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Background: Switchgrass (Panicum virgatum) is a herbaceous crop for the cellulosic biofuel feedstock development in the USA and Europe. As switchgrass is a naturally outcrossing species, accurate identification of selfed progeny is important to producing inbreds, which can be used in the production of heterotic hybrids. Development of a technically reliable, time-saving and easily used marker system is needed to quantify and characterize breeding origin of progeny plants of targeted parents. Results: Genome-wide screening of 915 mapped microsatellite (simple sequence repeat, SSR) markers was conducted, and 842 (92.0%) produced clear and scorable bands on a pooled DNA sample of eight switchgrass varieties. A total of 166 primer pairs were selected on the basis of their relatively even distribution in switchgrass genome and PCR amplification quality on 16 tetraploid genotypes. Mean polymorphic information content value for the 166 markers was 0.810 ranging from 0.116 to 0.959. From them, a core set of 48 loci, which had been mapped on 17 linkage groups, was further tested and optimized to develop 24 sets of duplex markers. Most of (up to 87.5%) targeted, but non-allelic amplicons within each duplex were separated by more than 10-bp. Using the established duplex PCR protocol, selfing ratio (i.e., selfed/all progeny x100%) was identified as 0% for a randomly selected open-pollinated 'Kanlow' genotype grown in the field, 15.4% for 22 field-grown plants of bagged inflorescences, and 77.3% for a selected plant grown in a growth chamber. Conclusions: The study developed a duplex SSR-based PCR protocol consisting of 48 markers, providing ample choices of non-tightly-linked loci in switchgrass whole genome, and representing a powerful, time-saving and easily used method for the identification of selfed progeny in switchgrass. The protocol should be a valuable tool in switchgrass breeding efforts. [ABSTRACT FROM AUTHOR]

Heading date (HD) is a key trait for the adaptation of rice cultivar to a specific growing region. Here, we report conventional and marker-assisted breeding strategies using genetic information related to the determination of HD, where the breeding objectives were to avoid the delayed heading common in indica × japonica hybrids, to increase the efficiency in selecting hybrid rice combinations having a suitable growth duration, and to develop cultivars with target growth duration by quantitative trait locus (QTL) pyramiding. The allelic constitution at the major HD loci was determined for a set of 109 leading Chinese rice cultivars by crossing them with HD tester lines. It was shown that the late heading in indica × japonica hybrids can be overcome by replacing the strong photoperiod-sensitivity allele Se-1 n with Se-1 e. A breeding strategy to enable the selection of hybrid combinations with suitable growth duration was proposed, based on HD genotypic information in rice. Meanwhile, a QTL analysis for HD was conducted over five years based on a recombinant inbred line population, derived from two parents Asominori ( japonica) and IR24 ( indica). Four QTLs, located on chromosomes 2, 3, 6, and 8, respectively, could be detected in all five years, indicating they were stably expressed QTL. According to this QTL information, and taking Asominori as an example, the HD genotypes for improving the growth duration were designed, and the best breeding selection schemes were determined by use of a genetic breeding simulation tool. Results obtained in this study demonstrate that genetic information related to HD can make a significant contribution to rice breeding. [ABSTRACT FROM AUTHOR]

Ubiquitination and deubiquitination are reversible processes that play crucial roles in regulating organ size in plants. However, information linking deubiquitination and seed size in rice (Oryza sativa) is limited. Here, we characterized a dominant large-grain mutant, large grain1-D (lg1-D), with a 30.8% increase in seed width and a 34.5% increase in 1,000-grain weight relative to the wild type. The lg1-D mutant had more cells oriented in the lateral direction of the spikelet hull compared with the wild type. Map-based cloning showed that LG1 encodes a constitutively expressed ubiquitin-specific protease15 (OsUBP15) that possesses deubiquitination activity in vitro. Loss-of-function and down-regulated expression of OsUBP15 produced narrower and smaller grains than the control. A set of in vivo experiments indicated that the mutant Osubp15 had enhanced protein stability relative to wild-type OsUBP15. Further experiments verified that OsDA1 directly interacted with OsUBP15. Genetic data indicated that OsUBP15 and GRAIN WIDTH 2 (GW2) were not independent in regulating grain width and size. In summary, we identified OsUBP15 as a positive regulator of grain width and size in rice and provide a promising strategy for improvement of grain yield by pyramiding OsUBP15 and gw2. [ABSTRACT FROM AUTHOR]

