Genetic and physical mapping of AvrPi7, a novel avirulence gene of Magnaporthe oryzae using physical position-ready markers

Rice blast, caused by the fungal pathogen Magnaporthe oryzae, is one of the most devastating crop diseases worldwide. The avirulence gene corresponding to rice blast resistance gene Pi7 in field isolate CHL346 was inherited as a single gene, designated AvrPi7, in a segregating population consisting of 189 ascospore progenies derived from a cross between field isolates CHL346 and CHL42. In order to determine the chromosomal location of the AvrPi7 locus, a total of 121 simple sequence repeat (SSR) markers were developed based on the whole-genome sequence of reference isolate 70-15 of M. oryzae. Linkage analysis of the locus with these SSR markers showed that eight SSR markers on chromosome 1 were linked to the locus, among which the closest flanking markers MS1-9 and MS1-15 were 3.2 and 16.4 cM from the locus, respectively. For fine mapping, additional PCR-based makers including eight SSR markers and three candidate avirulence gene (CAG) markers were developed in the region flanking both markers. The AvrPi7 locus was genetically delimited within a 1.6-cM region flanked by markers MS1-21 and MS1-22, and co-segregated with the marker CAG2. To construct a physical map of the AvrPi7 locus, molecular markers linked to the Avr gene were mapped on the supercontigs of the ref-erence isolate 70-15 through bioinformation analysis (BIA). Consequently, the AvrPi7 locus was delim-ited to a 75-kb interval flanked by markers MS1-21 and MS1-22 based on the reference sequence. Merodiploids observed in this study are also discussed.     

Characterization and molecular marker screening of a rice bacteria-resistant gene Xa-min(t)

To test the resistant spectrum of the Xa-min(t) gene introgressed from Oryza minuta, thirty-four isolates of different bacterial blight pathogen, Xanthomonas oryzae pv. oryzae (Xoo), from 11 countries were used to inoculate the Xa-min(t) introgression line 78-15. Four rice cultivars, IR24, C64 (IRBB21), Nipponbare and Zhonghua 11 were used as controls. The results showed that the Xa-min(t) gene was broad-spectrum and highly resistant to diverse Xoo isolates. The methods of bulk segregant analysis (BSA), randomly amplified polymorphic DNA (RAPD) and sequence characterized amplified regions (SCAR) were used to analyze F2 individuals of the hybrid IR24×78-15 and molecular genetic markers linked to Xa-min(t) gene were identified. A total of 800 arbitrary decamer oligonucleotide primers were used for RAPD analysis. Two RAPD markers, BE05300 and BE061400, produced by primers BE05 and BE06 respectively, were closely linked to the Xa-min(t) gene. Based on the sequences of these two markers, sequence specific primers were designed and used to screen all F2 plants. One RAPD marker, BE05300, was converted into a stable SCAR marker (ScBE05300). Linkage analysis was carried out using markers ScBE05300 and BE061400 on 948 and 719 F2 individuals of the hybrid IR24×78-15. Our results indicate that the genetic distances from Xa-min(t) to ScBE05300 and BE061400 are 2.2 cM and 3.7 cM respectively on the same side. This study may facilitate the construction of the fine physical map of the Xa-min(t) gene.     

Development of simple sequence repeats (SSR) markers of ramie and comparison of SSR and inter-SSR marker systems

Ramie (Boehmeria nivea L. ) is an important bast fiber crop. To study genetic background of this species, we isolated and characterized microsatellite markers of ramie. A genomic library containing inserts of rapid amplification of polymorphic DNA (RAPD)fragments was constructed, and screened by PCR amplification using anchored simple sequence repeats as primers. A total of 26 clones were identified as positives, and 13 microsatellite loci were found after sequencing. The polymorphism of these 13 microsatellite loci was examined and the utility of simple sequence repeats (SSR) and inter-SSR (ISSR) marker systems for genetic characterization compared using 19 selected ramie cultivars. Both approaches successfully discriminated the 19 cultivars which differed in the amount of polymorphism detected. The level of polymorphism detected by SSR was 95.0 %, higher than that by ISSR (72.3 % ), but the average polymorphism information content (PIC) of ISSR (0. 651) was higher than that of SSR (0. 441). The higher PIC value of ISSR suggests that ISSR is more efficient for fingerprinting ramie cultivars than SSR markers. However, because the SSR loci are codominant, they are more suitable for determining the homozygosity levels of ramie, constructing linkage map, quantitative trait loci study of complex traits and marker-as-sisted selection.     

