Characterisation of a novel quantitative trait locus, <Emphasis Type="Italic">GN4</Emphasis>-<Emphasis Type="Italic">1</Emphasis>, for grain number and yield in rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.)

Key message

A novel QTL for grain number, GN4-1, was identified and fine-mapped to an ~ 190-kb region on the long arm of rice chromosome 4.

Abstract

Rice grain yield is primarily determined by three components: number of panicles per plant, grain number per panicle and grain weight. Among these traits, grain number per panicle is the major contributor to grain yield formation and is a crucial trait for yield improvement. In this study, we identified a major quantitative trait locus (QTL) responsible for rice grain number on chromosome 4, designated GN4-1 (a QTL for Grain Number on chromosome 4), using advanced segregating populations derived from the crosses between an elite indica cultivar ‘Zhonghui 8006’ (ZH8006) and a japonica rice ‘Wuyunjing 8’ (WYJ8). GN4-1 was delimited to an ~ 190-kb region on chromosome 4. The genetic effect of GN4-1 was estimated using a pair of near-isogenic lines. The GN4-1 gene from WYJ8 promoted accumulation of cytokinins in the inflorescence and increased grain number per panicle by ~ 17%. More importantly, introduction of the WYJ8 GN4-1 gene into ZH8006 increased grain yield by ~ 14.3 and ~ 11.5% in the experimental plots in 2014 and 2015, respectively. In addition, GN4-1 promoted thickening of the culm and may enhance resistance to lodging. These results demonstrate that GN4-1 is a potentially valuable gene for improvement of yield and lodging resistance in rice breeding.

     

Characterization of an IAA-glucose hydrolase gene <Emphasis Type="Italic">TaTGW6</Emphasis> associated with grain weight in common wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis> L.)

In rice, the TGW6 gene determines grain weight and encodes a protein with indole-3-acetic acid (IAA)-glucose hydrolase activity. Its homolog in wheat, TaTGW6, is considered as a candidate gene related to grain development. To amplify this gene, we designed primers based on a homologous conserved domain of the rice TGW6 gene. Sequence analysis indicated that TaTGW6 comprises only one exon, with 1656 bp in total and an open reading frame of 1035 bp. Three alleles at TaTGW6 locus detected by the primer pair TG23 were designated as TaTGW6-a, TaTGW6-b and TaTGW6-c, respectively. Compared with TaTGW6-a, TaTGW6-b had a 6-bp InDel at the position 170 downstream of initiation codon, and TaTGW6-c was a null mutant. Both TaTGW6-b and TaTGW6-c could significantly increase grain size and weight other than TaTGW6-a; however, the former two alleles showed a low frequency distribution in modern varieties. TaTGW6 was located on chromosome 4AL using a recombinant inbred line population and a set of Chinese Spring nullisomic-tetrasomic lines. It was linked to the SSR locus Xbarc1047 with a genetic distance of 6.62 cM and explained 15.8–21.0 % of phenotypic variation of grain weight in four environments. Association analysis using a natural population and Chinese wheat mini-core collections additionally validated the relationship of TaTGW6 with grain weight; the gene could explain 7.7–12.4 % of phenotypic variation in three environments. Quantitative real-time PCR revealed that TaTGW6-b showed relatively lower expression than TaTGW6-a in immature grain at 20 and 30 days post-anthesis and in mature grain. The low expression of TaTGW6 generally associated with low IAA content, but with high grain weight. The novel functional marker, designated as TG23, can be used for marker-assisted selection to improve grain weight in wheat and also provides insights into the regulatory mechanism underlying grain weight.     

