Assessment of molecular markers and marker-assisted selection for drought tolerance in barley(Hordeum vulgare L.)

This review updates the present status of the field of molecular markers and marker-assisted selection(MAS),using the example of drought tolerance in barley.The accuracy of selected quantitative trait loci(QTLs),candidate genes and suggested markers was assessed in the barley genome cv.Morex.Six common strategies are described for molecular marker development,candidate gene identification and verification,and their possible applications in MAS to improve the grain yield and yield components in barley under drought stress.These strategies are based on the following five principles:(1)Molecular markers are designated as genomic‘tags’,and their‘prediction’is strongly dependent on their distance from a candidate gene on genetic or physical maps;(2)plants react differently under favourable and stressful conditions or depending on their stage of development;(3)each candidate gene must be verified by confirming its expression in the relevant conditions,e.g.,drought;(4)the molecular marker identified must be validated for MAS for tolerance to drought stress and improved grain yield;and(5)the small number of molecular markers realized for MAS in breeding,from among the many studies targeting candidate genes,can be explained by the complex nature of drought stress,and multiple stress-responsive genes in each barley genotype that are expressed differentially depending on many other factors.

Marker-assisted selection to pyramid Fusarium head blight resistance loci Fhb1 and Fhb2 in the high-quality soft wheat cultivar Yangmai 15

Fusarium head blight(FHB)is one of the most detrimental wheat diseases which greatly decreases the yield and grain quality,especially in the middle and lower reaches of the Yangtze River of China.Fhb1 and Fhb2 are two major resistance loci against Fusarium graminearum.Yangmai 15(YM15)is one of the most popular varieties in the middle and lower reaches of the Yangtze River,and it has good weak gluten characters but poor resistance to FHB.Here we used Fhb1 and Fhb2 to improve the FHB resistance of YM15 by a molecular marker-assisted selection(MAS)backcrossing strategy.The selection of agronomic traits was performed for each generation.We successfully selected seven introgressed lines which carry homozygous Fhb1 and Fhb2 with significantly higher FHB resistance than the recurrent parent YM15.Three of the introgressed lines had agronomic and quality characters that were similar to YM15.This study demonstrates that the pyramiding of Fhb1 and Fhb2 could significantly improve the FHB resistance in wheat using the MAS approach.

Supporting Nursing Technology for the Marker-Assisted Selection of Castanea mollissima Blume

[Objective] The aim was to study the supporting raising technology of Castanea mollissima Blume molecular marker-assisted selection to reduce the cost and time for chestnut breeding.[Method] To find the best cutting method,10 treatments were designed according to transverse diameter and vertical diameter of the chestnut including different cutting ratios in 1/2,1/3,1/4 and different directions about cross cutting,vertical cutting,bevel cutting and two side-cutting.The germination rate,seedling survival rate and morphological indicators of the treatments were measured;significant difference among them was also analyzed.[Result] 1/3 bevel cutting was the best,which not only could give high germination rate and survival rate,but also could guarantee high quality of nursery stock,which did not grow so differently with the untreated seedlings.[Conclusion] This way can not only reduce too much cost and time for seedling breeding,but also promote the germination of chestnut in advance,which could do much good to production.

Fine mapping and marker-assisted selection (MAS) of a low glutelin content gene in rice