崇明水仙(Narcissus tazetta var.chinensis)盆栽褐枯病病原的鉴定

依据柯赫氏法则对崇明水仙盆栽中叶褐枯病病原菌进行分离,得到3个可造成鳞茎和叶不同程度腐烂的致病真菌菌株.应用形态学鉴定法和真菌rDNA-ITS序列分析及ISSR分子标记技术进行鉴定,结果1株为丛赤壳属的丛赤壳菌(Nectria gliocladioides),2株为镰刀菌属的串珠镰刀菌胶胞变种(Fusarium moniliforme var.subglutinans).其中Nectria gliocladioides为引起崇明水仙盆栽枯萎病的主要病原真菌,该菌的最适生长温度为25℃,低于5℃和高于40℃菌丝均不能生长.研究结果证实ISSR技术可用于区分菌株间的种属差异,可作为病原真菌鉴定的分子辅助手段.     

Fine mapping of a semidwarf gene sd-g in indica rice（Oryza sativa L.）

The semidwarf gene sd-g which has been usedin indiea rice breeding in southern China is a new one, non-allelic to sd-1. To map sd-g, an F2 population derived fromthe cross between Xinguiaishuangai and 02428 was con-structed. The sd-g was roughly mapped between two mi-crosatellite markers RM440 and RM163, with genetic dis-tances of 0.5 and 2.5 cM, respectively. Then nine new poly-morphic microsatellite markers were developed in this region.The sd-g was further mapped between two microsatellitemarkers SSR5-1 and SSR5-51, with genetic distances of 0.1and 0.3 cM, respectively, while cosegregated with SSR418. ABAC contig was found to span the sd-g locus, the region be-ing delimited to 85 kb. This result was very useful for cloningof the sd-g gene.     

河北省和黑龙江省马铃薯晚疫病菌SSR基因型分析

用SSR分子标记,对致病疫霉群体结构进行分析,完善了中国研究晚疫病菌的分子标记体系。利用两个SSR引物Pi4B和Pi4G对来自河北和黑龙江两个省份的58个马铃薯晚疫病菌株基因组DNA进行SSR基因型鉴定,分析其遗传多样性。河北省的菌株共鉴定出4个SSR基因型:D-03,F-01,G-02和F-06。其中,F-06是首次报道的新的SSR基因型。黑龙江的菌株共鉴定出2个SSR基因型。在两省的菌株中,基因型F-01所占比例都最大,且占绝对优势。不同交配型可存在于同一SSR基因型中,同一SSR基因型的菌株对甲霜灵的敏感性存在差异。聚类结果还显示占被测菌株群体最大比例的F-01基因型与国外的对照菌株的亲缘关系较远。     

Mining,characterization, and exploitation of EST-derived microsatellites in <Emphasis Type="Italic">Gossypium barbadense</Emphasis>

Simple sequence repeats (SSRs) have been widely applied as molecular markers in genetic studies. However, the number of ex-pressed sequence tags (ESTs) and SSR markers from Gossypium barbadense is fewer than those from other cotton species. In this study, EST-SSR distribution from G. barbadense was characterized and new G. barbadense-derived EST-SSR markers were de-termined on the basis of the ESTs obtained by randomly sequencing 2 cDNA libraries associated with fiber development in G. barbadense. By mining 9697 non-redundant ESTs, a total of 638 SSR loci derived from 595 ESTs were observed. In G. barba-dense, the frequency of ESTs containing SSRs was 6.13%, with an average of 1 SSR in every 10.4 kb of EST sequence. Further-more, trinucleotide was found to be the most abundant repeat type among 2–6-nucleotide repeat types. It accounted for 26.6% of the total, followed by the hexanucleotide (26.0%) and pentanucleotide repeats (25.9%). Among all the repeat motifs, (AAG)n accounted for the highest proportion. EST-SSR primer pairs were developed using the Primer3 program, and the redundant primers were removed using the virtual PCR approach. As a result, 380 non-redundant EST-SSR primer pairs were developed and used to detect polymorphisms between the mapping parents G. hirsutum ‘TM-1’ and G. barbadense ‘Hai7124’ for constructing linkage groups in cultivated allotetraploid cotton. Out of these, 98 (25.8%) primer pairs detected polymorphisms. Finally, 95 polymorphic loci from 82 primer pairs were integrated into the backbone genetic map; of these, 42 were mapped into the A subgenome and 53 into the D subgenome. The present work provided the foundation for constructing saturated genetic maps and conducting comparative genomic studies on different cotton species.     

