Fine mapping and characterization of BPH27, a brown planthopper resistance gene from wild rice (Oryza rufipogon Griff.)

The brown planthopper (Nilaparvata lugens Stål; BPH) is one of the most serious rice pests worldwide. Growing resistant varieties is the most effective way to manage this insect, and wild rice species are a valuable source of resistance genes for developing resistant cultivars. BPH27 derived from an accession of Guangxi wild rice, Oryza rufipogon Griff. (Accession no. 2183, hereafter named GX2183), was primarily mapped to a 17-cM region on the long arm of the chromosome four. In this study, fine mapping of BPH27 was conducted using two BC₁F₂ populations derived from introgression lines of GX2183. Insect resistance was evaluated in the BC₁F₂ populations with 6,010 individual offsprings, and 346 resistance extremes were obtained and employed for fine mapping of BPH27. High-resolution linkage analysis defined the BPH27 locus to an 86.3-kb region in Nipponbare. Regarding the sequence information of rice cultivars, Nipponbare and 93-11, all predicted open reading frames (ORFs) in the fine-mapping region have been annotated as 11 types of proteins, and three ORFs encode disease-related proteins. Moreover, the average BPH numbers showed significant differences in 96–120 h after release in comparisons between the preliminary near-isogenic lines (pre-NILs, lines harboring resistance genes) and BaiR54. BPH growth and development were inhibited and survival rates were lower in the pre-NIL plants compared with the recurrent parent BaiR54. The pre-NIL exhibited 50.7 % reductions in population growth rates (PGR) compared to BaiR54. The new development in fine mapping of BPH27 will facilitate the efforts to clone this important resistant gene and to use it in BPH-resistance rice breeding.     

High-resolution mapping of brown planthopper (BPH) resistance gene Bph27(t) in rice (Oryza sativa L.)

Brown planthopper (BPH), Nilaparvata lugens Stål, is a destructive insect pest of rice (Oryza sativa L.). Identification and utilization of resistance genes is an efficient strategy for controlling this insect. BPH-resistant indica cultivars Balamawee, Kaharamana and Pokkali were previously reported to have the same dominant gene Bph9 on chromosome 12. Our studies of BPH feeding performance showed that Balamawee had higher levels of antixenosis and antibiosis against BPH than Kaharamana and Pokkali. In order to identify the BPH resistance gene in Balamawee, an F2 population was derived by crossing Balamawee and susceptible japonica cultivar 02428. A single major resistance gene was identified and mapped to the long arm of chromosome 4. Further recombination analysis showed that the gene was located in an interval of about 63 kb between InDel markers Q52 and Q20. This new BPH resistance locus was designated Bph27(t).     

Comparison of the genetic diversity of common wild rice (Oryza rufipogon Griff.) and cultivated rice (O. sativa L.) using RFLP markers

Forty fourth single-copy RFLP markers were used to evaluate the genetic diversity of 122 accessions of common wild rice (CWR, Oryza rufipogon Griff.) and 75 entries of cultivated rice (Oryza sativa L. ) from more than ten Asian countries. A comparison of the parameters showing genetic diversity, including the percentage of polymorphic loci (P), the average number of alleles per locus (A), the number of genotypes (Ng), the average heterozygosity (Ho) and the average genetic multiplicity (Hs) of CWR and indica and japonica subspecies of cultivated rice from different countries and regions, indicated that CWR from China possesses the highest genetic diversity, followed by CWR from South Asia and Southeast Asia. The genetic diversity of CWR from India is the second highest. Although the average gene diversity (Hs)of the South Asian CWR is higher than that of the Southeast Asian CWR, its percentage of polymorphic loci (P), number of alleles (Na) and number of genotypes (Ng) are all smaller. It was also found that the genetic diversity of cultivated rice is obviously lower than that of CWR. At the 44 loci investigated, the number of polymorphic loci of cultivated rice is only 3/4 that of CWR, while the number of alleles, 60%, and the number of genotypes is about 1/2 that of CWR. Of the two subspecies studied, the genetic diversity of indica is higher than that of japonica. The average heterozygosity of the Chinese CWR is the highest among all the entries studied. The average heterozygosity of CWR is about two-times that of cultivated rice. It is suggested that during the course of evolution from wild rice to cultivated rice, many alleles were lost through natural and human selection, leading to the lower heterozygosity and genetic diversity of the cultivated rice. Received: 19 May 1999 / Accepted: 26 April 2000     

