Identification of Rice Accessions Having Cold Tolerance at the Seedling Stage and Development of Novel Genotypic Assays for Predicting Cold Tolerance.

Rice is susceptible to cold stress at the seedling stage, which can delay growth and decrease yield. We evaluated 187 rice accessions for cold tolerance at the seedling stage and developed genotypic assays for three markers. All japonica (20/20) and 20/140 indica accessions were highly cold tolerant. Two SNP markers specific for COLD1 and LOC_Os10g34840 were practical to use by normal agarose gel. The SNP marker specific for COLD1 was highly specific for predicting cold tolerance. However, the sensitivity of this marker was low as several cold-tolerant indica accessions lacked the cold-tolerant allele. The LOC_Os10g34840 marker was slightly more sensitive than the COLD1 marker for predicting highly cold-tolerant accessions. An insertion/deletion variant in the NAC6 gene was identified as a novel cold tolerance marker. The NAC6 marker predicted more highly cold-tolerant accessions compared with the other two markers. The SNP marker specific for LOC_Os10g34840 and the NAC6 marker were present in several tested subgroups, suggesting their wide effects and distribution. The three markers combined predicted the most highly cold-tolerant accessions, indicating that the marker combination is superior for applications such as marker-assisted breeding. The cold-tolerant accessions and the genotypic marker assays will be useful for future rice breeding.

RNA Sequencing Reveals Rice Genes Involved in Male Reproductive Development under Temperature Alteration.

Rice (Oryza sativa L.) is one of the most important food crops, providing food for nearly half of the world population. Rice grain yields are affected by temperature changes. Temperature stresses, both low and high, affect male reproductive development, resulting in yield reduction. Thermosensitive genic male sterility (TGMS) rice is sterile at high temperature and fertile at low temperature conditions, facilitating hybrid production, and is a good model to study effects of temperatures on male development. Semithin sections of the anthers of a TGMS rice line under low (fertile) and high (sterile) temperature conditions showed differences starting from the dyad stage, suggesting that genes involved in male development play a role during postmeiotic microspore development. Using RNA sequencing (RNA-Seq), transcriptional profiling of TGMS rice panicles at the dyad stage revealed 232 genes showing differential expression (DEGs) in a sterile, compared to a fertile, condition. Using qRT-PCR to study expression of 20 selected DEGs using panicles of TGMS and wild type rice plants grown under low and high temperature conditions, revealed that six out of the 20 selected genes may be unique to TGMS, while the other 14 genes showed common responses to temperatures in both TGMS and wild-type rice plants. The results presented here would be useful for further investigation into molecular mechanisms controlling TGMS and rice responses to temperature alteration.

Rice ORMDL controls sphingolipid homeostasis affecting fertility resulting from abnormal pollen development.

The orosomucoids (ORM) are ER-resisdent polypeptides encoded by ORM and ORMDL (ORM-like) genes. In humans, ORMDL3 was reported as genetic risk factor associated to asthma. In yeast, ORM proteins act as negative regulators of sphingolipid synthesis. Sphingolipids are important molecules regulating several processes including stress responses and apoptosis. However, the function of ORM/ORMDL genes in plants has not yet been reported. Previously, we found that temperature sensitive genetic male sterility (TGMS) rice lines controlled by tms2 contain a deletion of about 70 kb in chromosome 7. We identified four genes expressed in panicles, including an ORMDL ortholog, as candidates for tms2. In this report, we quantified expression of the only two candidate genes normally expressed in anthers of wild type plants grown in controlled growth rooms for fertile and sterile conditions. We found that only the ORMDL gene (LOC_Os07g26940) showed differential expression under these conditions. To better understand the function of rice ORMDL genes, we generated RNAi transgenic rice plants suppressing either LOC_Os07g26940, or all three ORMDL genes present in rice. We found that the RNAi transgenic plants with low expression of either LOC_Os07g26940 alone or all three ORMDL genes were sterile, having abnormal pollen morphology and staining. In addition, we found that both sphingolipid metabolism and expression of genes involved in sphingolipid synthesis were perturbed in the tms2 mutant, analogous to the role of ORMs in yeast. Our results indicated that plant ORMDL proteins influence sphingolipid homeostasis, and deletion of this gene affected fertility resulting from abnormal pollen development.

Genetic structure of Thai rice and rice accessions obtained from the International Rice Research Institute.

