Haplotype analysis and marker development of five salt-tolerant-related genes in rice (Oryza sativa L.).

Salinity stress is a great threat to the growth and productivity of crops, and development of salt-tolerant crops is of great necessity to ensure food security. Although a few genes with natural variations that confer salt tolerance at germination and seedling stage in rice have been cloned, effective intragenic markers for these genes are awaited to be developed, which hinder the use of these genes in genetic improvement of salt tolerance in rice. In this study, we first performed haplotype analysis of five rice salt-tolerant-related genes using 38 rice accessions with reference genome and 4,726 rice germplasm accessions with imputed genotypes and classified main haplotype groups and haplotypes. Subsequently, we identified unique variations for elite haplotypes reported in previous studies and developed 11 effective intragenic makers. Finally, we conducted genotyping of 533 of the 4,726 rice accessions from worldwide and 70 approved temperate geng/japonica cultivars in China using the developed markers. These results could provide effective donors and markers of salt-tolerant-related genes and thus could be of great use in genetic improvement of salt tolerance in rice.

Development and Application of Intragenic Markers for 14 Nitrogen-Use Efficiency Genes in Rice (Oryza sativa L.).

Asian cultivated rice consists of two main subspecies, xian/indica (XI) and geng/japonica (GJ), and GJ accessions have significantly lower nitrogen-use efficiency (NUE) than XI accessions. In order to facilitate genetic improvement of NUE in GJ accessions, we conducted haplotype analysis of 14 cloned NUE genes using 36 rice germplasm accessions with high-quality reference genome and developed 18 intragenic markers for elite haplotypes, which were then used to evaluate NUE genes in another 41 genetically diverse germplasm accessions from 12 countries and 71 approved GJ cultivars from northern provinces of China. Our results show that elite haplotypes of 12 NUE genes are mainly existed in XI accessions, but few is distributed in GJ accessions. The number of elite haplotypes carried by an XI accession can reach 10, while that carried by a GJ accession is less than 3. Surprisingly, the elite haplotype of gene DEP1 is nearly fixed in approved GJ cultivars, and elite haplotypes of gene MYB61 and NGR5 have been introduced into some approved GJ cultivars. The developed intragenic markers for NUE genes and evaluated 77 genetically diverse rice accessions could be of great use in the improvement of NUE in GJ cultivars.

An S-domain receptor-like kinase, OsESG1, regulates early crown root development and drought resistance in rice.

Plant receptor-like kinase (RLKs) are serine/threonine protein kinases that play fundamental roles in development, innate immunity, and abiotic stress response. Here, we identified an S-domain receptor-like kinase OsESG1 from rice (Oryza sativa), and identified its involvement in early crown root (CR) development and drought response. The OsESG1 kinase domain possessed auto-phosphorylation activity and was able to phosphorylate MBP and His proteins. OsESG1 was expressed ubiquitously in all tissues that were examined, with relatively higher expression in the embryo. And it could be induced to express by treating with PEG, NaCl and ABA. Transgenic plants carrying anti-sense (AS) OsESG1 were generated by knockdown of OsESG1 expression. At the early seedling stage, AS lines had fewer CRs and shorter shoot compared with wild type (WT) plants. IAA flux and the genes' expressions of the auxin responsive and efflux carrier were infected in the AS lines. These results indicated that auxin signaling and polar auxin transport (PAT) were disrupted. The AS lines were more sensitive to osmotic stress compared to WT, and showed excessive accumulation of reactive oxygen species (ROS) and malondialdehyde (MDA), lower activities of antioxidant enzymes, and impaired expressions of stress-related genes under PEG treatment. Results above suggested that OsESG1 may regulate CR initiation and development by controlling auxin response and distribution, and participate in stress response by regulating the activities of antioxidants and expressions of stress-regulated genes.

Genetic dissection of QTLs and differentiation analysis of alleles for heading date genes in rice.

Heading date is an important agronomic trait in rice (Oryza sativa L.); it determines the geographical and seasonal adaptability of the crop. Single segment substitution lines (SSSLs) have become the preferred experimental materials in mapping functional genetic variations as the particular chromosome segments from donor genotypes can be evaluated for their impact on the phenotype in a recurrent recipient background. The phenotypic differences can be attributed to the control of quantitative trait loci (QTLs). Here, we evaluated a library consisting of 1,123 SSSLs in the same genetic background of an elite rice variety, Huajingxian74 (HJX74), and revealed four SSSLs, W05-1-11-2-7-6 (W05), W08-16-3-2 (W08), W12-28-58-03-19-1 (W12), and W22-9-5-2-4-9-3 (W22), which had a significantly different heading date compared to HJX74. To further genetically dissect the QTLs controlling heading date on chromosomes 3, 6, and 10, four SSSLs were used to develop 15 secondary SSSLs with the smaller substituted segments. The qHD-3 heading date QTL detected in W05 and W08 was delimited to an interval of 4.15 cM, whereas qHD-6-1 and qHD-6-2 heading date QTLs dissected from the substituted segments in W12 were mapped to the intervals of 2.25-cM and 2.55-cM, respectively. The qHD-10 QTL detected on the substituted segment in W22 was mapped to an interval of 6.85-cM. The nucleotide and amino acid sequence changes for those genes in the secondary SSSLs were also revealed. The allele variations of those genes might contribute to the heading date QTLs on chromosome 3 (DTH3, OsDof12, and EHD4), chromosome 6 (Hd3a, Hd17, and RFT1), and chromosome 10 (Ehd1 and Ehd2). These sequence variations in heading date genes would be useful resources for further studying the function of genes, and would be important for rice breeding. Overall, our results indicate that secondary SSSLs were powerful tools for genetic dissection of QTLs and identification of differentiation in the genes.

