稻瘟病抗病基因Pi15的精细定位

稻瘟病抗病基因Pi15曾被作者鉴定为与已知抗病基因Pii具有连锁关系,但是,Pii基因究竟位于染色体6还是9上存在争议.为了确定Pi15基因的染色体位置,利用分子标记在由15个抗病个体和141个感病个体组成的F2群体中,通过混合群体分离法(BSA)与隐性群体分析法(RCA)相结合的手段,对目标基因进行了连锁分析.首先,从染色体6和9分别选择10个微卫星标记进行了分析,结果表明,只有位于染色体9的RM316与目标基因连锁,重组率为(19.1±3.7)%.为了进一步确定这种连锁关系,从染色体9选择了4个序列标定位点(STS)标记进行分析,结果表明,只有G103与目标基因连锁,重组率为(5.7±2.1)%.为了获得与目标基因更加紧密连锁的分子标记,对目标基因进行了RAPD)分析.在筛选、分析了1 000个随机引物之后,从中获得了3个目标基因紧密连锁的分子标记BAPi15486、BAPi15782、BAPi15844.它们与目标基因的重组率分别为0.35%、0.35%和1.1%.这些紧密连锁的分子标记可作为分子标记辅助基因聚合和克隆的出发点.     

稻瘟病抗病基因Pi15的精细定位

稻瘟病抗病基因Pi15曾被作者鉴定为与已知抗病基因Pii具有连锁关系,但是,Pii基因究竟位于染色体6还是9上存在争议。为了确定Pi15基因的染色体位置,利用分子标记在由15个抗病个体和141个感病个体组成的F_2群体中,通过混合群体分离法(BSA)与隐性群体分析法(RCA)相结合的手段,对目标基因进行了连锁分析。首先,从染色体6和9分别选择10个微卫星标记进行了分析,结果表明,只有位于染色体9的RM316与目标基因连锁,重组率为(19.1±3.7)％。为了进一步确定这种连锁关系,从染色体9选择了4个序列标定位点(STS)标记进行分析,结果表明,只有G103与目标基因连锁,重组率为(5.7±2.1)％。为了获得与目标基因更加紧密连锁的分子标记,对目标基囚进行了RAPD分析。在筛选、分析了1000个随机引物之后,从中获得了3个目标基因紧密连锁的分子标记BAPi15_(486)、BAPi15_(782)、BAPi15_(844)。它们与目标基因的重组率分别为0.35％、0.35％和1.1％。这些紧密连锁的分子标记可作为分子标记辅助基因聚合和克隆的出发点。     

Fine Mapping of the Friend Retrovirus Resistance Gene, Rfv3, on Mouse Chromosome 15

Rfv3 is a host resistance gene that operates through an unknown mechanism to control the development of the virus-neutralizing antibody response required for recovery from infection with Friend retrovirus. The Rfv3 gene was previously mapped to an approximately 20-centimorgan (cM) region of chromosome 15. More refined mapping was not possible, due to a lack of microsatellite markers and leakiness in the Rfv3 phenotype, which prevented definitive phenotyping of individual recombinant mice. In the present study, we overcame these difficulties by taking advantage of seven new microsatellite markers in the Rfv3 region and by using progeny tests to accurately determine the Rfv3 phenotype of recombinant mice. Detailed linkage analysis of relevant crossovers narrowed the location of Rfv3 to a 0.83-cM region. Mapping of closely linked genes in an interspecific backcross panel allowed us to exclude two previous candidate genes, Ly6 and Wnt7b. These studies also showed for the first time that the Hsf1 gene maps to the Rfv3-linked cluster of genes including Il2rb, Il3rb, and Pdgfb. This localization of Rfv3 to a region of less than 1 cM now makes it feasible to attempt the cloning of Rfv3 by physical methods.     

