Fine mapping of <Emphasis Type="Italic">qSTV11</Emphasis><Superscript><Emphasis Type="Italic">KAS</Emphasis></Superscript>, a major QTL for rice stripe disease resistance

Rice stripe disease, caused by rice stripe virus (RSV), is one of the most serious diseases in temperate rice-growing areas. In the present study, we performed quantitative trait locus (QTL) analysis for RSV resistance using 98 backcross inbred lines derived from the cross between the highly resistant variety, Kasalath, and the highly susceptible variety, Nipponbare. Under artificial inoculation in the greenhouse, two QTLs for RSV resistance, designated qSTV7 and qSTV11 ^KAS , were detected on chromosomes 7 and 11 respectively, whereas only one QTL was detected in the same location of chromosome 11 under natural inoculation in the field. The stability of qSTV11 ^KAS was validated using 39 established chromosome segment substitution lines. Fine mapping of qSTV11 ^KAS was carried out using 372 BC₃F_2:3 recombinants and 399 BC₃F_3:4 lines selected from 7,018 BC₃F₂ plants of the cross SL-234/Koshihikari. The qSTV11 ^KAS was localized to a 39.2 kb region containing seven annotated genes. The most likely candidate gene, LOC_Os11g30910, is predicted to encode a sulfotransferase domain-containing protein. The predicted protein encoded by the Kasalath allele differs from Nipponbare by a single amino acid substitution and the deletion of two amino acids within the sulfotransferase domain. Marker-resistance association analysis revealed that the markers L104-155 bp and R48-194 bp were highly correlated with RSV resistance in the 148 landrace varieties. These results provide a basis for the cloning of qSTV11 ^KAS , and the markers may be used for molecular breeding of RSV resistant rice varieties.     

Genetic dissection of the resistance to Rice stripe virus present in the indica rice cultivar 'IR24'

Rice stripe disease, caused by Rice stripe virus (RSV) and transmitted by the small brown planthopper (Laodelphax striatellus Fallen), is one of the most serious viral diseases of rice in temperate East Asian production regions. Prior quantitative trait loci (QTL) mapping has established that Oryza sativa L. subsp. indica 'IR24' carries positive alleles at the three loci qSTV3, qSTV7, and qSTV11-i. Here, we report an advanced backcross analysis based on three selected chromosome segment substitution lines (CSSLs), each predicted to carry one of these three QTL. Three sets of BC(4)F(2:3) populations were bred from a cross between the critical CSSL and its recurrent parent Oryza sativa L. subsp. japonica 'Asominori'. Both qSTV3 and qSTV11-i were detected in their respective population, but qSTV7 was not. An allelic analysis based on a known carrier of the major RSV resistance gene Stvb-i, which is located on chromosome 11, showed that qSTV11-i was not allelic with Stvb-i. A large mapping population was used to delimit the location of qSTV11-i to a 73.6-kb region. The de novo markers developed for this purpose will be useful as marker-assisted selection tools in efforts to introduce qSTV11-i into breeding programmes aiming to improve the level of RSV resistance.     

Detection and fine mapping of two quantitative trait loci for partial resistance to stripe virus in rice (Oryza sativa L.)

Rice stripe virus (RSV) is one of the most destructive pathogens of rice (Oryza sativa L.) in East Asia. Development of resistant varieties offers a more economical and efficient way to control this disease. In the present study, tests using four inoculation methods were used on 85 backcross inbred lines of Sasanishiki (japonica)/Habataki (indica) to map quantitative trait loci (QTL) conferring resistance to RSV. One QTL on chromosome 3 and two on chromosome 11 were detected, jointly explaining 18?C47?% of the trait variance. The QTL (qSTV11 ^HAB -1 and qSTV11 ^HAB -2) on chromosome 11 were closely linked, and mapped in the intervals G257-RM457 and RM457-RM187, respectively. The stabilities of qSTV11 ^HAB -1 and qSTV11 ^HAB -2 were validated using a set of 38 established chromosome segmental substitution lines. The two QTL, when combined, showed higher resistance than either of them alone in both field and mass inoculation tests, indicating additivity. Fine mapping of the two genes was carried out using 147 recombined F2:3 lines selected from 2,750 secondary F2 plants of the cross Sasanishiki/SL437. Four SSR (simple sequence repeat) and eight InDel (insertion?Cdeletion) markers newly developed to fine-map the two loci. According to the Nipponbare genomic sequence, qSTV11 ^HAB -1 was localized to a 333.2-kb interval which was about 230?kb from the well-known Stvb-i. The other locus, qSTV11 ^HAB -2, which appears to be a new QTL for RSV resistance, was delimited to a 203.9-kb region. Four flanking markers (R15, RM209, R69 and R73) can be used in marker-assisted selection. These results provide an opportunity for map-based cloning of qSTV11 ^HAB -1 and qSTV11 ^HAB -2, thereby promoting the breeding program of RSV resistance.     

