Oryza Rufipogon Griff Research Articles

Transcriptome analysis of phosphorus stress responsiveness in the seedlings of Dongxiang wild rice (Oryza rufipogon Griff.)

BackgroundLow phosphorus availability is a major factor restricting rice growth. Dongxiang wild rice (Oryza rufipogon Griff.) has many useful genes lacking in cultivated rice, including stress resistance to phosphorus deficiency, cold, salt and drought, which is considered to be a precious germplasm resource for rice breeding. However, the molecular mechanism of regulation of phosphorus deficiency tolerance is not clear.ResultsIn this study, cDNA libraries were constructed from the leaf and root tissues of phosphorus stressed and untreated Dongxiang wild rice seedlings, and transcriptome sequencing was performed with the goal of elucidating the molecular mechanisms involved in phosphorus stress response. The results indicated that 1184 transcripts were differentially expressed in the leaves (323 up-regulated and 861 down-regulated) and 986 transcripts were differentially expressed in the roots (756 up-regulated and 230 down-regulated). 43 genes were up-regulated both in leaves and roots, 38 genes were up-regulated in roots but down-regulated in leaves, and only 2 genes were down-regulated in roots but up-regulated in leaves. Among these differentially expressed genes, the detection of many transcription factors and functional genes demonstrated that multiple regulatory pathways were involved in phosphorus deficiency tolerance. Meanwhile, the differentially expressed genes were also annotated with gene ontology terms and key pathways via functional classification and Kyoto Encyclopedia of Gene and Genomes pathway mapping, respectively. A set of the most important candidate genes was then identified by combining the differentially expressed genes found in the present study with previously identified phosphorus deficiency tolerance quantitative trait loci.ConclusionThe present work provides abundant genomic information for functional dissection of the phosphorus deficiency resistance of Dongxiang wild rice, which will be help to understand the biological regulatory mechanisms of phosphorus deficiency tolerance in Dongxiang wild rice.

Morphological and Molecular Data Reveal Three Distinct Populations of Indian Wild Rice Oryza rufipogon Griff. Species Complex.

Wild relatives of crops possess adaptive mutations for agronomically important traits, which could play significant role in crop improvement for sustainable agriculture. However, global climate change and human activities pose serious threats to the natural habitats leading to erosion of genetic diversity of wild rice populations. The purpose of this study was to explore and characterize India’s huge untapped wild rice diversity in Oryza rufipogon Griff. species complex from a wide range of ecological niches. We made strategic expeditions around diversity hot spots in 64 districts of nine different agro-climatic zones of the country and collected 418 wild rice accessions. Significant variation was observed among the accessions for 46 morphological descriptors, allowing classification into O. nivara, O. rufipogon, and O. sativa f. spontanea morpho-taxonomic groups. Genome-specific pSINE1 markers confirmed all the accessions having AA genome, which were further classified using ecotype-specific pSINE1 markers into annual, perennial, intermediate, and an unknown type. Principal component analysis revealed continuous variation for the morphological traits in each ecotype group. Genetic diversity analysis based on multi-allelic SSR markers clustered these accessions into three major groups and analysis of molecular variance for nine agro-climatic zones showed that 68% of the genetic variation was inherent amongst individuals while only 11% of the variation separated the zones, though there was significant correlation between genetic and spatial distances of the accessions. Model based population structure analysis using genome wide bi-allelic SNP markers revealed three sub-populations designated ‘Pro-Indica,’ ‘Pro-Aus,’ and ‘Mid-Gangetic,’ which showed poor correspondence with the morpho-taxonomic classification or pSINE1 ecotypes. There was Pan-India distribution of the ‘Pro-Indica’ and ‘Pro-Aus’ sub-populations across agro-climatic zones, indicating a more fundamental grouping based on the ancestry closely related to ‘Indica’ and ‘Aus’ groups of rice cultivars. The Pro-Indica population has substantial presence in the Eastern Himalayan Region and Lower Gangetic Plains, whereas ‘Pro-Aus’ sub-population was predominant in the Upper Gangetic Plains, Western Himalayan Region, Gujarat Plains and Hills, and Western Coastal Plains. In contrast ‘Mid-Gangetic’ population was largely concentrated in the Mid Gangetic Plains. The information presented here will be useful in the utilization of wild rice resources for varietal improvement.

