trait loci (QTLs) controlling cold tolerance is important for rice breeding. Recent studies have suggested that bulked segregant analysis (BSA) combined with next-generation sequencing (NGS) can be an efficient and cost-effective way for QTL mapping. In this study, we employed NGS-assisted BSA to map QTLs conferring cold tolerance at the seedling stage in rice. By deep sequencing of a pair of large DNA pools acquired from a very large F3 population (10,800 individuals), we obtained ∼450,000 single nucleotide polymorphisms (SNPs) after strict screening. We employed two statistical methods for QTL analysis based on these SNPs, which yielded consistent results. Six QTLs were mapped on chromosomes 1, 2, 5, 8 and 10. The three most significant QTLs on chromosomes 1, 2 and 8 were validated by comparison with previous studies. Two QTLs on chromosomes 2 and 5 were also identified previously, but at the booting stage rather than the seedling stage, suggesting that some QTLs may function at different developmental stages, which would be useful for cold tolerance breeding in rice. Compared with previously reported QTL mapping studies for cold tolerance in rice based on the traditional approaches, the results of this study demonstrated the advantages of NGS-assisted BSA in both efficiency and statistical power.

Highlights

Many traits of agronomic importance in crops, including those related to abiotic stress tolerance, are quantitatively inherited
10,800 F3 seedlings from the cross between rice varieties Nipponbare and LPBG were tested for cold tolerance, from which 430 extremely sensitive (ES) and 385 extremely tolerant (ET) individuals were selected and a pair of DNA pools was obtained
Based on the uniquely mapped reads, a total of 456,777 single nucleotide polymorphisms (SNPs) met the chosen requirements for QTL analysis. These SNPs showed an approximately symmetric unimodal distribution of the Nipponbare allele frequency in the whole pool with an average of,45%, which was close to the expected value 50%, suggesting that the genetic segregation of these SNPs was approximately normal

Summary

Introduction

Many traits of agronomic importance in crops, including those related to abiotic stress tolerance, are quantitatively inherited. QTL mapping is one of the most common approaches for the genetic study of quantitative traits, which provides the basis for map-based cloning of related genes and marker-assisted selection (MAS) in crop breeding. Since 2000, high-throughput genotyping technologies based on microarray [2] and generation sequencing (NGS) [3] have developed very quickly. Using these technologies, BSA can identify large numbers of markers linked to the target genes or QTLs. Using these technologies, BSA can identify large numbers of markers linked to the target genes or QTLs Based on these linked markers, the target genes or QTLs can be directly mapped by referring to reference genome sequences. With the availability of highthroughput genotyping technologies and with reference genome sequences from more and more species, BSA is becoming an increasingly useful approach for gene or QTL mapping

Methods

Results

Conclusion