Characterization of the Aspergillus niger pelB gene: structure and regulation of expression

Summary Nearly isogenic lines (NILs) of rice (Oryza sativa) differing at a locus conferring resistance to the pathogen Xanthomonas oryzae pv. oryzae were surveyed with 123 DNA markers and 985 random primers using restriction fragment length plymorphism (RFLP) and random amplified polymorphic DNA (RAPD) analysis. One chromosome 11 marker (RG103) detected polymorphism between the NILs that cosegregated with Xa21. All other chromosome 11 DNA markers tested were monomorphic between the NILs, localizing the Xa21 introgressed region to an 8.3 cM interval on chromosome 11. Furthermore, we identified two polymerase chain reaction (PCR) products (RAPD2148 and RAPD818) that detected polymorphisms between the NILs. Genomic sequences hybridizing with RAPD818, RAPD248 and RG103 were duplicated specifically in the Xa21 NIL. All three markers cosegregated with the resistance locus, Xa21, in a F₂ population of 386 progeny. Based on the frequency with which we recovered polymorphic Xa21-linked markers, we estimated the physical size of the introgressed region to be approximately 800 kb. This estimation was supported by physical mapping (using pulsed field gel electrophoresis) of the sequences hybridizing with the three Xa21-linked DNA markers. The results showed that the three Xa21-linked markers are physically close to each other, with one copy of the RAPD818 sequences located within 60 kb of RAPD248 and the other copy within 270 kb of RG103. None of the enzymes tested generated a DNA fragment that hybridized with all three of the markers indicating that the introgressed region containing the resistance locus Xa21 is probably larger than 270 kb.     

Fine Mapping of Xa2, a Bacterial Blight Resistance Gene in Rice

The rice bacterial blight resistance gene, Xa2, confers resistance to T7147 of the bacterial blight pathogen Xanthomonas oryzae pv. oryzae. It is located on the long arm of chromosome 4. Here, we report the fine mapping of Xa2 by genetic recombination analysis with simple sequence repeat (SSR) markers according to the genome sequence. Two F₂ populations are constructed to localize Xa2. In a primary analysis with 136 random F₂ plants of Zhenzhuai/IRBB2, it was found that Xa2 was located in approximately 20 cM region. To accurately determine the locus of Xa2, 120 new SSR markers were developed in this region by screening the sequence. Twelve new SSR markers were successfully used in genetic recombination analysis in IR24/IRBB2 population, while 20 in ZZA/IRBB2 population. We found that the nearest SSR markers to Xa2 are HZR950-5 and HZR970-4, which cover approximately 190-kb region. The sequence analysis of this 190-kb region revealed the presence of a homologous sequence of leucine rich repeat (LRR)-kinase. These results are very useful for transferring or pyramiding Xa2 by molecular marker-assistant selection in rice breeding programs and for cloning Xa2 by map-based cloning in combination with a long-range PCR strategy. Electronic supplementary material Electronic supplementary material is available for this article at and accessible for authorised users.     

High-resolution mapping and gene prediction of <Emphasis Type="Italic">Xanthomonas Oryzae</Emphasis> pv. <Emphasis Type="Italic">Oryzae</Emphasis> resistance gene <Emphasis Type="Italic">Xa7</Emphasis>

Bacterial blight (BB) caused by Xanthomonas oryzae pv. oryzae (Xoo) is a devastating disease in rice worldwide. The resistance gene Xa7, which provides dominant resistance against the pathogen with avirulence (Avr) gene AvrXa7, has proved to be durably resistant to BB. A set of SSR markers were selected from the “gramene” database based on the Xa7 gene initial mapping region on chromosome 6. These markers were used to construct a high-resolution genetic map of the chromosomal region surrounding the Xa7 gene. An F₂ mapping population with 721 highly susceptible individuals derived from a cross between the near isogenic lines (NILs) IRBB7 and IR24 were constructed to localize the Xa7 gene. In a primary analysis with eleven polymorphic SSR markers, Xa7 was located in approximately the 0.28-cM region. To walk closer to the target gene, recombinant F₂ individuals were tested using newly developed STMS (sequence tagged microsatellite) markers. Finally, the Xa7 gene was mapped to a 0.21-cM interval between the markers GDSSR02 and RM20593. The Xa7-linked markers were landed on the reference sequence of cv. Nipponbare through bioinformatics analysis. A contig map corresponding to the Xa7 gene was constructed. The target gene was assumed to span an interval of approximately 118.5-kb which contained a total of fourteen genes released by the TIGR Genome Annotation Version 5.0. Candidate-gene analysis of Xa7 revealed that the fourteen genes encode novel domains that have no amino acid sequence similar to other cloned Xa(xa) genes. Shen Chen and Zhanghui Huang are contributed equally to this work.     

