用AFLP标记快速构建遗传连锁图谱并定位一个新基因tms5

报导了一个分子标记连锁图的快速构建方法.通过对水稻(Oryza sativa L.)"安农S-1"和"南京11"的F2分离群体的AFLP分析找到了142个AFLP标记,用这142个AFLP标记以及已定位的25个SSR标记和5个RFLP标记构建了水稻12个染色体的分子标记连锁图,该图覆盖水稻基因组的1 537.4 cM,相邻标记间的平均间距为9.0 cM,这是在国内建立的第一张AFLP标记连锁图.在建立连锁图谱的同时把一个新基因tms5 (水稻温敏核不育基因)定位在第2染色体上.     

甘蓝型油菜花瓣缺失基因的图谱定位

在无花瓣品系APT02和正常有花瓣品种中双4号构建的的F2分离群体中,运用AFLP和SRAP两种标记技术对甘蓝型油菜花瓣缺失基因进行分子标记和图谱定位。在两亲本间筛选20对AFLP引物和170对SRAP 引物,进一步通过BSA法筛选,获得了与甘蓝型油菜花瓣缺失基因WHB连锁的1个SRAP标记e8m3_4（600bp）和1个AFLP标记E3247_15（150bp）,标记与基因WHB之间的遗传距离分别为5 cM和13.5cM;构建了一个甘蓝型油菜(Brassica napus.L )的分子标记遗传连锁图谱,该图谱共包含213个AFLP标记、56个SRAP标记和1个形态标记,分布于17个主要连锁群、两个三联体和4个连锁对中,遗传图距总长2487.1cM,标记间平均距离为10.09 cM。通过图谱定位,控制花瓣缺失性状的基因WHB被定位到第4连锁群(LG4)上。     

野败型水稻细胞质雄性不育恢复基因Rf-4的分子标记定位

为了用分子标记准确定位野败型水稻细胞质雄性不育恢复基因Rf-4,将日本水稻基因组项目（Rice Genome Program,RGP)构建的水稻遗传连锁图谱第10染色体分子遗传图上的分子标记R1877和G2155之间对应区域YAC物理图上的6个YAC克隆进行了亚克隆,获得119个片段,对这些探针进行多态性探查,获得了2个多态分子标记,用珍汕97A和恢复基因近等基因系的杂种F2分离群体中的117完全不育株进行连锁分析表明,从YAC4892获得的亚克隆Y3－8与Rf－4座位的连锁距离为0．9cM,从YAC4630获得的亚克隆Y1－10与Rf－4座位的连锁距离为3．2cM,根据以上结果把Rf－4座位定位于第10染色体的特定位置,为该基因的分子标记辅助选择和定位克隆打下了基础。     

水稻中大麦Mlo和玉米Hm1抗病基因同源序列的分析和定位

大麦抗病基因Mlo和玉米抗病基因Hm1编码的产物不具有绝大多数植物抗病基因产物所含有的保守结构域。这两个抗病基因的作用机理也不符合基因对基因学说。从水稻中分离克隆了Mlo基因的同源序列OsMlo-1和玉米Hm1基因的同源序列DFR－1。利用水稻分子标记遗传连锁图,将OsMlo-1定位于水稻第六染色体的两俱RZ667和RG424之间;Osmlo-1距离这两个分子标记分别为20．6和6．0cM(centi-Morgan)。将DFR－1定位于水稻第一染色体两个分子标记R2635和RG462之间;DFR－1距离这两个分子标记分别为11．3和23．9cM。参照已发表的水稻分子标记连锁图,发现OsMlo-1和DFR－1的染色体位点分别与两个报道的水稻抗稻瘟病数量性状位点（QTL）有较好的对应关系。结果提示,水稻中与大麦Mlo 和玉米Hml同源的基因可能也参于抗病反应的调控。     