Rice with low glutelin content is suitable as functional food for patients affected with diabetes and kidney failure. The fine mapping of the gene(s) responsible for low glutelin content will provide information regarding the distribution of glutelin related genes in rice genome and will generate markers for the selection of low glutelin rice varieties. Following an SDS-PAGE screen of rice germplasm from Taihu Valley of China, Japonica selection W3660 is identified to be a novel mutant characterized with low glutelin content. For fine mapping the mutant gene for low glutelin content, F2 and F3 populations were derived from a cross between W3660 and Jingrennuo. SDS-PAGE analysis of the total endosperm protein showed that the low glutelin content trait was controlled by a single dominant nuclear gene. Genetic mapping, using SSRs, located this gene to chromosome 2, in the region between SSR2-001/SSR2-004 and RM1358. The dis- tances of the two markers to the target gene were 1.1 cM and 3.8 cM respectively. By semi-quantitative RT-PCR analysis, the transcripts of GluB4/GluB5 genes located within the region do not change. However, GluB5 gene located proximal to SSR2-001/SSR2-004 was specifically reduced. SSR profiles of seven Japonica varieties were compared with that of W3660 for loci in the relevant genetic region. The markers SSR2-004 and RM1358 were used for marker- assisted selection. The selection efficiencies of SSR2-004 and RM1358 were 96.8% and 92.7% respectively. This provides a standard starting point for the breeding of low glutelin content rice varieties in China.

Marker-assisted pyramiding of soybean resistance genes R_(SC4),R_(SC8),and R_(SC14Q) to soybean mosaic virus

Soybean mosaic virus （SMV） is one of the major viral pathogens affecting soybean crops worldwide. Three SMV resistance genes, Rsc4, Rsc8, and Rsc14Q, have been identified and mapped on soybean chromosomes 14, 2, and 13 from Dabaima, Kefeng 1, and Qihuang 1 cultivars, respectively. Soybean cultivar Nannong 1138-2 is widely grown in the Yangtze River Valley of China. In this study, crosses were made between Qihuang l^Kefeng 1 and DabaimaxNannong 1138-2. Ten simple sequence repeat （SSR） markers linked to three resistance loci （Rsc4, Rsc8, and Rsc^4Q） were used to assist pyramided breeding. Pyramided families containing three resistance loci （Rsc4, Rsc8, and Rsc14Q） were evaluated by inoculating them with 21 SMV strains from China. Results indicated that the 10 markers can be used effectively to assist the selection of resistant individuals containing Rsc4, Rsc8, and Rsc14Q. A total of 53 F6 plants were confirmed to contain three homozygous alleles conferring resistance to SMV. Five F7 homozygous pyramided families exhibited resistance to 21 strains of SMV and showed desirable agronomic traits using dual selection. The strategy of pyramiding resistance gene derived from different varieties has practical breeding value in providing broad-spectrum resistance against the existing strains of SMV in China.

Transferring Translucent Endosperm Mutant Gene Wx-mq and Rice Stripe Disease Resistance Gene Stv-bi by Marker-Assisted Selection in Rice (Oryza sativa)

A high-yielding japonica rice variety, Wuyunjing 7, bred in Jiangsu Province, China as a female parent was crossed with a Japanese rice variety Kantou 194, which carries a rice stripe disease resistance gene Stv-b＇ and a translucent endosperm mutant gene Wx-mq. From F2 generations, a sequence characterized amplified region （SCAR） marker tightly linked with Stv-b＇ and a cleaved amplified polymorphic sequence （CAPS） marker for Wx-mq were used for marker-assisted selection. Finally, a new japonica rice line, Ning 9108, with excellent agronomic traits was obtained by multi-generational selection on stripe disease resistance and endosperm appearance. The utilization of the markers from genes related to rice quality and disease resistance was helpful not only for establishing a marker-assisted selection system of high-quality and disease resistance for rice but also for providing important intermediate materials and rapid selection method for good quality, disease resistance and high yield in rice breeding.

