Search

Anthocyanins play an important role in the growth of plants, and are beneficial to human health. In plants, the MYB-bHLH-WD40 (MBW) complex activates the genes for anthocyanin biosynthesis. However, in rice, the WD40 regulators remain to be conclusively identified. Here, a crucial anthocyanin biosynthesis gene was fine mapped to a 43.4-kb genomic region on chromosome 2, and a WD40 gene OsTTG1 (Oryza sativa TRANSPARENT TESTA GLABRA1) was identified as ideal candidate gene. Subsequently, a homozygous mutant (osttg1) generated by CRISPR/Cas9 showed significantly decreased anthocyanin accumulation in various rice organs. OsTTG1 was highly expressed in various rice tissues after germination, and it was affected by light and temperature. OsTTG1 protein was localized to the nucleus, and can physically interact with Kala4, OsC1, OsDFR and Rc. Furthermore, a total of 59 hub transcription factor genes might affect rice anthocyanin biosynthesis, and LOC_Os01g28680 and LOC_Os02g32430 could have functional redundancy with OsTTG1. Phylogenetic analysis indicated that directional selection has driven the evolutionary divergence of the indica and japonica OsTTG1 alleles. Our results suggest that OsTTG1 is a vital regulator of anthocyanin biosynthesis, and an important gene resource for the genetic engineering of anthocyanin biosynthesis in rice and other plants.

Low-temperature stress significantly affects rice growth and causes serious loss of yield in temperate and high-altitude areas of the world. Rice lacks cold tolerance (CT) at germination stage, which reduces seedling vigor, hinders crop establishment and crop growth, and even affect direct-seeded rice. A chromosome segment substitution line (CSSL) population, including 271 lines, was derived from cold-tolerant donor wild rice Y11 (Oryza rufipogon Griff.) crossed with cold-sensitive rice variety GH998 to explore new genetic resource with CT and further investigate quantitative trait loci (QTLs) responsible for germination properties under low temperature. The germination rates of Y11 and GH998 were 95% and 73.33%, respectively, under cold stress. The range of variance of the germination rate in the CSSLs was between 0% and 98.33%. In addition, the CSSLs and parents were sequenced via whole genome resequencing. Results showed 33.46, 33.36, and 475.65 Gbp of clean data of GH998, Y11, and CSSLs, respectively. In 12 linkage groups, the general map distance was 941.49 cM, while the average map distance was 0.63 cM. On thebasis of 1484 bins, a high-density linkage map of the CSSLs was developed. The average distance of the linkage map ranged from 0.42 cM to 1.39 cM. The phenotype of CSSLs under low temperature and 615,466 single nucleotide polymorphisms (SNPs) between Y11 and GH998 were used for QTL analysis. Two QTLs, namely, low-temperature germination 8 (qLTG8) and qLTG11, were responsible for the germination ability under low temperature in rice. qLTG8 was mapped on chromosome 8, and it explained 14.55% of the total phenotypic variation explained (PVE) during the germination stage. qLTG8 was in 195.5 kb, and 32 genes were predicted based on the Rice Genome Annotation Project. qLTG11 was located on chromosome 11, and it explained 14.31% of the total PVE during the germination stage. qLTG11 was mapped at a narrow distance in 78.83 kb, and only 12 genes fall within this range according to the Rice Genome Annotation Project. The expression patterns of these 32 genes in the qLTG8 region demonstrated that LOC_Os08g01120, LOC_Os08g01140, LOC_Os08g01390, LOC_Os08g01170, and LOC_Os08g01380 were highly induced by cold stress in Y11 compared with GH998. The expression patterns of 12 genes in the qLTG11 region suggested that LOC_Os11g32880 and LOC_Os11g32940 were highly induced by cold stress in Y11 compared with GH998. This study provides an effective method, i.e., constructing CSSLs of wild rice, to explore excellent genes of wild rice and create new genetic resources. The results also provide a basis for identifying the genes underlying qLTG8 and qLTG11, indicating that QTL could be used for genetic improvement of CT in cultivar rice.

