与小麦白粉病抗性基因Pm2紧密连锁PAPD标记的筛选研究

以２５６个随机引物对含小麦抗白粉病基因Ｐｍ２近等基因系进行ＲＡＰＤ分析,发现１７个随机引物的扩增产物在抗、感ＮＩＬｓ材料间表现多态性,且其中５个引物经４次以上重复,均获相同结果,其多态性标记分别为ＯＰＭ０８１６００、ＯＰＩ０４１７００、ＯＰＨ１９９００及ＯＰＭ１６８５０。当以这５个随机引物对１４个已知含Ｐｍ２基因的抗病材料及９个不含Ｐｍ２基因的感病材料进行检测时,只有标记ＯＰＩ０４１７００在１２个     

小麦抗白粉病基因Pm6的RAPD标记

从提莫菲维小麦转移到普通小麦中的小麦白粉病抗性基因Ｐｍ６是小麦白粉病（Ｅｒｙｓｉｐｈｅ ｈｒａｍｉｎｉｓ ｆ ｓｐ．ｔｒｉｔｉｃｉ）的有效抗性基因。用７００个随机引物对Ｐｍ６近等基因系进行ＲＡＰＤ分析,发现引物ＯＰＶ２０可在抗病近等基因系中产生大小为２ｋｂ的稳定的多态片段。用该引物检测１０个其他携Ｐｍ６的渐渗系材料,均可稳定扩增出该２ｋｂ的多态片段。理一步用ＯＰＶ２０对Ｐｍ６Ｆ２（ＩＧＶ１－４６３     

甘蓝型油菜Pol CMS育性恢复基因的PCR标记

采用恢、保回交群体和集团混合分析法,筛选了１０４０个１０－ｍｅｒ随机引物,找到了与甘蓝型油菜波里马细胞质雄性不育系（Ｐｏｌ ＣＭＳ）育性恢复基因（Ｒｆｐ）连锁的两个ＲＡＰＤ标记Ｓ１０１９７２０和Ｓ１０３６８１０。它们位于Ｒｆｐ的一侧,与该基因的遗传图距分别为５．８ｃＭ和１２．３ｃＭ。随后,克隆并测序这２个多态性片段,根据其２端序列设计了２对２０～２４－ｍｅｒ的特异引物,它们在１３８株的回交群体中Ｐ     

利用分子标记定位农垦58S的光敏核不育基因

对农垦５８Ｓ（Ｏｒｙｚａｓａｔｉｖａｓｐ．ｊａｐｏｎｉｃａ）／大黑矮生标记基因系ＦＬ２组合组建可育集团和不育集团,并以亲本为对照进行了ＲＦＬＰ、ＲＡＰＤ和双引物ＲＡＰＤ分析,结果第１２染色体上的一个单拷贝标记Ｇ２１４０与光敏核不育基因连锁遗传,二者间的遗传图距为１４．１ｃＭ（ｃｅｎｔｉｍｏｒｇａｎ）。在筛选过的１０４０个随机单引物和１９０个双引物中,仅引物ＯＰＡＵ１０扩增出与光敏核不育基因连锁的１．５ｋｂＤＮＡ片段,回收、克隆该ＤＮＡ片段并制备探针,将其转换成共显性的ＲＦＬＰ标记并命名为ＯＰＡＵ１０１５００。分离群体连锁分析表明该标记与标记Ｇ２１４０紧密连锁,将农垦５８Ｓ的一对光敏核不育基因定位于第１２染色体上。     

与小麦抗白粉病基因Pm6紧密连锁的分子标记筛选

以Ｐｒｉｎｓ＊ＰＩ１７０９１４／７＊ＰｉｎｓＦ２分离群体对在Ｐｒｉｎｓ与其Ｐｍ６近等基因系ＰＩ１７０９１４／７＊Ｐｒｉｎｓ之间呈现多态性的ＲＦＬＰ标记进行遗传和图,发现８个多态性标中有３个与转称到普通小麦２Ｂ染色体长臂上的来源于。     

用新的分子标记方法（RAPD）分析小麦抗白粉病基因Pm4α的近等基因系

ＲＡＰＤ是一种新发展的分子标记技术,本实验利用这种技术对小麦抗白粉病基因Ｐｍ４ａ的近等基因系进行分析。在１００个随机引物中找到了３个引物在这对抗白粉病的近等基因系中所扩增出的带型出现差异。并根据理论计算所找到的差异与抗生基因Ｐｍ４ａ连锁的概率是０．７,即３个差异中应有２个标记与抗性基因连锁。     