RFLP and RAPD mapping of the rice gm2 gene that confers resistance to biotype 1 of gall midge (Orseolia oryzae)

Gm2 is dominant gene conferring resistance to biotype 1 of gall midge (Orseolia oryzae Wood-Mason), the major dipteran pest of rice. The gene was mapped by restriction fragment length polymorphism (RFLP) analysis of a set of 40 recombinant inbred lines derived from a cross between the resistant variety Phalguna and the susceptible landrace ARC 6650. The gene is located on chromosome 4 at a position 1.3 cM from marker RG329 and 3.4 cM from RG476. Since the low (28%) polymorphism of this indica x indica cross hindered full coverage of the genome with RFLP markers, the mapping was checked by random amplified polymorphic DNA (RAPD)/bulked segregant analysis. Through the use of 160 RAPD primers, the number of polymorphic markers was increased from 43 to 231. Two RAPD primers amplified loci that co-segregated with resistance/susceptibility. RFLP mapping of these loci showed that they are located 0.7 cM and 2.0 cM from RG476, confirming the location of Gm2 in this region of chromosome 4. Use of these DNA markers will accelerate breeding for gall midge resistance by permitting selection of the Gm2 gene independently of the availability of the insect.     

Breeding bacterial blight-resistant hybrid rice with the cloned bacterial blight resistance gene Xa21

The cloned bacterial blight (BB) resistance gene Xa21 was transferred into Minghui63, a widely used restorer line of indica hybrid rice in China, through an Agrobacterium-mediated system. Molecular and resistance analyses revealed that the Xa21 gene was integrated in the genomes of transgenic plants and their progeny inherited resistance stably. For the purpose of hybrid breeding, Xa21 transgenic homozygous restorer lines were selected through `within-lane' dosage comparison of hybridization signal in combination with PCR and resistance analyses. The selected transgenic restorer lines were then crossed with a commonly used sterile line, Zhenshan97A, to produce Xa21 transgenic hybrid rice, Shanyou63-Xa21. The hybrid rice plants with Xa21 displayed high broad-spectrum resistance to Xanthomonas oryzae pv. oryzae (Xoo) races and maintained elite agronomic characters of Shanyou63. The propagation of this BB-resistant hybrid variety with Xa21 will benefit rice production.     

Genetic and physical mapping of xa13, a recessive bacterial blight resistance gene in rice

 The recessive gene, xa13, confers resistance to Philippine race 6 (PXO99) of the bacterial blight pathogen Xanthomonas oryzae pv oryzae. Fine genetic mapping and physical mapping were conducted as initial steps in an effort to isolate the gene. Using nine selected DNA markers and two F₂ populations of 132 and 230 plants, xa13 was fine-mapped to a genomic region <4 cM on the long arm of rice chromosome 8, flanked by two RFLP markers, RG136 and R2027. Four DNA markers, RG136, R2027, S14003, and G1149, in the target region were used to identify bacterial artificial chromosome (BAC) clones potentially harboring the xa13 locus from a rice BAC library. A total of 11 BACs were identified, forming four separate contigs including a single-clone contig, 29I3, associated with the RG136 STS marker, the S14003 contig consisting of four clones (44F8, 41O2, 12A16, and 12F20), the G1149 contig with two clones, 23D11 and 21H18, and the R2027 contig consisting of four overlapping clones, 42C23, 30B5, 6B7 and 21H14. Genetic mapping indicated that the xa13 locus was contained in the R2027 contig. Chromosomal walking on the R2027 contig resulted in two more clones, 33C7 and 14L3. DNA fingerprinting showed that the six clones of the R2027 contig were overlapping. Clone 44F8 hybridized with a single fragment from the clone 14L3, integrating the R2027 and S14003 contigs into a single contig consisting of ten BAC clones with a total size of approximately 330 kb. The physical presence of the xa13 locus in the contig was determined by mapping the ends of the BAC inserts generated by TAIL-PCR. In an F₂ population of 230 plants, the BAC-end markers 42C23R and 6B7F flanked the xa13 locus. The probes 21H14F and 21H14R derived from BAC clone 21H14 were found to flank xa13 at a distance of 0.5 cM on either side, using a second F₂ population of 132 plants. Thus, genetic mapping indicated that the contig and the 96-kb clone, 21H14, contained the xa13 locus. Received: 15 August 1998 / Accepted: 29 September 1998     