Natural Variation of Pto and Fen Genes and Marker-Assisted Selection for Resistance to Bacterial Speck in Tomato

The resistance in tomato plants to bacterial speck caused by Pseudomonas syringae pv. tomato is triggered by the interactions between the plant resistance protein Pto and the pathogen avirulence proteins AvrPto or AvrPtoB. Fen is a gene encoding closely related functional protein kinases as the Pto gene. To investigate the status of resistance to the pathogen and natural variation of Pto and Fen genes in tomato, 67 lines including 29 growing in China were subject to disease resistance evaluation and fenthion-sensitivity test. Alleles of Pto and Fen were amplified from genomic DNA of 25 tomato lines using polymerase chain reaction （PCR） and sequences were determined by sequencing the PCR products. The results indicated that none of the 29 cultivars/hybrids growing in China were resistant to bacterial speck race 0 strain DC3000. Seven of eight tomato lines resistant to DC3000 were also fenthion-sensitive. Analysis of deduced amino acid sequences identified three novel residue substitutions between Pto and pto, and one new substitution identified between Fen and fen. A PCR-based marker was developed and successfully used to select plants with resistance to DC3000.

Marker-Assisted Breeding of Thermo-Sensitive Genic Male Sterile Line 1892S for Disease Resistance and Submergence Tolerance

Rice line 1892S is an elite thermo-sensitive genic male sterile(TGMS)line for two-line hybrid rice production.However,1892S is susceptible to rice blast,bacterial blight and submergence.Here we reported the introduction of blast resistance(R)gene Pi9,bacterial blight R gene Xa21 and submergence tolerance gene Sub1A into 1892S genetic background through backcrossing and marker-assisted selection.The improved TGMS line 31892S and its hybrids conferred disease resistance to rice blast and bacterial blight,and showed submergence tolerance for over 14 d without significant loss of viability.The sterility-fertility conversion of 31892S was similar to that of 1892S.31892S and its derived hybrid rice had similar agronomic traits and grain quality with 1892S and the control hybrid rice,respectively.The newly developed 31892S provided an improved TGMS line for two-line hybrid rice production with disease resistance to rice blast and bacterial blight,and submergence tolerance with no yield penalty or change in grain quality.

Marker-Assisted Selection of Xa21 Conferring Resistance to Bacterial Leaf Blight in indica Rice Cultivar LT2

Bacterial leaf blight of rice (BLB), caused by Xanthomonas oryzae pv. oryzae, is one of the most destructive diseases in Asian rice fields. A high-quality rice variety, LT2, was used as the recipient parent.IRBB21, which carries the Xa21 gene, was used as the donor parent. The resistance gene Xa21 was introduced into LT2 by marker-assisted backcrossing. Three Xoo races were used to inoculate the improved lines following the clipping method. Eleven BC_3F_3 lines carrying Xa21 were obtained based on molecular markers and agronomic performance. The 11 lines were then inoculated with the three Xoo races. All the 11 improved lines showed better resistance to BLB than the recipient parent LT2. Based on the level of resistance to BLB and their agronomic performance, five lines (BC_3F_3 5.1.5.1, BC_3F_3 5.1.5.12, BC_3F_3 8.5.6.44, BC_3F_3 9.5.4.1 and BC_3F_3 9.5.4.23) were selected as the most promising for commercial release. These improved lines could contribute to rice production in terms of food security.

Marker-Assisted Introgression of Quantitative Resistance Gene pi21 Confers Broad Spectrum Resistance to Rice Blast

The quantitative resistance gene pi21 from Sensho was introgressed to an indica breeding line IR63307-4B-13-2,a pyramiding line IRBB4/5/13/21,and a tropical japonica line Kinandang Patong by marker-assisted backcrossing.A total of 192 improved lines at the BC4F3 and BC4F4 generations were developed and confirmed to have the gene introgression via genotyping using a pi21-specific InDel marker.Thirteen randomly selected improved lines,representing all the three genetic backgrounds,demonstrated resistance against leaf blast composites in the field and a broader spectrum resistance against individual isolates compared to the recurrent parents in the glasshouse.Specifically,the tested lines exhibited pi21-acquired resistance against 11 leaf blast isolates that elicited susceptible reactions from the recurrent parents.All the tested lines maintained a comparative heading date,and similar or improved panicle length,number of primary branches per panicle and number of total grains per panicle relative to the recurrent parents.The physical grain characteristics of the recurrent parents were also maintained in the 13 lines tested,although variability in the amylose content and chalkiness degree was observed.The successful marker-assisted introgression of pi21 in diverse genetic backgrounds and the resulting broader spectrum resistance of improved lines against leaf blast indicate the potential of pi21 for deployment in cultivars grown across other rice growing regions in Asia.

