QTL analysis on rice grain appearance quality,as exemplifying the typical events of transgenic or backcrossing breeding

Rice grain shape and yield are usually controlled by multiple quantitative trait loci (QTL). This study used a set of F_9–10 recombinant inbred lines (RILs) derived from a cross of Huahui 3 (Bt/Xa21) and Zhongguoxiangdao, and detected 27 QTLs on ten rice chromosomes. Among them, twelve QTLs responsive for grain shape/ or yield were mostly reproducibly detected and had not yet been reported before. Interestingly, the two known genes involved in the materials, with one insect-resistant Bt gene, and the other disease-resistant Xa21 gene, were found to closely link the QTLs responsive for grain shape and weight. The Bt fragment insertion was firstly mapped on the chromosome 10 in Huahui 3 and may disrupt grain-related QTLs resulting in weaker yield performance in transgenic plants. The introgression of Xa21 gene by backcrossing from donor material into receptor Minghui 63 may also contain a donor linkage drag which included minor-effect QTL alleles positively affecting grain shape and yield. The QTL analysis on rice grain appearance quality exemplified the typical events of transgenic or backcrossing breeding. The QTL findings in this study will in the future facilitate the gene isolation and breeding application for improvement of rice grain shape and yield.     

A new gene <Emphasis Type="Italic">Bph33</Emphasis>(<Emphasis Type="Italic">t</Emphasis>) conferring resistance to brown planthopper (BPH), <Emphasis Type="Italic">Nilaparvata lugens</Emphasis> (Stål) in rice line RP2068-18-3-5

The rice brown planthopper (BPH) Nilaparvata lugens (Stål) is one of the major pests of rice across Asia. Host-plant resistance is the most ecologically acceptable means to manage this pest. A rice breeding line RP2068-18-3-5 (RP2068) derived from the land race Velluthacheera is reported to be resistant to BPH populations across India. We identified a new R gene [Bph33(t)] in this line using advanced generation RILs derived from TN1 × RP2068 cross through phenotyping at two locations and linkage analysis with 99 polymorphic SSR markers. QTL analysis through IciMapping identified at least two major QTL on chromosome 1 influencing seedling damage score in seed box screening, honey dew excretion by adults and nymphal survival. Since no BPH R gene has been reported on chromosome 1, we designate this locus as a new gene Bph33(t) which accounted for over 20% of phenotypic variance. Scanning the region for candidate gene suggested two likely candidates a leucine rich repeat (LRR) gene and a heat shock protein (HSP) coding gene. Expression profiling of the two genes in the two contrasting parents and RILs showed induction of the HSP gene (LOC_Os01g42190.1) at 6 h after infestation while LRR gene did not show such induction. It is likely that the HSP represented Bph33(t).     

Role of the rice blast resistance gene Pi-cd in rice (Oryza sativa) breeding programmes

A series of DNA markers for various agronomic traits may accelerate the success of marker-assisted selection in practical plant breeding programmes. Here, we developed DNA markers for the blast resistance gene Pi-cd. In this study, we examined the effects of the Pi-cd locus on not only blast resistance but also agronomic traits in agriculture. We developed three pyramiding lines (PLs) coupling Pi-cd with three blast resistance genes, pi21, Pi35 and Pi39. The effect of Pi-cd on blast resistance was dependent on the coupled resistance genes. Then, we evaluated the effects of Pi-cd on 13 agronomic traits. Amylose content and 1,000-grain weight showed significant differences between the PLs and current commercial varieties, which had no negative effects on agronomic trait values. Furthermore, we investigated the distribution of genotype for the Pi-cd locus among varieties of upland rice. The KT genotype specific to rice blast resistance may be predominant in the varieties. The results suggested that Pi-cd has the potential to be useful for improving blast resistance in rice breeding programmes.     