Improvement of seedling and panicle blast resistance in <Emphasis Type="Italic">Xian</Emphasis> rice varieties following <Emphasis Type="Italic">Pish</Emphasis> introgression

Rice blast is a serious disease caused by the filamentous ascomycetous fungus Magnaporthe oryzae. Incorporating disease resistance genes in rice varieties and characterizing the distribution of M. oryzae isolates form the foundation for enhancing rice blast resistance. In this study, the blast resistance gene Pish was observed to be differentially distributed in the genomes of rice sub-species. Specifically, Pish was present in 80.5% of Geng varieties, but in only 2.3% of Xian varieties. Moreover, Pish conferred resistance against only 23.5% of the M. oryzae isolates from the Geng-planting regions, but against up to 63.2% of the isolates from the Xian-planting regions. Thus, Pish may be an elite resistance gene for improving rice blast resistance in Xian varieties. Therefore, near-isogenic lines (NILs) with Pish and the polygene pyramid lines (PPLs) PPL^Pish/Pi1, PPL^Pish/Pi54, and PPL^Pish/Pi33 in the Xian background Yangdao 6 were generated using a molecular marker-assisted selection method. The results suggested that (1) Pish significantly improved rice blast resistance in Xian varieties, which exhibited considerably improved seedling and panicle blast resistance after Pish was introduced; (2) PPLs with Pish were more effective than the NILs with Pish regarding seedling and panicle blast resistance; (3) the PPL seedling and panicle blast resistance was improved by the complementary and overlapping effects of different resistance genes; and (4) the stability of NIL and PPL resistance varied under different environmental conditions, with only PPL^Pish/Pi54 exhibiting highly stable resistance in three natural disease nurseries (Jianyang, Jinggangshan, and Huangshan). This study provides new blast resistance germplasm resources and describes a novel molecular strategy for enhancing rice blast resistance.     

A new <Emphasis Type="Italic">Em</Emphasis>-like protein from <Emphasis Type="Italic">Lactuca sativa</Emphasis>, <Emphasis Type="Italic">LsEm1</Emphasis>, enhances drought and salt stress tolerance in <Emphasis Type="Italic">Escherichia coli</Emphasis> and rice

Late embryogenesis abundant (LEA) proteins are closely related to abiotic stress tolerance of plants. In the present study, we identified a novel Em-like gene from lettuce, termed LsEm1, which could be classified into group 1 LEA proteins, and shared high homology with Cynara cardunculus Em protein. The LsEm1 protein contained three different 20-mer conserved elements (C-element, N-element, and M-element) in the C-termini, N-termini, and middle-region, respectively. The LsEm1 mRNAs were accumulated in all examined tissues during the flowering and mature stages, with a little accumulation in the roots and leaves during the seedling stage. Furthermore, the LsEm1 gene was also expressed in response to salt, dehydration, abscisic acid (ABA), and cold stresses in young seedlings. The LsEm1 protein could effectively reduce damage to the lactate dehydrogenase (LDH) and protect LDH activity under desiccation and salt treatments. The Escherichia coli cells overexpressing the LsEm1 gene showed a growth advantage over the control under drought and salt stresses. Moreover, LsEm1-overexpressing rice seeds were relatively sensitive to exogenously applied ABA, suggesting that the LsEm1 gene might depend on an ABA signaling pathway in response to environmental stresses. The transgenic rice plants overexpressing the LsEm1 gene showed higher tolerance to drought and salt stresses than did wild-type (WT) plants on the basis of the germination performances, higher survival rates, higher chlorophyll content, more accumulation of soluble sugar, lower relative electrolyte leakage, and higher superoxide dismutase activity under stress conditions. The LsEm1-overexpressing rice lines also showed less yield loss compared with WT rice under stress conditions. Furthermore, the LsEm1 gene had a positive effect on the expression of the OsCDPK9, OsCDPK13, OsCDPK15, OsCDPK25, and rab21 (rab16a) genes in transgenic rice under drought and salt stress conditions, implying that overexpression of these genes may be involved in the enhanced drought and salt tolerance of transgenic rice. Thus, this work paves the way for improvement in tolerance of crops by genetic engineering breeding.     

Identification of a novel <Emphasis Type="Italic">SPLIT</Emphasis>-<Emphasis Type="Italic">HULL</Emphasis> (<Emphasis Type="Italic">SPH</Emphasis>) gene associated with hull splitting in rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.)

Key message

The split-hull phenotype caused by reduced lemma width and low lignin content is under control of SPH encoding a type-2 13-lipoxygenase and contributes to high dehulling efficiency.