东乡普通野生稻与栽培稻苗期抗旱性的比较

干旱影响水稻生长发育,不论什么时期发生最终都导致产量损失。研究水稻资源抗旱性有助于水稻抗旱改良和稳定干旱胁迫下水稻的产量。东乡普通野生稻被公认为是栽培稻的祖先,对增强水稻抗旱性可能十分重要。对4份来自3个仅存的居群的东乡野生稻与15份栽培稻进行苗期抗旱性比较,考察了3次重复的盆栽土培试验中8个抗旱指标。表明东乡普通野生稻比栽培稻更为抗旱,表现在最大根长、茎长、根干重、根鲜重、根干鲜重比及抗旱指数等6个性状,而不表现在根数及根茎长比;其中茎长、最长根长、根干重、根鲜重及根系相对含水量对水稻苗期抗旱性影响更大。采用抗旱指数和抗旱总级别值法对水稻抗旱性进行评定,结果表明4份东乡野生稻材料间的抗旱性存在很大差异,且来水桃树下居群的抗性最高,东乡野生稻抗旱性可能与其原生境状况有关。结果认为东乡普通野生稻可作为栽培稻抗旱改良的遗传资源。     

Construction of introgression lines carrying wild rice (Oryza rufipogon Griff.) segments in cultivated rice (Oryza sativa L.) background and characterization of introgressed segments associated with yield-related traits

Introgression lines (ILs) are useful tools for precise mapping of quantitative trait loci (QTLs) and the evaluation of gene action or interaction in theoretical studies. A set of 159 ILs carrying variant introgressed segments from Chinese common wild rice (Oryza rufipogon Griff.), collected from Dongxiang county, Jiangxi Province, in the background of Indica cultivar (Oryza sativa L.), Guichao 2, was developed using 126 polymorphic simple sequence repeats (SSR) loci. The 159 ILs represented 67.5% of the genome of O. rufipogon. All the ILs have the proportions of the recurrent parent ranging from 92.4 to 99.9%, with an average of 97.4%. The average proportion of the donor genome for the BC₄F₄ population was about 2.2%. The mean numbers of homozygous and heterozygous donor segments were 2 (ranging 0–8) and 1 (ranging 0–7), respectively, and the majority of these segments had sizes less than 10 cM. QTL analysis was conducted based on evaluation of yield-related traits of the 159 ILs at two sites, in Beijing and Hainan. For 6 out of 17 QTLs identified at two sites corresponding to three traits (panicles per plant, grains per panicle and filled grains per plant, respectively), the QTLs derived from O. rufipogon were usually associated with an improvement of the target trait, although the overall phenotypic characters of O. rufipogon were inferior to that of the recurrent parent. Of the 17 QTLs, 5 specific QTLs strongly associated with more than one trait were observed. Further analysis of the high-yielding and low-yielding ILs revealed that the high-yielding ILs contained relatively less introgressed segments than the low-yielding ILs, and that the yield increase or decrease was mainly due to the number of grain. On the other hand, low-yielding ILs contained more negative QTLs or disharmonious interactions between QTLs which masked trait-enchancing QTLs. These ILs will be useful in identifying the traits of yield, tolerance to low temperature and drought stress, and detecting favorable genes of common wild rice.     

Plant regeneration from protoplasts of wild rice (Oryza rufipogon Griff.)

