Nipponbare Genome Sequence Research Articles

Genome-wide association mapping and gene expression analysis identify OsCPS1 as a new candidate gene controlling early seedling length in rice.

High seedling vigor can improve the ability to compete against weeds and flooding at the seedling stage and is essential for the direct seeding of rice. Early shoot length is an important performance index in seedling vigor evaluation. However, information on the identity of rice germplasm with high seedling vigor, and the genetic basis of seedling vigor are not well understood. In this study, we have conducted a genome-wide association study using 302 international diverse rice accessions from the Rice Diversity Panel 2. Six quantitative trait loci (QTLs) were found to associate with shoot length (SL). The locus qSL2 was further analyzed for candidate gene characterization. We identified OsCPS1, which encodes CDP synthase and functions in GA (Gibberellins) biosynthesis in rice, exhibits differential expression between long and short SL accessions. Using the Nipponbare genome sequence as the reference, we identified a 36 bp deletion in the 5’ UTR of OsCPS1 in long SL accessions, which is absent in short SL accessions. GA content analysis showed that the levels of bioactive GA1 and GA4 are considerably higher in long SL accessions than in short SL accessions. Genome-wide gene expression analysis indicated the expression of some photosynthesis genes is higher in long SL accessions than in short SL accessions. In contrast, genes involved in ABA (Abscisic Acid)-activated signal pathway showed lower expression in long SL accessions. Population analysis across wild rice, indica and japonica, suggested that OsCPS1 may be under selection in japonica during domestication. The results suggest that OsCPS1 is a candidate gene underlying qSL2. These data provide a promising source for candidate genetic variation associated with seedling vigor, with practical applications in rice breeding.

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.

Fine mapping of a major quantitative trait locus, qLG-9, that controls seed longevity in rice (Oryza sativa L.).

We fine-mapped a quantitative trait locus, qLG - 9, for seed longevity detected between Japonica-type and Indica-type cultivars. qLG - 9 was mapped in a 30-kb interval of the Nipponbare genome sequence. A quantitative trait locus, qLG-9, for seed longevity in rice has previously been detected on chromosome 9 by using backcross inbred lines derived from a cross between Japonica-type (Nipponbare) and Indica-type (Kasalath) cultivars. In the present study, the chromosomal location of qLG-9 was precisely determined by fine-scale mapping. Firstly, allelic difference in qLG-9 was verified by QTL analysis of an F2 population derived from a cross between Nipponbare and NKSL-1, in which a segment of Kasalath chromosome 9 was substituted in Nipponbare genetic background. Then, we selected F2 plants in which recombination had occurred near qLG-9 and performed F3 progeny testing on these plants to determine the genotype classes of qLG-9. Eventually, qLG-9 was mapped in a 30-kb interval (defined by two markers, CAPSb and CHPa12) of the Nipponbare genome sequence. This allowed us to nominate positional candidate genes of qLG-9. Additionally, we developed near-isogenic lines (NIL) for qLG-9 by marker-assisted selection. qLG-9 NIL showed significantly higher seed longevity than isogenic control of Nipponbare. These results will facilitate cloning of the gene(s) underlying qLG-9 as well as marker-assisted transfer of desirable genes for seed longevity improvement in rice.

Fine Mapping of a Quantitative Trait Loci Controlling the Number of Spikelets per Panicle in Rice

Spikelets per panicle (SPP) is one of the most important traits associated with rice yield. In this study, IL28, a near isogenic line (NIL) developed by introgressing chromosomal segments from ‘Moroberekan’ into ‘Ilpumbyeo’ showed significantly higher number of spikelets per panicle than the recurrent parent, ‘Ilpumbyeo’. Quantitative trait locus (QTL) analysis in 243 F2 plants derived from a cross between IL28 and Ilpumbyeo indicated that a QTL for spikelets per panicle, qSPP6 was located in the interval RM3430 - RM20580. The Moroberekan allele increased SPP. The fact that QTLs for panicle length and the number of secondary branches were mapped in the same interval as qSPP6 appears to indicate that this locus was associated with panicle structure. To map the QTL more precisely, substitution mapping of qSPP6 using F3 lines was conducted. Substitution mapping with 41 F3 lines further narrowed the interval containing not only qSPP6 for spikelets per panicle but also qNDW6 for node width to about 680-kb between markers RM20521 and RM20572 based on Nipponbare genome sequence. The locus, qSPP6 is of particular interest because of its independence from undesirable height and flowering time. SSR markers tightly linked to the qSPP6 will facilitate cloning of the gene underlying this QTL as well as marker assisted selection for variation in SPP in the breeding program.