家蚕高密度AFLP连锁图谱的构建

为了进行家蚕Bombyx mori数量性状的QTL定位研究,以白色茧系品种C₁₀₀ (♀)和近交系大造(P50)(♂)杂交得到F₁,用F₁(♂)与双隐性标记的C₁₀₀ (♀)回交,得到回交一代(BC₁),用改进的AFLP分子标记方法,经96组选择性扩增引物扩增,获得分离比为1∶1(P≤0.05)的1 744个AFLP位点。用Map Manager QTXb19（Version 0.29）连锁图谱构建软件,构建了具有814个标记,36个连锁群的家蚕高密度AFLP分子标记连锁图谱。该连锁图谱覆盖的家蚕基因组长度为13 005 cM,连锁群长度变化范围为109.0～1 573.7 cM,连锁群的平均长度为361.25 cM,其标记间平均图距15.98 cM,最小图距2.3 cM,最大图距47.7 cM,标记间大于30 cM的gap共有39个。该连锁图平均每个连锁群23个标记,最多一个连锁群有92个标记,最少8个标记。该连锁图谱确定了与经典实验遗传图谱第15连锁群和W染色体连锁群相对应的两个连锁群。     

普通小麦Qz180中一个抗条锈病基因的分子作图(英文)

普通小麦(Triticum aestivum L.)材料Qz180具有良好的抗条锈病特性,经基因推导发现其含有一个优良的抗条锈病的基因,暂定名为YrQz。用Qz180与感病材料铭贤169和WL1分别杂交构建了两个F_2群体,用条中30号条锈菌小种对这两个群体进行的抗性测验表明,YrQz为显性单基因遗传。通过SSR和AFLP结合BSA的方法对这个基因进行了分子作图,结果鉴定出与YrQz连锁的2个SSR标记和2个AFLP标记。根据SSR标记的染色体位置,该基因被定位在2B染色体的长臂上,位于两个SSR位点Xgwm388和Xgwm526之间;两个AFLP标记P35M48(452)和P36M61(163)分别位于该基因的两侧,遗传距离分别为3.4cM和4.1cM。     

普通小麦Qz180中一个抗条锈病基因的分子作图

普通小麦(Triticum aestivum L.)材料Qz180具有良好的抗条锈病特性,经基因推导发现其含有一个优良的抗条锈病的基因,暂定名为YrQz.用Qz180与感病材料铭贤169和WL1分别杂交构建了两个F2群体,用条中30号条锈菌小种对这两个群体进行的抗性测验表明,YrQz为显性单基因遗传.通过SSR和AFLP结合BSA的方法对这个基因进行了分子作图,结果鉴定出与YrQz连锁的2个SSR标记和2个AFLP标记.根据SSR标记的染色体位置,该基因被定位在2B染色体的长臂上,位于两个SSR位点Xgwm388和Xgwm526之间;两个AFLP标记P35M48(452)和P36M61(163)分别位于该基因的两侧,遗传距离分别为3.4 cM和4.1cM.     

水稻顶节间长度控制基因(EUI)的精细定位

通过对一水稻顶节间特异伸长突变体Mh-1进行经典遗传学和基因定位分析,认为该表型是一个核基因隐性突变所致。利用Mh-1与正常的T65-sd1杂交的F2群体对该位点定位研究分析,发现其与水稻第5条染色体长臂STS标记E30531和CAPS标记C903连锁,遗传距离分别为6．7cM和2．8cM。经进一步发展新的分子标记,将该基因精确定位在0．3cM的区域,为进一步克隆和研究该基因的分子机制奠定基础。     

大粒裸燕麦(Avena nuda L.)遗传连锁图谱的构建

以“元莜麦”和“555”杂交得到的281个F2单株为作图群体,利用20对AFLP引物、3对SSR引物和1个穗型性状构建了一张大粒裸燕麦遗传连锁图。该图谱全长1544.8cM,包含19个连锁群,其上分布有92个AFLP标记、3个SSR标记和1个穗型形态标记,不同连锁群标记数为2-14个,长度在23.7-276.3cM之间,平均长度为81.3cM,标记间平均距离为20.1cM。穗型标记分离比符合3：1,11个AFLP标记表现为偏分离,偏分离比为11.5%。该图谱符合遗传连锁框架图的要求,为今后大粒裸燕麦的QTL定位、分子标记辅助育种和比较基因组学等研究奠定基础。     