Embryogenic callus initiated from basal segments of micropropagated shoots of Oryza rufipogon were used to initiate cell suspension cultures. After approximately 3 months these cultures were capable of yielding large numbers of protoplasts which underwent sustained division in agarose-solidified medium at a frequency comparable to that observed with Japonica rice protoplasts in previous studies. O. rufipogon plants were reproducibly regenerated from the protoplast-derived callus and are currently being grown to maturity. This is the first report of plant regeneration from protoplasts of a wild species of Oryza.Abbreviations BAP 6-benzyalamino purine - 2,4-D 2,4-dichlorophenoxyacetic acid - MES 2[N-morpholino] ethanesulphonic acid - NAA -naphthalene acetic acid - PAR photosynthetically active radiation - SCV settled-cell volume     

High-resolution mapping of a new brown planthopper (BPH) resistance gene, Bph18(t), and marker-assisted selection for BPH resistance in rice (Oryza sativa L.)

Brown planthopper (BPH) is a destructive insect pest of rice in Asia. Identification and the incorporation of new BPH resistance genes into modern rice cultivars are important breeding strategies to control the damage caused by new biotypes of BPH. In this study, a major resistance gene, Bph18(t), has been identified in an introgression line (IR65482-7-216-1-2) that has inherited the gene from the wild species Oryza australiensis. Genetic analysis revealed the dominant nature of the Bph18(t) gene and identified it as non-allelic to another gene, Bph10 that was earlier introgressed from O. australiensis. After linkage analysis using MapMaker followed by single-locus ANOVA on quantitatively expressed resistance levels of the progenies from an F₂ mapping population identified with marker allele types, the Bph18(t) gene was initially located on the subterminal region of the long arm of chromosome 12 flanked by the SSR marker RM463 and the STS marker S15552. The corresponding physical region was identified in the Nipponbare genome pseudomolecule 3 through electronic chromosome landing (e-landing), in which 15 BAC clones covered 1.612 Mb. Eleven DNA markers tagging the BAC clones were used to construct a high-resolution genetic map of the target region. The Bph18(t) locus was further localized within a 0.843-Mb physical interval that includes three BAC clones between the markers R10289S and RM6869 by means of single-locus ANOVA of resistance levels of mapping population and marker-gene association analysis on 86 susceptible F₂ progenies based on six time-point phenotyping. Using gene annotation information of TIGR, a putative resistance gene was identified in the BAC clone OSJNBa0028L05 and the sequence information was used to generate STS marker 7312.T4A. The marker allele of 1,078 bp completely co-segregated with the BPH resistance phenotype. STS marker 7312.T4A was validated using BC₂F₂ progenies derived from two temperate japonica backgrounds. Some 97 resistant BC₂F₂ individuals out of 433 screened completely co-segregated with the resistance-specific marker allele (1,078 bp) in either homozygous or heterozygous state. This further confirmed a major gene-controlled resistance to the BPH biotype of Korea. Identification of Bph18(t) enlarges the BPH resistance gene pool to help develop improved rice cultivars, and the PCR marker (7312.T4A) for the Bph18(t) gene should be readily applicable for marker-assisted selection (MAS). K. K. Jena and J. U. Jeung contributed equally to this study.     

Molecular tagging of genes for brown planthopper resistance and earliness introgressed from Oryza australiensis into cultivated rice, O. sativa.

Restriction fragment length polymorphism analysis was carried out to tag the alien genes for brown planthopper (BPH) resistance and earliness introgressed from wild species Oryza australiensis into cultivated rice, O. sativa L. One introgression line (IR65482-4-136-2-2), resistant to biotypes 1, 2, and 3 of BPH and early in flowering, was selected from BC2F4 of the cross between O. sativa (IR31917-45-3-2) and O. australiensis (accession 100882). Recurrent parent, O. australiensis, and introgression line were surveyed for RFLP using probes of chromosomes 10 and 12. Two probes, RG457 and CDO98, detected introgression from O. australiensis. Cosegregation between introgressed characters and molecular markers was studied in F2 derived from the cross between the introgression line and recurrent parent. The gene for BPH resistance is linked with RG457 of chromosome 12 at a distance of 3.68 +/- 1.29 cM, and the gene for earliness is linked with CDO98 of chromosome 10 at a distance of 9.96 +/- 3.28 cM. Such close linkage is useful in marker-based selection while transferring BPH resistance from introgression line into other elite breeding lines. Introgression at the molecular level indicates that the mechanism of alien gene transfer is probably genetic recombination through crossing over rather than substitution of whole or large segment of chromosomes of wild species.     