Lesion-mimic mutants are useful to dissect programmed cell death and defense-related pathways in plants. Here we identified a new rice lesion-mimic mutant, spotted leaf 33 (spl33) and cloned the causal gene by a map-based cloning strategy. SPL33 encodes a eukaryotic translation elongation factor 1 alpha (eEF1A)-like protein consisting of a nonfunctional zinc finger domain and three functional EF-Tu domains. spl33 exhibited programmed cell death-mediated cell death and early leaf senescence, as evidenced by analyses of four histochemical markers, namely H2O2 accumulation, cell death, callose accumulation and TUNEL-positive nuclei, and by four indicators, namely loss of chlorophyll, breakdown of chloroplasts, down-regulation of photosynthesis-related genes, and up-regulation of senescenceassociated genes. Defense responses were induced in the spl33 mutant, as shown by enhanced resistance to both the fungal pathogen Magnaporthe oryzae and the bacterial pathogen Xanthomonas oryzae pv. oryzae and by upregulation of defense response genes. Transcriptome analysis of the spl33 mutant and its wild type provided further evidence for the biological effects of loss of SPL33 function in cell death, leaf senescence and defense responses in rice. Detailed analyses showed that reactive oxygen species accumulation may be the cause of cell death in the spl33 mutant, whereas uncontrolled activation of multiple innate immunity-related receptor genes and signaling molecules may be responsible for the enhanced disease resistance observed in spl33. Thus, we have demonstrated involvement of an eEF1A-like protein in programmed cell death and provided a link to defense responses in rice. [ABSTRACT FROM AUTHOR]

Intersubspecific hybrid sterility is a common form of reproductive isolation in rice (Oryza sativa L.), which significantly hampers the utilization of heterosis between indica and japonica varieties. Here, we elucidated the mechanism of S7, which specially causes Aus-japonica/indica hybrid female sterility, through cytological and genetic analysis, map-based cloning, and transformation experiments. Abnormal positioning of polar nuclei and smaller embryo sac were observed in F1 compared with male and female parents. Female gametes carrying S7cp and S7i were aborted in S7ai/S7cp and S7ai/S7i, respectively, whereas they were normal in both N22 and Dular possessing a neutral allele, S7n. S7 was fine mapped to a 139-kb region in the centromere region on chromosome 7, where the recombination was remarkably suppressed due to aggregation of retrotransposons. Among 16 putative open reading frames (ORFs) localized in the mapping region, ORF3 encoding a tetratricopeptide repeat domain containing protein was highly expressed in the pistil. Transformation experiments demonstrated that ORF3 is the candidate gene: downregulated expression of ORF3 restored spikelet fertility and eliminated absolutely preferential transmission of S7ai in heterozygote S7ai/S7cp; sterility occurred in the transformants Cpslo17-S7ai. Our results may provide implications for overcoming hybrid embryo sac sterility in intersubspecific hybrid rice and utilization of hybrid heterosis for cultivated rice improvement. [ABSTRACT FROM AUTHOR]

Meiosis is essential for gametogenesis in sexual reproduction in rice (Oryza sativa L.). We identified a MutS-homolog (MSH) family gene OsMSH4 in a trisomic plant. Cytological analysis showed that developments of both pollen and embryo sacs in an Osmsh4 mutant were blocked due to defective chromosome pairing. Compared with the wild type, the Osmsh4 mutant displayed a significant ~21.9% reduction in chiasma frequency, which followed a Poisson distribution, suggesting that class I crossover formation in the mutant was impaired. Temporal and spatial expression pattern analyses showed that OsMSH4 was preferentially expressed in meiocytes during their meiosis, indicating a critical role in gametogenesis. Subcellular localization showed that OsMSH4-green fluorescent protein was predominantly located in the nucleus. OsMSH4 could interact with another MSH member (OsMSH5) through the N-terminus and C-terminus, respectively. Direct physical interaction between OsMSH5, OsRPA1a, OsRPA2b, OsRPA1c, and OsRPA2c was identified by yeast two-hybrid assays and further validated by pull-down assays. Our results supported the conclusion that the OsMSH4/5 heterodimer plays a key role in regulation of crossover formation during rice meiosis by interaction with the RPA complex. [ABSTRACT FROM AUTHOR]