Fine mapping of <Emphasis Type="Italic">qSTV11</Emphasis><Superscript><Emphasis Type="Italic">TQ</Emphasis></Superscript>, a major gene conferring resistance to rice stripe disease

The indica rice cultivar, Teqing, shows a high level of resistance to rice stripe virus (RSV). It is believed that this resistance is controlled by the gene, qSTV11 ^TQ . For positional cloning of the resistance gene, a set of chromosome single segment substitution lines (CSSSLs) was constructed, all of which had the genetic background of the susceptible japonica cultivar, Lemont, with different single substituted segments of Teqing on chromosome 11. By identifying the resistance of the CSSSLs-2006 in a field within a heavily diseased area, the resistance gene qSTV11 ^TQ was mapped between the markers Indel7 and RM229. Furthermore, in that region, six new markers were developed and 52 subregion CSSSLs (CSSSLs-2007) were constructed. The natural infection experiment was conducted again at different sites, with two replicates used in each site in order to identify the resistance phenotypes of the CSSSLs-2007 and resistant/susceptible controls in 2007. Through the results of 2007, qSTV11 ^TQ was localized in a region defined by the markers, CAPs1 and Indel4. In order to further confirm the position of qSTV11 ^TQ , another set of subregion CSSSLs (CSSSLs-2009) was constructed. Finally, qSTV11 ^TQ was localized to a 55.7 kb region containing nine annotated genes according to the genome sequence of japonica Nipponbare. The relationship between qSTV11 ^TQ and Stvb-i (Hayano-Saito et al. in Theor Appl Genet 101:59–63, 2000) and the reliability of the markers used on both sides of qSTV11 ^TQ for marker-assisted breeding of resistance to rice stripe disease are discussed.     

Fine Mapping and Candidate Gene Analysis of qSTL3, a Stigma Length-Conditioning Locus in Rice (Oryza sativa L.)

The efficiency of hybrid seed production can be improved by increasing the percentage of exserted stigma, which is closely related to the stigma length in rice. In the chromosome segment substitute line (CSSL) population derived from Nipponbare (recipient) and Kasalath (donor), a single CSSL (SSSL14) was found to show a longer stigma length than that of Nipponbare. The difference in stigma length between Nipponbare and SSSL14 was controlled by one locus (qSTL3). Using 7,917 individuals from the SSSL14/Nipponbare F₂ population, the qSTL3 locus was delimited to a 19.8-kb region in the middle of the short arm of chromosome 3. Within the 19.8-kb chromosome region, three annotated genes (LOC_Os03g14850, LOC_Os03g14860 and LOC_Os03g14880) were found in the rice genome annotation database. According to gene sequence alignments in LOC_Os03g14850, a transition of G (Nipponbare) to A (Kasalath) was detected at the 474-bp site in CDS. The transition created a stop codon, leading to a deletion of 28 amino acids in the deduced peptide sequence in Kasalath. A T-DNA insertion mutant (05Z11CN28) of LOC_Os03g14850 showed a longer stigma length than that of wild type (Zhonghua 11), validating that LOC_Os03g14850 is the gene controlling stigma length. However, the Kasalath allele of LOC_Os03g14850 is unique because all of the alleles were the same as that of Nipponbare at the 474-bp site in the CDS of LOC_Os03g14850 among the investigated accessions with different stigma lengths. A gene-specific InDel marker LQ30 was developed for improving stigma length during rice hybrid breeding by marker-assisted selection.     

RNAi-directed down-regulation of RSV results in increased resistance in rice (Oryza sativa L.)

Rice stripe disease (RSD), caused by rice stripe virus (RSV), is a serious disease in temperate rice-growing areas. We have created an RNAi construct containing coat protein gene (CP) and disease specific protein gene (SP) sequences from RSV. The RNAi construct was transformed into two susceptible japonica varieties, Suyunuo and Guanglingxiangjing, to develop resistance against RSD. The homozygous progeny of rice plants in the T(5) and T(7) generations containing RNAi constructs, after self-fertilization were strongly resistant to viral infection. RT-PCR indicated that viral replication of SP and CP in the transgenic plants was significantly inhibited. There were no obvious morphological or developmental differences between the transgenic and wild-type plants from seedling stage to maturity. The excellent agronomic traits of these two varieties, such as high yield and good quality were maintained. Suppression of virus genes using RNAi is therefore a practical and effective strategy for controlling viral infection in crops.     

