国外引进水稻种质资源的稻瘟病抗性基因检测与评价

为了筛选出福建省水稻稻瘟病重发区育种中可利用的新抗性资源,在福建省上杭县对156份外引水稻种质资源进行了2年田间自然诱发鉴定,并对Pi2、Pi9、Pi5、Pi54、Pikm、Pita、Pia和Pib等8个稻瘟病抗性基因做了分子检测。结果表明:156份资源对苗瘟、叶瘟、穗颈瘟和综合抗性表现抗病的分别有10份、14份、29份和26份,且苗瘟抗性级别与叶瘟抗性级别(r=0.816,P<0.01)、苗瘟抗性级别与穗颈瘟抗性级别(r=0.347,P<0.01)、以及叶瘟抗性级别与穗颈瘟抗性级别(r=0.344,P<0.01),均呈极显著正相关。分子标记检测到携带稻瘟病抗性基因Pi9、Pi2、Pi54、Pikm、Pi5、Pib、Pia和Pita的水稻资源分别有1、6、20、22、37、88、101和106份,其中携带稻瘟病抗性基因Pi9和Pi2的水稻资源的抗性表现较好,表现抗病的超过60%,携带其他稻瘟病抗性基因的水稻资源表现抗病的均在50%以下;水稻资源携带0~6个稻瘟病抗性基因,随着携带抗性基因数目增加,抗病率呈上升趋势,综合抗性等级呈下降趋势。进一步研究发现,携带Pi9+Pi5+Pikm+Pia、Pi5+Pib+Pita+Pikm+Pia和Pi2+Pi54+Pib+Pita+Pikm+Pia等3个基因型的水稻资源,稻瘟病抗性较好。最后,筛选了8份稻瘟病抗性较好的材料,提供育种者参考、利用。     

黄淮区粳稻抗稻瘟病基因Pi-ta、Pi-b、Pi54、Pikm的分子检测

利用水稻稻瘟病抗病基因Pi-ta、Pi-b、Pi54 和Pikm的功能标记对2016年山东省水稻中晚熟组区试、机插秧组区试的32个参试品系及连云港农业科学院科企水稻联合体黄淮粳稻区试16个品系进行了分子标记检测,结合稻瘟病抗性接种鉴定,对基因型与表型进行相关性分析。结果表明48个品种中携带Pi-ta、Pi-b、Pi54和Pikm抗性基因的品种数分别为15个、25个、26个和21个,其中鲁资稻7号、连粳14JD24含有4个基因的抗性等位基因,YS-6-6、济稻1号、D400等13个品种分别含有3个基因的抗性等位基因,临稻10号、丰稻2号、天和糯303等8个品种不含抗性等位基因。稻瘟病鉴定结果表明,48份品种中,济稻1号、圣稻072、连粳14JD24等4个品种表现中抗（MR）;临稻10、YS-6-6、圣稻053等26个品种表现中感（MS）;晶稻180、临13-105、圣稻504等15个品种表现感病（S）;H11-15、润农9号、晶稻160表现高感（HS）。Pi-ta、Pi-b、Pi54、Pikm 4个抗性基因已在黄淮区粳稻抗稻瘟病育种中得到广泛应用。其中Pikm与稻瘟病抗性综合指数存在显著相关性（r=0.477 5,P<0.01）。     

Development of pre-isogenic lines for rice blast-resistance by marker-aided selection from a recombinant inbred population

To increase the available set of near-isogenic lines (NILs) for blast-resistance in rice, we have developed a general method for establishing NILs from populations of fixed recombinants that have been used for gene mapping. We demonstrated the application of this method by the selection of lines carrying genes from the rice cultivar Moroberekan. Moroberekan is a West African japonica cultivar that is considered to have durable resistance to rice blast. Multiple genes from Moroberekan conferring complete and partial resistance to blast have previously been mapped using a recombinant inbred (RI) population derived from a cross between Moroberekan and the highly and broadly susceptible indica cultivar CO39. To analyze individual blast-resistance genes, it is desirable to transfer them individually into a susceptible genetic background. This RI population, and the associated data sets on blast reaction and restriction fragment length polymorphism (RFLP) genotypes, were used for selection of lines likely to carry individual blast-resistance genes and a minimum number of chromosomal segments from Moroberekan. Because skewed segregation in the RI population favored CO39 (indica) alleles, resistant lines carrying 8.7–17.5% of Moroberekan alleles (the proportion expected after two or three backcrosses) could be selected. We chose three RI lines carrying different complete resistance genes to blast and two RI lines carrying partial resistance genes to blast as potential parents for the development of NILs. These lines were subjected to genetic analysis, which allowed clarification of some issues that could not be resolved during the initial gene-mapping study.     