New PCR-based sequence-tagged site marker for bacterial blight resistance gene Xa38 of rice

Bacterial blight (BB), caused by Xanthomonas oryzae pv. oryzae, is a major disease of rice managed largely through the deployment of resistance genes. Xa38, a BB resistance gene identified from Oryza nivara acc. IRGC 81825, was mapped on chromosome 4L in a 38.4-kb region. The closely linked markers for this gene, identified earlier, were simple sequence repeat marker RM17499 and sequence-tagged site markers developed from loci Os04g53060 and Os04g53120. Marker Os04g53060 is dominant while the other two markers show smaller size differences difficult to resolve accurately on agarose gel. Based on gene annotation, three nucleotide binding site?Cleucine-rich repeat genes present in the target region were cloned from O. nivara and sequenced. One of the loci, LOC_Os04g53050, had a 48-base-pair deletion in O. nivara acc. IRGC 81825 compared to the cultivated rice. Primers were designed around the deletion and the resulting marker is codominant and easy to score in agarose gel. The newly designed marker co-segregated with Xa38, amplifying products of 269?bp in O. nivara and 317?bp in cultivated rice. This marker could be more useful for marker-assisted selection than ones reported earlier.     

Chalcone synthase in rice (Oryza sativa L.): Detection of the CHS protein in seedlings and molecular mapping of the chs locus

The chalcone synthase is a key enzyme that catalyses the first dedicated reaction of the flavonoid pathway in higher plants. The chs gene and its protein product in rice has been investigated. The presence of a chalcone synthase (CHS) protein in rice seedlings and its developmental stage-specific expression has been demonstrated by western analysis. The chalcone synthase of rice was found to be immunologically similar to that of maize. A rice cDNA clone, Os-chs cDNA, encoding chalcone synthase, isolated from a leaf cDNA library of an indica rice variety Purpleputtu has been mapped to the centromeric region of chromosome 11 of rice. It was mapped between RFLP markers RG2 and RG103. RG2 is the nearest RFLP marker located at a genetic distance of 3.3 cM. Some segments of chromosome 11 of rice including chs locus are conserved on chromosome 4 of maize. The markers, including chs locus on chromosome 11 of rice are located, though not in the same order, on chromosome 4 of maize. Genetic analysis of purple pigmentation in two rice lines, Abhaya and Shyamala, used in the present mapping studies, indicated the involvement of three genes, one of which has been identified as a dominant inhibitor of leaf pigmentation. The Os-chs cDNA shows extensive sequence homology, both for DNA and protein (deduced), to that of maize, barley and also to different monocots and dicots.     

The semidwarf gene,sd-1, of rice (Oryza sativa L.). II. Molecular mapping and marker-assisted selection

To establish the location of the semidwarf gene, sd-1, the anthocyanin activator (A), purple node (Pn), purple auricle (Pau), and the isozyme locus, EstI-2, in relation to DNA markers on the molecular linkage map of rice, 20 RFLP markers, previously mapped to the central region of chromosome 1 (McCouch et al. 1988), were mapped onto an F₂ population derived from the cross Taichung 65 (A,Pn,Pau)/Taichung 65 (sd-1). sd-1 and EstI-2 were determined to be linked most tightly to RFLP markers RG 109 and RG 220, which cosegregated with each other. The distance between these RFLP markers and sd-1 was estimated to be 0.8 cM, based on an observed recombination value of 0.8%. The order of genes and markers in this region of chromosome 1 was determined to be sd-1 — (EstI-2 — RG220 — RG109) — RG381 — A — Pn — Pau. To test the efficacy of selection for sd-1 based on these linked markers, 50-day-old F₂ seedlings derived from another cross, Milyang 23/Gihobyeo, were analyzed for marker genotype. At this age, the semidwarf character could not be clearly detected based on phenotype. In addition, plant height was normally distributed in this population, making it difficult to unambiguously identify plants carrying sd-1. Thirteen seedlings homozygous for the sd-1-associated allele at EstI-2, RG220 and RG109, and 13 seedlings homozygous for the Sd-1-associated allele at all three marker loci were selected for further genetic analysis. At 20 days after heading, the culm lengths of these 26 plants were measured and the expected phenotype was confirmed in every case. These 26 plants were then selfed for four generations and F₆ lines were again evaluated to determine whether any recombination among the three molecular markers, or between these markers and the sd-1 gene, could be detected. No recombinants were identified, confirming the tight linkage of these loci and the usefulness of genotypic selection for this recessive semidwarf character prior to the time when it can be evaluated based on phenotype.     