Fine mapping of Grh1, a major gene associated with antibiosis to green rice leafhopper in rice

Green rice leafhopper (GRH, Nephotettix cincticeps Uhler) is one of the insect pests that damage cultivated rice in East Asia. GRH also transmits viruses such as rice dwarf virus. The mortality of GRH nymphs is high in rice cultivar Shingwang, indicating that Shingwang is resistant to GRH. Genetic analyses were performed to map GRH resistance in Shingwang using F2 and F3 populations derived from a cross between a GRH-resistant near-isogenic line (NIL-IS60) from Shingwang and recurrent parent Ilpum. Resistance to GRH in Shingwang was found to be controlled by a single dominant gene (Grh1) mapped within an approximately 670-kb region between 8.10 and 8.77 Mb on the short arm of chromosome 5. Genotypes with three simple sequence repeat markers (RM18166, RM516, and RM18171) and one indel marker (Indel 15040) co-segregated with GRH resistance controlled by the Grh1 locus. A detailed map of the Grh1 locus will facilitate marker-assisted selection of resistance to GRH in rice breeding.     

Candidate genes and molecular markers associated with brown planthopper (<Emphasis Type="Italic">Nilaparvata lugens</Emphasis> Stål) resistance in rice cultivar Rathu Heenati

Brown planthopper (BPH) is a destructive insect pest of rice and causes severe yield loss. In attempts to develop a BPH-resistant rice variety, Rathu Heenati (RH), a rice cultivar with a strong BPH resistance, has been used as the donor in breeding programs. Quantitative trait loci analysis was conducted for the area under the curve of BPH damage scores of a backcross (BC₃F₅) population infested by six different BPH populations. Single nucleotide polymorphism (SNP) markers on chromosome 4, i.e., LecRK2-SNP and LecRK3-SNP, and markers on chromosome 6, i.e., Bph32-SNP and SSR23, were identified to be associated with resistance against five BPH populations. To identify genes on chromosome 6 that are involved in BPH resistance, expression analysis was conducted for genes located in the genomic region of Bph32-SNP and SSR23. Genes that showed differential expression ofRH at 24 h after BPH infestation, when compared to an RH control, were identified. Those that encode proteins putatively involved in the BPH resistance mechanism are LOC_Os06g03240, LOC_Os06g03380, LOC_Os06g03486, LOC_Os06g03514, LOC_Os06g03520, LOC_Os06g03610, LOC_Os06g03676, and LOC_Os06g03890. SNP markers were developed from several differentially expressed genes and were validated by genotyping in the backcross population. The SNP marker developed from LOC_Os06g03514 showed the highest association with BPH resistance and the gene may be involved in the BPH resistance mechanism. This SNP marker will be useful in breeding programs for BPH resistance.     

Identification and characterization of BGL11(t), a novel gene regulating leaf-color mutation in rice (Oryza sativa L.)

A novel bright-green leaf mutant, bgl11, derived from Nipponbare (Oryza sativa L. ssp. japonica) treated by ethyl methanesulfonate (EMS), exhibited a distinct bright-green leaf phenotype throughout development. Chlorophyll contents of bgl11 decreased significantly than that of its wild-type parent. Genetic analysis suggested that the bright-green leaf trait was controlled by a single recessive nuclear gene, which was tentatively designed as BGL11(t). To isolate the BGL11(t) gene, a map-based cloning strategy was employed, and the gene was finally mapped in a 94.7 kb region between marker InDel11-5 and InDel11-9 on the long arm of chromosome 11, in which no gene leaded to leaf-color mutation had been mapped or cloned. Cloning and sequencing analysis revealed that, LOC_Os11g38040, which was predicted to encode an expressed protein, had a 9 bp segment deletion in the coding region of bgl11. Furthermore, the transgenic plants with wild-type gene LOC_Os11g38040 were restored to normal phenotype. Accordingly, the gene (LOC_Os11g38040) was identified as the BGL11(t) gene. These results are very valuable for further study on BGL11(t) gene and illuminating the mechanism of chloroplast development in rice.     