Abstract

Rice hulls consist of two bract-like structures, the lemma and palea. The hull is an important organ that helps to protect seeds from environmental stress, determines seed shape, and ensures grain filling. Achieving optimal hull size and morphology is beneficial for seed development. We characterized the split-hull (sph) mutant in rice, which exhibits hull splitting in the interlocking part between lemma and palea and/or the folded part of the lemma during the grain filling stage. Morphological and chemical analysis revealed that reduction in the width of the lemma and lignin content of the hull in the sph mutant might be the cause of hull splitting. Genetic analysis indicated that the mutant phenotype was controlled by a single recessive gene, sph (Os04g0447100), which encodes a type-2 13-lipoxygenase. SPH knockout and knockdown transgenic plants displayed the same split-hull phenotype as in the mutant. The sph mutant showed significantly higher linoleic and linolenic acid (substrates of lipoxygenase) contents in spikelets compared to the wild type. It is probably due to the genetic defect of SPH and subsequent decrease in lipoxygenase activity. In dehulling experiment, the sph mutant showed high dehulling efficiency even by a weak tearing force in a dehulling machine. Collectively, the results provide a basis for understanding of the functional role of lipoxygenase in structure and maintenance of hulls, and would facilitate breeding of easy-dehulling rice.

     

<Emphasis Type="Italic">Rf5</Emphasis> is able to partially restore fertility to Honglian-type cytoplasmic male sterile <Emphasis Type="Italic">japonica</Emphasis> rice (<Emphasis Type="Italic">Oryza sativa</Emphasis>) lines

Three-line japonica hybrids have been developed mainly on Chinsurah Boro II (BT)-type cytoplasmic male sterile (CMS) lines of Oryza sativa L., but the unstable sterility of some BT-type CMS lines, and the threat of genetic vulnerability when using a single cytoplasm source, have inhibited their use in rice cultivation. Previously, the sterility of Honglian (HL)-type japonica CMS lines derived from common red-awned wild rice (Oryza rufipogon) has been proven to be more stable than that of BT-type japonica CMS lines. Here, we genetically characterized HL-type japonica CMS lines and the restorer-of-fertility (Rf) gene for breeding HL-type japonica hybrids. HL-type japonica CMS lines displayed stained abortive pollen grains, unlike HL-type indica CMS lines. The BT-type japonica restorer lines, which contain Rf, had different capabilities to restore HL-LiuqianxinA (HL-LqxA), an HL-type japonica CMS line, and the restorers for the HL-type japonica CMS lines could be selected from the preexisting BT-type japonica restorers in rice production. A genetic analysis showed that the restoration of normal fertility to HL-LqxA was controlled by a major gene and was affected by minor effector genes and/or modifiers. The major Rf in SiR2982, a BT-type japonica restorer, was mapped to a ~100-kb physical region on chromosome 10, and was demonstrated to be Rf5 (Rf1a) by sequencing. Furthermore, Rf5 partially restored fertility and had a dosage effect on HL-type japonica CMS lines. These results will be helpful for the development of HL-type japonica hybrids.     

Semi-dwarf barley (<Emphasis Type="Italic">Hordeum vulgare</Emphasis> L.) <Emphasis Type="Italic">brh2</Emphasis> and <Emphasis Type="Italic">ari-l</Emphasis> mutants are deficient in a U-box E3 ubiquitin ligase

Lodging is the process where crop plants fall over and lie on the ground due to strong winds and heavy precipitation. This problem reduces yield and increases the risk of fungal infections and pre-harvest germination. In order to avoid lodging, plant breeders utilize short-culm mutants, which often have a robust culm that can support the weight of a heavy spike. In barley (Hordeum vulgare L.), thousands of short-culm mutants have been isolated in breeding programs around the world. Our long-term goal is to reveal the genetic network underlying culm length, with the objective to provide an enlarged repertoire of genes and alleles suitable for future breeding of lodging resistant barley. In the present work we studied a group of allelic brh2 and ari-l mutants, which have a relatively strong semi-dwarf phenotype and are phenotypically similar to previously identified mutants deficient in brassinosteroid signalling or metabolism. The Brh2 gene is located in the centromeric region of chromosome 4H and we applied a candidate gene approach to identify the gene. Brh2 is orthologous to TUD1 in rice (Orysa sativa L.), which encodes a U-box E3 ubiquitin ligase. We identified one missense mutation, one nonsense mutation and four deletions of the complete Brh2 gene. The mutants could respond to exogenously applied brassinolide, which suggests that the apparent brassinosteroid deficient phenotype of barley brh2 and ari-l mutants is related to brassinosteroid metabolism rather than signalling.     

