Haplotype-resolved gapless genome and chromosome segment substitution lines facilitate gene identification in wild rice.

The abundant genetic variation harbored by wild rice (Oryza rufipogon) has provided a reservoir of useful genes for rice breeding. However, the genome of wild rice has not yet been comprehensively assessed. Here, we report the haplotype-resolved gapless genome assembly and annotation of wild rice Y476. In addition, we develop two sets of chromosome segment substitution lines (CSSLs) using Y476 as the donor parent and cultivated rice as the recurrent parents. By analyzing the gapless reference genome and CSSL population, we identify 254 QTLs associated with agronomic traits, biotic and abiotic stresses. We clone a receptor-like kinase gene associated with rice blast resistance and confirm its wild rice allele improves rice blast resistance. Collectively, our study provides a haplotype-resolved gapless reference genome and demonstrates a highly efficient platform for gene identification from wild rice.

A wild rice CSSL population facilitated identification of salt tolerance genes and rice germplasm innovation.

Salt stress is one of the major factors that limits rice production. Therefore, identification of salt-tolerant alleles from wild rice is important for rice breeding. In this study, we constructed a set of chromosome segment substitution lines (CSSLs) using wild rice as the donor parent and cultivated rice Nipponbare (Nip) as the recurrent parent. Salt tolerance germinability (STG) was evaluated, and its association with genotypes was determined using this CSSL population. We identified 17 QTLs related to STG. By integrating the transcriptome and genome data, four candidate genes were identified, including the previously reported AGO2 and WRKY53. Compared with Nip, wild rice AGO2 has a structure variation in its promoter region and the expression levels were upregulated under salt treatments; wild rice WRKY53 also has natural variation in its promoter region, and the expression levels were downregulated under salt treatments. Wild rice AGO2 and WRKY53 alleles have combined effects for improving salt tolerance at the germination stage. One CSSL line, CSSL118 that harbors these two alleles was selected. Compared with the background parent Nip, CSSL118 showed comprehensive salt tolerance and higher yield, with improved transcript levels of reactive oxygen species scavenging genes. Our results provided promising genes and germplasm resources for future rice salt tolerance breeding.

Comparative Genomic and Expression Analysis Insight into Evolutionary Characteristics of PEBP Genes in Cultivated Peanuts and Their Roles in Floral Induction.

Phosphatidyl ethanolamine-binding proteins (PEBPs) are involved in regulating flowering time and various developmental processes. Functions and expression patterns in cultivated peanuts (Arachis hypogaea L.) remain unknown. In this study, 33 PEBP genes in cultivated peanuts were identified and divided into four subgroups: FT, TFL, MFT and FT-like. Gene structure analysis showed that orthologs from A and B genomes in cultivated peanuts had highly similar structures, but some orthologous genes have subgenomic dominance. Gene collinearity and phylogenetic analysis explain that some PEBP genes play key roles in evolution. Cis-element analysis revealed that PEBP genes are mainly regulated by hormones, light signals and stress-related pathways. Multiple PEPB genes had different expression patterns between early and late-flowering genotypes. Further detection of its response to temperature and photoperiod revealed that PEBPs ArahyM2THPA, ArahyEM6VH3, Arahy4GAQ4U, ArahyIZ8FG5, ArahyG6F3P2, ArahyLUT2QN, ArahyDYRS20 and ArahyBBG51B were the key genes controlling the flowering response to different flowering time genotypes, photoperiods and temperature. This study laid the foundation for the functional study of the PEBP gene in cultivated peanuts and the adaptation of peanuts to different environments.

Identification of candidate genes and clarification of the maintenance of the green pericarp of weedy rice grains.

The weedy rice (Oryza sativa f. spontanea) pericarp has diverse colors (e.g., purple, red, light-red, and white). However, research on pericarp colors has focused on red and purple, but not green. Unlike many other common weedy rice resources, LM8 has a green pericarp at maturity. In this study, the coloration of the LM8 pericarp was evaluated at the cellular and genetic levels. First, an examination of their ultrastructure indicated that LM8 chloroplasts were normal regarding plastid development and they contained many plastoglobules from the early immature stage to maturity. Analyses of transcriptome profiles and differentially expressed genes revealed that most chlorophyll (Chl) degradation-related genes in LM8 were expressed at lower levels than Chl a/b cycle-related genes in mature pericarps, suggesting that the green LM8 pericarp was associated with inhibited Chl degradation in intact chloroplasts. Second, the F2 generation derived from a cross between LM8 (green pericarp) and SLG (white pericarp) had a pericarp color segregation ratio of 9:3:4 (green:brown:white). The bulked segregant analysis of the F2 populations resulted in the identification of 12 known genes in the chromosome 3 and 4 hotspot regions as candidate genes related to Chl metabolism in the rice pericarp. The RNA-seq and sqRT-PCR assays indicated that the expression of the Chl a/b cycle-related structural gene DVR (encoding divinyl reductase) was sharply up-regulated. Moreover, genes encoding magnesium-chelatase subunit D and the light-harvesting Chl a/b-binding protein were transcriptionally active in the fully ripened dry pericarp. Regarding the ethylene signal transduction pathway, the CTR (encoding an ethylene-responsive protein kinase) and ERF (encoding an ethylene-responsive factor) genes expression profiles were determined. The findings of this study highlight the regulatory roles of Chl biosynthesis- and degradation-related genes influencing Chl accumulation during the maturation of the LM8 pericarp.