水稻广亲和基因（WCG）近等基因系的随机引物PCR分析

应用随机引物ＰＣＲ（ＲａｎｄｏｍＰｒｉｍｅｒＰＣＲ）技术分别在水稻广亲和基因（ＷＣＧ）的近等基因系和色素原基因（Ｃ）的分离群体库中寻找与ＷＣＧ和Ｃ基因连锁的分子标记。对于ＷＣＧ近等基因系,在２２６个随机引物中初筛到２２个显示多态性片段的引物。根据理论值计算,在２２个多态性片段中预期有２０个与ＷＣＧ连锁。在这些连锁标记中距ＷＣＧ最近的可达０．５ｃＭ。同样在分离群体库的筛选中有１０个扩增产物与Ｃ基因连锁。     

CD3ε和PMA对fas基因转录调控作用的研究

研究了ＣＤ３ε和ＰＭＡ对细胞凋亡基因ｆａｓ的转录调控作用．根据已知的人ｆａｓ基因５＇上游序列设计引物,用ＰＣＲ法从人胸腺细胞基因组ＤＮＡ中扩增出ｆａｓ５＇上游长为４４６ｂｐ的启动子片段．将此片段定向克隆到以虫荧光素酶为报告基因的真核细胞表达载体ｐＸＰ２中．经限制性内切酶酶切鉴定及序列测定分析表明此重组表达质粒ＤＮＡ的结构和序列正确．用此质粒（ｐＸＰ２－ｆａｓｕｐ）瞬时转染人ＪｕｒｋａｔＴ淋巴细胞,分析报告基因的表达水平．结果表明ｆａｓ基因上游－５０６到－６０的区域有弱启动子活性,约是阴性对照的１．５倍．抗ＣＤ３ε抗体和ＰＭＡ处理均可增强ｆａｓ启动子的活性,促进报告基因的表达,其虫荧光素酶活性分别是未经处理的ｐＸＰ２－ｆａｓｕｐ转染细胞的１．７倍和３．３倍,但二者没有协同作用．ＰＫＣ的抑制剂ｓｔａｕｒｏｓｐｏｒｉｎｅ和ＰＴＫ的抑制剂ｈｅｒｂｉｍｙｃｉｎＡ可抑制ＰＭＡ诱导的ｆａｓ基因转录,使虫荧光素酶基因的表达降至未用ＰＭＡ处理的水平．但ＰＴＫ的另一种抑制剂ｇｅｎｉｓｔｅｉｎ可与ＰＭＡ发挥协同作用,上调ｆａｓ基因的转录,使虫荧光素酶活性增加到５倍．这些结果为阐明ＣＤ３ε及ＰＭＡ介导Ｔ淋巴细胞激活和凋亡的分子机制     

与西瓜野生种质抗枯萎病基因连锁的RAPD标记

运用ＲＡＰＤ技术,采用混合分组分析（ｂｕｌｋｅｄｓｅｇｒｅｇａｎｔａｎａｌｙｓｉｓ,ＢＳＡ）方法进行了西瓜（Ｃｉｔｒｕｌｌｕｓｌａｎａｔｕｓ（Ｔｈｕｎｂ．）Ｍａｎｓｆｅｌｄｖａｒ． ｃｉｔｒｏｉｄｅｓ） 野生种质ＰＩ２９６３４１ 抗枯萎病基因连锁的分子标记研究。研究结果表明：西瓜野生种质Ｐ１２９６３４１ 抗枯萎病生理小种１ 的抗性由单显性基因控制,ＲＡＰＤ标记ＯＰＰＯＬ／７００ 与其抗病基因连锁,其遗传距离为３０ ｃＭ（ｃｅｎｔｉｍｏｒｇａｎ）。这为进行抗病分子标记辅助选择,以及最终定位与克隆其抗病基因打下了良好基础。     

鼠伤寒沙站氏菌嘌呤生物合成调控研究

在鼠伤寒沙门氏菌中,由嘌呤从头合成途径合成ＩＭＰ经１０步酶促反应,涉及１０个结构基因,其中ＰｕｒＪ ｐｕｒＨ和ｐｕｒＤ构成１个操纵子,除ｐｕｒＢ外均肥反式调节基因ｐｕｒＲ的负调控。本文以ｐｕｒＪＨＤＯ＾＋和Ｏ＾ｃ突变体为材料,通过体内克隆法分别克隆到Ｏ＾＋ｐｕｒＪＨＫ（Ｐ２－９）和Ｏ＾ｃｐｕｒＪＨＤ（Ｐ＾ｃ－１２）操纵子;遗传互补和限制性内切酶分析作出了Ｐ２－９和Ｐ＾ｃ－１２的物理谱。经２次亚克克     

Identification of a STS marker linked to the Aegilops speltoides-derived leaf rust resistance gene Lr28 in wheat