Molecular cloning and structural analysis of a novel Rac gene osRACB in rice (Oryza sativa L.)

Rac is a subfamily of small GTP-binding protein family. Its molecular weight is between 20 and 30 kilodaltons. As a signal protein, Rac directly or indirectly participates in many physiological processes, such as the regulation of cytoskeleton and the transduction of stress-induced signal. So Rac is also named ?molecular switch? The switch is based on the cycle from a GTP-bound 憃n?to a GDP-bound 憃ff?state[1]. In the superfamily of GTP-binding protein, only heterotrimeric G protein, Ra…     

Genetic analysis and molecular mapping of a novel gene for zebra mutation in rice （Oryza sativa L.）

A novel zebra mutant, zebra-15, derived from the restorer line JinhuilO （Oryza sativa L. ssp. indica） treated by EMS, displayed a distinctive zebra leaf from seedling stage to jointing stage. Its chlorophyll content decreased （55.4%） and the ratio of Chla/Chlb increased （90.2%） significantly in the yellow part of the zebra-15, compared with the wild type. Net photosynthetic rate and fluorescence kinetic parameters showed that the decrease of chlorophyll content significantly influenced the photosynthetic efficiency of the mutant. Genetic analysis of F2 segregation populations derived from the cross of XinonglA and zebra-15 indicated that the zebra leaf trait is controlled by a single recessive nuclear gene. Ninety-eight out of four hundred and eighty pairs of SSR markers showed the diversity between the XinonglA and the zebra-15, their F2 population was then used for gene mapping. Zebra-15 （Z-15） gene was primarily restricted on the short arm of chromosome 5 by 150 F2 recessive individuals, 19.6 cM from marker RM3322 and 6.0 cM from marker RM6082. Thirty-six SSR markers were newly designed in the restricted location, and the Z-15 was finally located between markers nSSR516 and nSSR502 with the physical region 258 kb by using 1,054 F2 recessive individuals.     

Fine mapping of a quantitative trait locus for grain number per panicle from wild rice (Oryza rufipogon Griff.)

SIL040, an introgression line (IL) developed by introgressing chromosomal segments from an accession of Oryza rufipogon into an indica cultivar Guichao 2, showed significantly less grains per panicle than the recurrent parent Guichao 2. Quantitative trait locus (QTL) analysis in F₂ and F₃ generations derived from the cross between SIL040 and Guichao 2 revealed that gpa7, a QTL located on the short arm of chromosome 7, was responsible of this variation. Alleles from O. rufipogon decreased grains per panicle. To fine mapping of gpa7, a high-resolution map with 1,966 F₂ plants derived from the cross between SIL040 and Guichao 2 using markers flanking gpa7 was constructed, and detailed quantitative evaluation of the structure of main panicle of each of F₃ families derived from recombinants screened was performed. By two-step substitution mapping, gpa7 was finally narrowed down to a 35-kb region that contains five predicted genes in cultivated rice. The fact that QTLs for five panicle traits (length of panicle, primary branches per panicle, secondary branches per panicle, grains on primary branches and grains on secondary branches) were all mapped in the same interval as that for gpa7 suggested that this locus was associated with panicle structure, showing pleiotropic effects. The characterizing of panicle structure of IL SIL040 further revealed that, during the domestication from common wild allele to cultivated rice one at gpa7, not only the number of branches and grains per panicle increased significantly, more importantly, but also the ratio of secondary branches per panicle to total branches per panicle and the ratio of grains on secondary branches per panicle to total grains per panicle increased significantly. All these results reinforced the idea that gpa7 might play an important role in the regulation of grain number per panicle and the ratio of secondary branches per panicle during the domestication of rice panicle.Feng Tian and Zuo Feng Zhu contributed equally to this work.     