Global whole-genome comparison and analysis to classify subpopulations and identify resistance genes in weedy rice relevant for improving crops.

Common weedy rice plants are important genetic resources for modern breeding programs because they are the closest relatives to rice cultivars and their genomes contain elite genes. Determining the utility and copy numbers of WRKY and nucleotide-binding site (NBS) resistance-related genes may help to clarify their variation patterns and lead to crop improvements. In this study, the weedy rice line LM8 was examined at the whole-genome level. To identify the Oryza sativa japonica subpopulation that LM8 belongs to, the single nucleotide polymorphisms (SNPs) of 180 cultivated and 23 weedy rice varieties were used to construct a phylogenetic tree and a principal component analysis and STRUCTURE analysis were performed. The results indicated that LM8 with admixture components from japonica (GJ) and indica (XI) belonged to GJ-admixture (GJ-adm), with more than 60% of its genetic background derived from XI-2 (22.98%), GJ-tropical (22.86%), and GJ-subtropical (17.76%). Less than 9% of its genetic background was introgressed from weedy rice. Our results also suggested LM8 may have originated in a subtropical or tropical geographic region. Moreover, the comparisons with Nipponbare (NIP) and Shuhui498 (R498) revealed many specific structure variations (SVs) in the LM8 genome and fewer SVs between LM8 and NIP than between LM8 and R498. Next, 96 WRKY and 464 NBS genes were identified and mapped on LM8 chromosomes to eliminate redundancies. Three WRKY genes (ORUFILM02g002693, ORUFILM05g002725, and ORUFILM05g001757) in group III and one RNL [including the resistance to powdery mildew 8 (RPW8) domain, NBS, and leucine rich repeats (LRRs)] type NBS gene (ORUFILM12g000772) were detected in LM8. Among the NBS genes, the RPW8 domain was detected only in ORUFILM12g000772. This gene may improve plant resistance to pathogens as previously reported. Its classification and potential utility imply LM8 should be considered as a germplasm resource relevant for rice breeding programs.

High-Quality Genomes and High-Density Genetic Map Facilitate the Identification of Genes From a Weedy Rice.

Genes have been lost or weakened from cultivated rice during rice domestication and breeding. Weedy rice (Oryza sativa f. spontanea) is usually recognized as the progeny between cultivated rice and wild rice and is also known to harbor an gene pool for rice breeding. Therefore, identifying genes from weedy rice germplasms is an important way to break the bottleneck of rice breeding. To discover genes from weedy rice germplasms, we constructed a genetic map based on w-hole-genome sequencing of a F2 population derived from the cross between LM8 and a cultivated rice variety. We further identified 31 QTLs associated with 12 important agronomic traits and revealed that ORUFILM03g000095 gene may play an important role in grain length regulation and participate in grain formation. To clarify the genomic characteristics from weedy rice germplasms of LM8, we generated a high-quality genome assembly using single-molecule sequencing, Bionano optical mapping, and Hi-C technologies. The genome harbored a total size of 375.8 Mb, a scaffold N50 of 24.1 Mb, and originated approximately 0.32 million years ago (Mya) and was more closely related to Oryza sativa ssp. japonica. and contained 672 unique genes. It is related to the formation of grain shape, heading date and tillering. This study generated a high-quality reference genome of weedy rice and high-density genetic map that would benefit the analysis of genome evolution for related species and suggested an effective way to identify genes related to important agronomic traits for further rice breeding.

Conservation recommendations for Oryza rufipogon Griff. in China based on genetic diversity analysis.

Over the past 30 years, human disturbance and habitat fragmentation have severely endangered the survival of common wild rice (Oryza rufipogon Griff.) in China. A better understanding of the genetic structure of O. rufipogon populations will therefore be useful for the development of conservation strategies. We examined the diversity and genetic structure of natural O. rufipogon populations at the national, provincial, and local levels using simple sequence repeat (SSR) markers. Twenty representative populations from sites across China showed high levels of genetic variability, and approximately 44% of the total genetic variation was among populations. At the local level, we studied fourteen populations in Guangxi Province and four populations in Jiangxi Province. Populations from similar ecosystems showed less genetic differentiation, and local environmental conditions rather than geographic distance appeared to have influenced gene flow during population genetic evolution. We identified a triangular area, including northern Hainan, southern Guangdong, and southwestern Guangxi, as the genetic diversity center of O. rufipogon in China, and we proposed that this area should be given priority during the development of ex situ and in situ conservation strategies. Populations from less common ecosystem types should also be given priority for in situ conservation.

A Combination of Long-Day Suppressor Genes Contributes to the Northward Expansion of Rice.