Characterization of Rice Blast Isolates by the Differential System and their Application for Mapping a Resistance Gene,Pi19(t)

To facilitate resistance gene characterization in the present study, the pathogenicities of newly collected blast isolates from rice fields in the Philippines were characterized using international blast differential varieties consisting of 31 monogenic lines that target 24 resistance genes. To classify and designate the blast isolates, we used a new international blast designation system, which has been proposed as a suitable naming system for comparing blast races among different studies. A total of 23 rice blast isolates collected from the Philippines were classified into 16 pathotypes, which showed reaction patterns different from those seen in the standard isolates. Among the blast pathotypes, 11 had differentiating ability for four Pik alleles (Pik, Pik‐m, Pik‐h, and Pik‐p) and Pi1, whereas the standard blast isolates from the Philippines were not able to differentiate these genes. In addition, several blast isolates were avirulent to IRBLt‐K59, IRBL19‐A, and Lijiangxintuanheigu, although the standard differential blast isolates were virulent to these lines. Moreover, two blast isolates were virulent to a monogenic line, IRBL9‐W, which harbours Pi9 and was resistant to all standard differential blast isolates. By using the isolates avirulent to IRBL19‐A, Pi19(t) was successfully mapped in the centromeric region on chromosome 12 with simple sequence repeat markers RM27937 and RM1337. These markers are useful for marker‐assisted Pi19(t) introgression worldwide.     

水稻品种‘沈农606’抗稻瘟病基因定位和连锁的SRAP片段分析

选用抗稻瘟病水稻品种‘沈农606’为抗病亲本与感病品种‘丽江新团黑谷’配制杂交组合.鉴定亲本、F_1正反交及其F_2群体的抗病性的结果表明,‘沈农606’的抗性受一对显性基因控制.采用相关序列扩增多态性(SRAP)和简单序列重复(SSR)标记,以及分离体分组混合分析法(BSA)将该基因定位于8号染色体上,其与SRAP标记m5e1-500的遗传距离为2.8 cM,与SSR标记RM25的遗传距离为9.8 cM,暂命名为Pi-SN606.m5e1-500序列位于8号染色体上,它能编码大于40个氨基酸的阅读框有2个,在NCBI网站上没有比对到同源性序列。     

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.     

Fine Mapping of RppP25, a Southern Rust Resistance Gene in Maize

Southern rust (Puccinia polysora Underw.) is a major disease that can cause severe yield losses in maize (Zea mays L.). In our previous study, a major gene RppP25 that confers resistance to southern rust was identified in inbred line P25. Here, we report the fine mapping and candidate gene analysis of RppP25 from the near-isogenic line F939, which harbors RppP25 in the genetic background of the susceptible inbred line F349. The inheritance of resistance to southern rust was investigated in the BC₁F₁ and BC₃F₁ populations, which were derived from a cross between F939 and F349 (as the recurrent parent). The 1:1 segregation ratio of resistance to susceptible plants in these two populations indicated that the resistance is controlled by a single dominant gene. Ten markers, including three simple sequence repeat (SSR) markers and seven insertion/deletion (InDel) markers, were developed in the RppP25 region. RppP25 was delimited to an interval between P091 and M271, with an estimated length of 40 kb based on the physical map of B73. In this region, a candidate gene was identified that was predicted to encode a putative nucleotide-binding site leucine-rich repeat (NBS-LRR) protein. Two co-segregated markers will aid in pyramiding diverse southern rust resistance alleles into elite materials, and thereby improve southern rust resistance worldwide.     

Molecular Mapping of Rice Blast Resistance Gene Pi-k h in the Rice Variety Tetep

We have used rice line Tetep as a resistant donor with the aim of mapping a durable blast resistance gene Pi-k ^h using RAPD and AFLP techniques in conjunction with bulk segregant analysis. An F₂ mapping population consisting of 205 plants was generated by crossing Tetep with HP2216, a highly susceptible cultivar. Inoculation with specific isolate (PLP-1) of Magnaporthe grisea at seeding stage showed that the Pi-k ^h gene inherited as a single dominant gene in F2 population. RAPD analysis was performed with 240 primers to detect polymorphism between resistant and susceptible parents. Of these, 48 primers produced polymorphic banding pattern between resistant and susceptible parents. Bulk segregant analysis was performed with 48 primers of which 5 showed polymorphism between resistant and susceptible bulks. A 700 bp DNA band was obtained in resistant F2 plants with primer 5-129 indicating its linkage to the resistance gene. Out of 64 AFLP primer combinations used for polymorphism survey between HP 2216 and Tetep, 11 AFLP primer combinations were able to distinguish the resistant and susceptible bulks. An AFLP band of 75 bp obtained with primer combination, E-TAlM-CTC co-segregated with the resistance gene. The RAPD marker 5-129₇₀₀ and AFLP₇₅ were placed on the linkage map at a distance of 2.1 eM and 15.1 eM flanking to Pi-k ^hgene, respectively. The RAPD band closely linked to Pi-k ^h gene was sequenced and used for the development of CAPs markers which also co-segregated with resistant phenotype in the mapping population. On sequence analysis and homology search of RAPD fragment with whole rice genome sequence database and the information available on physical, genetic and sequence maps of rice, the co-segregating CAPs marker was placed at long arm of rice chromosome 11. CAPs marker developed in this study showed polymorphism in different rice cultivars grown in North-Western Himalayan region and is being used for the pyramiding of Pi-k ^h gene along with other blast resistance genes using marker-assisted selection.     

