Genome Analysis of a Cultivar Tongil and Isolation of the yellow embryo lethal (yel) Gene in Rice

Abstract

Asian cultivated rice (Oryza sativa L.), divided into sub-species, Oryza sativa. indica and Oryza sativa. japonica has been cultivated in all over the world including Asia. Recent evidence suggests an ancient indica and japonica divided between 200,000 and 440,000 years ago based on nuclear genome sequence comparisons (Ma and Bennetzen, 2004

Tang et al., 2004

Vitte et al., 2004). During cultivation and domestication, these subspecies have developed the unique morphologies and characteristic agronomic traits. Many phenotypic differences are obvious between O. sativa indica and japonica and the beneficial traits from each subspecies are useful to develop a new varieties. However, various obstacles such as reproductive barriers, prevent gene exchange or gene flow between two subspecies. Although several studies have tried to explain the differences between indica and japonica at a certain developmental stage or molecular and physiological level, data from these studies are quite limited to explain general differences between indica and japonica. Whole genome analysis to elucidate the differences between indiica and japonica genome will be useful to explain the genome structure that led to their distinct features. In this study, we analyzed the nucleotide-level genome structure of Tongil rice which is a high-yielding rice variety derived from a three-way cross between indica and japonica and compared it to those of the parental varieties. Sequence data were obtained by whole-genome resequencing using the Illumina Hiseq. A total of 17.3 billion reads, 47× genome coverage, were generated for Tongil rice. Three parental varieties of Tongil rice, two indica types and one japonica type, were also sequenced at approximately 30x genome coverage. Indica-japonica genome composition was determined based on SNP data by comparing Tongil with three parental genome sequences using the sliding window approach. Analyses revealed that 91.8% of the Tongil genome originated from the indica parents and 7.9% from the japonica parent. Copy number of SSR motifs, ORF gene distribution throughout the whole genome, gene ontology (GO) annotation, and yield-related QTLs or genes variations were also comparatively analyzed between Tongil and parents using sequence-based tools. These results indicated that each genetic factor was transferred from parents into Tongil in proportion to the whole-genome composition. The yellow colored pericarp and embryo lethal mutant was derived from chemical mutagenesis using N-methyl-N-nitrosourea (MNU) on a japonica rice cultivar, Hwacheong. In this study, we cloned the gene responsible for yellow pericarp and lethal embryo phenotype using a map-based approach. Fine mapping revealed that the mutant gene, yel was located on the long arm of chromosome 2 and sequencing of candidate genes identified the gene responsible for the yellow pericarp and embryo lethal phenotype, OsCOP1 (Constitutive Photomorphogenic 1), orthologs of Arabidopsis COP1 which encodes a protein of comprising RING-finger, coiled-coil, and WD40 domains. A 706-bp deletion in the first exon including 5UTR and start codon of yel gene, LOC_Os02g53140 was found thus, it is conceivable that the yellow pericarp phenotype may have been the result of loss of function in rice yel gene. HPLC-MS analysis of the four candidate standards with extracts of embryo and endosperm of the mutant grain showed that extremely high level of orientin, luteolin-8-C-glucoside accumulated in mutant embryo and endosperm and was the major pigment both in embryo and endosperm. In addition, the antioxidant activities and total phenolic contents of embryo and endosperm of the mutant seed were significantly higher than those of wild-type. Fatty acid analysis showed that the content of saturated fatty acid was increased in mutant embryo on the other hand, the content of unsaturated fatty acid was decreased than that of wild-type. These results indicated that yellow embryo lethal (yel) gene might be involved in the flavonoids biosynthetic pathway and other ingredient characteristics of rice seed.