Breeding Rice Restorer Lines with High Resistance to Bacterial Blight by Using Molecular Marker-Assisted Selection

Two bacterial blight （BB） resistance genes, Xa21 and Xa4, from IRBB24 were introduced into hybrid rice restorer line Mianhui 725, which is highly susceptible to BB, by using hybridization and molecular marker-assisted selection technology. Four homologous restorer lines were obtained through testing the R target genes with molecular markers and analyzing parental genetic background. Inoculation of the four lines and their hybrids with the specific strains of Xanthomonas oryzae pv. oryzae, P1, P6 and seven representative strains of Chinese pathotype, C Ⅰ -CⅦ, showed that all of the four lines and their hybrids were highly resistant and presented broad resistance-spectrum to BB. The hybrids of G46A / R207-2 displayed good agronomic characters and high yield potential, and R207-2 was named Shuhui 207.

Pyramiding Breeding by Marker-Assisted Recurrent Selection in Upland Cotton: Selected Effects on Resistance to Helicoverpa armigera

The coincidence rates were more than 96% among the instar-weighted average of bioassaysin the lab, the percentage of resistance to Km in the field and the percentage of plantscontaining Bt gene. So, the performance of resistance to Km in the field can be used torepresent the transgenic Bt gene for selecting the resistance to bollworm. The instar-weighted averages were 30.585, 24.182, 16.615, 10.601, 10.123, 7.440 and 7.215 for theC0, P1, M1, M2, MP1, P2 and MP2 populations, respectively. The variance analysisindicated that the instar-weighted average in C0 was greatly significantly higher thanthat in all other populations, i.e., the performance of resistance to bollworm in C0 washighly significantly lower than all other populations. And the resistance in P1 wasgreatly lower than that of M1, M2, MP1, P2 and MP2, and M1 greatly lower than that of M2,MP1, P2 and MP2. There were no significant differences among M2, MP1, P2 and MP2. Withinthe populations of the first cycle selection, MP1 and M1 were greatly significantlyhigher than P1, and MP1 significantly higher than M1. The populations of the second cycleselection were significantly higher than their initial population M1, but no significantdifference among them. The boll size, seed index, the percent of the first harvest yield,fiber length, strength and elongation of the resistant plants to bollworm were significantlylower than that of sensitive plants to bollworm. And the yield of seed and lint cottonof the resistant plant to bollworm were lower than that of the sensitive to bollworm, butno significant difference between them. The boll numbers per plant, lint percent andmicronaire of the resistant plants to bollworm were significantly higher than that of thesensitive plant to bollworm.

Marker-assisted Selection of ZmC_4Ppc in Rice Breeding and Yield Trait Performances of Advanced Lines

The full-length of intact Zea mays gene for phosphoenolpyruvate carboxylase gene （ZmC4Ppc） is 6 781 bp. The products of PCR for this gene were not clear with poor repeatability, resulting in that it was difficult for marker-assisted selection （MAS） both in rice and maize. For selecting the markers for MAS, sequences presented only in maize rather than in rice were identified by BLAST, and used for primer design using Primer Premier 5.0. A pair of specific primer termed MRpc （Forward： 5＇ AAGCAGGGAAGCGAGACG 3＇, Reverse： 5＇ GATTGCCGCCAGCAGTAG 3＇） was used for selection of transformed rice, and ZmC4Ppc could be highly and constitutively expressed at each tested developmental stages in the transformed rice selected by using MRpc. Thus, MRpc was used for MAS of progenies carrying ZmC4Ppc gene in rice and some restorer lines with ZmC4Ppc （e.g. FPM881） derived from ZmC4Ppc-transformed Kitaake backcrossed with a restorer line Shuhui 881 were obtained. The analyses on genetic background, PEPCase activity, net photosynthetic rate, general combining ability （GCA） and specific combining ability （SCA） of FPM881 showed that similarity of genetic background reached above 95%, the PEPCase and net photosynthetic rate were higher than those of the control, and some of the progenies carrying ZmC4Ppc gene had better GCA and SCA for grain yield per plant, number of panicles per plant, and 1000-grain weight than those of the control. This suggested that the introduction of maize ZmC4Ppc gene via MAS and its stable expression could increase grain yield of rice and would likely provide a pathway for rice varietal improvement.