Genetic characterization and fine mapping of the blast resistance locus Pigm(t) tightly linked to Pi2 and Pi9 in a broad-spectrum resistant Chinese variety

The identification and utilization of broad-spectrum resistance genes have been proven the most effective and economical approach to control rice blast disease. To understand the molecular mechanism of broad-spectrum resistance to rice blast, we conducted genetic and fine mapping analysis of the blast resistance gene in a Chinese rice variety: Gumei 4 (GM4) identified with broad-spectrum resistance and used in rice breeding for blast resistance for more than 20 years. Genetic and mapping analysis indicated that blast resistance to nine isolates of different Chinese races in GM4 was controlled by the same dominant locus designated as Pigm(t) that was finely mapped to an approximately 70-kb interval between markers C5483 and C0428 on chromosome 6, which contains five candidate NBS--LRR disease resistance genes. The allelism test showed that Pigm(t) was either tightly linked or allelic to Pi2 and Pi9, two known blast resistance genes. Mapping information also indicated that another blast resistance gene Pi26(t) might also be located at the same region. Candidate genes were identified by sequence analysis of the Nipponbare and Pi9 locus and the corresponding region in GM4. Sequence divergence of candidate genes was observed between GM4 and model varieties Nipponbare and 9311, and Pi9. Our current study provides essential information and new genetic resource for the cloning of functional resistance gene(s) and for marker-assisted selection in rice breeding for broad-spectrum blast resistance.Yiwen Deng and Xudong Zhu contributed equally to this work.     

The in silico map-based cloning of Pi36, a rice coiled-coil nucleotide-binding site leucine-rich repeat gene that confers race-specific resistance to the blast fungus

The indica rice variety Kasalath carries Pi36, a gene that determines resistance to Chinese isolates of rice blast and that has been located to a 17-kb interval on chromosome 8. The genomic sequence of the reference japonica variety Nipponbare was used for an in silico prediction of the resistance (R) gene content of the interval and hence for the identification of candidate gene(s) for Pi36. Three such sequences, which all had both a nucleotide-binding site and a leucine-rich repeat motif, were present. The three candidate genes were amplified from the genomic DNA of a number of varieties by long-range PCR, and the resulting amplicons were inserted into pCAMBIA1300 and/or pYLTAC27 vectors to determine sequence polymorphisms correlated to the resistance phenotype and to perform transgenic complementation tests. Constructs containing each candidate gene were transformed into the blast-susceptible variety Q1063, which allowed the identification of Pi36-3 as the functional gene, with the other two candidates being probable pseudogenes. The Pi36-encoded protein is composed of 1056 amino acids, with a single substitution event (Asp to Ser) at residue 590 associated with the resistant phenotype. Pi36 is a single-copy gene in rice and is more closely related to the barley powdery mildew resistance genes Mla1 and Mla6 than to the rice blast R genes Pita, Pib, Pi9, and Piz-t. An RT-PCR analysis showed that Pi36 is constitutively expressed in Kasalath.     

Two broad-spectrum blast resistance genes,<Emphasis Type="Italic"> Pi9</Emphasis>(<Emphasis Type="Italic">t</Emphasis>) and<Emphasis Type="Italic"> Pi2</Emphasis>(<Emphasis Type="Italic">t</Emphasis>), are physically linked on rice chromosome 6

To understand the molecular basis of broad-spectrum resistance to rice blast, fine-scale mapping of the two blast resistance (R) genes, Pi9( t) and Pi2( t), was conducted. These two genes were introgressed from different resistance donors, previously reported to confer resistance to many blast isolates in the Philippines, and were mapped to an approximately 10-cM interval on chromosome 6. To further test their resistance spectrum, 43 blast isolates collected from 13 countries were used to inoculate the Pi2( t) and Pi9( t) plants. Pi9( t)-bearing lines were highly resistant to all isolates tested, and lines carrying Pi2( t) were resistant to 36 isolates, confirming the broad-spectrum resistance of these two genes to diverse blast isolates. Three RAPD markers tightly linked to Pi9( t) were identified using the bulk segregant analysis technique. Twelve positive bacterial artificial chromosome (BAC) clones were identified and a BAC contig covering about 100 kb was constructed when the Pi9( t) BAC library was screened with one of the markers. A high-resolution map of Pi9( t) was constructed using BAC ends. The Pi2( t) gene was tightly linked to all of the Pi9( t) markers in 450 F(2) plants. These data suggest that Pi9( t) and Pi2( t) are either allelic or tightly linked in an approximately 100-kb region. The mapping results for Pi9( t) and Pi2( t) provide essential information for the positional cloning of these two important blast resistance genes in rice.     

