<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.     

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.     

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.     

Fine mapping of <Emphasis Type="Italic">RYMV3</Emphasis>: a new resistance gene to <Emphasis Type="Italic">Rice yellow mottle virus</Emphasis> from <Emphasis Type="Italic">Oryza glaberrima</Emphasis>

Key message

A new resistance gene against Rice yellow mottle virus was identified and mapped in a 15-kb interval. The best candidate is a CC-NBS-LRR gene.

Abstract

Rice yellow mottle virus (RYMV) disease is a serious constraint to the cultivation of rice in Africa and selection for resistance is considered to be the most effective management strategy. The aim of this study was to characterize the resistance of Tog5307, a highly resistant accession belonging to the African cultivated rice species (Oryza glaberrima), that has none of the previously identified resistance genes to RYMV. The specificity of Tog5307 resistance was analyzed using 18 RYMV isolates. While three of them were able to infect Tog5307 very rapidly, resistance against the others was effective despite infection events attributed to resistance-breakdown or incomplete penetrance of the resistance. Segregation of resistance in an interspecific backcross population derived from a cross between Tog5307 and the susceptible Oryza sativa variety IR64 showed that resistance is dominant and is controlled by a single gene, named RYMV3. RYMV3 was mapped in an approximately 15-kb interval in which two candidate genes, coding for a putative transmembrane protein and a CC-NBS-LRR domain-containing protein, were annotated. Sequencing revealed non-synonymous polymorphisms between Tog5307 and the O. glaberrima susceptible accession CG14 in both candidate genes. An additional resistant O. glaberrima accession, Tog5672, was found to have the Tog5307 genotype for the CC-NBS-LRR gene but not for the putative transmembrane protein gene. Analysis of the cosegregation of Tog5672 resistance with the RYMV3 locus suggests that RYMV3 is also involved in Tog5672 resistance, thereby supporting the CC-NBS-LRR gene as the best candidate for RYMV3.

     

Overexpression of rice <Emphasis Type="Italic">LRK1</Emphasis> restricts internode elongation by down-regulating <Emphasis Type="Italic">OsKO2</Emphasis>

Rice (Oryza sativa) has the potential to undergo rapid internodal elongation which determines plant height. Gibberellin is involved in internode elongation. Leucine-rich repeat receptor-like kinases (LRR-RLKs) are the largest subfamily of transmembrane receptor-like kinases in plants. LRR-RLKs play important functions in mediating a variety of cellular processes and regulating responses to environmental signals. LRK1, a PSK receptor homolog, is a member of the LRR-RLK family. In the present study, differences in ectopic expression of LRK1 were consistent with extent of rice internode elongation. Analyses of gene expression demonstrated that LRK1 restricts gibberellin biosynthesis during the internode elongation process by down-regulation of the gibberellin biosynthetic gene coding for ent-kaurene oxidase.     

Genome-Wide Analysis in Wild and Cultivated <Emphasis Type="Italic">Oryza</Emphasis> Species Reveals Abundance of NBS Genes in Progenitors of Cultivated Rice

NBS-encoding genes play a critical role in the plant defense system. Wild relatives of crop plants are rich reservoirs of plant defense genes. Here, we performed a stringent genome-wide identification of NBS-encoding genes in three cultivated and eight wild Oryza species, representing three different genomes (AA, BB, and FF) from four continents. A total of 2688 NBS-encoding genes were identified from 11 Oryza genomes. All the three progenitor species of cultivated rice, namely O. barthii, O. rufipogon, and O. nivara, were the richest reservoir of NBS-encoding genes (214, 313, and 307 respectively). Interestingly, the two Asian cultivated species showed a contrasting pattern in the number of NBS-encoding genes. While indica subspecies maintained nearly equal number of NBS genes as its progenitor (309 and 313), the japonica subspecies had retained only two third in the course of evolution (213 and 307). Other major sources for NBS-encoding genes could be (i) O. longistaminata since it had the highest proportion of NBS-encoding genes and (ii) O. glumaepatula as it clustered distinctly away from the rest of the AA genome species. The present study thus revealed that NBS-encoding genes can be exploited from the primary gene pool for disease resistance breeding in rice.     