Rapid mapping of candidate genes for cold tolerance in Oryza rufipogon Griff. by QTL-seq of seedlings

Cold stress is a major problem in rice production. To rapidly identify genes for cold tolerance in Dongxiang wild rice (DWR, Oryza rufipogon Griff.), sequencing-based bulked segregant analysis of QTL-seq method was used to resequence the extremely resistant (R) and susceptible (S) bulks of a backcross inbred lines (BILs) population (derived from Oryza sativa×O. rufipogon) and their parents. Single nucleotide polymorphisms (SNP)-index graphs and corresponding ρ(SNP-index) graphs (at 99 and 95% confidence levels) for R- and S-bulks detected a total of 2 609 candidate SNPs, including 58 candidate cold-tolerance genes. Quantitative real-time PCR analysis revealed that 5 out of the 58 candidate genes had significant differences in expression between O. sativa and O. rufipogon. Structural variation and functional annotations of the 5 candidate genes were also analyzed, and allowed us to identify 2 insertion-deletion (InDel) markers (12-7 and 12-16) that were linked with candidate genes on chromosome 12 in DWR. These results are helpful for cloning and using cold tolerance genes from common wild rice in cultivated rice.

Identification of quantitative trait loci and candidate genes associated with ABA sensitivity in common wild rice (Oryza rufipogon Griff.)

Abscisic acid (ABA), as one of the foremost signaling molecules in plants, is an important hormone which plays versatile functions in regulating developmental process and adaptive stress process. A set of introgression lines were previously generated via a backcrossing program using an elite indica cultivar rice Teqing (O. sativa L.) as recipient and an accession of Yuanjiang common wild rice (O. rufipogon Griff.) as donor. In this study, the previously developed introgression lines were evaluated for ABA sensitivity. Here we reported that a total of 14 quantitative trait loci (QTLs) associated with ABA sensitivity were identified. An ABA sensitive introgression line, YIL53, was identified and characterized. Physiological characterization, including chlorophyll content, malondialdehyde content, soluble sugar content, and stomata movement, demonstrated that YIL53 exhibited the characteristics associated with ABA sensitivity. Genotypic analysis revealed that YIL53 harbored one QTL related to ABA sensitivity, qASS1-2, which was located on chromosome 1 within one introgressed segment derived from the Yuanjiang common wild rice. Furthermore, the qASS1-2 was finally narrowed down to a 441-kb region between simple sequence repeats (SSR) marker RM212 and single nucleotide polymorphism (SNP) marker M3 using the segregation population derived from the cross between Teqing and YIL53, and three candidate genes associated with ABA sensitivity were identified using a strategy combined gene expression analysis with QTL mapping. Identification of the QTLs related to ABA sensitivity and characterization of the ABA sensitive line YIL53 would provide a helpful basis for isolating novel genes related to ABA sensitivity.

Genetic diversity of cultivated rice (Oryza sativa L.) and wild rice (Oryza rufipogon Griff.) in Asia, especially in Myanmar, as revealed by organelle markers