Broad-spectrum Blast Resistance Gene Pi-k h Cloned from Rice Line Tetep Designated as Pi54

Blast disease caused by Magnaporthe oryzae is one of the important biotic stresses of rice. So far more than 85 blast resistance genes have been identified of these more than 14 have already been cloned. A broad spectrum rice blast resistance gene Pi-k ^h was cloned from the rice line Tetep. The gene was named Pi-k ^h based on the earlier reports on its genetic analysis in various rice lines. However, with the advances in molecular genetics and genomics of rice, the Pik locus has now been mapped more precisely. Since there are two reports on the mapping of Pi-k ^h gene from different rice lines, there is some confusion in the naming of this gene. In this report the name of Pi-k ^h gene cloned from the rice line Tetep has been designated as per the standard guidelines of Committee on Gene Symbolization, Nomenclature and Linkage (CGSNL) and its physical location on rice chromosome 11, which is ～2.5 Mbp away from the Pik locus mapped recently. Hence Pi-k ^h gene cloned from Tetep is now designated as Pi54.     

Development of Genetic Markers Linked to Straighthead Resistance through Fine Mapping in Rice (Oryza sativa L.)

Straighthead, a physiological disorder characterized by sterile florets and distorted spikelets, causes significant yield losses in rice, and occurs in many countries. The current control method of draining paddies early in the season stresses plants, is costly, and wastes water. Development of resistant cultivar is regarded as the most efficient way for its control. We mapped a QTL for straighthead resistance using two recombinant inbred line (RIL) F₉ populations that were phenotyped over two years using monosodium methanearsonate (MSMA) to induce the symptoms. One population of 170 RILs was genotyped with 136 SSRs and the other population of 91 RILs was genotyped with 159 SSRs. A major QTL qSH-8 was identified in an overlapping region in both populations, and explained 46% of total variation in one and 67% in another population for straighthead resistance. qSH-8 was fine mapped from 1.0 Mbp to 340 kb using 7 SSR markers and further mapped to 290 kb in a population between RM22573 and InDel 27 using 4 InDel markers. SSR AP3858-1 and InDel 11 were within the fine mapped region, and co-segregated with straighthead resistance in both RIL populations, as well as in a collection of diverse global accessions. These results demonstrate that AP3858-1 and InDel 11 can be used for marker-assisted selection (MAS) for straighthead resistant cultivars, which is especially important because there is no effective way to directly evaluate straighthead resistance.     

精细定位甜瓜白粉病抗性基因Pm-M

以抗白粉病甜瓜品种MR1与感白粉病新疆地方品种新密1号为亲本,构建BC1P2和F2群体,研究白粉病菌Px1B(P.xanthii race 1B)的抗性遗传规律.以BC1P2与F2群体为试验材料,利用BSA(Bulked segregation analysis)结合分子标记技术发掘多态性信息,并开发分子标记进行抗性基...     

Candidate Gene Identification with SNP Marker-Based Fine Mapping of Anthracnose Resistance Gene Co-4 in Common Bean

Anthracnose, caused by Colletotrichum lindemuthianum, is an important fungal disease of common bean (Phaseolus vulgaris). Alleles at the Co–4 locus confer resistance to a number of races of C. lindemuthianum. A population of 94 F_4:5 recombinant inbred lines of a cross between resistant black bean genotype B09197 and susceptible navy bean cultivar Nautica was used to identify markers associated with resistance in bean chromosome 8 (Pv08) where Co–4 is localized. Three SCAR markers with known linkage to Co–4 and a panel of single nucleotide markers were used for genotyping. A refined physical region on Pv08 with significant association with anthracnose resistance identified by markers was used in BLAST searches with the genomic sequence of common bean accession G19833. Thirty two unique annotated candidate genes were identified that spanned a physical region of 936.46 kb. A majority of the annotated genes identified had functional similarity to leucine rich repeats/receptor like kinase domains. Three annotated genes had similarity to 1, 3-β-glucanase domains. There were sequence similarities between some of the annotated genes found in the study and the genes associated with phosphoinositide-specific phosphilipases C associated with Co-x and the COK–4 loci found in previous studies. It is possible that the Co–4 locus is structured as a group of genes with functional domains dominated by protein tyrosine kinase along with leucine rich repeats/nucleotide binding site, phosphilipases C as well as β-glucanases.     