大豆遗传图谱的构建和分析

利用大豆栽培品种科丰1号和南农1138－2杂交得到的重组近交系NJRIKY,通过RFLP,SSR,RAPD和AFLP4种分子标记的遗传连锁分析,构建了包含24个连锁群,由792个遗传标记组成的大豆较高密度连锁图谱,该图谱覆盖2320．7cM,平均图距2．9cM,SSR标记的多态性较高,在基因组中的位置相对稳定,可以作为锚定标记,有利于连锁群的归并和不同图谱的比较整合;而AFLP标记对于增加图谱密度效率较高,但其容易出现聚集现象,从而造成连锁群上有很大的空隙（gap),另外,在连锁群中有21．7％的分子标记出现偏分离,该图谱为基因定位,比较基因组学和重要农艺性状的QTL定位等研究打下了基础。     

Linkage mapping of AFLP markers in a wild population of great reed warblers: importance of heterozygosity and number of genotyped individuals

Amplified fragment length polymorphisms (AFLP) are dominant markers frequently used to build linkage maps where heterozygosity could be inferred by a backcross breeding strategy. In the present study, we describe the utilization of an unmanipulated great reed warbler, Acrocephalus arundinaceus pedigree to infer heterozygous genotypes of AFLP markers in order to map these markers to a partial linkage map previously based on microsatellites. In total, 50 of the 83 autosomal AFLPs (60%) and 4 of 5 Z-linked AFLPs (80%) were mapped. For each marker, on average, 88% of the expected number of heterozygote parents was detected. The likelihood of map assignment was to a large extent due to the number and density of microsatellite markers already in the map. The 'parsimonious linkage map', that is the map based on the most parsimonious location of all significantly linked markers, consisted of 21 autosomal linkage groups with 2 to 15 markers and had a total map size of 552 cM in males and 858 cM in females. The Z-chromosome linkage group with 12 markers had a size of 155 cM. The autosomal 'framework linkage map', that is the map based only on markers with an unambiguous position, had a total size of 237 cM in males and 440 cM in females, respectively. The inclusion of AFLPs enlarged the previous map substantially (e.g. the autosomal parsimonious linkage map became 441 cM and 621 cM larger for male and female recombination, respectively). The probability that an AFLP became mapped increased with increasing level of heterozygosity, whereas the probability of mapping into a framework position increased with both heterozygosity and number of genotyped individuals. Our results suggest that AFLP provides a fast and inexpensive means of enlarging genetic maps already composed of markers with high polymorphism, also in wild populations with unmanipulated pedigrees.     

花椰菜遗传图谱的构建及NBS-LRR类抗性同源基因在图谱中的定位

利用AFLP和NBS profiling技术, 以花椰菜自交系“AD白花”与高代自交不亲和系“C-8”杂交得到的F1代自交产生的F2代分离群体为材料, 构建了第一个花椰菜遗传连锁图谱。该图谱由234个AFLP标记和21个NBS标记构成了9个连锁群, 总图距为668.4 cM, 标记间平均距离为2.9 cM。每个连锁群包含的位点数从12到47个, 相邻两标记之间的距离范围是0~14.9 cM。NBS标记分布在8个连锁群中, 这些标记大部分聚在一起。本研究为今后的基因定位及重要农艺性状的分析提供框架图。此外, 研究NBS profiling 方法在花椰菜中的稳定性和有效性以及NBS-LRR类RGA在花椰菜基因组中的分布和特点。     

A Preliminary Genetic Linkage Map of the Pacific Abalone Haliotis discus hannai Ino