Modern elite rice varieties of the 'Green Revolution' have retained a large introgression from wild rice around the Pi33 rice blast resistance locus

During the breeding process of cultivated crops, resistance genes to pests and diseases are commonly introgressed from wild species. The size of these introgressions is predicted by theoretical models but has rarely been measured in cultivated varieties. By combining resistance tests with isogenic strains, genotyping and sequencing of different rice accessions, it was shown that, in the elite rice variety IR64, the resistance conferring allele of the rice blast resistance gene Pi33 was introgressed from the wild rice Oryza rufipogon (accession IRGC101508). Further characterization of this introgression revealed a large introgression at this locus in IR64 and the related variety IR36. The introgressed fragment represents approximately half of the short arm of rice chromosome 8. This is the first report of a large introgression in a cultivated variety of rice. Such a large introgression is likely to have been maintained during backcrossing only if a selection pressure was exerted on this genomic region. The possible traits that were selected are discussed.     

Identification of major quantitative trait loci qSBR11-1 for sheath blight resistance in rice

Sheath blight caused by Rhizoctonia solani Kühn is one of the important diseases of rice, resulting in heavy yield loss in rice every year. No rice line resistant to sheath blight has been identified till date. However, in some rice lines a high degree of resistance to R. solani has been observed. An indica rice line, Tetep, is a well documented source of durable and broad spectrum resistance to rice blast as well as quantitative resistance to sheath blight. The present study identified genetic loci for quantitative resistance to sheath blight in rice line Tetep. A mapping population consisting of 127 recombinant inbred lines derived from a cross between rice cultivars HP2216 (susceptible) and Tetep (resistant to sheath blight) was evaluated for sheath blight resistance and other agronomic traits for 4 years across three locations. Based on sheath blight phenotypes and genetic map with 126 evenly distributed molecular markers, a quantitative trait loci (QTLs) contributing to sheath blight resistance was identified on long arm of chromosome 11. Two QTL mapping approaches i.e., single marker analysis and composite interval mapping in multi environments were used to identify QTLs for sheath blight resistance and agronomical traits. The QTL qSBR11-1 for sheath blight resistance was identified between the marker interval RM1233 (26.45 Mb) to sbq33 (28.35 Mb) on chromosome 11. This region was further narrowed down to marker interval K39516 to sbq33 (~0.85 Mb) and a total of 154 genes were predicted including 11 tandem repeats of chitinase genes which may be responsible for sheath blight resistance in rice line Tetep. A set of 96 varieties and a F₂ population were used for validation of markers linked to the QTL region. The results indicate that there is very high genetic variation among varieties at this locus, which can serve as a starting point for allele mining of sheath blight resistance.     

Suppression of NS3 and MP is important for the stable inheritance of RNAi-mediated rice stripe virus (RSV) resistance obtained by targeting the fully complementary RSV-CP gene

Rice stripe virus (RSV) is a viral disease that seriously impacts rice production in East Asia, most notably in Korea, China, and Japan. Highly RSV-resistant transgenic japonica rice plants were generated using a dsRNAi construct designed to silence the entire sequence region of the RSV-CP gene. Transgenic rice plants were inoculated with a population of viruliferous insects, small brown planthoppers (SBPH), and their resistance was evaluated using ELISA and an infection rate assay. A correlation between the expression of the RSV-CP homologous small RNAs and the RSV resistance of the transgenic rice lines was discovered. These plants were also analyzed by comparing the expression pattern of invading viral genes, small RNA production and the stable transmission of the RSV resistance trait to the T3 generation. Furthermore, the agronomic trait was stably transmitted to the T4 generation of transgenic plants.     

实时荧光定量PCR检测RSV胁迫下抗病、感病水稻中与脱落酸相关基因的差异表达

采用实时荧光定量PCR方法测定了水稻条纹病毒(Rice stripe virus,RSV)胁迫下抗性不同品种水稻中与脱落酸相关基因的mRNA转录水平变化.结果表明:感病品种武育梗3号中WGPI、OsGASA2、Polcalcin、OsCBIA、Myb和OsCIPK15基因表达水平均上调,上调比率分别为4.96、5.17、2.01、5.17、12.04和7.84.而抗病品系KT 95-418中,OsGASA2和OsCIPK15基因表达水平下调,下调比率分别为1/5.40和1/2.08;Polcalcin和Myb基因表达水平上调,上调比率分别为4.20和3.86;WGPI和OsCBIA表达量变化不明显.这些结果表明,RSV胁迫能诱导脱落酸相关基因表达量的变化,并且在抗病、感病水稻品种中的表达特征不同,从而提示植物激素脱落酸可能调控了RSV胁迫条件下相关基因的表达.     