Fine mapping and candidate gene analysis of the quantitative trait locus <Emphasis Type="Italic">gw8.1</Emphasis> associated with grain length in rice

A quantitative trait locus (QTL) gw8.1 was detected in the population derived from a cross between the elite japonica cultivar, ‘Hwaseong’ and Oryza rufipogon (IRGC 105491). Near isogenic lines (NILs) harboring the O. rufipogon segment on chromosome 8 showed increased grain length and weight compared to those of the recurrent parent, Hwaseong. This QTL was mapped to a 175.3-kb region containing 28 genes, of which four were considered as candidates based on the presence of mutations in their coding regions and as per the RNA expression pattern during the inflorescence stage. Leaves and panicles obtained from plants harvested 5 days after heading showed differences in gene expression between Hwaseong and gw8.1-NILs. Most genes were upregulated in O. rufipogon and gw8.1-NIL than in Hwaseong. Scanning electron microscopy analysis of the lemma inner epidermal cells indicated that cell length was higher in gw8.1 NIL than in Hwaseong, indicating that gw8.1 might regulate cell elongation. Among the candidate genes, LOC_Os08g34380 encoding a putative receptor-like kinase and LOC_Os08g34550 encoding putative RING-H2 finger protein were considered as possible candidates based on their functional similarity.     

Identification and fine mapping of <Emphasis Type="Italic">qWBPH11</Emphasis> conferring resistance to whitebacked planthopper (<Emphasis Type="Italic">Sogatella furcifera</Emphasis> Horvath) in rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.)

The whitebacked planthopper (WBPH), Sogatella furcifera Horvath, is one of the most destructive pests in rice (Oryza sativa L.) production. Host-plant resistance has been considered as an efficient and eco-friendly strategy to reduce yield losses caused by WBPH. In this study, we found that an indica rice cultivar IR54751-2-44-15-24-2 (IR54751) displayed high resistance to WBPH at both seedling and tillering stages. The resistance of IR54751 was mainly contributed by antixenosis and tolerance rather than antibiosis. An F₂ population derived from a cross between IR54751 and a susceptible japonica cultivar 02428 was constructed to detect the quantitative trait loci (QTLs) conferring the resistance to WBPH. In total, four QTLs including qWBPH3.1, qWBPH3.2, qWBPH11, and qWBPH12 were identified and distributed on three different chromosomes. The four QTLs had LOD scores of 3.8, 8.2, 5.8, and 3.9, accounting for 8.2, 21.5, 13.9, and 10.4% of the phenotypic variation, respectively. Except for qWBPH3.1, the resistance alleles of the other three QTLs were all from IR54751. Further, a secondary population harboring only single qWBPH11 locus was developed from the F₂ population by marker-assisted selection. Finally, qWBPH11 was delimited in a 450-kb region between markers DJ53973 and SNP56. The identification of WBPH resistance QTLs and the fine mapping of qWBPH11 will be helpful for cloning resistance genes and breeding resistant rice cultivars.     

Microsatellite based linkage disequilibrium analyses reveal <Emphasis Type="Italic">Saltol</Emphasis> haplotype fragmentation and identify novel QTLs for seedling stage salinity tolerance in rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.)

A set of 84 diverse rice genotypes were assessed for seedling stage salt tolerance and their genetic diversity using 41 polymorphic SSR markers comprising of 19 Saltol QTL linked and 22 random markers. Phenotypic screening under hydroponics identified three indica landraces (Badami, Shah Pasand and Pechi Badam), two Oryza rufipogon accessions (NKSWR2 and NKSWR17) and one each of Basmati rice (Seond Basmati) and japonica cultivars (Tompha Khau) as salt tolerant, having similar tolerance as of Pokkali and FL478. Among the salt tolerant genotypes, biomass showed positive correlation with shoot fresh weight and negative association with root and shoot Na⁺ content. The results indicated repression of Na⁺ loading within the tolerant plants. Linkage disequilibrium (LD) of the Saltol linked markers was weak, suggestive of high fragmentation of Pokkali haplotype, a result of evolutionary active recombination events. Poor haplotype structure of the Saltol region, may reduce its usefulness in marker assisted breeding programmes, if the target foreground markers chosen are wide apart. LD mapping identified eight robust marker-trait associations (QTLs), of which RM10927 was found linked to root and shoot Na⁺ content and RM10871 with shoot Na⁺/K⁺ ratio. RM271 on chromosome 10, an extra Saltol marker, was found associated to root Na⁺/K⁺ ratio. This marker showed a distinct allele among O. rufipogon accessions. There were also other novel loci detected on chromosomes 2, 5 and 10 influencing salt tolerance in the tested germplasm. Although Saltol remained as the key locus, the role of other genomic regions cannot be neglected in tailoring seedling stage salt tolerance in rice.     