A physical map of the human Y chromosome

The non-recombining region of the human Y chromosome (NRY), which comprises 95% of the chromosome, does not undergo sexual recombination and is present only in males. An understanding of its biological functions has begun to emerge from DNA studies of individuals with partial Y chromosomes, coupled with molecular characterization of genes implicated in gonadal sex reversal, Turner syndrome, graft rejection and spermatogenic failure. But mapping strategies applied successfully elsewhere in the genome have faltered in the NRY, where there is no meiotic recombination map and intrachromosomal repetitive sequences are abundant. Here we report a high-resolution physical map of the euchromatic, centromeric and heterochromatic regions of the NRY and its construction by unusual methods, including genomic clone subtraction and dissection of sequence family variants. Of the map's 758 DNA markers, 136 have multiple locations in the NRY, reflecting its unusually repetitive sequence composition. The markers anchor 1,038 bacterial artificial chromosome clones, 199 of which form a tiling path for sequencing.     

Construction of the primary physical map of rice chromosome 12

A primary physical map of rice chromosome 12 was constructed using marker-based chromosome landing and chromosome walking. A BAC library from IR64 was screened using 84 RFLP markers, 4 STS markers and 6 microsatellite markers on chromosome 12 by colony hybridization and polymerase chain reaction (PCR) amplification. A total of 59 contigs consisting of 419 BAC clones including 5 single-clones were physically aligned on rice chromosome 12 with the largest BAC contig covering 855 kb. The whole physical map had a size of ∼16 Mb and covered about 52% of rice chromosome 12. This physical map will be certainly helpful for map-based gene cloning of agronomically and biological important genes and understanding the genome structure of the chromosome. Foundation item: Supported by Rockefeller Foundation Biography: FU Bin-Ying (1965-), male, Ph. D. candidate, Reseach direction: plant molecular genetics.     

Genome-wide identification of R genes and exploitation of candidate RGA markers in rice

By scanning the whole genomic sequence of japonica rice using 45 known plant disease resistance (R) genes, we identified 2119 resistance gene homologs or analogs (RGAs) and verified that RGAs are not randomly distributed but tend to cluster in the rice genome. The RGAs were classified into 21 families according to their functional domain based on Hidden Markov model (HMM). By comparing the RGAs of japonica rice with the whole genomlc sequence of indica rice, we found 702 RGAs allelic between the two subspecies and revealed that 671 (95.6%) of them have length difference (InDels) in their genomic sequences (including coding and non-coding regions) between the two subspecies, suggesting that RGAs are highly polymorphic between the two subspecies in rice. We also exploited 402 PCR-based and co-dominant candidate RGA markers by designing primer pairs on the regions flanking the lnDels and validating them via e-PCR. The length differences of the candidate RGA markers between the two subspecies are from 1 to 742 hp, with an average of 10.26 hp. All related information of the RGAs is available from our web site(http://ibi.zju.edu.cn/RGAs/index.html).     

湿地松转录组SSR分析及EST-SSR标记开发

【目的】湿地松是南方地区优先推广的优质产脂树种。但湿地松分子遗传基础薄弱,基因组序列信息匮乏,影响了湿地松基因组学的深入研究。目前,湿地松分子研究所用的SSR标记主要来自其他近缘种或利用公共数据库中有限的基因序列资源开发的SSR标记,其多态性和通用性较差。为解决这一问题, 笔者根据湿地松转录组测序数据开发EST-SSR位点,并揭示其在转录组序列中的分布类型及特征,为湿地松分子标记辅助育种奠定基础。【方法】利用MISA软件对转录组序列进行SSRs查找和分布特征分析。查找标准参数设置为: 单核苷酸重复>10次,二核苷酸重复>6次,三、四、五、六核苷酸重复>5次。根据SSR位点两端的保守区域,利用Primer3.0设计并随机挑选120对SSR引物,通过琼脂糖电泳和毛细管电泳对来自美国和吉安的113份家系个体进行遗传多样性分析,确定引物多态性。【结果】79 574条unigenes序列中搜索到3 818个SSR位点,出现频率为4.80%,平均18.27 kb出现1个SSR位点,3 373个unigenes含有SSR位点,SSR发生频率(含SSR位点的序列数/搜索序列总数)为4.24%,其中2 980条序列含1个SSR位点,含1个以上SSR位点的序列有393条。在检测到的3 818个SSR标记中,单核苷酸分布最多,其次是二核苷酸和三核苷酸,SSR数量分别占总数的63.54%、19.15%和16.27%,而四、五、六核苷酸重复类型所占比例较小,分别为0.52%、0.13%和0.31%。SSR重复单元的重复次数分布在5~22次之间,除单核苷酸重复外的1 391个SSR中,重复5次的数量最多,为498个(35.80%);重复6次和7次的次之,分别为417个(29.98%)和198个(14.23%);重复10次以上的仅有38个(2.73%)。在检测到的731个二核苷酸重复SSR中,最常出现的重复单元为AT/AT,数量为491个(12.86%),AG/CT和AC/GT两种类型的重复单元出现的次数次之,分别为156(4.09%)和81个(2.12%)。在检测到的621个三核苷酸重复中,AAT/ATT是出现频率最高的单元,共139个(3.64%),其次是AAG/CTT,共122个(3.20%)。3 818个SSR中有24.59%的位置未知,其余的SSR则分布在非编码区域(untranslated region,UTR)或者编码区(coding sequence,CDS)上,分布数量表现为3'UTR>5'UTR>CDS。参试的120对SSR引物,有92对扩增成功(76.78%),其中24对呈现多态(20%)。24对引物(13个二核苷酸重复、7个三核苷酸重复和4个四核苷酸重复)共检测出81个等位基因,每个位点的等位基因数为2~9,平均为3.38个。多态性信息含量(polymorphism information content,PIC)为0.103~0.726,平均为0.349。【结论】通过对湿地松转录组数据的挖掘,共获得 3 818个SSR位点,主要重复单元为AT/AT和AAT/ATT,可扩增出多态性位点的引物重复单元以二、三核苷酸重复为主。基于湿地松转录组序列的SSR标记开发是可行的。     