Growing cultivated rice with a moderate heading date is the key to expanding its cultivation area and maintaining stable yields. The genes that regulate heading date are largely cloned; however, it remains unclear how genetic mutations and their combinations affect the heading date and adaptability of cultivated rice. Here, we report the analysis of genetic variation in eight long-day flowering suppressor genes (Hd1, DTH8, Ghd7, OsCOL4, DTH7, Hd6, Se5, and PhyB) and the phylogenetic relationship of eight genes. Genetic variations in DTH8, Ghd7, Hd1, DTH7, PhyB, and OsCOL4 are correlated with differences in heading date and the correlation between the genetic diversity of Hd6 and Se5 and rice heading data are weak. One group of haplotypes of DTH8, Ghd7, Hd1, DTH7, PhyB, and OsCOL4 are associated with earlier heading dates and appear to have accumulated during the northward expansion of rice cultivation. A minimum of four group A alleles of DTH8, Ghd7, Hd1, DTH7, PhyB, and OsCOL4 are required for the growth of cultivated rice at latitudes above 30°N. This study presents a preliminary investigation of the genetic patterns and adaptation mechanisms of long-day flowering suppressor genes and provides a useful reference for the molecular breeding of rice cultivars for various environments and farming systems.

Identification and genetic analysis of qCL1.2, a novel allele of the "green revolution" gene SD1 from wild rice (Oryza rufipogon) that enhances plant height.

BACKGROUND: The exploitation of novel alleles from wild rice that were lost during rice cultivation could be very important for rice breeding and evolutionary studies. Plant height (PH) was a target of artificial selection during rice domestication and is still a target of modern breeding. The "green revolution" gene semi-dwarf 1 (SD1) were well documented and used in the past decades, allele from wild rice could provide new insights into the functions and evolution of this gene. RESULTS: We identified a PH-related quantitative trait locus, qCL1.2,from wild riceusing a set of chromosome segment substitution lines. qCL1.2encodesa novel allele of SD1 gene. The wild allele of SD1 is a dominant locus that can significantly promote rice internode length by regulating the expression levels of genes involved in gibberellin biosynthesis and signal transduction. Nucleotide diversity and haplotype network analyses of the SD1 gene were performed using 2822 rice landraces. Two previously reported functional nucleotide polymorphisms clearly differentiated japonica and indica rice; however, they were not associated with PH selection. Other new functional nucleotide polymorphisms in the coding, but not promoter, regions were involved in PH selection during rice domestication. Our study increasesunderstanding of the rice SD1 gene and provides additional evidence of this gene's selection during rice domestication. CONCLUSIONS: Our findings provide evidence thatSD1 gene from wild rice enhances plant height and new functional nucleotide polymorphisms of this gene were artificially selected during cultivated rice differentiation.

Fine mapping and identification of a novel locus qGL12.2 control grain length in wild rice (Oryza rufipogon Griff.).

KEY MESSAGE: A wild rice QTL qGL12.2 for grain length was fine mapped to an 82-kb interval in chromosome 12 containing six candidate genes and none was reported previously. Grain length is an important trait for yield and commercial value in rice. Wild rice seeds have a very slender shape and have many desirable genes that have been lost in cultivated rice during domestication. In this study, we identified a quantitative trait locus, qGL12.2, which controls grain length in wild rice. First, a wild rice chromosome segment substitution line, CSSL41, was selected that has longer glume and grains than does the Oryza sativa indica cultivar, 9311. Next, an F2 population was constructed from a cross between CSSL41 and 9311. Using the next-generation sequencing combined with bulked-segregant analysis and F3 recombinants analysis, qGL12.2 was finally fine mapped to an 82-kb interval in chromosome 12. Six candidate genes were found, and no reported grain length genes were found in this interval. Using scanning electron microscopy, we found that CSSL41 cells are significantly longer than those of 9311, but there is no difference in cell widths. These data suggest that qGL12.2 is a novel gene that controls grain cell length in wild rice. Our study provides a new genetic resource for rice breeding and a starting point for functional characterization of the wild rice GL gene.

Nonfunctional alleles of long-day suppressor genes independently regulate flowering time.

Due to the remarkable adaptability to various environments, rice varieties with diverse flowering times have been domesticated or improved from Oryza rufipogon. Detailed knowledge of the genetic factors controlling flowering time will facilitate understanding the adaptation mechanism in cultivated rice and enable breeders to design appropriate genotypes for distinct preferences. In this study, four genes (Hd1, DTH8, Ghd7 and OsPRR37) in a rice long-day suppression pathway were collected and sequenced in 154, 74, 69 and 62 varieties of cultivated rice (Oryza sativa) respectively. Under long-day conditions, varieties with nonfunctional alleles flowered significantly earlier than those with functional alleles. However, the four genes have different genetic effects in the regulation of flowering time: Hd1 and OsPRR37 are major genes that generally regulate rice flowering time for all varieties, while DTH8 and Ghd7 only regulate regional rice varieties. Geographic analysis and network studies suggested that the nonfunctional alleles of these suppression loci with regional adaptability were derived recently and independently. Alleles with regional adaptability should be taken into consideration for genetic improvement. The rich genetic variations in these four genes, which adapt rice to different environments, provide the flexibility needed for breeding rice varieties with diverse flowering times.