Improving Blast Resistance of a Thermo-Sensitive Genic Male Sterile Rice Line GD-8S by Molecular Marker-Assisted Selection

The broad-spectrum blast resistance gene Pi-1, from donor line BL122, was introduced into a thermo-sensitive genic male sterile rice line GD-8S, which possessed good grain quality but high susceptibility to rice blast, by using backcross breeding and molecular marker-assisted selection. Five elite improved male sterile lines, RGD8S-1, RGD8S-2, RGD8S-3, RGD8S-4 and RGD8S-5, were selected based on the results of molecular marker analysis, spikelet sterility, recovery rate of genetic background and agronomic traits. Thirty-three representative blast isolates collected from Guangdong Province, China were used to inoculate the improved lines and the original line GD-8S artificially. The resistance frequencies of the improved lines ranged from 76.47% to 100%, much higher than that of the original line GD-8S （9.09%）. On the agronomic characters, there were no significant differences between the improved lines and GD-8S except for flag leaf length and panicle number per plant. The improved lines could be used for breeding hybrid rice with high blast resistance.

Improvement of three popular Indian groundnut varieties for foliar disease resistance and high oleic acid using SSR markers and SNP array in marker-assisted backcrossing

Foliar fungal diseases(rust and late leaf spot)incur large yield losses,in addition to the deterioration of fodder quality in groundnut worldwide.High oleic acid has emerged as a key market trait in groundnut,as it increases the shelf life of the produce/products in addition to providing health benefits to consumers.Marker-assisted backcrossing(MABC)is the most successful approach to introgressing or pyramiding one or more traits using traitlinked markers.We used MABC to improve three popular Indian cultivars(GJG 9,GG 20,and GJGHPS 1)for foliar disease resistance(FDR)and high oleic acid content.A total of 22 BC3F4 and 30 BC2F4 introgression lines(ILs)for FDR and 46 BC3F4 and 41 BC2F4 ILs for high oleic acid were developed.Recurrent parent genome analysis using the 58 K Axiom_Arachis array identified several lines showing upto 94%of genome recovery among second and third backcross progenies.Phenotyping of these ILs revealed FDR scores comparable to the resistant parent,GPBD 4,and ILs with high(~80%)oleic acid in addition to high genome recovery.These ILs provide further opportunities for pyramiding FDR and high oleic acid in all three genetic backgrounds as well as for conducting multi-location yield trials for further evaluation and release for cultivation in target regions of India.

Development and application of marker-assisted reverse breeding using hybrid maize germplasm