五个WRKY转录因子在水稻叶片生长和抗病反应中的表达研究

WRKY是植物基因组中最大的转录因子家族之一,它们在抗病及其他信号转导途径中发挥着重要的调控作用．为了解水稻WRKY的功能,我们选择了5个WRKY转录因子,用免疫印迹技术调查了它们在水稻叶片生长和在Xa21介导的白叶枯病抗性反应中的表达丰度变化．结果表明,OsWRKY13、23和71在叶片中表达,且随叶片生长而逐步增加,至成熟期略有下降,但在叶片中检测不到OsWRKY45和OsWRKY53的表达信号．在Xa21介导的白叶枯病抗性反应中,OsWRKY45、53和71均受诱导表达,而OsWRKY13和 OsWRKY23蛋白质的表达没有可见的变化．进一步比较OsWRKY45、OsWRKY53和OsWRKY71在抗、感和对照(Mock)反应中的表达,发现它们在抗、感反应中均发生相似变化．上述结果说明,OsWRKY13和OsWRKY23可能在叶片正常生长过程中发挥作用,OsWRKY45和OsWRKY53可能在水稻-白叶枯病菌互作过程中发挥作用,而OsWRKY71在二种条件下均有功能．     

RAPD and RFLP mapping of the bacterial blight resistance gene xa-13 in rice

Bacterial blight (BB) caused by Xanthomonas oryzae pv oryzae (Xoo) is one of the most serious diseases of rice. The recessive gene xa-13 confers resistance to Philippine race 6 of Xoo. To tag xa-13 with molecular markers, RAPD analysis was conducted with the combined use of near-isogenic lines and bulked segregant analysis. From the survey of 260 arbitrary 10-nucleotide primers, one primer (OPAC05) was detected to amplify specifically a 0.9-kb band from the DNA of susceptible plants. The distance between the RAPD marker OPAC05-900 and xa-13 was estimated to be 5.3 cM. The RAPD marker was then mapped on chromosome 8 using a mapping population of doubled haploid lines derived from the cross of IR64/Azucena. The linkage between RFLP markers and the RAPD marker was analyzed using an F₂ population of 135 plants derived from a cross between a near-isogenic line for xa-13, IR66699-5-5-4-2, and IR24. No recombinants were found between RZ28 and CDO116 and their distance from xa-13 was estimated to be 4.8 cM. RG136 was located at 3.7 cM on the other side of xa-13. The mapping of xa-13 with closely linked DNA markers provides the basis for marker-aided selection for rice improvement.Department of Agronomy, South China Agricultural University, Guangzhou, China     

Tagging genes for blast resistance in rice via linkage to RFLP markers

Summary Both Pi-2(t) and Pi-4(t) genes of rice confer complete resistance to the blast fungal pathogen Pyricularia oryzae Cav. As economically important plant genes, they have been recently characterized phenotypically, yet nothing is known about their classical linkage associations and gene products. We report here the isolation of DNA markers closely linked to these blast resistance genes in rice. The DNA markers were identified by testing 142 mapped rice genomic clones as hybridization probes against Southern blots, consisting of DNA from pairs of nearly isogenic lines (NILs) with or without the target genes. Chromosomal segments introgressed from donor genomes were distinguished by restriction fragment length polymorphisms (RFLPs) between the NILs. Linkage associations of the clones with Pi-2(t) and Pi4(t) were verified using F₃ segregating populations of known blast reaction. Cosegregation of the resistant genotype and donor-derived allele indicated the presence of linkage between the DNA marker and a blast resistance gene. RFLP analysis showed that Pi-2(t) is closely linked to a single-copy DNA clone RG64 on chromosome 6, with a distance of 2.8+1.4(SE) cMorgans. Another blast resistance gene, Pi-4(t), is 15.3+4.2(SE) cMorgans away from a DNA clone RG869 on chromosome 12. These chromosomal regions can now be examined with additional markers to define the precise locations of Pi-2(t) and Pi-4(t). Tightly linked DNA markers may facilitate early selection for blast resistance genes in breeding programs. These markers may also be useful to map new genes for resistance to blast isolates. They may ultimately lead to the cloning of those genes via chromosome walking. The gene tagging approach demonstrated in this paper may apply to other genes of interest for both monogenic and polygenic traits.     