水稻品种IR24抗条纹叶枯病相关QTL的检测

为探明籼稻品种IR24是否携有新的抗条纹叶枯病基因,利用衍生于Asominori/IR24的重组自交系（RIL）群体和以Asominori为遗传背景IR24插入片段的染色体片段置换系（CSSL）群体,进行抗条纹叶枯病相关QTL的检测。利用疫区田间自然条件鉴定的方法,在RIL群体中共检测到4个控制条纹叶枯病的QTL,分别位于第3、5、7、11染色体上（qSTV3、qSTV5、qSTV7、qSTV11）, 其中qSTV3、qSTV7和qSTV11增强抗性的等位基因来自抗性亲本IR24。采用图示基因型比较法,在CSSL群体中将4个抗条纹叶枯病相关基因位点分别定位在染色体片段置换系CSSL4、L17、L39、L61、L62的IR24插入片段上。对比分析RIL群体和CSSL群体的分子连锁图谱,发现qSTV3所在的标记区间与CSSL17的IR24片段相吻合,qSTV7所在的标记区间与CSSL4的杂合片段、CSSL39的IR24片段相吻合,qSTV11所在的标记区间与 CSSL61的IR24片段以及CSSL62的杂合片段相吻合,表明确实存在这3个位点。与前人的研究结果相比较,发现位于第3染色体上的qSTV3区域存在抗刺吸性害虫的基因簇,是一个表达稳定的抗灰飞虱基因座;位于第7染色体上的qSTV7不同于已报道的抗性基因座,表明IR24携有新的抗性基因,这些基因不同于主基因Stvb-i,为防止广泛使用单一基因而造成的遗传脆弱性提供了新的抗性基因源,并且为利用分子标记辅助选择,聚合不同抗性基因培育抗性稳定的条纹叶枯病抗性品种创造了条件。     

Detection of downy and powdery mildew resistance QTL in a ‘Regent’ × ‘RedGlobe’ population

One hundred and eighty six F₁ plants from a ‘Regent’ × ‘RedGlobe’ cross were used to generate a partial linkage map with 139 microsatellite markers spanning all 19 chromosomes. Phenotypic scores for downy mildew, taken over two years, confirmed a major resistance QTL (Rpv3) against downy mildew in the interval VVIN16-cjvh to UDV108 on chromosome 18 of ‘Regent’. This locus explained up to 62 % of the phenotypic variance observed. Additionally a putative minor downy mildew resistance locus was observed on chromosome 1 in one season. A major resistance locus against powdery mildew (Ren3) was also identified on chromosome 15 of ‘Regent’ in the interval UDV116 to VChr15CenGen06. This study established the efficacy of and validated the ‘Regent’-derived downy and powdery mildew major resistance genes/QTL under South African conditions. Closely linked SSR markers for marker-assisted selection and gene pyramiding strategies were identified.     

A new locus for resistance to brown planthopper identified in the indica rice variety DV85

The brown planthopper (BPH) is one of the most destructive insect pests of rice. Resistant varieties have proved to be one of the most economic and effective measures for BPH management. In this study, an indica rice ‘DV85’ showed resistance to biotype 2 of BPH by bulked seedling test, and a recombinant inbred line (RIL) population derived from a cross between a susceptible rice ‘Kinmaze’ and ‘DV85’ was phenotyped to map genetic factors conferring BPH resistance in ‘DV85′. Composite interval mapping revealed that one quantitative trait locus (QTL) with a LOD score of 10.1 was detected between XNpb202 and C1172 on chromosome 11. This QTL was designated as Qbph11. Qbph11 explained 68.4% of the phenotypic variance of BPH resistance in this population. The allele from the resistant parent ‘DV85’ at Qbph11 reduced the damage caused by BPH feeding and would be very useful in breeding resistant rice varieties via marker‐assisted selection.     

Identification of microsatellite markers linked to the blast resistance gene <Emphasis Type="Italic">Pi-1(t)</Emphasis> in rice

The present work was conducted to identify microsatellite markers linked to the rice blast resistance gene Pi-1(t) for a marker-assisted selection program. Twenty-four primer pairs corresponding to 19 microsatellite loci were selected from the Gramene database (www. gramene.org) considering their relative proximity to Pi-1(t) gene in the current rice genetic map. Progenitors and DNA bulks of resistant and susceptible families from F₃ segregating populations of a cross between the near-isogenic lines C101LAC (resistant) and C101A51 (susceptible) were used to identify polymorphic microsatellite markers associated to this gene through bulked segregant analysis. Putative molecular markers linked to the blast resistance gene Pi-1(t) were then used on the whole progeny for linkage analysis. Additionally, the diagnostic potential of the microsatellite markers associated to the resistance gene was also evaluated on 17 rice varieties planted in Latin America by amplification of the specific resistant alleles for the gene in each genotype. Comparing with greenhouse phenotypic evaluations for blast resistance, the usefulness of the highly linked microsatellite markers to identify resistant rice genotypes was evaluated. As expected, the phenotypic segregation in the F₃ generation agreed to the expected segregation ratio for a single gene model. Of the 24 microsatellite sequences tested, six resulted polymorphic and linked to the gene. Two markers (RM1233*I and RM224) mapped in the same position (0.0 cM) with the Pi-1(t) gene. Other three markers corresponding to the same genetic locus were located at 18.5 cM above the resistance gene, while another marker was positioned at 23.8 cM below the gene. Microsatellite analysis on elite rice varieties with different genetic background showed that all known sources of blast resistance included in this study carry the specific Pi-1(t) allele. Results are discussed considering the potential utility of the microsatellite markers found, for MAS in rice breeding programs aiming at developing rice varieties with durable blast resistance based on a combination of resistance genes. Centro Internactional de Agricultura Tropical (CIAT) institute where the research was carried out     