Asian rice (Oryza sativa L.) is widely cultivated in Asia, where it has been classified into Indica and Japonica Group, the latter is further classified into Tropical and Temperate Japonica Subgroup. O. rufipogon is believed to be the closest ancestor to O. sativa, but it remains unclear whether the two groups arose from a single ancestor or different ancestors. Therefore, here, we investigated the matrilineal ancestors of O. sativa using markers for organelle (chloroplast and mitochondrial) genomes, and 119 O. sativa landraces, 10 O. glaberrima Steud., 115 O. rufipogon Griff. from Asia, and 39 accessions from other wild rice species with AA genomes. We screened 18 organelle markers developed based on polymorphic loci in the organelle genomes. In addition, we used the open reading frame 100 of a chloroplast marker. The results indicated that O. rufipogon first differentiated into two lineages and then further differentiated into Indica and Japonica Group, respectively. Accessions of O. rufipogon (R-1f and R-2d types) from Myanmar appear to be the closest ancestors of Tropical Japonica Subgroup and Indica Group, respectively. Therefore, these wild strains may have made a strong contribution to the domestication of rice landraces in Myanmar.

Comparison of the Genetic Structure betweenIn Situ and Ex Situ Populations of DongxiangWild Rice (Oryza rufipogon Griff.)

In situ and ex situ conservation are two main wild rice (Oryza rufipogon Griff.) protection strategies. Few studies have compared the genetic diversity and genetic structure of wild rice between ex situ and in situ populations. Thus in this study, 278 individuals collected from three in situ and nine ex situ populations of Dongxiang wild rice (DXWR) were genotyped using 32 microsatellite loci to compare their population genetic structure and genetic diversity. Model‐based grouping, neighbor‐joining tree, and principal coordinate analyses showed that there were significant differences in the population structure between in situ and ex situ populations. The in situ populations clustered into three major groups, which were in accordance with their geographical distribution (Anjiashan, Zhangtang, and Shuitaoshu). However, the nine ex situ populations of DXWR showed no differences among populations, and there was a high heterozygosity for each individual. In addition, one of the in situ populations (Zhangtang) was not represented in the ex situ conservation garden. Given the above results, we concluded that the germplasm of both in situ and ex situ populations are precious, and that the combination of these two conservation strategies is necessary to protect the germplasm resources of DXWR, but special emphasis should be placed on in situ conservation to better maintain their genetic identities. In addition, it is necessary to recollect genetic resources from in situ populations for ex situ conservation, especially from the Zhangtang population.

Integrated RNA Sequencing and QTL Mapping to Identify Candidate Genes from Oryza rufipogon Associated with Salt Tolerance at the Seedling Stage.

Soil salinity is a common abiotic stress affecting crop productivity. To identify favorable alleles from wild rice (Oryza rufipogon Griff.) that enhance salinity tolerance of rice (O. sativa L.), a set of introgression lines (ILs) were developed. The ILs were derived from an O. rufipogon accession collected from Chaling (Hunan Province, China) as the donor, and a widely grown O. sativa indica cultivar 93-11 as the recipient. Through evaluating the salt tolerance of 285 ILs at the seedling stage, a total of 10 quantitative trait loci (QTLs) related to salt tolerance were identified on chromosomes 1, 5, 7 and 9–12, with individual QTLs explaining 2–8% of phenotypic variance. The O. rufipogon-derived alleles at four QTLs improved salt tolerance in the 93-11 background. At the same time, a salt-tolerant IL, 9L136, was identified and characterized. Compared with the recipient parent 93-11, a total of 1,391 differentially expressed genes (DEGs) were detected specifically in 9L136 between salt stress and normal condition through genome-wide expression analysis. Of these, four DEGs located in the QTL regions carried by 9L136, suggesting that the four genes might be candidates associated with salt tolerance. Both the highly salt-tolerant ILs and the favorable O. rufipogon-derived QTLs identified in the present study will provide new genetic resources for improving the resistance of cultivated rice against salinity stress using molecular breeding strategies in the future.

Cloning and Functional Analysis of Green Tissue-specific Expression Promoter of Common Wild Rice(Oryza rufipogon Griff.)

Cloning and Functional Analysis of Green Tissue-specific Expression Promoter of Common Wild Rice(Oryza rufipogon Griff.)

Molecular identification of the cytoplasmic male sterile source from Dongxiang wild rice (Oryza rufipogon Griff.)