Identification and Fine Mapping of Heading Date Related Mutant Gene in Rice

A rice heading-date-related mutant was isolated from a 60Co-γ-ray-induced mutation pool of Zhejing 22, a conventional japonica cultivar in Zhejiang Province, China. The mutant was characterized by a delayed heading date of almost 20 d longer than the wild type plant. Genetic analysis revealed that the mutation was controlled by a single nuclear-encoded recessive gene that was designed as HD(t) (heading date tentatively). To isolate the HD(t) gene, a map-based cloning approach was employed using 479 F2 mutant individual plants derived from the cross between the hd(t) mutant (japonica) × Zhenshan 97 (indica). Finally, the HD(t) gene was mapped to an approximate 53 kb region between the insertion and deletion (InDel) markers of 10-61W and 10-66W on chromosome 10. According to the genome sequence of Nipponbare, the target region contains 11 annotated genes. It is helpful for future cloning of HD(t) gene based on this fine mapping results.

Identification and gene mapping of a narrow and upper-albino leaf mutant in rice (Oryza sativa L.)

The leaf blade consists of color and shape traits. Studies of leaf-blade development are important for improvement of rice yield and quality because it is an essential organ for photosynthesis. A narrow and upper-albino leaf mutant (nul1) was identified from among progeny of the indica restorer line Jinhui10 raised from seeds treated with ethyl methane sulfonate. Under field conditions, the mutant displayed narrow and upper-albino leaf blades with significantly decreased photosynthetic pigment contents throughout their development. The narrow-leaf trait is caused by a decreased number of small veins. In contrast to the wild type, the growth period was extended by approximately 8 d and agronomic traits, such as effective panicle number, percentage seed set and 1000-grain weight, declined significantly in the nul1 mutant. Genetic analysis suggested that the narrow and upper-albino leaf characteristics showed coseparation and were controlled by one recessive gene. The Nul1 gene was mapped onto chromosome 7 between the Indel marker Ind07-1 and the Simple Sequence Repeat marker RM21637. The physical distance between the markers was 75 kb and eight genes were annotated in this region based on the rice Nipponbare genome sequence. These results provide a foundation for cloning and function analysis of Nul1.

Genetic and physical mapping of qHY-8, a pleiotropic QTL for heading date and yield-related traits in rice

A QTL, qHY-8, has been resolved into a single Mendelian locus controlling heading date and yield-related traits in rice. The trait-performance of the three genotypes for the QTL in the BC5F2 or BC4F2 populations (with cultivar Teqing or YuetaiB as recurrent parents) indicated that the Lemont (donor parent) allele at qHY-8 locus showed a positive and pleiotropic additive effect on heading date (HD), plant height (PH), panicle length (PL), and spikelets per panicle (SPP).The QTL’s additive effects varied for different traits according to its heritabilities, it showed a largest effect on HD with a heritability of 98%, secondly on PL with a heritability of 70–87%, then on PH with a heritability of 56–61%, and lastly on SPP with a heritability of 45–47%. In addition, the QTL showed a weaker dominant effect, significant only on HD and PL. With an advanced BC5F2 population, qHY-8 was firstly mapped to a 299 kb genomic region by recessive-class analysis. Then, with a set of segment substitution lines, substitution mapping located qHY-8 within a chromosomal region between markers ID3468 and ID4891, corresponding to 31.4 kb on the genome sequence of Nipponbare. Sequence analysis of an ORF Os08g07740 which encodes a histone-like CCAAT-box binding transcription factor suggested that the ORF would be tentatively referred to as the first strong candidate gene. All these results demonstrated that qHY-8 is a pleiotropic QTL for heading date and multiple yield-related traits and would be of great value to the improvement of rice productivity.