多年生长雄蕊野生稻感光性的遗传分析

非洲的长雄蕊野生稻(Oryza longistaminata)具有多年生、抗病、抗虫、耐寒、耐旱等优良特性,可用于栽培稻的遗传改良。但其感光性强,长日照下不抽穗。为了在分子标记辅助育种过程中排除不需要的强感光性,须先找到长雄蕊野生稻中控制感光性的QTL。感光性是指水稻受日照长短的影响而改变其抽穗期的特性。抽穗期作为水稻重要的农艺性状,与产量有着密切的联系。本研究以长雄蕊野生稻(Oryza longistaminata)为父本,栽培稻巴利拉(Balilla)为母本杂交构建的F2群体为研究材料。对F2群体的抽穗期性状进行QTL定位分析,共得到6个控制水稻抽穗期的QTL,这些QTL分别位于第2号、第4号、第8号和第9号染色体上。在两年长日照下的F2群体中都检测到第8号染色体上的qDTH-8-1位点,但短日照下未检测到该位点,说明其跟感光性相关。qDTH-8-1是一个主效位点,对抽穗期表型贡献率最大,其中包含已被克隆的DTH8/GHD8基因。此外,我们还检测到另外4个已被定位但未被克隆的位点qDTH-4-1、qDTH-8-2、qDTH-9-1和q DTH-9-2,他们在本群体中的效应不大。结果表明,长雄蕊野生稻的感光性主要由qDTH-8-1控制,育种后代中要注意检测该位点的基因型。通过检测长雄蕊野生稻中的抽穗期QTL并进一步克隆到基因,这有助于深入了解野生稻强感光性的遗传机理,为水稻品种改良提供基因资源。     

QTL analysis for brown spot resistance in American rice cultivar ‘Dawn’

Rice brown spot (BS), caused by Bipolaris oryzae, causes yield loss and deterioration of grain quality. Using single-nucleotide polymorphism (SNP) markers, we conducted quantitative trait locus (QTL) analysis of BS resistance in backcross inbred lines (BILs) from a cross between an American rice cultivar, ‘Dawn’ (resistant), and ‘Koshihikari’ (susceptible). Four QTLs for BS resistance were detected in a three-year field evaluation, and ‘Dawn’ contributed the resistance alleles at all QTLs. The QTL with the greatest effect, qBSR6-kd, explained 15.1% to 20.3% of the total phenotypic variation. Although disease score and days to heading (DTH) were negatively correlated in all three years, qBSR6-kd was located near a QTL for DTH at which the ‘Dawn’ allele promoted heading. Another BS resistance QTL (qBSR3.1-kd) was unlinked to the QTLs for DTH. Therefore, these two QTLs are likely to be useful for breeding BS-resistant varieties without delaying heading. The other two BS resistance QTLs (qBSR3.2-kd and qBSR7-kd) were located near DTH QTLs at which the ‘Dawn’ alleles delayed heading. The QTLs reported here will be good candidates for developing BS-resistant cultivars.     

水稻抗旱机理和抗旱育种研究进展

水资源短缺正成为制约中国水稻生产持续发展的重要因素,培育抗旱性强的水稻品种,不但能够节约水资源,而且有利于稳产增产、节约能源。日益深入的干旱胁迫响应基因及QTL定位研究的积累为进行抗旱育种提供了依据和基础。对水稻在干旱胁迫下的伤害机理和抗旱的分子机制以及水稻抗旱育种研究进展等方面进行了总结和综述,以期为今后进一步深入研究抗旱机理及抗旱育种提供理论依据。今后应加强对稻属抗旱基因资源的有效发掘、评价、创新和利用,进一步发挥抗旱分子标记辅助选择在抗旱育种实践中的作用,改良水稻根系,结合常规育种技术和现代生物技术,提高栽培稻的抗旱性。     

Variation in cooking and eating quality traits in Japanese rice germplasm accessions