拟南芥AST基因的精细作图

拟南芥(Arabidopsis thaliana(L.)Heynh.)ast(anthocyanin spottedtesta)突变体是由碳离子辐射诱导产生的与花青苷生物合成有关的基因突变体,受单隐性核基因控制.根据拟南芥数据库中的SNPs(single nucleotide polv-mophisms)序列和插入/缺失多态性(insertion/deletion polymorphisms)序列,设计了一系列分子标记.采用图位克隆策略,应用这些分子标记完成了对拟南芥AST基因的精细作图,成功地将AST基因定位到BAC克隆T13M11上,初步认为该BAC克隆中的基因T13M11.8可能是AST基因.该基因的DNA序列长1432bp,含有6个外显子和5个内含子,编码的蛋白与花青苷生物合成途径中的二氢黄酮醇-4-还原酶有较高的同源性.将进一步通过功能互补实验验证图位克隆的结果.     

Fine Mapping of Gene Regions Regulating Neurodegeneration

Background

Damage to nerve cells and axons leading to neurodegeneration is a characteristic feature of many neurological diseases. The degree of genetic influence on susceptibility to axotomy-induced neuronal death has so far been unknown. We have examined two gene regions, Vra1 and Vra2, previously linked to nerve cell loss after ventral root avulsion in a rat F2 intercross between the DA and PVG inbred rat strains.

Methodology/Principal Findings

In this study, we use two generations (G8 and G10 cohorts) of an advanced intercross line between DA and PVG^av1 to reproduce linkage to Vra1 and to fine-map this region. By isolating the effect from Vra1 in congenic strains, we demonstrate that Vra1 significantly regulates the loss of motoneurons after avulsion. The regulatory effect mediated by Vra1 thus resides in a congenic fragment of 9 megabases. Furthermore, we have used the advanced intercross lines to give more support to Vra2, originally detected as a suggestive QTL.

Conclusions/Significance

The results demonstrated here show that naturally occurring allelic variations affect susceptibility to axotomy-induced nerve cell death. Vra1 and Vra2 represent the first quantitative trait loci regulating this phenotype that are characterized and fine mapped in an advanced intercross line. In addition, congenic strains provide experimental evidence for the Vra1 effect on the extent of injury-induced neurodegeneration. Identification of the underlying genetic variations will increase our understanding of the regulation and mechanisms of neurodegeneration.     

拟南芥AST基因的精细作图

拟南芥（Arabidopsis thaliana(L.)Heynh)ast(anthocyanin spotted testa)突变体是由碳离子辐射诱导产生的与花青苷生物合成有关的基因突变体。受单隐性核基因控制。根据拟南芥数据库中的SNPs(single nucleotide poly-mophisms)序列和插入/缺失多态性（insertion/deletion polymorphisms)序列,设计了一系列分子标记,采用图位克隆策略。应用这些分子标记完成了对拟南芥AST基因的精细作图,成功地将AST基因定位到BAC克隆T13M11上,初步认为该BAC克隆中的基因T13M11.8可能是AST基因。该基因的DNA序列长1432bp。含有6个外显子和5个内含子,编码的蛋白与花青苷生物合成途径中的二氢黄酮醇-4-还原酶有较高的同源性。将进一步通过功能互补实验验证图位克隆的结果。     

Cloning and in silico Mapping of Resistance Gene Analogues Isolated from Rice Lines Containing Known Genes for Blast Resistance

We amplified resistance gene analogues (RGAs) from the genomic DNA of 10 rice lines having varying degree of resistance to Magnaporthe grisea by using degenerate primers and various RGAs were mapped in silico on different rice chromosomes. The amplified products were grouped into 3–8 restriction fragment length polymorphic classes by using Mbo1 and Alu1 restriction enzymes. Of 98 RGAs obtained in this study, 65 RGA clones showed more than 95% homology with various RGAs sequences present in the GenBank. Phylogenetic analysis of these RGAs formed 11 groups. Using sequence homology approach, RGAs isolated in this study were physically mapped on 23 loci on chromosomes 1, 2, 3, 4, 5, 6, 7, 8, 10, 11 and 12. Twenty RGAs were mapped near to the chromosomal regions containing known genes/QTLs for rice blast, bacterial leaf blight and sheath blight resistance. Thirty‐nine RGA sequences also contained open reading frame representing signature of potential disease resistance genes.     