A yeast DNA repair gene partially complements defective excision repair in mammalian cells.

The RAD10 gene of Saccharomyces cerevisiae is required for nucleotide excision repair of DNA. Expression of RAD10 mRNA and Rad10 protein was demonstrated in Chinese hamster ovary (CHO) cells containing amplified copies of the gene, and RAD10 mRNA was also detected in stable transfectants without gene amplification. Following transfection with the RAD10 gene, three independently isolated excision repair-defective CHO cell lines from the same genetic complementation group (complementation group 2) showed partial complementation of sensitivity to killing by UV radiation and to the DNA cross-linking agent mitomycin C. These results were not observed when RAD10 was introduced into excision repair-defective CHO cell lines from other genetic complementation groups, nor when the yeast RAD3 gene was expressed in cells from genetic complementation group 2. Enhanced UV resistance in cells carrying the RAD10 gene was accompanied by partial reactivation of the plasmid-borne chloramphenicol acetyltransferase (cat) gene following its inactivation by UV radiation. The phenotype of CHO cells from genetic complementation group 2 is also specifically complemented by the human ERCC1 gene, and the ERCC1 and RAD10 genes have similar amino acid sequences. The present experiments therefore indicate that the structural homology between the yeast Rad10 and human Ercc1 polypeptides is reflected at a functional level, and suggest that nucleotide excision repair proteins are conserved in eukaryotes.     

Different Mechanisms of Four Aluminum (Al)-Resistant Transgenes for Al Toxicity in Arabidopsis

We have characterized the mechanism of action of four transgenes (AtBCB [Arabidopsis blue copper-binding protein], parB [tobacco (Nicotiana tabacum) glutathione S-transferase], NtPox [tobacco peroxidase], and NtGDI1 [tobacco GDP dissociation inhibitor]) that independently Al resistance on transgenic Arabidopsis. All four transgenic lines showed lower deposition of callose after Al treatment than the Landsberg erecta ecotype of Arabidopsis, confirming that the four genes function to ameliorate Al toxicity. Influx and efflux experiments of Al ions suggested that the AtBCB gene may suppress Al absorption, whereas expression of the NtGDI1 gene promotes a release of Al in the root tip region of Arabidopsis. The total enzyme activities of glutathione S-transferases or peroxidases in transgenic lines carrying either the parB or NtPox genes were significantly higher than in the Landsberg erecta ecotype of Arabidopsis, and these enzyme activities were maintained at higher levels during Al stress. Furthermore, lipid peroxidation caused by Al stress was repressed in these two transgenic lines, suggesting that overexpression of these two genes diminishes oxidative damage caused by Al stress. Al-treated roots of transgenic plants were also stained by 4',6-diamino-2-phenylindole to monitor cell death caused by Al toxicity. The result suggested that cell death is repressed in the NtPox line. Analysis of F(1) hybrids between the four transgenic lines suggests that more resistant transgenic plants can be constructed by combinations of these four genes.     

Dm3 is one member of a large constitutively expressed family of nucleotide binding site-leucine-rich repeat encoding genes

The major cluster of resistance genes in lettuce cv. Diana contains approximately 32 nucleotide binding site-leucine-rich repeat encoding genes. Previous molecular dissection of this complex region had identified a large gene, RGC2B, as a candidate for encoding the downy mildew resistance gene, Dm3. This article describes genetic and transgenic complementation data that demonstrated RGC2B is necessary and sufficient to confer resistance with Dm3 specificity. Ethylmethanesulphonate was used to induce mutations to downy mildew susceptibility in cv. Diana (Dm1, Dm3, Dm7, and Dm8). Nineteen families were identified with a complete loss of resistance in one of the four resistance specificities. Sequencing revealed a variety of point mutations in RGC2B in the six dm3 mutants. Losses of resistance were due to single changes in amino acid sequence or a change in an intron splice site. These mutations did not cluster in any particular region of RGC2B. A full-length genomic copy of RGC2B was isolated from a lambdaphage library and introduced into two genotypes of lettuce. Transgenics expressing RGC2B exhibited resistance to all isolates expressing Avr3 from a wide range of geographical origins. In a wildtype Dm3-expressing genotype, many of the RGC2 family members are expressed at low levels throughout the plant.     