Fine mapping of qHUS6.1, a quantitative trait locus for silicon content in rice (Oryza sativa L.)

Silicon is essential for optimal growth of rice (Oryza sativa L.). This study was conducted to fine map qHUS6.1, a quantitative trait locus (QTL) for rice hull silicon content previously located in the interval RM510-RM19417 on the short arm of chromosome 6, and to analyze the effect of this QTL on the silicon content in different organs of rice. Selfed progenies of a residual heterozygous line of rice were detected using 13 microsatellite markers in the vicinity of qHUS6.1. Three plants with overlapping heterozygous segments were selected. Three sets of near isogenic lines (NILs) were developed from the selfed progenies of the 3 plants. They were grown in a paddy field and the silicon contents of the hull, flag leaf, and stem were measured at maturity. Based on analyses of the phenotypic distribution and variance among different genotypic groups in the same NIL set, a significant genotypic effect was shown in the NIL set that was heterogenous in the interval RM19410-RM5815, whereas a significant effect was not found in the remaining 2 NIL sets that were heterogenous in either of the intervals RM4923-RM19410 or RM19417-RM204. On comparison among the physical positions of the 3 heterogenous segments, qHUS6.1 was delimited to a 64.2-kb region flanked by RM19410 and RM19417 that contains nine annotated genes according to the genome sequence of Nipponbare. This QTL showed strong effects on all of the three traits tested, and the enhancing alleles were always derived from the paternal line Milyang 46. The present study will facilitate the cloning of qHUS6.1 and the exploration of new genetic resources for QTL fine mapping.

Fine mapping and identification of tightly linked DNA markers of blast resistance gene Pia by using an introgression line

An introgression line (INL) for a major rice blast resistance gene, Pia, was developed, with the genetic background of a blast susceptible variety, US-2. The reaction pattern of the INL was characterized by using 20 standard blast isolates from the Philippines. The introgression of the Pia gene was confirmed by DNA markers on the short arm of chromosome 11 where Pia was previously mapped. A genome-wide DNA marker survey revealed that most of the chromosomal regions were US-2 type. By using an F2 population derived from a cross between the INL and US-2, the chromosomal location of the Pia locus was mapped between RM26281 and RM3701. For fine mapping of the Pia locus, five additional markers were developed based on the genomic sequence of the corresponding region of a japonica-type variety, Nipponbare. The candidate region of Pia was delimited between two DNA markers, RM26281 and 82N19365, corresponding to a 140 kb region on the Nipponbare genome sequence. We obtained three DNA markers within this region. The developed INL, information on the map position of Pia, and DNA markers developed in the candidate region of the Pia locus are useful tools for blast resistance studies and a marker-aided breeding strategy.

Fine mapping of a gene for low-tiller number, Ltn, in japonica rice (Oryza sativa L.) variety Aikawa 1

Tillering is one of the most important agronomic traits related to grain production in rice (Oryza sativa L.). A japonica-type variety, Aikawa 1, is known to have low-tiller number. The detailed location of a low-tillering gene, Ltn, which has been localized on chromosome 8 in Aikawa 1, was confirmed by molecular mapping. Using BC5F2 individuals derived from a cross between IR64 and Aikawa 1, the low-tillering gene was mapped to an interval defined by SSR markers ssr5816-3 and A4765. This was designated as Ltn because there was no reported gene for tillering in the region of chromosome 8. Through high-resolution linkage analysis, the candidate region of Ltn was located between DNA markers ssr6049-23 and ind6049-1 corresponding to 38.6 kbp on the Nipponbare genome sequence. These DNA markers, which were tightly linked to Ltn, are useful for marker-assisted selection in breeding studies.