Tagging and combining bacterial blight resistance genes in rice using RAPD and RFLP markers

Four genes of rice,Oryza sativa L., conditioning resistance to the bacterial blight pathogenXanthomonas oryzae pv.oryzae (X. o. pv.oryzae), were tagged by restriction fragment length polymorphism (RFLP) and random amplified polymorphic DNA (RAPD) markers. No recombinants were observed betweenxa-5 and RFLP marker lociRZ390, RG556 orRG207 on chromosome 5.Xa-3 andXa-4 were linked to RFLP locusXNpb181 at the top of chromosome 11, at distances of 2.3 cM and 1.7 cM, respectively. The nearest marker toXa-10, also located on chromosome 11, was the RAPD locusO07 ₂₀₀₀ at a distance of 5.3 cM. From this study, the conventional map [19, 28] and two RFLP linkage maps of chromosome 11 [14, 26] were partially integrated. Using the RFLP and RAPD markers linked to the resistance genes, we selected rice lines homozygous for pairs of resistance genes,Xa-4 +xa-5 andXa-4 +Xa-10. Lines carryingXa-4 +xa-5 andXa-4 +Xa-10 were evaluated for reaction to eight strains of the bacterial blight pathogen, representing eight pathotypes and three genetic lineages. As expected, the lines carrying pairs of genes were resistant to more of the isolates than their single-gene parental lines. Lines carryingXa-4 +xa-5 were more resistant to isolates of race 4 than were either of the parental lines (quantitative complementation). No such effects were seen forXa-4 +Xa-10. Thus, combinations of resistance genes provide broader spectra of resistance through both ordinary gene action expected and quantitative complementation.     

High-resolution genetic mapping of bacterial blight resistance gene <Emphasis Type="Italic">Xa10</Emphasis>

Bacterial blight of rice, caused by Xanthomonas oryzae pv. oryzae (Xoo), is the most devastating disease of rice (Oryza sativa L). Rice lines that carry resistance (R) gene Xa10 confer race-specific resistance to Xoo strains harboring avirulence (Avr) gene avrXa10. Here we report on genetic study, disease evaluation and fine genetic mapping of the Xa10 gene. The inheritance of Xa10-mediated resistance to PXO99^A(pHM1avrXa10) did not follow typical Mendelian inheritance for single dominant gene in F₂ population derived from IR24 × IRBB10. A locus might be present in IRBB10 that caused distorted segregation in F₂ population. To eliminate this locus, an F₃ population (F₃-65) was identified, which showed normal Mendelian segregation ratio of 3:1 for resistance and susceptibility. A new near-isogenic line (F₃-65-1743) of Xa10 in IR24 genetic background was developed and designated as IRBB10A. IRBB10A retained similar resistance specificity as that of IRBB10 and provided complete resistance to PXO99^A(pHM1avrXa10) from seedling to adult stages. Linkage analysis using existing RFLP markers and F₂ mapping population mapped the Xa10 locus to the proximal side of E1981S with genetic distance at 0.93 cM. With five new RFLP markers developed from the genomic sequence of Nipponbare, Xa10 was finely mapped at genetic distance of 0.28 cM between proximal marker M491 and distal marker M419 and co-segregated with markers S723 and M604. The physical distance between M491 and M419 on Nipponbare genome is 74 kb. Seven genes have been annotated from this 74-kb region and six of them are possible Xa10 candidates. The results of this study will be useful in Xa10 cloning and marker-assisted breeding.     