Humankind has been through different periods of agricultural improvement aiming at enhancing our food supply and the performance of food crops. In recent years, whole genome sequencing and deep understanding of genetic and epigenetic mechanisms have facilitated new plant breeding approaches to meet the challenge of growing population, dwindling resources, and changing climate. Here we proposed a simple and fast molecular breeding method, marker-assisted reverse breeding（MARB）, which will revert any maize hybrid into inbred lines with any level of required similarity to its original parent lines. Since all the pericarp DNA of a hybrid is from the maternal parent, whereas one half of the embryo DNA is from the maternal parent and the other half from the paternal parent, so we firstly extract DNA from seed embryo and pericarp of a selected elite hybrid separately and then we derived the genotypes of the two parents with high-density single nucleotide polymorphism（SNP） chips. The following marker-assisted selection was performed based on an Illumina low-density SNP chip designed with 192 SNPs polymorphic between the two parental genotypes, which were uniformly distributed on 10 maize chromosomes. This method has the advantages of fast speed, fixed heterotic mode, and quick recovery of beneficial parental genotypes compared to traditional pedigree breeding using elite hybrids. Meanwhile, MARB has the advantage of not requiring sophisticated transformation and double haploid（DH） technologies over RNA interference（RNAi）-mediated reverse breeding. In addition, MARB can also be used with feed corn harvested from big farms, which is often similar to F_2 populations, and the relevant transgenes in the population can be eliminated by marker-assisted selection. As a result, the whole global commercial maize hybrids can be utilized as germplasm for breeding with MARB technology. Starting with an F_2 population derived from an elite hybrid, our experiment indicates that with three cycles of marker-assisted selection, selected lines could recover over 80% of the parental genotypes and associated beneficial genes in a fixed heterotic mode. The success application of MARB in maize suggests that this technology is applicable to any hybrid crop to breed new inbreds with improved hybrid performance but the same heterotic mode. As chip technology becomes cheap, it would be expected that polymorphism screening and following marker-assisted selection could be done with one all-purpose high density chip. Several issues associated with MARB were discussed, including its rationale, efficiency and advantages, along with food/feed and environmental safety issues and applications of MARB in variety protection and marker-assisted plant breeding.

A Co-Dominant Marker BoE332 Applied to Marker-Assisted Selection of Homozygous Male-Sterile Plants in Cabbage (Brassica oleracea var.capitata L.)

The dominant genic male sterility （DGMS） gene CDMs399-3 derived from a spontaneous mutation in the line 79-399-3 of spring cabbage （Brassica oleracea var. capitata L.）, has been successfully applied in hybrid seed production of several cabbage cultivars in China. During the development of dominant male sterility lines in cabbage, the conventional identification of homozygous male-sterile plants （CDMs399-3/CDMs399-3） is a laborious and time-consuming process. For marker-assisted selection （MAS） of the gene CDMs399-3 transferred into key spring cabbage line 397, expressed sequence tag-simple sequence repeats （EST-SSR） and SSR technology were used to identify markers that were linked to CDMs399-3 based on method of bulked segregant analysis （BSA）. By screening a set of 978 EST-SSRs and 395 SSRs, a marker BoE332 linked to the CDMs399-3 at a distance of 3.6 cM in the genetic background of cabbage line 397 were identified. 7 homozygons male-sterile plants in population P1170 with 20 plants were obtained finally via MAS of BoE332. Thus, BoE332 will greatly facilitate the transferring of the gene CDMs399-3 into the key spring cabbage line 397 and improve the application of DGMS in cabbage hybrid breeding.

Molecular mapping of a novel wheat powdery mildew resistance gene Ml92145E8-9 and its application in wheat breeding by marker-assisted selection

