Combining a New Exome Capture Panel With an Effective varBScore Algorithm Accelerates BSA-Based Gene Cloning in Wheat.

Chunhao Dong,Jirui Wang,Zhongxu Chen,Lichao Zhang,Chuan Xia,Xu Liu,Danping Li,Zhencheng Xie,Lixuan Gui,Yongqiang Gu,Qiang Zhang,Xueying Zhang,Xiuying Kong

doi:10.3389/fpls.2020.01249

Abstract

The discovery of functional genes underlying agronomic traits is of great importance for wheat improvement. Here we designed a new wheat exome capture probe panel based on IWGSC RefSeq v1.0 genome sequence information and developed an effective algorithm, varBScore, that can sufficiently reduce the background noise in gene mapping and identification. An effective method, termed bulked segregant exome capture sequencing (BSE-Seq) for identifying causal mutations or candidate genes was established by combining the use of a newly designed wheat exome capture panel, sequencing of bulked segregant pools from segregating populations, and the robust algorithm varBScore. We evaluated the effectiveness of varBScore on SNP calling using the published dataset for mapping and cloning the yellow rust resistance gene Yr7 in wheat. Furthermore, using BSE-Seq, we rapidly identified a wheat yellow leaf mutant gene, ygl1, in an ethyl methanesulfonate (EMS) mutant population and found that a single mutation of G to A at 921 position in the wild type YGL1 gene encoding magnesium-chelatase subunit chlI caused the leaf yellowing phenotype. We further showed that mutation of YGL1 through CRISPR/Cas9 gene editing led to a yellow phenotype on the leaves of transgenic wheat, indicating that ygl1 is the correct causal gene responsible for the mutant phenotype. In summary, our approach is highly efficient for discovering causal mutations and gene cloning in wheat.

Highlights

Wheat (Triticum aestivum L., 2n = 6x = 42, AABBDD) is an important staple crop, providing 20% of all calories consumed by the world population
The yellow-green leaf mutant ygl1 originated from mutagenesis of 6,000 seeds of the elite wheat variety YZ4110, which was treated with 1.2% ethyl methanesulfonate (EMS); the germination rates of the EMS-mutagenized seeds were 65% (Zhao et al, 2009), and the yellow-green leaf phenotype was inherited stably after four generations of selfpollination
A total of 299,387,477 bp regions were selected in the probe design, mainly including two aspects: (1) the 107,891 High Confidence (HC) and 161,537 Low Confidence (LC) genes containing the sequences from the 5′untranslated regions (UTR) to the 3′UTR, which come from RefSeq Annotation v1.1, and (2) novel transcripts annotated from 2,600 RNA-seq data downloaded from the Sequence Read Archive (SRA) database of the National Center for Biotechnology Information (NCBI); these transcripts were highly expressed but beyond the scope of RefSeq Annotation v1.1

Summary

Introduction

Identification of genes underlying desirable agronomic traits is of great significance for genetic improvement of wheat. Screening of induced mutant populations is an effective approach for discovering new genes underlying phenotypic variations in plants (Schneeberger, 2014; Krasileva et al, 2017). Map-based gene cloning usually requires multiple steps, including generating mapping populations, fine mapping to narrow the target gene region to identify genetic markers cosegregating with the target phenotype, candidate gene screening and gene identification by sequencing. This process is often timeconsuming and costly, especially in wheat

Methods

Results

Conclusion