Marker-assisted improvement of bacterial blight resistance in parental lines of Pusa RH10, a superfine grain aromatic rice hybrid

Pusa RH10, the widely cultivated superfine grain aromatic rice hybrid, and its parental lines Pusa6B and PRR78 are susceptible to bacterial blight (BB) disease caused by Xanthomonas oryzae pv. oryzae. Pusa1460, a Basmati rice variety, was utilized as the donor for introgressing BB resistance genes xa13 and Xa21 into Pusa6B and PRR78 using a marker-assisted backcross breeding program. The markers RG136 and pTA248 linked to BB resistance genes xa13 and Xa21, respectively, were used for foreground selection. Seventy-four STMS markers polymorphic between Pusa6B and Pusa1460, and 54 STMS markers polymorphic between PRR78 and Pusa1460, were utilized for background selection to recover the recurrent parent genome ranging from 85.14 to 97.30% and 87.04 to 92.81% in the 10 best BC₂F₅ families of Pusa6B and PRR78, respectively. RM6100, an STMS marker linked to fertility restorer gene (Rf), was used for marker-assisted selection of Rf gene in an improved version of PRR78. The extent of donor segments in the improved version of Pusa6B was estimated to be <0.97 and <2.15 Mb in the genomic regions flanking xa13 and Xa21, respectively, whereas in improved PRR78, it was estimated to be <2.07 and <3.45 Mb in the corresponding genomic regions. Improved lines of Pusa6B and PRR78 showed yield advantages of up to 8.24 and 5.23%, respectively. The performance of the BB-resistant version of Pusa RH10 produced by intercrossing the improved parental lines was on a par with or superior to the original Pusa RH10.     

Microsatellite and sequence-tagged site markers diagnostic for the rice bacterial leaf blight resistance gene xa-5

 Microsatellite and sequence-tagged site (STS) markers tightly linked to the bacterial leaf blight (BLB) resistance gene xa-5 were identified in this study. A survey was conducted to find molecular markers that detected polymorphisms between the resistant (IRBB5) and susceptible (‘IR24’) nearly isogenic lines for xa-5, and between Chinsurah Boro II (CBII), an alternative source of xa-5, and a widely planted variety (‘IR64’) that lacks xa-5. Two F₂ populations, from the crosses ‘IR24’×IRBB5 and CBIIבIR64’, were used to estimate linkage based on marker genotype and reaction to disease inoculation with Xanthomonas oryzae pv. oryzae. Two RFLP clones, RZ390 and RG556, were found to co-segregate with xa-5 and were converted into STS markers. A microsatellite marker, RM390, was developed based on a simple sequence repeat in the 5′ untranslated region of the cDNA probe, RZ390, and found to co-segregate with resistance. Two other microsatellites, RM122 and RM13, were located 0.4 cM and 14.1 cM away from xa-5. A germplasm survey of diverse lines containing BLB resistance genes using automated fluorescent detection indicated the range of allelic diversity for each of the microsatellite loci linked to xa-5 and confirmed their usefulness in following genes through the narrow crosses typical of a breeding program. The limited number of alleles observed at the microsatellite loci linked to the resistance gene in 35 xa-5-containing accessions suggested either a single ancestral origin or a few independent origins of the xa-5 gene. PCR-based markers, like the ones developed in this study, are economical and easy to use, and have applicability in efforts to pyramid the recessive xa-5 gene with other BLB resistance genes. Received: 27 September 1996/Accepted: 7 February 1997     

Molecular mapping of gene Gm-6(t) which confers resistance against four biotypes of Asian rice gall midge in China

The Chinese rice cultivar Duokang #1 carries a single dominant gene Gm-6(t) that confers resistance to the four biotypes of Asian rice gall midge (Orseolia oryzae Wood-Mason) known in China. Bulked segregant analysis was performed on progeny of a cross between Duokang #1 and the gall midge-susceptible cultivar Feng Yin Zhan using the RAPD method. The RAPD marker OPM06₍₁₄₀₀₎ amplified a locus linked to Gm-6(t). The locus was subsequently mapped to rice chromosome 4 in a region flanked by cloned RFLP markers RG214 and RG163. Fine mapping of Gm-6(t) revealed that markers RG214 and RG476 flanked the gene at distances of 1.0 and 2.3 cM, respectively. Another gall midge resistance gene, Gm-2, mapped previously to chromosome 4, is located about 16 cM from Gm-6(t), to judge by data from a segregating population derived from a cross between Duokang #1 and the Indian cultivar Phalguna that carries Gm-2. We developed a PCR-based marker-assisted selection kit for transfer of the Gm-6(t) gene into Ming Hui 63 and IR50404, two parental lines commonly used in hybrid rice production in China. The kit contains PCR primer pairs based on the terminal sequences of the RG214 and RG476 clones. Polymorphism between Duokang #1 and the hybrid parental lines was found at these markers after digestion of the PCR products with specific restriction endonucleases. The kit will accelerate introduction of gall midge resistance into hybrid rice in China. Received: 18 May 2000 / Accepted: 9 March 2001     