Preliminary genetic linkage maps were constructed for the Pacific abalone (Haliotis discus hannai Ino) using amplified fragment length polymorphism (AFLP), randomly amplified polymorphic DNA (RAPD), and microsatellite markers segregating in a F₁ family. Nine microsatellite loci, 41 RAPD, and 2688 AFLP markers were genotyped in the parents and 86 progeny of the mapping family. Among the 2738 markers, 384 (including 365 AFLP markers, 10 RAPD markers, and 9 microsatellite loci) were polymorphic and segregated in one or both parents: 241 in the female and 146 in the male. The majority of these markers, 232 in the female and 134 in the male, segregated according to the expected 1:1 Mendelian ratio (α = 0.05). Two genetic linkage maps were constructed using markers segregating in the female or the male parent. The female framework map consisted of 119 markers in 22 linkage groups, covering 1773.6 cM with an average intermarker space of 18.3 cM. The male framework map contained 94 markers in 19 linkage groups, spanning 1365.9 cM with an average intermarker space of 18.2 cM. The sex determination locus was mapped to the male map but not to the female map, suggesting a XY-male determination mechanism. Distorted markers showing excess of homozygotes were mapped in clusters, probably because of their linkage to a gene that is incompatible between two parental populations.     

A genetic map of the lettuce downy mildew pathogen,Bremia lactucae,constructed from molecular markers and avirulence genes

The genetic map of Bremia lactucae was expanded utilizing 97 F(1) progeny derived from a cross between Finnish and Californian isolates (SF5xC82P24). Genetic maps were constructed for each parent utilizing 7 avirulence genes, 83 RFLP markers, and 347 AFLP markers, and a consensus map was constructed from the complete data set. The framework map for SF5 contained 24 linkage groups distributed over 835cM; the map for C82P24 contained 21 linkage groups distributed over 606cM. The consensus map contained 12 linkage groups with markers from both parents and 24 parent-specific groups. Six avirulence genes mapped to different linkage groups; four were located at the ends of linkage groups. The closest linkages between molecular markers and avirulence genes were 3cM to Avr4 and 1cM to Avr7. Mating type seemed to be determined by a single locus, where the heterozygote determined the B(2) type and the homozygous recessive genotype determined the B(1) type.     

A detailed linkage map of lettuce based on SSAP, AFLP and NBS markers

Molecular markers based upon a novel lettuce LTR retrotransposon and the nucleotide binding site-leucine-rich repeat (NBS-LRR) family of disease resistance-associated genes have been combined with AFLP markers to generate a 458 locus genetic linkage map for lettuce. A total of 187 retrotransposon-specific SSAP markers, 29 NBS-LRR markers and 242 AFLP markers were mapped in an F₂ population, derived from an interspecific cross between a Lactuca sativa cultivar commonly used in Europe and a wild Lactuca serriola isolate from Northern Europe. The cross has been designed to aid efforts to assess gene flow from cultivated lettuce into the wild in the perspective of genetic modification biosafety. The markers were mapped in nine major and one minor linkage groups spanning 1,266.1 cM, with an average distance of 2.8 cM between adjacent mapped markers. The markers are well distributed throughout the lettuce genome, with limited clustering of different marker types. Seventy-seven of the AFLP markers have been mapped previously and cross-comparison shows that the map from this study corresponds well with the previous linkage map.     

Generation and mapping of SCAR and CAPS markers linked to the seed coat color gene in Brassica napus using a genome-walking technique.

The yellow seed coat trait in No. 2127-17, a resynthesized purely yellow Brassica napus line, is controlled by a single partially dominant gene, Y. A double-haploid population derived from the F1 of No. 2127-17 x 'ZY821' was used to map the seed coat color phenotype. A combination of AFLP analysis and bulked segregant analysis identified 18 AFLP markers linked to the seed coat color trait. The 18 AFLP markers were mapped to a chromosomal region of 37.0 cM with an average of 2.0 cM between adjacent markers. Two markers, AFLP-K and AFLP-H, bracketed the Y locus in an interval of 1.0 cM, such that each was 0.5 cM away from the Y locus. Two other markers, AFLP-A and AFLP-B, co-segregated with the seed color gene. For ease of use in breeding programs, these 4 most tightly linked AFLP markers were converted into reliable PCR-based markers. SCAR-K, which was derived from AFLP-K, was assigned to linkage group 9 (N9) of a B. napus reference map consisting of 150 commonly used SSR (simple sequence repeat) markers. Furthermore, 2 SSR markers (Na14-E08 and Na10-B07) linked to SCAR-K on the reference map were reversely mapped to the linkage map constructed in this study, and also showed linkage to the Y locus. These linked markers would be useful for the transfer of the dominant allele Y from No. 2127-17 to elite cultivars using a marker-assisted selection strategy and would accelerate the cloning of the seed coat color gene.     