Characterization and mapping of a lesion mimic mutant in rice （Oryza sativa L.）

A rice initiation-type lesion mimic mutant (lmi) was identified, which was isolated from an indica rice Zhongxian 3037 through γ radiation mutagenesis. Trypan blue staining and sterile culture revealed that the mutant spontaneously developed lesions on the leaves in a developmentally regulated and light-dependent manner. Genetic analysis indicated that the lesion mimic trait was controlled by a single resessive locus. Using public molecular markers and an F2 population derived from lmi and 93-11, we mapped the lmi locus to the short arm of chromosome 8, nearby the centromere, between two SSR markers RM547 and RM331. The genetic distance was 1.2 and 3.2 cM, respectively. Then according to the public rice genomic sequence between the two SSR markers, lmi was further finely tagged by three CAPS markers: C4135-8, C4135-9 and C4135-10. And lmi locus was a co-segregated with marker C4135-10, providing a starting point for lmi gene cloning.     

Genetic contribution of foreign germplasm to elite Chinese soybean （Glycine max） cultivars revealed by SSR markers

Among 651 soybean (Glycine max) cultivars re- leased from 1923 to 1995, most of the parents used in the breeding program of northeast China and Huang- huai-hai valley were cultivars well adapted to local ecologic areas. Narrow parentage and lack of geneti…     

Fine mapping of an incomplete recessive gene for leaf rolling in rice （Oryza sativa L.）

Genetic analysis and fine mapping of genes controlling leaf rolling were conducted using two backcrossed generations （BC4F2, BC4F3） derived from a cross between QMX, a non-rolled leaf cultivar as a recurrent parent, and JZB, a rolled leaf NIL of ZB as a donor parent. Results indicated that leaf rolling was mainly controlled by an incompletely recessive major gene, namely rl（t）, and at the same time, affected by quantitative trait loci （QTLs） and/or the environment. A genetic linkage map was constructed using MAPMAKER/EXP3.0 with eight polymorphic markers on chromosome 2, which were screened by BAS method from 500 SSR markers and 15 newly developed insertion/deletion （InDel） markers. The position of rl（t） was estimated with composite interval mapping （CIM） method using WinQTLcart2.5. Gene rl（t） was mapped between markers InDel 112 and RM3763, and 1.0 cM away from InDel 112 using 241 plants in BC4F2 population. To fine map r（t）, one BC4F3 line with 855 plants was generated from one semi-rolled leaf plant in BC4F2. Four new polymorphic InDel markers were developed, including InDel 112.6 and InDel 113 located between markers InDe1112 and RM3763. Based on the information of recombination offered by 191 rolled leaf plants and 185 non-rolled leaf plants from the BC4F3 line ,we mapped r（t） to a 137-kb region between markers InDel 112.6 and InDel 113. Homologous gene analysis suggested that r（t）was probably related to the process of leaf development regulated by microRNA.     