Evaluating the Effect of Expressing a Peanut Resveratrol Synthase Gene in Rice.

Resveratrol (Res) is a type of natural plant stilbenes and phytoalexins that only exists in a few plant species. Studies have shown that the Res could be biosynthesized and accumulated within plants, once the complete metabolic pathway and related enzymes, such as the key enzyme resveratrol synthase (RS), existed. In this study, a RS gene named PNRS1 was cloned from the peanut, and the activity was confirmed in E. coli. Using transgenic approach, the PNRS1 transgenic rice was obtained. In T3 generation, the Res production and accumulation were further detected by HPLC. Our data revealed that compared to the wild type rice which trans-resveratrol was undetectable, in transgenic rice, the trans-resveratrol could be synthesized and achieved up to 0.697 µg/g FW in seedlings and 3.053 µg/g DW in seeds. Furthermore, the concentration of trans-resveratrol in transgenic rice seedlings could be induced up to eight or four-fold higher by ultraviolet (UV-C) or dark, respectively. Simultaneously, the endogenous increased of Res also showed the advantages in protecting the host plant from UV-C caused damage or dark-induced senescence. Our data indicated that Res was involved in host-defense responses against environmental stresses in transgenic rice. Here the results describes the processes of a peanut resveratrol synthase gene transformed into rice, and the detection of trans-resveratrol in transgenic rice, and the role of trans-resveratrol as a phytoalexin in transgenic rice when treated by UV-C and dark. These findings present new outcomes of transgenic approaches for functional genes and their corresponding physiological functions, and shed some light on broadening available resources of Res, nutritional improvement of crops, and new variety cultivation by genetic engineering.

Domain dissection of AvrRxo1 for suppressor, avirulence and cytotoxicity functions.

AvrRxo1, a type III effector from Xanthomonas oryzae pv. oryzicola (Xoc) which causes bacterial leaf streak (BLS) in rice, can be recognised by non-host resistance protein Rxo1. It triggers a hypersensitive response (HR) in maize. Little is known regarding the virulence function of AvrRxo1. In this study, we determined that AvrRxo1 is able to suppress the HR caused by the non-host resistance recognition of Xanthomonas oryzae pv. oryzae (Xoo) by Nicotiana benthamiana. It is toxic, inducing cell death from transient expression in N. benthamiana, as well as in yeast. Among the four AvrRxo1 alleles from different Xoc strains, we concluded that the toxicity is abolished by a single amino acid substitution at residue 344 in two AvrRxo1 alleles. A series of truncations from the carboxyl terminus (C-terminus) indicate that the complete C-terminus of AvrRxo1 plays an essential role as a suppressor or cytotoxic protein. The C-terminus was also required for the avirulence function, but the last two residues were not necessary. The first 52 amino acids of N-terminus are unessential for toxicity. Point mutagenesis experiments indicate that the ATP/GTP binding site motif A is required for all three functions of AvrRxo1, and NLS is required for both the avirulence and the suppression of non-host resistance. The putative thiol protease site is only required for the cytotoxicity function. These results determine that AvrRxo1 plays a role in the complex interaction with host proteins after delivery into plant cells.

Delimitation of the PSH1(t) gene for rice purple leaf sheath to a 23.5 kb DNA fragment.

Leaf sheath color plays an important role as a marker for rice genetic improvement. A recombinant inbred line (RIL) population consisting of 220 individuals was developed from a cross between an Oryza sativa subsp. indica variety, IRBB60, and an Oryza sativa subsp. japonica variety, 9407. Within the RIL population, a line, RI51, was found to have purple leaf sheath (PSH). To map the gene governing PSH, RI51 was crossed with 9407 green leaf sheath (GSH) to develop an F2 segregating population. The distribution of F2 plants with PSH and GSH fitted a segregation ratio of 3:1, indicating that the PSH was controlled by a major dominant gene. The gene locus for PSH, tentatively designated as PSH1(t), was identified by surveying two bulks made of the respective 40 individuals with PSH and GSH with SSR markers covering the entire rice genome. The survey indicated that the PSH1(t) region was located on chromosome 1. Further confirmation was made using a large random sample of 360 individuals from the same F2 population and the PSH1(t) locus was then mapped on chromosome 1 between SSR markers RM3475 and RM7202 with genetic distances of 2.0 and 1.1 cM, respectively. For fine mapping of PSH1(t), a large F(2:3) segregating population with 3300 individuals from the seven heterozygous F2 plants in the RM3475-RM7202 region was constructed. Analysis of recombinants in the PSH1(t) region anchored the gene locus to an interval of 23.5 kb flanked by the left marker L03 and the right marker L05. Sequence analysis of this fragment predicted six open reading frames encoding a putative trans-sialidase, a putative Plastidic ATP/ADP-transporter, and four unknown proteins. The detailed genetic and physical maps of the PSH1(t) locus will be very useful in molecular cloning of the PSH1(t) gene.