 A sequence-tagged-site (STS) marker is reported linked to Lr28, a leaf rust resistance gene in wheat. RAPD (random amplified polymorphic DNA) analysis of near-isogenic lines (NILs) of Lr28 in eight varietal backgrounds was carried out using random primers. Genomic DNA enriched for low-copy sequences was used for RAPD analysis to overcome the lack of reproducibility due to the highly repetitive DNA sequences present in wheat. Of 80 random primers tested on the enriched DNA, one RAPD marker distinguished the NILs and the donor parent from the susceptible recurrent parents. The additional band present in resistant lines was cloned, sequenced, and STS primers specific for Lr28 were designed. The STS marker (Indian patent pending: 380 Del98) was further confirmed by bulk segregation analysis of F₃ families. It was consistently present in the NILs, the resistant F₃ bulk and the resistant F₃ lines, but was absent in recurrent parents, the susceptible F₃ bulk and the susceptible F₃ lines. Received: 20 February 1998 / Accepted: 4 March 1998     

RAPD based DNA markers linked to anthracnose disease resistance in Sorghum bicolor ( L.) Moench

Anthracnose caused by Colletotrichum graminicola is one of the major diseases of sorghum. The locus for disease resistance in sorghum [Sorghum biocolor (L.) Moench] accession G73 was found to segregate as a simple recessive trait in a cross to susceptible cultivar HC136. In order to identify molecular markers linked to the locus for disease resistance, random amplified polymorphic DNA (RAPD) analysis was coupled with bulk segregant analysis. DNA from the parental cultivars and the bulks were, screened by PCR amplification with 114 RAPD primers. Three RAPD primers amplified a sequence that consegregated with the recessive resistance allele, while another three amplified a band linked to the susceptible allele. The six disease linked markers were screened with individual resistant and susceptible genotypes to observe degree of linkage of identified RAPD markers with the gene for resistance. Two primer sequences (OPI 16 and OPD 12) were found to be closely linked to the locus for disease resistance.     

Identification, mapping, and application of polymorphic DNA associated with resistance gene Pm21 of wheat.

A new powdery mildew resistance gene designated Pm21, from Haynaldia villosa, a relative of wheat, has been identified and incorporated into wheat through an alien translocation line. Cytogenetic and biochemical analyses showed that chromosome arms 6VS and 6AL were involved in this translocation. Random amplified polymorphic DNA (RAPD) analysis was performed on recipient wheat cultivar Yangmai 5, the translocation line, and H. villosa with 180 random primers. Eight of the 180 primers amplified polymorphic DNA in the translocation line, and the same results were obtained in four replications. Furthermore, RAPD analysis was reported for substitution line 6V, seven addition lines (1V-7V), and the F1, as well as F2 plants of (translocation line x 'Yangmai 5'), using two of the eight random primers. One RAPD marker, specific to chromosome arm 6VS, OPH17-1900, could be used as a molecular marker for the detection of gene Pm21 in breeding materials with powdery mildew resistance introduced from H. villosa. Key words : RAPD analysis, 6VS-specific marker, Pm21, Erysiphe graminis f.sp. tritici, Triticum aestivum - Haynaldia villosa translocation.     

Screening and identification of random amplified polymorphic DNA (RAPD) markers linked to mungbean yellow mosaic virus (MYMV) resistance in mungbean (Vigna radiata (L.) Wilczek)

Bulk segregant analysis (BSA) and random amplified polymorphic DNA (RAPD) techniques were used to analyse the F2 individuals of susceptible VBN (Gg) 2 × resistant KMG 189 to screen and identify the molecular marker linked to mungbean yellow mosaic virus (MYMV) resistant gene in mungbean. Two DNA bulks namely resistant bulks and susceptible bulks were setup by pooling equal amount of DNA from five randomly selected plants of each disease response. A total of 72 random sequence decamer oligonucleotide primers were used for RAPD analysis. Primer OPBB 05 (5′-GGGCCGAACA-3′) generated OPBB 05 260 fragment in resistant parent and their bulks but not in the susceptible parent and their bulks. Co segregation analysis was performed in resistant and susceptible F2 individuals, it confirmed that OPBB 05 260 marker was tightly linked to mungbean yellow mosaic virus resistant gene in mungbean.     