AbstractThe use of cytoplasm male sterility (CMS) is crucial for three-line hybrid seed production. Two types of CMS have been discovered from Dongxiang wild rice, namely the wild abortion type CMS (CMS-WA) and the Dongxiang wild type CMS (CMS-DW). In this study, we show the molecular identification of the two types of CMS in Dongxiang wild rice. WA352, which conferred CMS-WA, was not detected in Dongxiang wild rice, implying Dongxiang wild rice does not carry the CMS-WA source. Further analysis of WA352 in DY1A, a CMS-DW line, by PCR amplification and sequencing, revealed two insertion-deletion polymorphisms occurred in CMS-DW compared to WA352 of CMS-WA. It was reported that WA352 was comprised of an unknown origin sequence and partial sequences of three open reading frames (ORFs), orf284, orf224 and orf288. The 42-bp insertion was located between the two segments of orf224 and orf288, which created a new chimeric ORF, orf216. This new ORF was also detected in CMS-HL. Based on the 9-bp deletion in orf284, a specific mitochondrial marker of DW-M1 was developed, which could be used to specifically distinguish the DW-type source. Moreover, semi-quantitative RT-PCR analysis preliminarily suggested that both orf216 and orf284 could be considered as candidates for CMS-DW. These findings present a preliminary understanding of CMS-DW at the molecular level.

Genome wide re-sequencing of newly developed Rice Lines from common wild rice (Oryza rufipogon Griff.) for the identification of NBS-LRR genes.

Common wild rice (Oryza rufipogon Griff.) is an important germplasm for rice breeding, which contains many resistance genes. Re-sequencing provides an unprecedented opportunity to explore the abundant useful genes at whole genome level. Here, we identified the nucleotide-binding site leucine-rich repeat (NBS-LRR) encoding genes by re-sequencing of two wild rice lines (i.e. Huaye 1 and Huaye 2) that were developed from common wild rice. We obtained 128 to 147 million reads with approximately 32.5-fold coverage depth, and uniquely covered more than 89.6% (> = 1 fold) of reference genomes. Two wild rice lines showed high SNP (single-nucleotide polymorphisms) variation rate in 12 chromosomes against the reference genomes of Nipponbare (japonica cultivar) and 93–11 (indica cultivar). InDels (insertion/deletion polymorphisms) count-length distribution exhibited normal distribution in the two lines, and most of the InDels were ranged from -5 to 5 bp. With reference to the Nipponbare genome sequence, we detected a total of 1,209,308 SNPs, 161,117 InDels and 4,192 SVs (structural variations) in Huaye 1, and 1,387,959 SNPs, 180,226 InDels and 5,305 SVs in Huaye 2. A total of 44.9% and 46.9% genes exhibited sequence variations in two wild rice lines compared to the Nipponbare and 93–11 reference genomes, respectively. Analysis of NBS-LRR mutant candidate genes showed that they were mainly distributed on chromosome 11, and NBS domain was more conserved than LRR domain in both wild rice lines. NBS genes depicted higher levels of genetic diversity in Huaye 1 than that found in Huaye 2. Furthermore, protein-protein interaction analysis showed that NBS genes mostly interacted with the cytochrome C protein (Os05g0420600, Os01g0885000 and BGIOSGA038922), while some NBS genes interacted with heat shock protein, DNA-binding activity, Phosphoinositide 3-kinase and a coiled coil region. We explored abundant NBS-LRR encoding genes in two common wild rice lines through genome wide re-sequencing, which proved to be a useful tool to exploit elite NBS-LRR genes in wild rice. The data here provide a foundation for future work aimed at dissecting the genetic basis of disease resistance in rice, and the two wild rice lines will be useful germplasm for the molecular improvement of cultivated rice.