Field assessments of gene flow from transgenic to cultivated rice (Oryza sativa L.) using a herbicide resistance gene as tracer marker

Development of plant genetic engineering has led to the deployment of transgenic crops and, simultaneously, to the need for a thorough assessment of the risks associated with their environmental release. This study investigated the occurrence of gene flow from transgenic rice to non-transgenic rice plants under agronomic conditions using a herbicide resistance gene as a tracer marker. Two field experiments were established in the paddy fields of two main Mediterranean rice-growing areas of Spain and Italy. In both locations analyses of phenotypic, molecular and segregation data showed that pollination of recipient plants with pollen of the transgenic source occurred at a significant frequency. A gene flow slightly lower than 0.1% was detected in a normal side-by-side plot design. Similar results were found in a circular plot when the plants were placed at 1-m distance from the transgenic central nucleus. A strong asymmetric distribution of the gene flow was detected among this circle and highest values (0.53%) were recorded following the direction of the dominant wind. A significant lowest value (0.01%) was found in the other circle (5 m from the transgenic plants) as was expected according to the characteristics of rice pollen. Such circular-field trial designs could also prove to be very useful in studying the gene flow to other commercial cultivars of rice with the aim of establishing strategies to prevent pollen dispersal from commercial transgenic fields to the neighbouring conventional fields. Received: 23 February 2001 / Accepted: 31 March 2001     

Molecular mapping of genes conferring aluminum tolerance in rice (Oryza sativa L.)

Crop productivity on acid soil is restricted by multiple abiotic stress factors. Aluminum (Al) tolerance seems to be a key to productivity on soil with a pH below 5.0, but other factors such as Mn toxicity and the deficiency of P, Ca and Mg also play a role. The development of Al-tolerant genotypes of rice is an urgent necessity for improving crop productivity in developing countries. Inhibition of root growth is a primary and early symptom of Al toxicity. The present study was conducted to identify genetic factors controlling the aluminum tolerance of rice. Several parameters related to Al tolerance, most importantly the relative root growth under Al stress versus non-stress conditions, were scored in 188 F₃ selfed families from a cross between an Al-tolerant Vietnamese local variety, Chiembau, and an Al-susceptible improved variety, Omon269–65. The two varieties are both Oryza sativa ssp. indica, but showed a relatively high level of DNA polymorphism, permitting the assembly of an RFLP map consisting of 164 loci spanning 1,715.8 cM, and covering most of the rice genome. A total of nine different genomic regions on eight chromosomes have been implicated in the genetic control of root and shoot growth under aluminum stress. By far the greatest effects on aluminum tolerance were associated with the region near WG110 on chromosome 1. This region does not seem to correspond to most of the genes that have been mapped for aluminum tolerance in other species, nor do they correspond closely to one another. Most results, both from physiological studies and from molecular mapping studies, tend to suggest that aluminum tolerance is a complex multi-genic trait. The identification of DNA markers (such as WG110) that are diagnostic for aluminum tolerance in particular gene pools provides an important starting point for transferring and pyramiding genes that may contribute to the sustainable improvement of crop productivity in aluminum-rich soils. The isolation of genes responsible for aluminum tolerance is likely to be necessary to gain a comprehensive understanding of this complex trait. Received: 29 March 2000 / Accepted: 16 August 2000     

Genome-wide searching of single-nucleotide polymorphisms among eight distantly and closely related rice cultivars (Oryza sativa L.) and a wild accession (Oryza rufipogon Griff.).