Localization of the rice stripe disease resistance gene, Stv-bi, by graphical genotyping and linkage analyses with molecular markers

 We used graphical genotyping and linkage analyses with molecular markers to determine the chromosomal location of the rice stripe disease resistance gene, Stv-b ⁱ . The stripe resistance gene from the indica rice (Oryza sativa) cv ‘Modan’ was introgressed into several Japanese rice varieties. We found 4 RFLP markers in ‘Modan’, five susceptible parental rice varieties (‘Norin No. 8’, ‘Sachihikari’, ‘Kanto No. 98’, ‘Hokuriku No.103’ and ‘Koganebare’) and four resistant progeny varieties (‘St. No. 1’, ‘Aichi No. 6’, ‘Aoisora’ and ‘Asanohikari’). Graphical genotyping of the resistant progeny revealed a chromosomal segment ascribable to ‘Modan’ and associated with stripe resistance. The chromosomal segment from ‘Modan’ was located at 35.85 cM on chromosome 11. Linkage analysis using 120 F₂ individuals from a cross between ‘Koshihikari’ (susceptible) and ‘Asanohikari’ (resistant) revealed another 8 RFLP markers in the same chromosome. We performed a bioassay for rice stripe resistance in F₃ lines of the F₂ individuals using infective small brown planthoppers and identified an 1.8-cM segment harboring the rice stripe disease resistance gene, Stv-b ⁱ , between XNpb220 and XNpb257/ XNpb254. Furthermore, Stv-b ⁱ was linked by 0.0 cM to a RFLP marker, ST10, which was developed on the basis of the results of RAPD analysis. These DNA markers near the Stv-b ⁱ locus may be useful in marker-assisted selection and map-based cloning of the Stv-b ⁱ gene. Received: 26 September 1997 / Accepted: 4 November 1997     

Fine mapping of a dominant minute-grain gene, Mi3, in rice

Grain weight is a major determinant of rice grain yield and is widely believed to be controlled by quantitative trait loci (QTL). We have previously reported a new major gene, Mi3, regulating grain length in rice, and that the Mi3 allele from Y34 functioned in a dominant manner. In this paper we report the fine mapping and candidate analysis of Mi3. By employing a chromosome walking strategy in the F₂ population of 9311/Y34, the Mi3 gene was finally narrowed to an interval of ~?41.6?kb between the markers RM6881 and LM9 in the pericentromeric region of rice chromosome 3. According to the rice genome annotations, five putative gene loci, LOC_Os03g_29614, LOC_Os03g_29630, LOC_Os03g_29650, LOC_Os03g_29660 and LOC_Os03g_29680, were located in this candidate region. Mi3 was also determined to be a new gene for grain size in rice by allelic analysis with the previously reported genes. Our results will facilitate the cloning and functional characterization of the Mi3 gene and targeted marker-assisted breeding.     

Differentiation of Indica-Japonica rice revealed by insertion/deletion (InDel) fragments obtained from the comparative genomic study of DNA sequences between 93-11 (Indica) and Nipponbare (Japonica)

DNA polymorphisms from nucleotide insertion/deletions (InDels) in genomic sequences are the basis for developing InDel molecular markers.To validate the InDel primer pairs on the basis of the comparative genomic study on DNA sequences between an Indica rice 93-11 and a Japonica rice Nipponbare for identifying Indica and Japonica rice varieties and studying wild Oryza species,we studied 49 Indica,43 Japonica,and 24 wild rice accessions collected from ten Asian countries using 45 InDel primer pairs.Results indicated that of the 45 InDel primer pairs,41 can accurately identify Indica and Japonica rice varieties with a reliability of over 80%.The scatter plotting data of the principal component analysis (PCA) indicated that:(i) the InDel primer pairs can easily distinguish Indica from Japonica rice varieties,in addition to revealing their genetic differentiation;(ii) the AA-genome wild rice species showed a relatively close genetic relationship with the Indica rice varieties;and (iii)the non-AA genome wild rice species did not show evident differentiation into the Indica and Japonica types.It is concluded from the study that most of the InDel primer pairs obtained from DNA sequences of 93-11 and Nipponbare can be used for identifying lndica and Japonica rice varieties,and for studying genetic relationships of wild rice species,particularly in terms of the Indica-Japonica differentiation.