The causal deletions in the second exon of An-3 closely associated with awn development and rice yield

Awn is one of important traits during rice domestication. To understand the development of rice awn and the roles it played in rice domestication, we preliminary mapped a major QTL An-3 for awn development using chromosome segment substitution line CSSL138 developed by introgressed genomic fragments of long-awned Guangxi common wild rice (GXCWR, Oryza rufipogon Griff.) into genetic background of short-awned indica cultivar 93–11. An-3 was then fine mapped to a 7-kb region of chromosome 8. An epidermal patterning factor-like protein gene was identified as the single candidate gene corresponding to this QTL. An-3 was showed to be an allele of RAE2 and GAD1, and negatively regulated 1000-grains weight, grain length, and length–width ratio. Comparing with the coding sequences of An-3 from CSSL138, a 2- and 4-bp frame-shift deletions in the second exon were identified in 93–11 and Nipponbare, respectively. Taken together, our results provide valuable natural variation in the alleles of An-3 between common wild rice and cultivated rice, which will be helpful in clarifying the mechanism of awn development and promoting the application of an-3 in genetic improvement of rice yield traits.

Chronology of ancient Dongxiang wild rice (Oryza rufipogon Griff.), and the morphologies of grains, double-peaked phytoliths, and starch, in the middle Yangtze river region, China

Common wild rice (Oryza rufipogon), the ancestor of Asian domesticated rice (Oryza sative), is used as a comparator for identifying Oryza species in archaeological records. However, little attention has been paid to the characteristics of the grain morphology, double-peaked phytoliths and starch granules in ancient wild rice. Dongxiang wild rice is believed to be the current northernmost extent of wild Oryza in Asia, but few studies have been reported on the history of its growth in Dongxiang. A stratigraphic, chronological analysis of the ancient wild rice remains discovered in the sediment core at the Anjiashan in situ conservation (ISC) site in Dongxiang proves that wild rice may have been present in Dongxiang since at least 500–300cal. BP, which includes the period of climatic variations commonly called the “Little Ice Age”. Furthermore, based on a comparison between ancient and modern wild rice, as well as some indica (Huixiang 9126), japonica (Bijing 37) cultivars, it is demonstrated that the grain size of wild rice, in contrast to the double-peaked phytolith and starch granule parameters, changed little during the “Little Ice Age” of the Ming and Qing Dynasties in China, and that in terms of grain shape analysis wild rice is easily confused with indica. This may be one of the reasons why traditional grain shape and grain size analysis usually does not work well in classifying the Oryza species, and why some rice remains from archaeological sites were mainly indica as judged from their shapes but japonica in terms of their morphological parameters of double-peaked phytoliths and spikelets. Our findings may not only assist in elucidating the spatiotemporal distribution of wild rice, but can also provide important reference parameters for the identification of Oryza species in archaeological records.

Linkage map construction and QTL identification of P-deficiency tolerance in Oryza rufipogon Griff. at early seedling stage

Low phosphorus availability is a major factor limiting rice productivity. In this study, a population of backcross recombinant inbred lines (BILs) derived from an inter-specific cross (Oryza sativa L. × O. rufipogon Griff.) was used for genetic linkage map construction and quantitative trait locus (QTL) mapping. The results showed that a linkage map consisting of 153 markers was constructed. Twenty-one out of 231 BILs were tolerant of low-phosphorus according to the index to P-deficiency tolerance. Twenty-three QTLs on chromosomes 1, 2, 3, 7, 8, 9 and 11 were detected, of which eight QTLs showed high (22.93–37.32%) contribution to phenotypic variation. In addition, most of QTLs in this study (18 out of 23 QTLs) were located and overlapped on the chromosome 1, 3 and 11, which individually explained 6.07–34.70% phenotypic variation, indicating that there might be multiple main effect QTLs related to P-deficiency tolerance in O. rufipogon, and these QTLs might cluster in the same region. These results would provide helpful information for cloning and utilizing the P-deficiency tolerance-responsive genes from O. rufipogon.

Identification of microRNAs in Response to Drought in Common Wild Rice (Oryza rufipogon Griff.) Shoots and Roots.