Characterization and fine mapping of <Emphasis Type="Italic">osh15</Emphasis>(<Emphasis Type="Italic">t</Emphasis>), a novel dwarf mutant gene in rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.)

Plant height is one of the most important agronomic traits of plant architecture, and also affects grain yield in rice. In this study, we obtained a novel dwarf rice mutant of japonica variety Shennong9816, designated Shennong9816d. Compared with wild-type, the Shennong9816d plant height was significantly reduced, and the tiller number significantly increased. Additionally, the mutant yield component, and the number of large and small vascular bundles were significantly decreased compared with wild-type. Genetic analysis indicated that the Shennong9816d dwarf phenotype was controlled by a recessive nuclear gene, while the plant was shown to be sensitive to gibberellic acid. Using a large F₂ population derived from a cross between Shennong9816d and the indica rice variety Habataki, the osh15(t) gene was fine mapped between RM20891 and RM20898, within a physical distance of 73.78 kb. Sequencing analysis showed that Shennong9816d carries a 1 bp mutation and a 30 bp insertion in the OSH15 region. These results suggest that osh15(t) is a novel allelic mutant originally derived from japonica variety Shennong9816, which may be useful for introducing the semi-dwarf phenotype to improve plant architecture in rice breeding practice.     

Mapping of the Ogura fertility restorer gene <Emphasis Type="Italic">Rfo</Emphasis> and development of <Emphasis Type="Italic">Rfo</Emphasis> allele-specific markers in canola (<Emphasis Type="Italic">Brassica napus</Emphasis> L.)

Ogura cytoplasmic male sterility (CMS) and its corresponding nuclear fertility restorer gene, Rfo, have been introduced from radish to Brassica species by interspecific crosses. Rfo restores male fertility by altering the translational expression of Orf138, a mitochondrial gene, whose expression results in the male sterile phenotype. This system has been extensively investigated and breeding restorer lines for the Ogura CMS has become a major objective for hybrid seed production in many canola breeding programs. In this study, we have sequenced genomic clones of Rfo amplified from a canola restorer line R2000, licensed from INRA, France, and a Dow AgroSciences non-restorer line Nexera 705 using primers designed from the radish Rfo sequence (GenBank accession AJ550021). Sequence alignment revealed three homologous sequences of Rfo. Two of the sequences were present in both R2000 and Nexera 705 but the third one was present only in R2000. These results suggested that the first two sequences could be the homoeologous sequences of Rfo already existing in the canola genome and the third one could be the radish Rfo introduced into canola. Based on the sequence differences between the restorer and non-restorer lines, Rfo allele-specific PCR markers were developed. We also developed a high throughput, Rfo allele-specific Invader^® assay through Third Wave Technologies. Linkage analysis revealed a co-segregation between the allele-specific marker and the phenotypes for fertility restoration. This allele-specific marker has been mapped in the linkage group N19 and proved to be very useful for direct selection of Rfo alleles for fertility restoration during marker-assisted introgression of the Ogura restorer for hybrid development in canola.     