Marker assisted selection of bacterial blight resistance genes in rice

Bacterial leaf blight caused by Xanthomonas oryzae pv. oryzae is one of the most important diseases affecting rice production in Asia. We were interested in surveying rice genotypes that are popularly used in the Indian breeding program for conferring resistance to bacterial blight, using 11 STMS and 6 STS markers. The basis of selection of these DNA markers was their close linkage to xa5, xa13, and Xa21 genes and their positions on the rice genetic map relative to bacterial blight resistance genes. Eight lines were found to contain the xa5 gene while two lines contained Xa21 gene and none of the lines contained the xa13 gene with the exception of its near-isogenic line. Using the polymorphic markers obtained in the initial survey, marker-assisted selection was performed in the F3 population of a cross between IR-64 and IET-14444 to detect lines containing multiple resistance genes. Of the 59 progeny lines analyzed, eight lines contained both the resistance genes, xa5 and Xa4.     

Low sequence similarity and gene content of symbiotic clusters of Bradyrhizobium sp. WM9 (Lupinus) indicate early divergence of “lupin” lineage in the genus Bradyrhizobium

Two sequenced nodulation regions of lupin Bradyrhizobium sp. WM9 carried the majority of genes involved in the Nod factor production. The nod region I harbored: nolA, nodD, nodA, nodB, nodC, nodS, nodI, nodJ, nolO, nodZ, fixR, nifA, fixA, nodM, nolK and noeL. This gene arrangement resembled that found in the nodulation region of Bradyrhizobium japonicum USDA110, however strain WM9 harbored only one nodD gene copy, while the nodM, nolK and noeL genes had no counterparts in the 410 kb symbiotic region of strain USDA110. Region II harbored nolL and nodW, but lacked an nodV gene. Both regions carried ORFs that lacked similarity to the published USDA110 sequences, though they had homologues in symbiotic regions of Rhizobium etli, Sinorhizobium sp. NGR234 and Mesorhizobium loti. These differences in gene content, as well as a low average sequence identity (70%) of symbiotic genes with respect to B. japonicum USDA110 were in contrast with the phylogenetic relationship of USDA110 and WM9 revealed by the analysis of 16S rDNA and dnaK sequences. This most likely reflected an early divergence of symbiotic loci, and possible co-speciation with distinct legumes. During this process the loss of a noeI gene and the acquisition of a nolL gene could be regarded as an adaptation towards these legumes that responded to Nod factors carrying 4-O-acetylfucose rather than 2-O-methylfucose. This explained various responses of lupins and serradella plants to infection by mutants in nodZ and nolL genes, knowing that serradella is a stringent legume while lupins are more promiscuous legumes. This revised version was published online in August 2006 with corrections to the Cover Date.     

Molecular breeding: marker-assisted selection combined with biolistic transformation for blast and bacterial blight resistance in Indica rice (cv. CO39)

Based on blast pathogen population dynamics and lineage exclusion assays, we found that the major blast resistance genes Pi-1 and Piz-5 confer resistance against most Magnaporthe grisea lineages. Near-isogenic rice lines C101LAC and C101A51 carrying these two major genes for blast resistance in the background of a most blast-susceptible genotype were used for developing the pyramids. The closely linked RFLP marker RZ536 and NBS-LRR r10 marker for Pi-1 and a PCR-based SAP marker RG64 for Piz-5 were used to identify the genes in the parents and in marker-assisted breeding of the pyramided populations. To achieve multiple resistance against blast and blight in this cultivar, these blast-resistant pyramids were transformed with the cloned bacterial blight resistance gene Xa21 known to confer resistance to all races of Xanthomonas oryzae pv. oryzae (Xoo). Bioassays with six independent transformants showed that transgenic CO39 plants were resistant to both pathogens, M. grisea and Xoo. We report here the stacking of three major genes (Pi-1 + Piz-5 + Xa21) into rice using two different approaches of molecular breeding: marker-assisted selection (MAS) and genetic transformation.