BACKGROUND: Although the genetic structure of rice germplasm has been characterized worldwide, few studies investigated germplasm from Thailand, the world's largest exporter of rice. Thailand and the International Rice Research Institute (IRRI) have diverse collections of rice germplasm, which could be used to develop breeding lines with desirable traits. This study aimed to investigate the level of genetic diversity and structures of Thai and selected IRRI germplasm. Understanding the genetic structure and relationships among these germplasm will be useful for parent selection used in rice breeding programs. RESULTS: From the 98 InDel markers tested for single copy and polymorphism, 19 markers were used to evaluate 43 Thai and 57 IRRI germplasm, including improved cultivars, breeding lines, landraces, and 5 other Oryza species. The Thai accessions were selected from all rice ecologies such as irrigated, deep water, upland, and rainfed lowland ecosystems. The IRRI accessions were groups of germplasm having agronomic desirable traits, including temperature-sensitive genetic male sterility (TGMS), new plant type, early flowering, and biotic and abiotic stress resistances. Most of the InDel markers were genes with diverse functions. These markers produced the total of 127 alleles for all loci, with a mean of 6.68 alleles per locus, and a mean Polymorphic Information Content (PIC) of 0.440. Genetic diversity of Thai rice were 0.3665, 0.4479 and 0.3972 for improved cultivars, breeding lines, and landraces, respectively, while genetic diversity of IRRI improved and breeding lines were 0.3272 and 0.2970, respectively. Cluster, structure, and differentiation analyses showed six distinct groups: japonica, TGMS, deep-water, IRRI germplasm, Thai landraces and breeding lines, and other Oryza species. CONCLUSIONS: Thai and IRRI germplasm were significantly different. Thus, they can be used to broaden the genetic base and trait improvements. Cluster, structure, and differentiation analyses showed concordant results having six distinct groups, in agreement with their development, and ecologies.

Identification of a deletion in tms2 and development of gene-based markers for selection.

Rice is one of the most important food crops. The temperature-sensitive genic male sterility (TGMS) system provides a great potential for improving food production by hybrids. The use of TGMS system is simple, inexpensive, effective, and eliminates the limitations of the conventional three-line system. A rice gene, tms2, generated by irradiation of a japonica variety has been reported to control TGMS in several rice lines. Previous studies reported genetic markers linked to this gene, and the gene was transferred to an aromatic Thai cultivar. Using information obtained from published databases, we located positions of the reported genetic markers flanking the gene in rice genomic sequences, and developed gene-based markers located inside the flanking markers for polymorphism detection. We found that inbred indica tms2 mutant plants contain about 1 Mb of japonica DNA, in which at least 70 kb was deleted. Using RT-PCR for expression analysis, four genes out of seven genes annotated as expressed proteins located inside the deletion showed expression in panicles. These genes could be responsible for TGMS phenotypes of tms2. In addition, we developed gene-based markers flanking and inside the deletion for selecting the tms2 gene in breeding populations. By genotyping 102 diverse rice lines including 38 Thai rice lines, 5 species of wild rice, and 59 exotic rice lines including TGMS lines and cultivars with desirable traits, a gene-based marker located inside the deletion and one flanking marker were shown to be highly specific for the tms2 mutant.

The reticulate history of Medicago (Fabaceae).

The phylogenetic history of Medicago was examined for 60 accessions from 56 species using two nuclear genes (CNGC5 and beta-cop) and one mitochondrial region (rpS14-cob). The results of several analyses revealed that extensive robustly supported incongruence exists among the nuclear genes, the cause of which we seek to explain. After rejecting several processes, hybridization and lineage sorting of ancestral polymorphisms remained as the most likely factors promoting incongruence. Using coalescence simulations, we rejected lineage sorting alone as an explanation of the differences among gene trees. The results indicate that hybridization has been common and ongoing among lineages since the origin of Medicago. Coalescence provides a good framework to test the causes of incongruence commonly seen among gene trees but requires knowledge of effective population sizes and generation times. We estimated the effective population size at 240,000 individuals and assumed a generation time of 1 year in Medicago (many are annual plants). A sensitivity analysis showed that our conclusions remain unchanged using a larger effective population size and/or longer generation time.

A novel dwarfing mutation in a green revolution gene from Brassica rapa.

Mutations in the biosynthesis or signaling pathways of gibberellin (GA) can cause dwarfing phenotypes in plants, and the use of such mutations in plant breeding was a major factor in the success of the Green Revolution. DELLA proteins are GA signaling repressors whose functions are conserved in different plant species. Recent studies show that GA promotes stem growth by causing degradation of DELLA proteins via the ubiquitin-proteasome pathway. The most widely utilized dwarfing alleles in wheat (Triticum aestivum; e.g. Rht-B1b and Rht-D1b) encode GA-resistant forms of a DELLA protein that function as dominant and constitutively active repressors of stem growth. All of the previously identified dominant DELLA repressors from several plant species contain N-terminal mutations. Here we report on a novel dwarf mutant from Brassica rapa (Brrga1-d) that is caused by substitution of a conserved amino acid in the C-terminal domain of a DELLA protein. Brrga1-d, like N-terminal DELLA mutants, retains its repressor function and accumulates to high levels, even in the presence of GA. However, unlike wild-type and N-terminal DELLA mutants, Brrga1-d does not interact with a protein component required for degradation, suggesting that the mutated amino acid causes dwarfism by preventing an interaction needed for its degradation. This novel mutation confers nondeleterious dwarf phenotypes when transferred to Arabidopsis (Arabidopsis thaliana) and oilseed rape (Brassica napus), indicating its potential usefulness in other crop species.