Contrasting modes of evolution acting on the complex N locus for rust resistance in flax

Three rust resistance specificities, N, N1 and N2, map to the complex N locus of flax. We used a degenerate PCR approach, with primers directed to the nucleotide binding site (NBS) domain characteristic of many plant resistance genes, to isolate resistance gene analogs (RGAs) from flax. One RGA clone detected RFLPs co-segregating with alleles of the N locus. With this probe we isolated four related genes that occur within a 30kbp region and encode proteins with NBS and leucine-rich repeat (LRR) domains and N-terminal Toll/Interleukin-1 Receptor homology (TIR) domains. One of these four genes was identified as the N resistance gene by sequence analysis of three mutant alleles and by transgenic expression. We isolated homologous genes from two flax lines containing the N1 or N2 specificities and from flax lines carrying no N locus resistance specificities. Analysis of shared polymorphisms among this set of 18 N locus sequences revealed three groups of genes with independent lineages. Sequence exchanges have only occurred between genes within each group, but not between groups. Two of the groups contain only one sequence from each haplotype and probably represent orthologous genes. However, the third group contains two genes from each haplotype. We suggest that the re-assortment of variation by recombination/gene conversion at this locus is limited by the degree of sequence identity between genes.     

Complexities of chromosome landing in a highly duplicated genome: toward map-based cloning of a gene controlling blackleg resistance in Brassica napus

The LmR1 locus, which controls seedling resistance to the blackleg fungus Leptosphaeria maculans in the Brassica napus cultivar Shiralee, was positioned on linkage group N7. Fine genetic mapping in a population of 2500 backcross lines identified three molecular markers that cosegregated with LmR1. Additional linkage mapping in a second population colocalized a seedling resistance gene, ClmR1, from the cultivar Cresor to the same genetic interval on N7 as LmR1. Both genes were located in a region that showed extensive inter- and intragenomic duplications as well as intrachromosomal tandem duplications. The tandem duplications seem to have occurred in the Brassica lineage before the divergence of B. rapa and B. oleracea but after the separation of Brassica and Arabidopsis from a common ancestor. Microsynteny was found between the region on N7 carrying the resistance gene and the end of Arabidopsis chromosome 1, interrupted by a single inversion close to the resistance locus. The collinear region in Arabidopsis was assayed for the presence of possible candidate genes for blackleg resistance. These data provided novel insights into the genomic structure and evolution of plant resistance loci and an evaluation of the candidate gene approach using comparative mapping with a model organism.     

<Emphasis Type="Italic">CaMi</Emphasis>, a root-knot nematode resistance gene from hot pepper (<Emphasis Type="Italic">Capsium annuum</Emphasis> L.) confers nematode resistance in tomato

Several root-knot nematode (Meloidogyne spp.) resistance genes have been discovered in different pepper (Capsium annuum L.) lines; however, none of them has yet been cloned. In this study, a candidate root-knot nematode resistance gene (designated as CaMi) was isolated from the resistant pepper line PR 205 by degenerate PCR amplification combined with the RACE technique. Expression profiling analysis revealed that this gene was highly expressed in roots, leaves, and flowers and expressed at a lower level in stems and was not detectable in fruits. To verify the function of CaMi, a sense vector containing the genomic DNA spanning the full coding region of CaMi was constructed and transferred into root-knot nematode susceptible tomato plants. Sixteen transgenic plants carrying one to five copies of T-DNA inserts were generated from two nematode susceptible tomato cultivars. RT-PCR analysis revealed that the expression levels of CaMi gene varied in different transgenic plants. Nematode assays showed that the resistance to root-knot nematodes was significantly improved in some transgenic lines compared to untransformed susceptible plants, and that the resistance was inheritable. Ultrastructure analysis showed that nematodes led to the formation of galls or root knots in the susceptible lines while in the resistant transgenic plants, the CaMi gene triggered a hypersensitive response (HR) as well as many necrotic cells around nematodes. Rugang Chen and Hanxia Li are contributed equally to this work.     

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.     

A Genetic and Molecular Analysis of the 46c Chromosomal Region Surrounding the Fmrfamide Neuropeptide Gene in Drosophila Melanogaster

We have analyzed the FMRFamide neuropeptide gene region of Drosophila melanogaster. This gene maps to the 46C region of chromosome 2R; this interval previously was not well characterized. For this genetic and molecular analysis, we have used X-ray mutagenesis, EMS mutagenesis, and the recently reported local P element transposition method. We identified four overlapping deletions, two of which have proximal breakpoints that define a 50-60-kb region surrounding the FMRFamide gene in 46C. To this small region, we mapped three lethal complementation groups; 10 additional lethal complementation groups were mapped to more distal regions of 46CD. One of these groups corresponds to even-skipped, the other 12 are previously unidentified. Using various lines of evidence we excluded the possibility that FMRFamide corresponds to any of the three lethal complementation groups mapping to its immediate 50-60-kb vicinity. The positions of two of the three lethal complementation groups were identified with P elements using a local transposition scheme. The third lethal complementation group was excluded as being FMRFamide mutants by sequence analysis and by immunocytochemistry with proFMRFamide precursor-specific antibodies. This analysis has (1) provided a genetic map of the 46CD chromosomal region and a detailed molecular map of a portion of the 46C region and (2) provided additional evidence of the utility of local transposition for targeting nearby genes.     