BackgroundDrought is the most important factor that limits rice production in drought-prone environments. Plant microRNAs (miRNAs) are involved in biotic and abiotic stress responses. Common wild rice (Oryza rufipogon Griff.) contains abundant drought-resistant genes, which provide an opportunity to explore these excellent resources as contributors to improve rice resistance, productivity, and quality.ResultsIn this study, we constructed four small RNA libraries, called CL and CR from PEG6000-free samples and DL and DR from PEG6000-treated samples, where ‘R’ indicates the root tissue and ‘L’ indicates the shoot tissue. A total of 200 miRNAs were identified to be differentially expressed under the drought-treated conditions (16% PEG6000 for 24 h), and the changes in the miRNA expression profile of the shoot were distinct from those of the root. At the miRNA level, 77 known miRNAs, which belong to 23 families, including 40 up-regulated and 37 down-regulated in the shoot, and 85 known miRNAs in 46 families, including 65 up-regulated and 20 down-regulated in the root, were identified as differentially expressed. In addition, we predicted 26 new miRNA candidates from the shoot and 43 from the root that were differentially expressed during the drought stress. The quantitative real-time PCR analysis results were consistent with high-throughput sequencing data. Moreover, 88 miRNAs that were differentially-expressed were predicted to match with 197 targets for drought-stress.ConclusionOur results suggest that the miRNAs of O. rufipogon are responsive to drought stress. The differentially expressed miRNAs that are tissue-specific under drought conditions could play different roles in the regulation of the auxin pathway, the flowering pathway, the drought pathway, and lateral root formation. Thus, the present study provides an account of tissue-specific miRNAs that are involved in the drought adaption of O. rufipogon.

Cytological analysis for meiotic patterns in wild rice (Oryza rufipogon Griff.)

The present report explores the chromosomal patterns during meiosis as a fundamental cell division study in wild rice (Oryza rufipogon Griff.). Cytological assays revealed normal meiosis in most cases but in some instances meiotic abnormalities such as weak desynapsis, univalent and quadrivalent formation, translocation, spindle abnormalities and precocious movement of chromosomes were noticed. Interestingly, this wild species also has the bi-nucleoli in first meiotic stages alike the cultivated species of Oryza (O. sativa). The present investigation emphatically addresses the questions of high adaptability of wild rice supported by high pollen fertility for their potential to strong fitness in nature.

The Photosynthetic Characteristics in Chaling Common Wild Rice (Oryza rufipogon Griff.) under the Cold Stress

Chaling common wild rice (CWR, Oryza rufipogon Griff.), distributed in Chaling county, Hunan province, China, is one of the only two wild rice populations grown northernmost in the world. Although the cold tolerance of Chaling CWR is extremely strong, its photosynthetic characteristics under the cold stress remain unknown. In this study, we determined the net photosynthetic rate (Pn), total chlorophyll (Chl) and total carotenoid (Car) contents, maximum quantum yield of PSII (Fv/Fm), apparent quantum yield (AQY), and carboxylation efficiency (CE) of leaves from Chaling CWR at tiller stage under cold stress (15 ℃) and normal temperature condition (28 ℃). These photosynthetic parameters were compared with standard rice cultivars including typical indica Guangluai 4, indica Y liangyou 1 (a Chinese super high grain yield hybrid), and typical japonica Nipponbare under the same conditions. All photosynthetic parameters in Chaling CWR were significantly (P £ 0.05 or P £ 0.01) higher than those in standard rice cultivars under the cold stress. The ratios of these parameters from Chaling CWR under the cold stress to those under the normal temperature were 47.9% ~ 84.9%, much higher than those from standard rice cultivars, which were 3.1% ~ 73.8%. Taken together, these data show the photosynthetic characteristics of Chaling CWR under the cold stress are excellent. The finding would generate an enormous benefit if the trait of the cold stress tolerance of Chaling CWR is transferred to the cultivated rice via the breeding or other genetic approaches.