Diversity of the Ty-1 copia retrotransposon Tos17 in rice (Oryza sativa L.) and the AA genome of the Oryza genus

Retrotransposons are mobile genetic elements, ubiquitous in Eukaryotic genomes, which have proven to be major genetic tools in determining phylogeny and structuring genetic diversity, notably in plants. We investigate here the diversity of the Ty1-copia retrotransposon Tos17 in the cultivated rice of Asian origin (Oryza sativa L.) and related AA genome species of the Oryza genus, to contribute understanding of the complex evolutionary history in this group of species through that of the element in the lineages. In that aim, we used a combination of Southern hybridization with a reverse transcriptase (RT) probe and an adapter-PCR mediated amplification, which allowed the sequencing of the genomic regions flanking Tos17 insertions. This analysis was carried out in a collection of 47 A-genome Oryza species accessions and 202 accessions of a core collection of Oryza sativa L. representative of the diversity of the species. Our Southern hybridization results show that Tos17 is present in all the accessions of the A-genome Oryza species, except for the South American species O. glumaepatula and the African species O. glaberrima and O. breviligulata. In O. sativa, the number of putative copies of Tos17 per accession ranged from 1 to 11 and multivariate analysis based on presence/absence of putative copies yielded a varietal clustering which is consistent with the isozyme classification of rice. Adapter PCR amplification and sequencing of flanking regions of Tos17 insertions in A-genome species other than O. sativa, followed by anchoring on the Nipponbare genome sequence, revealed 13 insertion sites of Tos17 in the surveyed O. rufipogon and O. longistaminata accessions, including one shared by both species. In O. sativa, the same approach revealed 25 insertions in the 6 varietal groups. Four insertion sites located on chromosomes 1, 2, 10, and 11 were found orthologous in O. rufipogon and O. sativa. The chromosome 1 insertion was also shared between O. rufipogon and O. longistaminata. The presence of Tos17 at three insertion sites was confirmed by retrotransposon-based insertion polymorphism (RBIP) in a sample of O. sativa accessions. The first insertion, located on chromosome 3 was only found in two japonica accessions from the Bhutan region while the second insertion, located on chromosome 10 was specific to the varietal groups 1, 2, and 5. The third insertion located on chromosome 7 corresponds to the only insertion shown active in rice so far, notably in cv. Nipponbare, where it has been extensively used for insertion mutagenesis. This copy was only found in a few varieties of the japonica group 6 and in one group 5 accession. Taken together, these results confirm that Tos17 was probably present in the ancestor of A-genome species and that some copies of the element remained active in some Oryza lineages--notably in O. rufipogon and O. longistaminata--as well as in the indica and japonica O. sativa L. lineages.

The qSD12 underlying gene promotes abscisic acid accumulation in early developing seeds to induce primary dormancy in rice

Seeds acquire primary dormancy during their development and the phytohormone abscisic acid (ABA) is known to play a role in inducing the dormancy. qSD12 is a major seed dormancy quantitative trait locus (QTL) identified from weedy rice. This research was conducted to identify qSD12 candidate genes, isolate the candidates from weedy rice, and determine the relation of the dormancy gene to ABA. A fine mapping experiment, followed by marker-assisted progeny testing for selected recombinants, narrowed down qSD12 to a genomic region of <75 kb, where there are nine predicted genes including a cluster of six transposon/retrotransposon protein genes and three putative (a PIL5, a hypothetic protein, and a bHLH transcription factor) genes based on the annotated Nipponbare genome sequence. The PIL5 and bHLH genes are more likely to be the QTL candidate genes. A bacterial artificial chromosome (BAC) library equivalent to 8-9 times of the haploid genome size was constructed for the weedy rice. One of the two BAC contigs developed from the library covers the PIL5 to bHLH interval. A pair of lines different only in the QTL-containing region of <200 kb was developed as isogenic lines for the qSD12 dormancy and non-dormancy alleles. The dormant line accumulated much higher ABA in 10-day developing seeds than the non-dormant line. In the QTL-containing region there is no predicted gene that has been assigned to ABA biosynthetic or metabolic pathways. Thus, it is concluded that the qSD12 underlying gene promotes ABA accumulation in early developing seeds to induce primary seed dormancy.