Cyanide, a coproduct of plant hormone ethylene biosynthesis, contributes to the resistance of rice to blast fungus

Rice (Oryza sativa) plants carrying the Pi-i resistance gene to blast fungus Magnaporthe oryzae restrict invaded fungus in infected tissue via hypersensitive reaction or response (HR), which is accompanied by rapid ethylene production and formation of small HR lesions. Ethylene biosynthesis has been implicated to be important for blast resistance; however, the individual roles of ethylene and cyanide, which are produced from the precursor 1-aminocyclopropane-1-carboxylic acid, remain unevaluated. In this study, we found that Pi-i-mediated resistance was compromised in transgenic rice lines, in which ethylene biosynthetic enzyme genes were silenced and then ethylene production was inhibited. The compromised resistance in transgenic lines was recovered by exogenously applying cyanide but not ethephon, an ethylene-releasing chemical in plant tissue. In a susceptible rice cultivar, treatment with cyanide or 1-aminocyclopropane-1-carboxylic acid induced the resistance to blast fungus in a dose-dependent manner, while ethephon did not have the effect. Cyanide inhibited the growth of blast fungus in vitro and in planta, and application of flavonoids, secondary metabolites that exist ubiquitously in the plant kingdom, enhanced the cyanide-induced inhibition of fungal growth. These results suggested that cyanide, whose production is triggered by HR in infected tissue, contributes to the resistance in rice plants via restriction of fungal growth.     

4个太湖流域粳稻地方品种抗稻瘟病性的遗传分析

以太湖流域粳稻地方品种薄稻、铁杆青、江南晚和缺儿糯等广谱、高抗稻瘟病为材料, 与高感稻瘟病品种苏御糯杂交, 获得杂交F1、F2 , 分别接种日本稻瘟病鉴别菌系北1和中国稻瘟病菌生理小种ZE3、ZG1, 根据P1、P2、F1和F2等不同世代植株的抗、感反应, 分析地方品种对不同稻瘟病菌生理小种(菌系)的抗性遗传机理。结果表明: 薄稻、铁杆青及缺儿糯对北1菌系的抗性均可能由一对显性基因控制, 江南晚对北1的抗性则可能由两对抑制基因互作控制; 铁杆青及缺儿糯对ZE3小种的抗性均可能由一对显性基因控制, 薄稻和江南晚对ZE3小种的抗性可能分别由两对显性基因和两对抑制基因互作控制; 铁杆青对ZG1小种的抗性可能是由一对显性主基因控制, 薄稻和江南晚对ZG1小种的抗性则可能由两对抑制基因互作控制。进一步将薄稻与12个日本稻瘟病菌鉴别品种杂交,用北1菌系接种不同组合的F1和F2 , 进行抗病基因的等位性测定。结果表明, 薄稻对北1菌系的抗性基因与12个鉴别品种所携带的已知抗稻瘟病基因是不等位, 将该基因暂定为Pi-bd1(t)。     

35S驱动Pib基因启动区和编码区的转基因初步分析

将包含Pib基因启动区及下游完整编码区的9.9 kb DNA片段克隆到双元载体pPZP2Ha3(+)中, 构建了35S驱动的正义表达载体pNAR701(20.3 kb); 同时将Pib基因编码区6 986~9 392 bp之间的DNA片段, 克隆到双元载体pPZP2Ha3(-)中, 构建了35S驱动的反义表达载体pNAR703(12.8 kb); 用农杆菌介导法转入中感稻瘟病水稻品种R109中。PCR、Southern blot鉴定以及转基因T0代种子的潮霉素抗性鉴定证明, 目的基因已经整合到R109基因组中, 并能在后代稳定遗传。Northern blot分析表明含有启动区及下游完整编码的Pib基因片段在35S驱动下能够在转基因后代中表达。对T1代苗期转基因植株和分蘖期离体叶片进行抗稻瘟病初步分析, 结果显示pNAR701转基因植株对稻瘟病生理小种ZD1和ZG1的抗性较对照增强, 而转反义片段的pNAR703转基因植株对稻瘟病的抗性较对照减弱。