Genome-wide identification and evolution of <Emphasis Type="Italic">TC1</Emphasis>/<Emphasis Type="Italic">Mariner</Emphasis> in the silkworm (<Emphasis Type="Italic">Bombyx mori</Emphasis>) genome

TC1/Mariner transposons belong to class II transposable elements (TEs) that use DNA-mediated “cut and paste” mechanism to transpose, and they have been identified in almost all organisms. Although silkworm (Bombyx mori) has a large amount of TC1/Mariner elements, the genome wide information of this superfamily in the silkworm is unknown. In this study, we have identified 2670 TC1/Mariner (Bmmar) elements in the silkworm genome. All the TEs were classified into 22 families by means of fgclust, a tool of repetitive sequence classification, seven of which was first reported in this study. Phylogenetic and structure analyses based on the catalytic domain (DDxD/E) of transposase sequences indicated that all members of TC1/Mariner were grouped into five subgroups: Mariner, Tc1, maT, DD40D and DD41D/E. Of these five subgroups, maT rather than Mariner possessed most members of TC1/Mariner (51.23%) in the silkworm genome. In particular, phylogenetic analysis and structure analysis revealed that Bmmar15 (DD40D) formed a new basal subgroup of TC1/Mariner element in insects, which was referred to as bmori. Furthermore, we concluded that DD40D appeared to intermediate between mariner and Tc1. Finally, we estimated the insertion time for each copy of TC1/Mariner in the silkworm and found that most of members were dramatically amplified during a period from 0 to 1 mya. Moreover, the detailed functional data analysis showed that Bmmar1, Bmmar6 and Bmmar9 had EST evidence and intact transposases. These implied that TC1/Mariner might have potential transpositional activity. In conclusion, this study provides some new insights into the landscape, origin and evolution of TC1/Mariner in the insect genomes.     

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.     

Map-based cloning and functional analysis of the chromogen gene <Emphasis Type="Italic">C</Emphasis> in rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.)

The chromogen gene C is critical for anthocyanin regulation in rice, and apiculus color is an important agronomic trait in selective breeding and variety purification. Mapbased cloning and in-depth functional analysis of the C gene will be useful for understanding the molecular mechanism of anthocyanin biosynthesis and for rice breeding. Japonica landrace Lijiangxintuanheigu (LTH) has red apiculi and purple stigmas. Genetic analysis showed that red apiculus and purple stigma in LTH co-segregated indicating control by a single dominant gene, or by two completely linked genes. Using 1,851 recessive individuals from two F₂ populations, the target gene OsC was delimited to a 70.8 kb interval on chromosome 6 that contains the rice homologue of the maize anthocyanin regulatory gene C1. When the entire OsC gene and its full-length cDNA cloned from LTH were transformed into japonica cultivar Kitaake with colorless apiculi and stigmas all positive transformants had red apiculi but non-colored stigmas, validating that OsC alone was responsible for the apiculus color and represented the functional C gene. OsC was constitutively expressed in all tissues examined, with strongest expression in leaf blades. These results set a foundation to clarify the regulatory mechanisms of OsC in the anthocyanin biosynthetic pathway.     