Seed storability in rice (Oryza sativa L.) is an important agronomic trait. Two segregating populations with N22 (indica) as a common parent, viz. a set of 122 backcross-inbred lines (BILs) derived from the backcross Nanjing35 (japonica)/N22//Nanjing35 and another population comprising 189 recombinant inbred lines (RILs) from the cross of USSR5 (japonica) and N22, were studied to detect quantitative trait loci (QTL) controlling seed storability. Germination percentage (GP) was used to evaluate seed storability after aging treated under three different conditions, viz. natural, artificial and combined aging treatments. A total of seven QTLs were identified on chromosomes 1, 2, 5, 6 and 9. Among them, a major QTL, qSSn-9, was common in the two populations. In contrast, four QTLs (qSSnj-2-1, qSSn-2-2, qSSn-5 and qSSn-6) were detected in BILs and the QTL qSSn-1 was identified in RILs, which was a new QTL for seed storability. The N22-derived alleles increased the seed storability at all the loci except qSSnj-2-1. We also investigated the effect of QTLs using five selected lines with high storability from BILs and verified qSSn-5 with a near-isogenic line (NIL). These results provide an opportunity for pyramiding or map-based cloning major QTLs for seed storability in rice. [ABSTRACT FROM AUTHOR]

During routine seed increase procedures in rice, semi-sterile plants are common; however, such semi-sterility mutants in rice varieties have been only rarely analyzed genetically. W207-2 is a semi-sterile selection from the japonica rice variety Nipponbare. In this report, we found the female gamete of W207-2 was normal, and its semi-sterility was unaffected by growth duration but was conditioned by a recessive nuclear gene whose action leads to pollen semi-sterility and anther indehiscence, and the gene was named as pss1 (pollen semi-sterile). Using an F2 population derived from the two parents W207-2 and Dular and a pooled DNA strategy, pss1 was mapped to an interval on chromosome 8 defined by the two SSR loci RM6356 and RS41. The position of pss1 was confirmed in another F2 population derived from the cross W207-2 × Nipponbare. Over 2,000 homozygous pss1 segregants from the large W207-2 × Dular F2 population were used to fine map pss1 to a 0.04 cM segment flanked by a CAPs marker L2 and a dCAPs L3 marker. Sequences for both markers are present on a single PAC clone, and the physical distance between them is about 28 kb. Analysis of the PAC sequence predicts the presence of five open reading frames, they are as follows: putative ribonuclease PH, putative avr9 elicitor response protein, kinesin1-like protein, putative protein RNP-D precursor and putative 40S ribosomal protein S13. This result would be helpful in cloning the pss1 gene. [ABSTRACT FROM AUTHOR]

Quantitative trait loci (QTL) analysis for Al tolerance was performed in rice using a mapping population of 98 BC1F10 lines (backcross inbred lines: BILs), derived from a cross of Al-tolerant cultivar of rice ( Oryza sativa L. cv. Nipponbare) and Al-sensitive cultivar (cv. Kasalath). Three characters related to Al tolerance, including root elongation under non-stress conditions (CRE), root elongation under Al stress (SRE) and the relative root elongation (RRE) under Al stress versus non-stress conditions, were evaluated for the BILs and the parents at seedling stage. A total of seven QTLs for the three traits were identified. Among them, three putative QTLs for CRE ( qCRE-6, qCRE-8 and qCRE-9) were mapped on chromosomes 6, 8 and 9, respectively. One QTL for SRE ( qSRE-4) was identified on chromosome 4. Three QTLs ( qRRE-5, qRRE-9 and qRRE-10) for RRE were detected on chromosomes 5, 9, 10 and accounted for 9.7–11.8% of total phenotypic variation. Interestingly, the QTL qRRE-5 appears to be syntenic with the genomic region carrying a major Al tolerance gene on chromosome 6 of maize. Another QTL, qRRE-9, appears to be similar among different rice populations, while qRRE-10 is unique in the BIL population. The common QTLs for CRE and RRE indicate that candidate genes conferring Al tolerance in the rice chromosome 9 may be associated with root growth rates. The existence of QTLs for Al tolerance was confirmed in substitution lines for corresponding chromosomal segments. These results also provide the possibilities of enhancing Al tolerance in rice through using marker-assisted selection (MAS) and pyramiding QTLs. [ABSTRACT FROM AUTHOR]