Screening and Insert Size Analysis of Candidate C-Genome BAC Clones from Brassica napus

Thirty-two C-genome specific candidate bacterial artificial chromosome (BAC) clones were successfully screened from the BAC library by four-dimensional PCR method with the primer pairs of 75 simple sequence repeat (SSR) markers located in the nine C-genome linkage groups of Brassica napus. The screened 32 BAC clones have an average insert size of 114.2 kb with a range of 30-190 kb. They are the first set of C-genome BAC clones screened from B. napus genomic BAC library. The average insert size of this set of BAC clones presented that the constructed BAC library had a high quality. This set of BAC clones can be used as markers to identify individual chromosomes of B. napus C-genome.     

A physical map of the mouse genome

A physical map of a genome is an essential guide for navigation, allowing the location of any gene or other landmark in the chromosomal DNA. We have constructed a physical map of the mouse genome that contains 296 contigs of overlapping bacterial clones and 16,992 unique markers. The mouse contigs were aligned to the human genome sequence on the basis of 51,486 homology matches, thus enabling use of the conserved synteny (correspondence between chromosome blocks) of the two genomes to accelerate construction of the mouse map. The map provides a framework for assembly of whole-genome shotgun sequence data, and a tile path of clones for generation of the reference sequence. Definition of the human-mouse alignment at this level of resolution enables identification of a mouse clone that corresponds to almost any position in the human genome. The human sequence may be used to facilitate construction of other mammalian genome maps using the same strategy.     

Analysis of simple sequence repeats markers derived from Phytophthora sojae expressed sequence tags

Five thousand and eight hundred publicly available expressed sequence tags (ESTs) of Phytophthora sojae were electronically searched and 415 simple sequence repeats (SSRs) were identified in 369 ESTs. The average density of SSRs was one SSR per 8.9 kb of EST sequence screened. The most frequent repeats were trinucleotide repeats (50.1%) and the least frequent were tetranucleotide repeats (8.2%). Forty primer pairs were designed and tested on 5 strains of P. sojae. Thirty-three primer pairs had successful PCR amplifications. Of the 33 functional primer pairs, 28 primer pairs produced characteristic SSR bands of the expected size, and 15 primer pairs (45.5%) detected polymorphism among 5 tested strains of P. sojae. Based on the polymorphisms detected with 20 EST-SSR markers, the 5 tested strains of P. sojae were clustered into 3 groups. In this study, the SSR markers of P. sojae were developed for the first time. These markers could be useful for identification, genetic variation study, and molecular mapping of P. sojae and its relative species.     

春小麦重组自交系及多种SSR标记构建遗传图谱

 以春小麦重组自交系(RIL)宁春4 号×宁春27 号为作图群体,利用SSR 标记构建小麦遗传连锁图谱。结果表明,用1001对SSR 引物选出亲本间表现多态性的引物307 对,多态性频率为30.7%。利用307 对多态性引物对RIL 群体进行分析,共检测到266 个多态性标记位点。通过χ²检测(P<0.05),有147 个SSR 标记表现为偏分离,偏分离率为55.3%,129 个偏向母本宁春4号,其偏分离位点主要分布在B 和D 基因组上。用Mapmaker 3.0 和Mapdraw 2.1 软件将266 个SSR 位点绘制在小麦遗传连锁图上,该图谱覆盖小麦基因组全长2187.79 cM,标记间的平均遗传距离为8.22 cM。     

利用小麦微卫星引物建立黑麦特异标记及其局限性

用50对小麦SSR引物对13种黑麦基因组DNA进行扩增,只有Xgwm232,Xgwm260,Xgwm644这3对小麦SSR引物能扩增出黑麦特异的片段．这些黑麦特异片段主要集中在500bp,600bp,800bp和1000bp左右,并包含了启动子区域．然而这3对SSR引物都不能从所有13种黑麦中扩增出黑麦特异条带．这一结果表明尽管利用小麦微卫星引物能够开发黑麦特异DNA标记,但这类标记不是对所有黑麦都适用．利用某一物种的微卫星引物开发其近缘种属的特异DNA标记虽然可行,但不一定具有通用性．     

A map of human genome sequence variation containing 1.42 million single nucleotide polymorphisms

We describe a map of 1.42 million single nucleotide polymorphisms (SNPs) distributed throughout the human genome, providing an average density on available sequence of one SNP every 1.9 kilobases. These SNPs were primarily discovered by two projects: The SNP Consortium and the analysis of clone overlaps by the International Human Genome Sequencing Consortium. The map integrates all publicly available SNPs with described genes and other genomic features. We estimate that 60,000 SNPs fall within exon (coding and untranslated regions), and 85% of exons are within 5 kb of the nearest SNP. Nucleotide diversity varies greatly across the genome, in a manner broadly consistent with a standard population genetic model of human history. This high-density SNP map provides a public resource for defining haplotype variation across the genome, and should help to identify biomedically important genes for diagnosis and therapy.