The eating quality of cooked rice is important and determines its market price and consumer acceptance. To comprehensively describe the variation of eating quality in 183 rice germplasm accessions, we evaluated 33 eating-quality traits including amylose and protein contents, pasting properties of rice flour, and texture of cooked rice grains. All eating-quality traits varied widely in the germplasm accessions. Principal-components analysis (PCA) revealed that allelic differences in the Wx gene explained the largest proportion of phenotypic variation of the eating-quality traits. In 146 accessions of non-glutinous temperate japonica rice, PCA revealed that protein content and surface texture of the cooked rice grains significantly explained phenotypic variations of the eating-quality traits. An allelic difference based on simple sequence repeats, which was located near a quantitative trait locus (QTL) on the short arm of chromosome 3, was associated with differences in the eating quality of non-glutinous temperate japonica rice. These results suggest that eating quality is controlled by genetic factors, including the Wx gene and the QTL on chromosome 3, in Japanese rice accessions. These genetic factors have been consciously selected for eating quality during rice breeding programs in Japan.     

Development of elite rice restorer lines in the genetic background of R022 possessing tolerance to brown planthopper, stem borer, leaf folder and herbicide through marker-assisted breeding

Rice leaf folder, stem borer and brown planthopper (BPH) are the most devastating rice insect pests. Developing and planting insect-resistant rice varieties is the most economical and effective measure for controlling these pests. BPH can be controlled with native BPH-resistance genes in rice, while at present rice leaf folder and stem borer can only be controlled through planting transgenic Bt rice. In this study, the breeding of a new restorer line KR022 possessing stacked BPH-resistance genes Bph14 and Bph15, Bt gene cry1C and glufosinate-resistance gene bar, is reported for the first time. A rice restorer line R022 with BPH-resistance genes Bph14 and Bph15 was used as a recurrent parent to cross with the transgenic rice T1C-19 of cry1C and bar genes during the breeding process. The restorer line KR022 was developed from the backcross populations of R022 and T1C-19 through molecular marker-assisted selection and glufosinate-resistance selection. The cry1C and bar genes were found to integrate on chromosome 11 of KR022, and the genome recovery of KR022 was up to 95.8 % of the R022 genome. The quantification of Cry1C protein expression showed that it was expressed at different levels in the leaf, stem, panicle, endosperm, and root of KR022 and its hybrid rice. The insect-resistance evaluation indicated that KR022 and its hybrid rice had good resistance to rice leaf folder and stem borer, both in laboratory settings and in the field. Furthermore, they exhibited increased resistance to BPH at both the seedling and mature stage. The field trial showed there was no significant difference in key agronomic traits between KR022 and its recurrent parent R022, and four hybrids from KR022 yield much higher than the control II-You 838. Moreover, KR022 and its hybrid rice were found to have resistance to the herbicide glufosinate. These results demonstrate that KR022 is effective as a rice restorer line for the breeding of “green super rice”, possessing multiple tolerances to rice BPH, stem borer, leaf folder and glufosinate.     

Fine mapping of stable QTLs related to eating quality in rice (Oryza sativa L.) by CSSLs harboring small target chromosomal segments

Amylose content (AC) and viscosity profile are primary indices for evaluating eating and cooking qualities of rice grain. Using chromosome segment substitution lines (CSSLs), previous studies identified a QTL cluster of genes for rice eating and cooking quality in the interval R727–G1149 on chromosome 8. In this study we report two QTLs for viscosity parameters, respectively controlling setback viscosity (SBV) and consistency viscosity (CSV), located in the same interval using rapid viscosity analyzer (RVA) profile as an indicator of eating quality. Previously reported QTL for AC was dissected into two components with opposite genetic effects. Of four QTLs, qCSV-8 and qAC-8-2 had stable genetic effects across three and four environments, respectively. qSBV-8, qCSV-8 and qAC-8-1 partly overlapped, but were separated from qAC-8-2. Based on data from an Affymetrix rice GeneChip, two genes related to starch biosynthesis at the qAC-8-2 locus were chosen for further quantitative expression analysis. Both genes showed enhanced expression in sub-CSSLs carrying the target qAC-8-2 allele, but not in sub-CSSLs without the target qAC-8-2 allele, indicating their possible role in rice quality determination. Molecular markers closely linked to the two stable QTL provide the opportunity for marker-assisted selection (MAS) in breeding high quality rice.     

Mapping and genetic analysis of two fertility restorer loci in the wild-abortive cytoplasmic male sterility system of rice (Oryza sativa L.)