Abscisic acid biosynthesis under water stress: anomalous behavior of the 9-<Emphasis Type="Italic">cis</Emphasis>-epoxycarotenoid dioxygenase1 (<Emphasis Type="Italic">NCED1</Emphasis>) gene in rice

The gene NCED1 encodes 9-cis-epoxycarotenoid dioxygenase, which catalyzes oxidative cleavage of 9-cis-epoxycarotenoids neoxanthin and violaxanthin to xanthoxin, a key step in the biosynthesis of abscisic acid (ABA) in higher plants. In the present study, the complete NCED1 of 1 917 bp was cloned and characterized from rice (Oryza sativa L. cv. N22) as no earlier reports were available for its characterization from the indica cultivar. The NCED1 had no intron and encoded a protein of 639 amino acids with a predicted molecular mass of 68.62 kD and pI of 6.07. The aliphatic index and grand average of hydropathicity were found to be 77.04 and -0.148, respectively. Multiple alignment analysis revealed that the sequence shared a high identity with the Oryza sativa japonica group (100 %) followed by Triticum aestivum (90 %), Hordeum vulgare (90 %), and Zea mays (89 %). The enzyme had a RPE65 domain of 476 amino acid residues. The RPE65 domain requires Fe(II) as a cofactor coordinated with 4 histidine residues and 3 glutamic acid residues. The phylogenic tree shows that NCED1 of japonica rice and NCED1 of indica rice were in the same group. They might have been evolved from a common ancestor. Analysis with a PSORT III tool shows that NCED is a chloroplastic protein. The real-time quantitative PCR and RNA-sequencing studies show that the expression of NCED1 was progressively reduced with increasing water stress, and a negative correlation between expression of OsNCED1 and severity of stress was established. Further, NCED1 expression negatively correlated with ABA accumulation under water stress whereas in some other species, its expression increased along with ABA accumulation. This might be due to feedback inhibition of the ABA biosynthesis in rice.     

Genomewide association analysis for awn length linked to the seed shattering gene <Emphasis Type="Italic">qSH1</Emphasis> in rice

Awn is one of the most important domesticated traits in rice (Oryza sativa). Understanding the genetic basis of awn length is important for grain harvest and production, because long awn length is disadvantageous for both grain harvest and milling. We investigated the awn length of 529 rice cultivars and performed a Genomewide association studies (GWAS) in the indica and japonica subpopulations, and the whole population. In total, we found 17 loci associated with awn length. Of these loci, seven were linked to previously reported quantitative trait loci, and one was linked to the awn gene An-1. Nine novel loci were repeatedly identified in different environments. One of the nine associations was identified in both the whole and japonica populations. Special interest was the detection of the most significant association SNP, sf0136352825, which was less than 95 kb from the seed shattering gene qSH1. These results may provide potentially favourable haplotypes for molecular breeding in rice.     

A high-density linkage map with 2560 markers and its application for the localization of the male-sterile genes <Emphasis Type="Italic">ms3</Emphasis> and <Emphasis Type="Italic">ms4</Emphasis> in <Emphasis Type="Italic">Cryptomeria japonica</Emphasis> D. Don

Cryptomeria japonica pollinosis is one of the most serious allergic diseases in Japan; this is a social problem because C. japonica is the most important Japanese forestry species. In order to reduce the amount of pollen dispersed, breeding programs using trees with male-sterile genes have been implemented. High-density linkage maps with stable ordering of markers facilitate the localization of male-sterile genes and the construction of partial linkage maps around them in order to develop markers for use in marker-assisted selection. In this study, a high-density linkage map for C. japonica with 2560 markers was constructed. The observed map length was 1266.2 cM and the mean distance between adjacent markers was 0.49 cM. Using information from this high-density map, we newly located two male-sterile genes (ms3 and ms4) on the first and fourth linkage groups, respectively, and constructed partial linkage maps around these loci. We also constructed new partial linkage maps around the ms1 and ms2 loci using additional SNP markers. The closest markers to the ms1, ms2, ms3, and ms4 male-sterile loci were estSNP04188 (1.8 cM), estSNP00695 (7.0 cM), gSNP05415 (3.1 cM), and estSNP01408 (7.0 cM) respectively. These results allowed us to develop SNP markers tightly linked to the male sterile genes for use in MAS; this will accelerate the future isolation of these genes by map-based cloning approaches.