Characterization of Rice Blast Resistance Genes in the Pik Cluster and Fine Mapping of the Pik-p Locus

Pik-p is carried by cv. K60, which is one of the Japanese differentials widely used in both Japan and China since the 1980s. Its utility and specificity was evaluated with a total of 612 isolates of Magnaporthe oryzae collected from various regions in China in combination with 16 main resistance genes being used in the breeding programs. Pik-p is an independently and dominantly acting gene in the Pik cluster, which conditions differential reactions against many isolates and contains higher resistance in Guangdong, Jiangsu, and Sichuan provinces, China, indicating that this gene could be still used in these regions. A high-resolution genetic map of Pik-p was constructed using genomic position-ready markers. A set of 47 recombinants out of 681 F(2) plants derived from the crosses cv. K60 (resistant) x cv. AS20-1 (susceptible) and x cv. Kasalath (susceptible) was identified in the genetic interval defined by the markers RM5926 and K37 which flank the Pik gene cluster. This set was then genotyped with seven markers known to reside within the interval. The closest markers to Pik-p were K28 (approximately 0.60 centimorgans [cM]) and K39 (approximately 0.07 cM). A further four markers in the K28-K39 interval were developed from an in silico analysis based on the cv. Nipponbare genome sequence, and these all co-segregated with Pik-p. This 0.67-cM region is equivalent to a physical separation in cv. Nipponbare of approximately 126 kb, plus an as-yet-unfilled genomic gap of unknown length. Four nucleotide-binding site leucine-rich repeat-type resistance genes are present in this interval, and these represent good candidates for Pik-p.

Transcriptional activity of rice autonomous transposable element Dart

The rice Dart/nDart transposon system belongs to the hAT superfamily of class II transposons. The nonautonomous element nDart is active in intact rice plants. The autonomous element Dart was identified based on sequence similarity to nDart. Because the rice genome sequence of Nipponbare contains at least 51 Dart elements, it is not clear whether Dart elements are expressed or whether they are transposable. This study characterized for the expression of the predicted ORF of Dart. RNA blotting analysis revealed three transcripts of different lengths. Only the longest transcript (2.5kb) corresponding to the predicted ORF of the Dart element produced a functional TPase. The other transcripts had a frame-shift generating a premature stop codon through alternative splicing. These transcripts were expressed from either of two potentially autonomous Dart elements, Dart01/28 and Dart02. The frequency of alternative splicing differed between the transcripts of the derivative elements. More than 90% of the transcripts from Dart02 were alternatively spliced, compared with only 3% from Dart01/28. The element-specific expression and alternative splicing may control the transposition of nDart.

SSR Markers Closely Linked to the Pi-z Locus are Useful for Selection of Blast Resistance in a Broad Array of Rice Germplasm

Pi-z is a disease resistance gene that has been effectively used to combat a broad-spectrum of races of the rice blast fungus Magnaporthe grisea. Although DNA markers have been reported for selection of the Pi2(t) and Pi-z resistance genes at the Pi-z locus, markers that are more tightly linked to the Pi-z locus would benefit rapid and effective cultivar development. Analysis of the publicly available genome sequence of Nipponbare near the Pi-z locus revealed numerous SSRs that could be converted into markers. Three SSRs on rice PAC AP005659 were found to be very tightly linked to the Pi-z locus, with one marker, AP5659-3, co-segregating with the Pi-z resistance reaction. The Pi-z factor conferring resistance to two races of blast was mapped to a 57 kb region on the physical map of Nipponbare in a location where the Pi2(t) gene was physically mapped. Two SSR marker haplotypes were unique for cultivars carrying the Pi-z gene, which indicates these markers are useful for selection of resistance genes at the Pi-z locus in rice germplasm.