Hybrid varieties developed by making use of the wild abortive cytoplasmic male sterility system account for 90% of hybrid rice produced. Previous inheritance studies have established that the fertility restoration in this system is controlled by two major loci, but the chromosomal locations of the fertility restorer (Rf) loci have yet to be resolved. In this study we determined the genomic locations of the two Rf loci by their linkage to molecular markers. The Rf gene containing regions were identified by surveying two bulks, made of 30 highly fertile and 46 highly sterile plants from a large F₂ population of the cross between Zhenshan 97A and Minghui 63, with RFLP markers covering the entire rice genome. The survey identified two likely Rf gene containing regions, located on chromosomes 1 and 10 respectively. This was confirmed by ANOVA using a large random sample from the same F₂ population and also with a genome-wide QTL analysis of a test-cross population. The results also showed that both loci have major effects of almost complete dominance on fertility restoration and the effect of the locus on chromosome 10 is larger than the one on chromosome 1. The two loci acted as a pair of classical duplicate genes; a single dominant allele at one of the two loci would suffice to restore the fertility to normal or nearly normal. Closely linked markers identified in this study may be used for marker assisted selection in hybrid rice breeding programs. This revised version was published online in July 2006 with corrections to the Cover Date.     

Detection of QTLs for traits associated with pre-harvest sprouting resistance in bread wheat (Triticum aestivum L.)

Pre-harvest sprouting (PHS) is one of the serious problems for wheat production, especially in rainy regions. Although seed dormancy is the most critical trait for PHS resistance, the control of heading time should also be considered to prevent seed maturation during unfavorable conditions. In addition, awning is known to enhance water absorption by the spike, causing PHS. In this study, we conducted QTL analysis for three PHS resistant related traits, seed dormancy, heading time and awn length, by using recombinant inbred lines from ‘Zenkouji-komugi’ (high PHS resistance) × ‘Chinese Spring’ (weak PHS resistance). QTLs for seed dormancy were detected on chromosomes 1B (QDor-1B) and 4A (QDor-4A), in addition to a QTL on chromosome 3A, which was recently cloned as TaMFT-3A. In addition, the accumulation of the QTLs and their epistatic interactions contributed significantly to a higher level of dormancy. QDor-4A is co-located with the Hooded locus for awn development. Furthermore, an effective QTL, which confers early heading by the Zenkouji-komugi allele, was detected on the short arm of chromosome 7B, where the Vrn-B3 locus is located. Understanding the genetic architecture of traits associated with PHS resistance will facilitate the marker assisted selection to breed new varieties with higher PHS resistance.     

Isolation and linkage mapping of disease-resistance-like sequences from various rice cultivars, containing different recognition specificities

Degenerated oligonucleotide primers identified from the nucleotide‐binding sites of known disease resistance (R) genes were used from rice cultivars harbouring different recognition specificities to amplify and clone homologous sequences of R genes. A total of 68 non‐redundant clones, which showed various degrees of sequence homology to R genes, were obtained from 18 rice cultivars. These clones had a high degree of sequence diversity both in the nucleotides and in the predicted amino acids, and were classified into five groups using clustal analysis. Fifteen of the 68 clones were mapped to 17 loci on chromosomes 3, 5, 11 and 12 in the rice molecular linkage map. The loci of the mapped clones correlated with the locations of known rice R genes for blast resistance and bacterial blight resistance on chromosomes 11 and 12. Other mapped loci occurred in cluster on chromosome 3, and correlated with the position of a quantitative trait locus for bacterial blight resistance. The mapping of the R gene homologues may aid the identification and isolation of R gene candidates.     

Characterization of near-isogenic lines carrying QTL for high spikelet number with the genetic background of an indica rice variety IR64 (Oryza sativa L.)

Total spikelet number per panicle (TSN) is one of the most important traits associated with rice yield potential. This trait was assessed in a set of 334 chromosomal segment introgression lines (ILs: BC₃-derived lines), developed from new plant type (NPT) varieties as donor parents and having the genetic background of an indica-type rice variety IR64. Among the 334 ILs, five lines which had different donor parents and showed significantly higher TSN than IR64 were used for genetic analysis. Quantitative trait locus (QTL) analysis was conducted using F₂ populations derived from crosses between IR64 and these ILs. As a result, a QTL for high TSN (one from each NPT donor variety) was detected on common region of the long arm of chromosome 4. The effect of the QTL was confirmed by an increase in TSN of five near-isogenic lines (NILs) developed in the present study. The variation in TSN was found among these NILs, attributing to the panicle architecture in the numbers of primary, secondary and tertiary branches. The NILs for TSN and the SSR markers linked to the TSN QTLs are expected to be useful materials for research and breeding to enhance the yield potential of rice varieties.     