黄瓜枯萎病抗性基因的连锁分子标记

黄瓜枯萎病是危害我国黄瓜的主要病害。本实验以黄瓜抗枯萎病亲本WIS2757和感枯萎病亲本津研2号及其F2代分离群体为试材,采用分离群体分组分析法(BSA)进行了与黄瓜抗枯萎病基因连锁的分子标记研究。AFLP分析表明:引物对P15M5扩增出的特异DNA片段P15M5-310与WIS2757黄瓜枯萎病抗性基因连锁,遗传距离为7cM。     

Genetic Mapping of Linked Antibiotic Resistance Loci in Neisseria gonorrhoeae

Loci for resistance to several antibiotics in laboratory-derived strains of Neisseria gonorrhoeae were mapped by genetic transformation. Genes for high-level resistance to streptomycin (str) and spectinomycin (spc) and for low-level resistance to tetracycline (tet) and chloramphenicol (chl) were linked. Also, a locus for high-level resistance to rifampin (rif) was linked to str and tet. The apparent order was rif... str... tet... chl... spc. Loci for resistance to other antibiotics (penicillin, erythromycin) were transferred independently of each other and were not linked to the cluster around str. Similar linkage relationships were found with str, tet, chl, and spc loci obtained from naturally occurring (clinical) isolates of N. gonorrhoeae.     

The Blast Resistance Gene Pi54of Cloned from Oryza officinalis Interacts with Avr-Pi54 through Its Novel Non-LRR Domains

The dominant rice blast resistance gene Pi54 cloned by map-based cloning approach from indica rice cultivar Tetep confers broad spectrum resistance to Magnaporthe oryzae. In this investigation, an orthologue of Pi54 designated as Pi54of was cloned from Oryza officinalis conferring high degree of resistance to M. oryzae and is functionally validated. We have also characterized the Pi54of protein and demonstrate its interaction with AVR-Pi54 protein. The Pi54of encoded ∼43 kDa small and unique cytoplasmic LRR family of disease resistance protein having unique Zinc finger domain overlapped with the leucine rich repeat regions. Pi54of showed Magnaporthe-induced expression. The phylogenetic and western blot analysis confirmed orthologous nature of Pi54 and Pi54of genes, whereas the identity of protein was confirmed through MALDI-TOF analysis. The in silico analysis showed that Pi54of is structurally more stable than other cloned Pi54 proteins. The molecular docking revealed that Pi54of protein interacts with AVR-Pi54 through novel non-LRR domains such as STI1 and RhoGEF. The STI1 and GEF domains which interact with AVR-Pi54 are also components of rice defensome complex. The Pi54of protein showed differential domain specificity while interacting with the AVR protein. Functional complementation revealed that Pi54of transferred in two rice lines belonging to indica and japonica background imparts enhanced resistance against three highly virulent strains of M. oryzae. In this study, for the first time, we demonstrated that a rice blast resistance gene Pi54of cloned from wild species of rice provides high degree of resistance to M. oryzae and might display different molecular mechanism involved in AVRPi54-Pi54of interaction.     

番茄抗青枯病基因的AFLP分子标记

用番茄高抗青枯病品种“T51A”与高感青枯病品种“T9230”配制杂交组合,接种鉴定其正反交Ｆ₁代及Ｆ₂代分离群体的青枯病发生情况。结果表明,T51A对青枯病的抗性属于细胞质遗传,受1对杂合基因加性控制。用64个EcoRＩ/ＭseＩ引物组合对“T51A”、“T9230”两个亲本及其Ｆ₂代抗病和感病基因池进行AFLP分析,共扩增出约4200条可分辨的带,其中2条为稳定的差异。用“T51A”和“T9230”杂交产生的Ｆ₂代分离群体对2个特异条带与目的基因的遗传连锁性进行分析,发现特异条带AAG/CAT与暂定名为RRS-342的抗青枯病基因紧密连锁,二者之间的遗传距离为6.7 cM。将AAG/CAT片段回收、克隆和测序,成功地将其转化为SCAR标记,可以更加方便地用于对番茄青枯病基因的标记辅助选择。