Linkage map construction and QTL mapping for cold tolerance in Oryza rufipogon Griff. at early seedling stage

Cold stress is one of the major restraints for rice production. Cold tolerance is controlled by complex genetic factor. In this study, a backcross inbred lines (BILs) population derived from an inter-specific cross (Oryza sativa L.×O. rufipogon Griff.) was used for genetic linkage map construction and quantitative trait locus (QTL) mapping. A linkage map consisting of 153 markers was constructed, spanning 1 596.8 cM with an average distance of 11.32 cM between the adjacent markers. Phenotypic evaluation of the parents and BILs under (6±1)°C cold stress revealed that the ability of cold tolerance in BILs at early seedling obeyed a skewed normal and continuous distribution. Fifteen QTLs on chromosomes 6, 7, 8, 11, and 12 were identified using survival percent (SP) and non death percent (NDP) as indicators of cold tolerance, which could explain 5.99 to 40.07% of the phenotypic variance, of which the LOD values ranged from 3.04 to 11.32. Four QTLs on chromosomes 3, 5 and 7 were detected using leaf conductivity (LC) and root conductivity (RC) as indicators of cold tolerance, ranging from 19.54 to 33.53% for the phenotypic variance explained and 2.54 to 6.12 for the LOD values. These results suggested that there might be multi major QTLs in O. rufipogon and some useful genes for cold tolerance have been transferred into cultivated rice, which would be helpful for cloning and utilizing the cold tolerance-responsive genes from wild rice.

Development and Evaluation of Chromosome Segment Substitution Lines Carrying Overlapping Chromosome Segments of the Whole Wild Rice Genome.

Common wild rice (Oryza rufipogon Griff.) represents an important resource for rice improvement. Genetic populations provide the basis for a wide range of genetic and genomic studies. In particular, chromosome segment substitution lines (CSSLs) are most powerful tools for the detection and precise mapping of quantitative trait loci (QTLs). In this study, 146 CSSLs were produced; they were derived from the crossing and back-crossing of two rice cultivars: Dongnanihui 810 (Oryza sativa L.), an indica rice cultivar as the recipient, and ZhangPu wild rice, a wild rice cultivar as the donor. First, a physical map of the 146 CSSLs was constructed using 149 molecular markers. Based on this map, the total size of the 147 substituted segments in the population was 1145.65 Mb, or 3.04 times that of the rice genome. To further facilitate gene mapping, heterozygous chromosome segment substitution lines (HCSSLs) were also produced, which were heterozygous in the target regions. Second, a physical map of the 244 HCSSLs was produced using 149 molecular markers. Based on this map, the total length of substituted segments in the HCSSLs was 1683.75 Mb, or 4.47 times the total length of the rice genome. Third, using the 146 CSSLs, two QTLs for plant height, and one major QTL for apiculus coloration were identified. Using the two populations of HCSSLs, the qPa-6-2 gene was precisely mapped to an 88 kb region. These CSSLs and HCSSLs may, therefore, provide powerful tools for future whole genome large-scale gene discovery in wild rice, providing a foundation enabling the development of new rice varieties. This research will also facilitate fine mapping and cloning of quantitative trait genes, providing for the development of superior rice varieties.