Inheritance of field resistance to Septoria tritici blotch in the wheat doubled-haploid population Solitär × Mazurka

Breeding for field resistance to Septoria tritici blotch (STB), caused by Mycosphaerella graminicola (anamorph: Septoria tritici), is the most suitable strategy for controlling this important disease of wheat. Although many Stb genes for resistance to single pathogen isolates have been identified in wheat, knowledge of their efficiency against natural fungal populations is lacking. In a quantitative-trait-locus (QTL) mapping approach in six environments and four locations, field resistance to STB was studied in a doubled-haploid population derived from a cross between the field-resistant cultivar Solitär and the susceptible cultivar Mazurka. After plant height as a disease escape trait was accounted for, five QTL with effects on STB response on chromosomes 5A, 6D and 7D explained 20 % of the genotypic variance; QTL × environment interactions were minor. Field resistance was conferred exclusively by alleles from Solitär, which was previously shown to carry the isolate-specific genes Stb6 and Stb11 as well as minor QTL detected with seven fungal isolates. Surprisingly, neither the Stb6 nor Stb11 isolate-specific genes nor minor QTL previously detected in Solitär were found to be involved in its field resistance. The study suggests that resistance breeding for STB should not rest solely on the deployment of Stb genes. Field tests are indispensable to show their efficacy and durability and to identify genes conferring partial field resistance to STB.     

Genetic and physical mapping of the QTLAR3 controlling blight resistance in chickpea (Cicer arietinum L)

Physical and genetic maps of chickpea a QTL related to Ascochyta blight resistance and located in LG2 (QTL_AR3) have been constructed. Single-copy markers based on candidate genes located in the Ca2 pseudomolecule were for the first time obtained and found to be useful for refining the QTL position. The location of the QTL_AR3 peak was linked to an ethylene insensitive 3-like gene (Ein3). The Ein3 gene explained the highest percentage of the total phenotypic variation for resistance to blight (44.3 %) with a confidence interval of 16.3 cM. This genomic region was predicted to be at the Ca2 physical position 32–33 Mb, comprising 42 genes. Candidate genes located in this region include Ein3, Avr9/Cf9 and Argonaute 4, directly involved in disease resistance mechanisms. However, there are other genes outside the confidence interval that may play a role in the blight resistance pathway. The information reported in this paper will facilitate the development of functional markers to be used in the screening of germplasm collections or breeding materials, improving the efficiency and effectiveness of conventional breeding methods.     

Genetic mapping of bph20(t) and bph21(t) loci conferring brown planthopper resistance to Nilaparvata lugens St?l in rice (Oryza sativa L.)

Brown planthopper(BPH) is one of the most serious and destructive insect pests of rice in most rice growing regions of the world. In this study, two major resistance genes against BPH have been identified in an Oryza rufipogon (Griff.) introgression rice line, RBPH54. Inheritance of the BPH resistance in RBPH54 was studied by screening the resistance in parents, F1, F2 and BC1 generations against BPH biotype 2. A population of BC3F2 lines was developed and SSR markers were employed for the gene mapping, and new markers were designed for fine mapping of the resistance genes, while sequence information of BAC/PAC clones was used to construct physical maps of the genes. The results showed that the BPH resistance in RBPH54 was governed by recessive alleles at two loci, tentatively designated as bph20(t) and bph21(t). The locus bph20(t) was fine mapped to the short arm of chromosome 6 about 2.7 cM to the upper marker RM435 and 2.5 cM to lower marker RM540 and in a 2.5 cM region flanked by two new SSR markers BYL7 and BYL8 which were developed in the present study. The other BPH resistance locus bph21(t) was initially mapped to a region 7.9 cM to upper marker RM222 and 4.0 cM to lower marker RM244 on the short arm of chromosome 10. For physical mapping, the bph20(t)-linked markers were landed on BAC/PAC clones of the reference cv., Nipponbare, released by the International Rice Genome Sequencing Project. The bph20(t) locus was physically defined to an interval of about 75 kb with clone P0514G1. Identification and location of these two genes in the present study have diversified the BPH resistance gene pool, which give benefit to the development of resistant rice cultivars, and the linkage PCR-based SSR markers for the bph20(t) and bph21(t) genes would help realize the application of the genes in rice breeding through marker-assisted selection.