Association analysis of the glutelin synthesis genes <Emphasis Type="Italic">GluA</Emphasis> and <Emphasis Type="Italic">GluB1</Emphasis> in a <Emphasis Type="Italic">Japonica</Emphasis> rice collection

Glutelin is the most significant seed storage protein and is regarded as an important nutrient quality trait in rice. Research on the genetic basis of the glutelin content distinction in rice will provide more choices for the diets of people with kidney disease and diabetes. The GluA and GluB1 genes play important roles in the process of glutelin synthesis. In this study, 128 Japonica rice accessions with wide geographic distributions were collected to construct the association panel. Among all the 128 accessions, both sequences of the GluA and GluB1 genes were obtained, and nucleotide polymorphisms were detected. A total of 46 SNPs and eight InDels, six SNPs and four InDels were found in the GluA and GluB1 gene sequences, respectively. Eight haplotypes and two haplotypes were classified based on the SNPs in the coding region of the GluA and GluB1 genes, respectively. Moreover, the association of the polymorphic sites in the two genes with glutelin content in the tested population was estimated. The results revealed that five SNPs in the GluA gene, one SNP and one InDel in the GluB1 gene were associated with glutelin content at a significant level (P < 0.01). Corresponding markers were also designed to check the alleles of GluA and GluB1 genes. These results suggested that polymorphisms in the GluA and GluB1 genes in rice could be utilized in molecular marker-assisted selection to improve the nutrient quality of rice breeding programmes.     

Development and characterization of <Emphasis Type="Italic">japonica</Emphasis> rice lines carrying the brown planthopper-resistance genes <Emphasis Type="Italic">BPH12</Emphasis> and <Emphasis Type="Italic">BPH6</Emphasis>

The brown planthopper (Nilaparvata lugens Stål; BPH) has become a severe constraint on rice production. Identification and pyramiding BPH-resistance genes is an economical and effective solution to increase the resistance level of rice varieties. All the BPH-resistance genes identified to date have been from indica rice or wild species. The BPH12 gene in the indica rice accession B14 is derived from the wild species Oryza latifolia. Using an F₂ population from a cross between the indica cultivar 93-11 and B14, we mapped the BPH12 gene to a 1.9-cM region on chromosome 4, flanked by the markers RM16459 and RM1305. In this population, BPH12 appeared to be partially dominant and explained 73.8% of the phenotypic variance in BPH resistance. A near-isogenic line (NIL) containing the BPH12 locus in the background of the susceptible japonica variety Nipponbare was developed and crossed with a NIL carrying BPH6 to generate a pyramid line (PYL) with both genes. BPH insects showed significant differences in non-preference in comparisons between the lines harboring resistance genes (NILs and PYL) and Nipponbare. BPH growth and development were inhibited and survival rates were lower on the NIL-BPH12 and NIL-BPH6 plants compared to the recurrent parent Nipponbare. PYL-BPH6 + BPH12 exhibited 46.4, 26.8 and 72.1% reductions in population growth rates (PGR) compared to NIL-BPH12, NIL-BPH6 and Nipponbare, respectively. Furthermore, insect survival rates were the lowest on the PYL-BPH6 + BPH12 plants. These results demonstrated that pyramiding different BPH-resistance genes resulted in stronger antixenotic and antibiotic effects on the BPH insects. This gene pyramiding strategy should be of great benefit for the breeding of BPH-resistant japonica rice varieties.     

Fine mapping and identification of a novel locus <Emphasis Type="Italic">qGL12.2</Emphasis> control grain length in wild rice (<Emphasis Type="Italic">Oryza rufipogon</Emphasis> Griff.)

Key message

A wild rice QTL qGL12.2 for grain length was fine mapped to an 82-kb interval in chromosome 12 containing six candidate genes and none was reported previously.

Abstract

Grain length is an important trait for yield and commercial value in rice. Wild rice seeds have a very slender shape and have many desirable genes that have been lost in cultivated rice during domestication. In this study, we identified a quantitative trait locus, qGL12.2, which controls grain length in wild rice. First, a wild rice chromosome segment substitution line, CSSL41, was selected that has longer glume and grains than does the Oryza sativa indica cultivar, 9311. Next, an F₂ population was constructed from a cross between CSSL41 and 9311. Using the next-generation sequencing combined with bulked-segregant analysis and F₃ recombinants analysis, qGL12.2 was finally fine mapped to an 82-kb interval in chromosome 12. Six candidate genes were found, and no reported grain length genes were found in this interval. Using scanning electron microscopy, we found that CSSL41 cells are significantly longer than those of 9311, but there is no difference in cell widths. These data suggest that qGL12.2 is a novel gene that controls grain cell length in wild rice. Our study provides a new genetic resource for rice breeding and a starting point for functional characterization of the wild rice GL gene.