Background: Nitrogen (N) is a major nutrient element for crop yield improvement, but it also causes environmental problems. Therefore, N use efficiency (NUE) enhancement is an important strategy to solve this problem. In plants, the members of the peptide transporter (PTR) gene family are involved in nitrate uptake and transport. In this study, we performed a genome-wide analysis on PTR genes in Nipponbare, R498, and Oryza glaberrima, and identified 96, 85, and 78 PTR genes respectively. Results: Phylogenetic tree showed that the 96 PTR genes could be classified into 8 groups, and the distributions of PTR genes in Asian cultivated riceand African cultivated rice were consistent. The number of PTR gene was higher in peanuts and soybeans, which were 125 and 127, respectively. The 521 PTR genes in rice, maize, sorghum, peanut, soybean and Arabidopsis could be classified into 4 groups, and the PTR gene distribution was different between monocots and dicots. In Nipponbare genome, the 25 PTR genes were distributed in 5 segmental duplication regions on chromosome 1, 2, 3, 4, 5, 7, 8, 9, and 10. Rice PTR genes have 0-11 introns and 1-12 exons , and 16 PTR genes have the NPF (NRT1/PTR family ) domain. The results of RNA-Seq showed that the number of differentially expressed genes (DEGs) between NIL15 and NIL19 were 928, 1467, and 1586 at three stages, respectively. Under low N conditions, the number of differentially expressed PTR genes increased significantly. The RNA-Seq data was analyzed using WGCNA to predict the potential interaction between genes. We classified the genes with similar expression pattern into one group, and obtained 25 target modules. Among these modules, three modules may be involved in rice nitrogen uptake and utilization, especially the brown module, in which hub genes were annotated as protein kinase that may regulate rice N metabolism. Conclusions: In this study, we comprehensively analyzed the PTR gene family in rice. Ninety-six PTR genes were identified in Nippobare genome and twenty-five genes locates on five large segmental duplication regions. The Ka/Ks ratio indicated that many PTR genes had nudergone positive selection. The RNA-seq results showed that many PTR genes were involved in rice UNE. Protein kinases may play an important role in regulating NUE in rice. These results will provide a fundamental basis for molecular breeding of NUE in rice.

Starch is the major component of milled rice, and amylose content (AC) affects eating quality. In this study, a genome-wide association study (GWAS) with specific-locus amplified fragment sequencing (SLAF-seq) data was performed for AC on a core collection of 419 rice landraces. Using the compressed mixed linear method based on the Q+K model, we identified a new gene, LAC6 (Chr6: 5.65–6.04 Mb), associated with AC in the low amylose content panel. The LAC6 candidate gene was detected by qRT-PCR in rice panicles. Results indicate that LOC_Os06g11130 was up-regulated, and LOC_Os06g11340 was significantly down-regulated, making it most likely a key candidate gene of LAC6. In conclusion, the findings provide a certain theoretiacal basis of molecular biology for genetic improvement of AC in rice and rice quality variety breeding.

The complex traits are derived from multiple genes and exhibit a large variety of phenotypes. High-throughput sequencing technologies have become the new strategies for mapping the important traits of crops. However, these methods have their own disadvantages and limitations. Here we introduced Pair-wise Comparison Analysis for Multiple Pool-seq (PCAMP) for mapping the candidate genomic regions involved in anthocyanin biosynthesis in rice pericarp. In this protocol, the second filial generation (F2) populations obtained by crossing two parents with different target traits were divided into n (n>=3) subpopulations according to their phenotypes. Thirty phenotypically identical individuals were selected from each subpopulation and DNA samples were extracted to form a pool for sequencing. Finally, we compared the SNP-index between every two Pool-seqs to map the candidate genomic regions. We applied PCAMP to analyse F2populations and successfully identified five known genes and five new candidate genomic regions for anthocyanin biosynthesis in rice pericarp. These results demonstrate that PCAMP is an efficient new method for dissecting the complex traits of crops.