Discovery, utilization and molecular mechanisms of CMS-WA in rice

The discovery and exploitation of the Wild Abortive type cytoplasmic male sterility (CMS-WA) is a key issue in hybrid rice breeding for utilization of heterosis, which is the best successful case for exploitation of wild plant resources in agriculture. In this review, we traced the history of discovery and research of CMS-WA, and introduced the latest progresses on study of the molecular basis of CMS-WA and its restoration as well as the evolution of the related genes. The Chinese scientist Yuan LongPing pioneered the study of male sterility in rice since 1964. In 1970s, Yuan’s group discovered a male sterile wild rice ( Oryza rufipogon Griff.) plant in Hainan Island of China, and designated this material as “Wild Abortive (WA)”. Genetic study showed that this male sterility is conferred by the cytoplasm, thus is called CMS-WA. Chinese breeders made great efforts to breed CMS lines by introgression of the CMS-WA cytoplasm into a number of rice cultivars via backcrossing. Accordingly, related maintainer lines and restorer lines were bred to successfully develop the “three-line” system for hybrid rice production. The commercial hybrid rice was released in 1976 in China, and has occupied about 50% of the total rice planting area since the late 1980s, which increased grain yield by 20%–30%. In recent years, scientists have made great progress on the molecular studies of CMS-WA and its restoration, and the evolution of the genes for this system. As representative in this field, LIU YaoGuang’s group isolated the CMS-WA gene WA352 from the mitochondrial genome, which is a new gene consisting of multiple mitochondrial genomic segments of unknown function. WA352 is expressed constitutively into three transcripts, but the WA352 protein accumulates specifically in the anther tapetal cells at the microspore mother cell stage, and interacts directly with a nucleus-encoded protein COX11, which is the assembly factor for cytochrome c oxidase and also has a role in scavenge of reactive oxygen species (ROS) and inhibition of programmed cell death (PCD). When WA352 arrests COX11 by the interaction, the metabolism of ROS is affected, leading to ROS burst and cytochrome c release to the cytosol and triggering premature PCD in the tapetum. This abnormal tapetal degeneration eventually causes pollen abortion. Two loci, Rf3 and Rf4 , were mapped on chromosome 1 and 10, respectively, as the restorer genes for CMS-WA. Rf4 was firstly cloned recently by Liu’s group. Rf4 encodes a pentatricopeptide repeat (PPR) protein (PPR9-782-M) possessing a mitochondrial transit signal and 18 PPR motifs, with high similarity to PPR3-791-M encoded by Rf1a of the CMS-BT system. The Rf4 protein decreases the mRNA level of WA352 , thereby restoring WA352 -mediated male sterility probably in a post-transcriptional mechanism. However, Rf3 does not affect the WA352 transcript abundance but impairs the production of WA352 protein. Thus, the studies indicate that plant CMS/restoration systems comprises multiple layers of cytoplasmic-nuclear gene interactions in the molecular level. Recently, LIU’s group further studied the evolutionary trajectory of WA352 by identification and characterization of a number of mitochondrial genomic recombinant structures related to WA352 in the wild rice. These structures originated and evolved through complex evolutionary routes by multiple rearrangements in the mitochondrial genome of O. rufipogon. The study revealed that functional CMS genes originated from non-CMS protogenes by sequence variation-based functionalization as well as substoichiometric shifting (i.e., copy number variation). Finally, this review discussed the key issues and future directions of the hybrid rice breeding programs.

Combining QTL mapping and expression profile analysis to identify candidate genes of cold tolerance from Dongxiang common wild rice (Oryza rufipogon Griff.)

Rice (Oryza sativa L.), a tropical and subtropical crop, is susceptible to low temperature stress during seedling, booting, and flowering stages, which leads to lower grain quality levels and decreasing rice yields. Cold tolerance is affected by multiple genetic factors in rice, and the complex genetic mechanisms associated with chilling stress tolerance remain unclear. Here, we detected seven quantitative trait loci (QTLs) for cold tolerance at booting stage and identified one cold tolerant line, SIL157, in an introgression line population derived from a cross between the indica variety Guichao 2, as the recipient, and Dongxiang common wild rice, as the donor. When compared with Guichao 2, SIL157 showed a stronger cold tolerance during different growth stages. Through an integrated strategy that combined QTL-mapping with expression profile analysis, six candidate genes, which were up-regulated under chilling stress at the seedling and booting developmental stages, were studied. The results may help in understanding cold tolerance mechanisms and in using beneficial alleles from wild rice to improve the cold tolerance of rice cultivars through molecular marker-assisted selection.