Powdery mildew, caused by Blumeria graminis f. sp. tritici(Bgt), is one of the most devastating diseases of common wheat(Triticum aestivum L.). The wheat line 92145 E8-9 is immune to Bgt isolate E09. Genetic analysis reveals that the powdery mildew resistance in 92145 E8-9 is controlled by a single dominant gene, temporarily designated Ml92145 E8-9. Bulkedsegregant analysis(BSA) with simple sequence repeat(SSR) markers indicates that Ml92145 E8-9 is located on chromosome 2 AL. According to the reactions of 92145 E8-9,VPM1(Pm4 b carrier), and Lankao 906(PmLK906 carrier) to 14 Bgt isolates, the resistance spectrum of 92145 E8-9 differs from those of Pm4 b and PmLK906, both of which were previously localized to 2 AL. To test the allelism among Ml92145 E8-9, Pm4 b and PmLK906, two F2 populations of 92145 E8-9 × VPM1(Pm4 b) and 92145 E8-9 × Lankao 906(PmLK906) were developed in this study. Screening of 784 F2 progeny of 92145 E8-9 × VPM1 and 973 F2 progeny of 92145 E8-9 × Lankao 906 for Bgt isolate E09 identified 37 and 19 susceptible plants, respectively. These findings indicated that Ml92145 E8-9 is non-allelic to either Pm4 b or PmLK906. Thus, Ml92145 E8-9 is likely to be a new powdery mildew resistance gene on2 AL. New polymorphic markers were developed based on the collinearity of genomic regions of Ml92145 E8-9 with the reference sequences of the International Wheat Genome Sequencing Consortium(IWGSC). Ml92145 E8-9 was mapped to a 3.6 c M interval flanked by molecular markers Xsdauk13 and Xsdauk682. This study also developed five powdery mildew-resistant wheat lines(SDAU3561, SDAU3562, SDAU4173, SDAU4174, and SDAU4175)using flanking marker-aided selection. The markers closely linked to Ml92145 E8-9 would be useful in marker-assisted selection for wheat powdery mildew resistance breeding.

Development of New Submergence Tolerant Rice Variety for Bangladesh Using Marker-Assisted Backcrossing

Submergence tolerant high yielding rice variety was developed using BR11 as a recipient parent applying foreground, phenotypic and background selection approaches. Recombinant selection was found essential to minimize linkage drag by BC2F2 generation. Without recombinant selection, the introgression size in the backcross recombinant lines （BRLs） was approximately 15 Mb on the carrier chromosome. The BRLs were found submergence tolerance compared to the check varieties under complete submergence for two weeks at Bangladesh Rice Research Institute, and produced higher yield compared to the isogenic Subl-line under controlled submerged condition. The BRL IR85260-66-654-Gaz2 was released as BRRI dhan52 in 2010, which was the first high yielding submergence tolerant variety in Bangladesh. BRRI dhan52 produced grain yield ranging from 4.2 to 5.2 t/hm2 under different flash flood prone areas of Bangladesh in three consecutive seasons. The study demonstrated the efficiency of recombinant selection and better adaptability of the newly released submergence tolerant high yielding variety in flash flood prone different areas of the country with respect to submergence tolerance and yield potential.

Breeding of R8012,a Rice Restorer Line Resistant to Blast and Bacterial Blight Through Marker-Assisted Selection

Genetic improvement is one of the most effective strategies to prevent rice from blast and bacterial blight （BB） diseases, the two most prevalent diseases jeopardizing rice production. Rice hybrids with dural resistance to blast and BB are needed for sustainable production of food. An incomplete diallele design resulted in 25 crosses between five blast and five BB resistant germplasm accessions. Only one pair of parents, DH146 ×TM487, showed polymorphism for all the markers to identify one blast resistance gene Pi25 and three BB resistance genes, Xa21, xa13 and xa5, thus it was used in the marker-assisted selection （MAS）. F2 individuals of DH146× TM487 were genotyped using flanking markers of RM3330 and sequence tagged site （STS） marker SA7 for Pi25. The resistant F2 plants with Pi25 were used for pyramiding BB resistance genes Xa21, xa13 and xa5 identified by the markers pTA248, RM264 and RM153, respectively in subsequent generations. Finally, after selection for agronomic traits and restoration ability among 12 pyramided lines, we acquired an elite restorer line, R8012 including all four target genes （Pi25＋Xa21＋xa13＋xa5）. Hybrid Zhong 9NR8012 derived from the selected line showed stronger resistance to blast and BB, and higher grain yield than the commercial checks uniformally in experimental plots, 2007 state-wide yield trial and 2008 nation-wide yield trial. This study provides a paradigmatic example to show that MAS is a practically feasible tool in effectively pyramiding multiple resistance genes. The resultant restoring line and its hybrid would play an important role in securing rice production in China.