The complex traits are derived from multiple genes and exhibit a large variety of phenotypes. High-throughput sequencing technologies have become the new strategies for mapping the important traits of crops. However, these methods have their own disadvantages and limitations. Here we introduced Pair-wise Comparison Analysis for Multiple Pool-seq (PCAMP) for mapping the candidate genomic regions involved in anthocyanin biosynthesis in rice pericarp. In this protocol, the second filial generation (F2) populations obtained by crossing two parents with different target traits were divided into n (n>=3) subpopulations according to their phenotypes. Thirty phenotypically identical individuals were selected from each subpopulation and DNA samples were extracted to form a pool for sequencing. Finally, we compared the SNP-index between every two Pool-seqs to map the candidate genomic regions. We applied PCAMP to analyse F2 populations and successfully identified five known genes and five new candidate genomic regions for anthocyanin biosynthesis in rice pericarp. These results demonstrate that PCAMP is an efficient new method for dissecting the complex traits of crops.

Rice is an important cereal in the world, uncovering the genetic basis of agronomic traits in rice landraces genes associated with agronomically important traits is indispensable for both understanding the genetic basis of phenotypic variation and efficient crop improvement. Gelatinization temperature, gel consistency and pericarp color are important indices of rice cooking and eating quality evaluation and potential nutritional importance, which attract wide attentions in the application of genetic and breeding. To dissect the genetic basis of gelatinization temperature (GT), gel consistency (GC) and pericarp color (PC), a total of 419 rice landraces core germplasm collections consisting of 330 indica lines, 78 japonica lines and 11 uncertain varieties were grown, collected, then GT, GC, PC were measured for two years, and sequenced using Specific Locus Amplified Fragment Sequencing (SLAF) technology. In this study, 261,385,070 clean reads and 56,768 polymorphic SLAF tags were obtained, which a total of 211,818 single nucleotide polymorphisms (SNPs) were discovered. With 208,993 SNPs meeting the criterion of minor allele frequency (MAF) > 0.05 and integrity> 0.5, the phylogenetic tree and population structure analysis were performed for all 419 rice landraces, and the whole panel mainly separated into six subpopulations based on population structure analysis. Genome-wide association study (GWAS) was carried out for the whole panel, indica subpanel and japonica subpanel with subset SNPs respectively. One quantitative trait locus (QTL) on chromosome 6 for GT was detected in the whole panel and indica subpanel, and one QTL associated with GC was located on chromosome 6 in the whole panel and indica subpanel. For the PC trait, 8 QTLs were detected in the whole panel on chromosome 1, 3, 4, 7, 8, 10 and 11, and 7 QTLs in the indica subpanel on chromosome 3, 4, 7, 8, 10 and 11. The loci on chromosome 3, 8, 10 and 11 have not been identified previously, and they may be the candidate genes of pericarp color. For the three traits, no QTL was detected in japonica subpanel probably because of the polymorphism repartition between the subpanel, or small population size of japonica subpanel. This paper provides new gene resources and insights into the molecular mechanisms of important agricultural trait of rice phenotypic variation and genetic improvement of rice quality variety breeding.

Nitrogen is a major nutritional element in rice production. However, excessive application of nitrogen fertilizer has caused severe environmental pollution. Therefore, development of rice varieties with improved nitrogen use efficiency (NUE) is urgent for sustainable agriculture. In this study, bulked segregant analysis (BSA) combined with whole genome re-sequencing (WGS) technology was applied to finely map quantitative trait loci (QTL) for NUE. A key QTL, designated as qNUE6 was identified on chromosome 6 and further validated by Insertion/Deletion (InDel) marker-based substitutional mapping in recombinants from F2 population (NIL-13B4×GH998). Forty-four genes were identified in this 266.5-kb region. According to detection and annotation analysis of variation sites, thirty-nine genes with large-effect single-nucleotide polymorphisms (SNPs) and large-effect InDels were selected as candidates and their expression levels were analyzed by qRT-PCR. Significant differences in the expression levels of LOC_Os06g15370 (peptide transporter PTR2) and LOC_Os06g15420 (asparagine synthetase) were observed between two parents (Y11 and GH998). Phylogenetic analysis in Arabidopsis thaliana identified two closely related homologues, AT1G68570 (AtNPF3.1) and AT5G65010 (ASN2), which share 72.3% and 87.5% amino acid similarity with LOC_Os06g15370 and LOC_Os06g15420, respectively. Taken together, our results suggested that qNUE6 is a possible candidate gene for NUE in rice. The fine mapping and candidate gene analysis of qNUE6 provide the basis of molecular breeding for genetic improvement of rice varieties with high NUE, and lay the foundation for further cloning and functional analysis.