Mapping QTLs for submergence tolerance in rice by AFLP analysis and selective genotyping

By combining the amplified fragment length polymorphism (AFLP) technique with selective genotyping, we constructed a linkage map for rice and assigned each linkage group to a corresponding chromosome. The AFLP map, consisting of 202 AFLP markers, was generated from 74 recombinant inbred lines (RIL) which were selected from both extremes of the population (250 lines) with respect to the response to complete submergence. Map length was 1756 cM, with an average interval size of 8.5 cM. To assign linkage groups to chromosomes, we used 50 previously mapped AFLP markers as anchor markers distributed over the 12 chromosomes. Other AFLP markers were then assigned to specific chromosomes based on their linkage to anchor markers. This AFLP map is equivalent to the RFLP/AFLP map constructed previously as the anchors were in the same order in both maps. Furthermore, tests with two restriction fragment length polymorphism (RFLP) markers and two sequence-tagged site (STS) markers showed that they mapped in the expected positions. Using this AFLP map, a major gene for submergence tolerance was localized on chromosome 9. Quantitative trait loci (QTL) associated with submergence tolerance were detected on chromosomes 6, 7, 11, and 12. We conclude that the combination of AFLP mapping and selective genotyping provides a much faster and easier approach to QTL identification than the use of RFLP markers. Received: 20 December 1996 / Accepted: 21 January 1997     

Tagging and mapping of rice sheath blight resistant gene

Sheath blight (Rhizoctonia solani Kühn) is one of the severe rice diseases worldwide. In this study, an F(2) population from a cross between "4011" and "Xiangzaoxian19" is used to identify molecular markers linked with the resistant trait. "4011" was a transgenic rice cultivar carrying a resistant gene to sheath blight, while "Xiangzaoxian19" is a highly susceptible one. As a result, five molecular markers, including three RFLP markers converted from RAPD and AFLP markers, and two SSR markers were identified to link with the sheath blight resistant gene. This dominant resistant gene was named as R sb 1 and mapped on rice chromosome 5. The linkage distance between the markers (E-AT:M-CAC(120), E-AT:M-CTA(230), OPN-16(2000), RM164(320) and RM39(300)) and R sb 1 was 1.6 cM, 9.9 cM, 1.6 cM, 15.2 cM and 1.6 cM, respectively.     

An interspecific (Capsicum annuum ×C. chinese) F2 linkage map in pepper using RFLP and AFLP markers

We have constructed a molecular linkage map of pepper (Capsicum spp.) in an interspecific F₂ population of 107 plants with 150 RFLP and 430 AFLP markers. The resulting linkage map consists of 11 large (206–60.3 cM) and 5 small (32.6–10.3 cM) linkage groups covering 1,320 cM with an average map distance between framework markers of 7.5 cM. Most (80%) of the RFLP markers were pepper-derived clones, and these markers were evenly distributed across the genome. By using 30 primer combinations, we were able to generate 444 AFLP markers in the F₂ population. The majority of the AFLP markers clustered in each linkage group, although PstI/MseI markers were more evenly distributed than EcoRI/MseI markers within the linkage groups. Genes for the biosynthesis of carotenoids and capsaicinoids were mapped on our linkage map. This map will provide the basis of studying secondary metabolites in pepper. Received: 20 October 1999 / Accepted: 3 July 2000