     

Map-based cloning of the dominant genic male sterile <Emphasis Type="Italic">Ms</Emphasis>-<Emphasis Type="Italic">cd1</Emphasis> gene in cabbage (<Emphasis Type="Italic">Brassica oleracea</Emphasis>)

Key message

Using map-based cloning, we delimited the Ms - cd1 gene responsible for the male sterile phenotype in B. oleracea to an approximately 39-kb fragment. Expression analysis suggests that a new predicted gene, a homolog of the Arabidopsis SIED1 gene, is a potential candidate gene.

Abstract

A dominant genic male sterile (DGMS) mutant 79-399-3 in Brassica oleracea (B. oleracea) is controlled by a single gene named Ms-cd1, which was genetically mapped on chromosome C09. The derived DGMS lines of 79-399-3 have been successfully applied in hybrid cabbage breeding and commercial hybrid seed production of several B. oleracea cultivars in China. However, the Ms-cd1 gene responsible for the DGMS has not been identified, and the molecular basis of the DGMS is unclear, which then limits its widespread application in hybrid cabbage seed production. In the present study, a large BC₉ population with 12,269 individuals was developed for map-based cloning of the Ms-cd1 gene, and Ms-cd1 was mapped to a 39.4-kb DNA fragment between two InDel markers, InDel14 and InDel24. Four genes were identified in this region, including two annotated genes based on the available B. oleracea annotation database and two new predicted open reading frames (ORFs). Finally, a newly predicted ORF designated Bol357N3 was identified as the candidate of the Ms-cd1 gene. These results will be useful to reveal the molecular mechanism of the DGMS and develop more practical DGMS lines with stable male sterility for hybrid seed production in cabbage.

     

<Emphasis Type="Italic">Sr33</Emphasis> and <Emphasis Type="Italic">Sr35</Emphasis> gene homolog identification in genomes of cereals related to <Emphasis Type="Italic">Aegilops tauschii</Emphasis> and <Emphasis Type="Italic">Triticum monococcum</Emphasis>

Using bioinformatics analysis, the homologs of genes Sr33 and Sr35 were identified in the genomes of Triticum aestivum, Hordeum vulgare, and Triticum urartu. It is known that these genes confer resistance to highly virulent wheat stem rust races (Ug99). To identify amino acid sites important for this resistance, the found homologs were compared with the Sr33 and Sr35 protein sequences. It was found that sequences S5DMA6 and E9P785 are the closest homologs of protein RGAle, a Sr33 gene product, and sequences M7YFA9 (CNL-C) and F2E9R2 are homologs of protein CNL9, a Sr35 gene product. It is assumed that the homologs of genes Sr33 and Sr35, which were obtained from the wild relatives of wheat and barley, can confer resistance to various forms of stem rust and can be used in the future breeding programs aimed at improvement of national wheat varieties.     

Tolerance to soil water stress by <Emphasis Type="Italic">Oryza sativa</Emphasis> cv. IR20 was improved by expression of <Emphasis Type="Italic">Wsi18</Emphasis> gene locus from <Emphasis Type="Italic">Oryza nivara</Emphasis>

Wild rice genotypes are rich in genetic diversity. This has potential to improve agronomic rice by allele mining for superior traits. Late embryogenesis abundant (LEA) proteins are often associated with desiccation tolerance and stress signalling. In the present study, a group 3 LEA gene, Wsi18 from the wild rice Oryza nivara was expressed under its own inducible promoter element in stress susceptible cultivated indica rice (cv. IR20). The resulting transgenic plants cultivated in a greenhouse showed enhanced tolerance to soil water deficit. Transgenic plants had higher grain yield, plant survival rate, and shoot relative water content compared to wild type (WT) IR20. Cell membrane stability index, proline and soluble sugar content were also greater in transgenic than WT plants under water stress. These results demonstrate the potential for improving SWS tolerance in agronomically important rice cultivar by incorporating Wsi18 gene from a wild rice O. nivara.