We searched the genomes of eight rice cultivars (Oryza sativa L. ssp. japonica and ssp. indica) and a wild rice accession (Oryza rufipogon Griffith) for nucleotide polymorphisms, and identified 7805 polymorphic loci, including single-nucleotide polymorphisms (SNPs) and insertions/deletions (InDels), in predicted intergenic regions. Polymorphisms are useful as DNA markers for genetic analysis or positional cloning with segregating populations of crosses. Pairwise comparison between cultivars and a neighbor-joining tree calculated from SNPs agreed very well with relationships between rice strains predicted from pedigree data or calculated with other DNA markers such as p-SINE1 and simple sequence repeats (SSRs), suggesting that whole-genome SNP information can be used for analysis of evolutionary relationships. Using multiple SNPs to identify alleles, we drew a map to illustrate the alleles shared among the eight cultivars and the accession. The map revealed that most of the genome is mono- or di-allelic among japonica cultivars, whereas alleles well conserved among modern japonica paddy rice cultivars were often shared with indica cultivars or wild rice, suggesting that the genome structure of modern cultivars is composed of chromosomal segments from various genetic backgrounds. Use of allele-sharing analysis and association analysis were also tested and are discussed.     

Genetic analysis and QTL mapping for grain chalkiness characteristics of brown rice (Oryza sativa L.)

Grain chalkiness is one of the important appearance qualities in rice marketing. But it is a complex trait, controlled by polygenes and easily influenced by the environment. Genetic analysis and QTL detection was carried out on six characteristics of grain chalkiness consisting of the percentage of chalkiness (PGC), white belly (PWB) and white core grains (PWC), and the area of chalkiness (CA), white belly (WBA) and white core (WCA) in brown rice. A total of 16 main-effect QTLs associated with chalkiness characteristics of brown rice were mapped on seven chromosomes over two years. Among them,qPGC7.1 andqPWB7.2 were simultaneously located on chromosome 7 flanked by 7038 and 7042 at LOD scores 4.34 and 3.76, whileqPWC2.1 andqWCA2.1 were simultaneously located on chromosome 2 flanked by RM492 and RM324 with LOD scores of 2.50 and 3.39. Twelve epistatic combinations were detected for five chalkiness characteristics except for CA. Results indicated that WBA was mainly influenced by the additive effects of main-effect QTLs. PGC and PWC were affected by the effects of epistatic QTLs and the interactions between additive-by-additive effects and the environment. The effects of epistatic QTLs and the main-effect QTLs played important roles on CA, PWB and WCA. For the genetic improvement of grain chalkiness in breeding system, more attention should be paid to epistatic effects and the additive effects of main-effect QTLs.     

东乡野生稻回交重组系的抗旱性评价体系

选用协青早B//东乡野生稻/协青早B回交重组系及其亲本共79份水稻材料,研究了水稻发芽期、苗期、孕穗期、成熟期的种子萌发抗旱指数等31个指标与东乡野生稻回交重组系抗旱性的关系.结果表明:15％ PEG-6000溶液处理的相对发芽势、20％ PEG-6000溶液处理的种子萌发抗旱指数和相对发芽势及水分胁迫后最长根长、苗高、根鲜质量、根干质量、根系相对含水量、萎蔫率、叶片可溶性糖含量、叶片脯氨酸含量、叶片丙二醛含量、叶片相对含水量、卷叶级别、株高、单株分蘖数、单株有效穗数、穗实粒数、着粒密度、结实率、千粒重等21个指标的相对值与抗旱系数或抗旱指数的相关性显著.通过逐步回归分析筛选出9个抗旱性鉴定指标:20％ PEG-6000溶液处理的相对发芽势及水分胁迫后根干质量、根系相对含水量、叶片可溶性糖含量、叶片脯氨酸含量、叶片丙二醛含量、单株有效穗、结实率和千粒重的相对值.根据这些指标和偏相关系数,建立了不同时期的抗旱性评价(D值)方程和评价体系,可对东乡野生稻回交重组系抗旱性进行较好的评价.