Identification of the Molecular Markers Linked to the Salt-resistance Locus in the Wheat Using RAPD-BSA Technique

Calli from mature embryo of “Jimai-24” wheat ( Triticum aestivum L.) were induced on medium containing Zhengdingmycin then continuously cultured on medium containing 0.5% NaCl till to regenerate plants named 8901-17 salt-tolerant mutant. “Jimai-24” was compared with 8901-17 by using the technique of RAPD. Thirty-five out of 280 random primers could detect DNA polymorphism. The similarity index was 0.978, indicating that they were NILs (near-isogenic lines). Two F2 populations (“Jimai-24”×8901-17 and 8901-17דZhongmai-9”) had been constructed using the method of half-division. The two relative DNA pools (salt tolerant DNA pool and susceptible DNA pool) which come from the two F2 populations, respectively, had been made according to the method of BSA (bulked segregant analysis). RAPD analysis between the two DNA relative pools was carried on with above 35 random primers which could detect DNA polymorphism definitely. The identical polymorphism between the two sets of DNA pools come from the two F2 populations could be determined only by OperonQ4 primer. This result implied that the polymorphic fragment amplified by OperonQ4 primer was the molecular marker of RAPD closely linked to the salt tolerant mutation locus.     

Deletion of a large genomic segment in tobacco varieties that are resistant to potato virus Y (PVY)

Recessive alleles (va, va ¹ , va ² , etc) of the tobacco Va locus confer resistance to potato virus Y (PVY). To elucidate the mechanism underlying this resistance, we attempted to identify randomly amplified polymorphic (RAPD) markers that reveal polymorphism between two nearly isogenic lines (NILs) that differ in their susceptibility to PVY. Using each of 500 primers and 800 pairs of primers, we identified over 100 RAPD fragments that differed between the NILs. We applied these RAPD primers or primer combinations to an F₂ population obtained from a cross between the susceptible line BY4 and the resistant va ² -bearing NIL, F55. It was found that only 10 RAPD markers were polymorphic between resistant and susceptible plants. Unexpectedly, these markers were all linked to Va. All 10 RAPD markers were present in all 8 susceptible varieties tested. At least one RAPD marker was not detected in 8 out of 10 resistant varieties. Southern analysis revealed that the sequences of markers were not present in the genomes of resistant varieties, and the markers were found in individually distinct positions on the chromosomes of susceptible tobacco varieties. These results strongly suggest that the resistance conferred by va is due to deletions at the Va locus governing susceptibility to PVY. Received: 20 May 1999 / Accepted: 17 August 1999     

Identification and localization of molecular markers linked to the Lr9 leaf rust resistance gene of wheat

Near-isogenic lines (NILs) for the leaf rust resistance gene Lr9 were screened for polymorphisms at the molecular level. RAPD (random amplified polymorphic DNA) primers as well as RFLP (restriction fragment length polymorphism) markers were used. Out of 395 RAPD primers tested, three showed polymorphisms between NILs, i.e., an additional band was found in resistant lines. One of these polymorphic bands was cloned and sequenced. Specific primers were synthesized, and after amplification only resistant lines showed an amplified product. Thus, these primers define a sequence-tagged site that is specific for the translocated fragment carrying the Lr9 gene. A cross between a resistant NIL and the spelt (Triticum spelta) variety Oberkulmer was made, and F₂ plants were analyzed for genetic linkage. All three polymorphisms detected by the PCR (polymerase chain reaction) and one RFLP marker (cMWG684) showed complete linkage to the Lr9 gene in 156 and 133 plants analyzed, respectively. A second RFLP marker (PSR546) was closely linked (8±2.4 cM) to the Lr9 gene and the other four DNA markers. As this marker maps to the distal part of the long arm of chromosome 6B of wheat, Lr9 and the other DNA markers also map to the distal region of 6BL. All three PCR markers detected the Lr9 gene in independently derived breeding lines and varieties, thus proving their general applicability in wheat breeding programs.     

Identification of RAPD markers linked to the Uvf-1 gene conferring hypersensitive resistance against rust (<Emphasis Type="Italic">Uromyces viciae-fabae</Emphasis>) in <Emphasis Type="Italic">Vicia faba</Emphasis> L.

Bulk segregant analysis was used to identify random amplified polymorphic DNA (RAPD) markers linked to a gene determining hypersensitive resistance in Vicia faba line 2N52 against race 1 of the rust fungus Uromyces viciae-fabae. The monogenic nature of the resistance was determined by analyzing the F(2) population from a cross between resistant line 2N52 and susceptible line VF-176, and further confirmed in the F(2:3)-derived families. Linkage of the RAPD markers was confirmed by screening 55 F(2) plants segregating for resistance. Three RAPD markers (OPD13(736), OPL18(1032) and OPI20(900)) were mapped in coupling phase to the resistance gene for race 1 ( Uvf-1). No recombinants between OPI20(900) and Uvf-1 were detected. Two additional markers (OPP02(1172) and OPR07(930)) were linked to the gene in repulsion phase at a distance of 9.9 and 11.5 cM, respectively. The application of marker-assisted selection to develop new faba bean varieties with rust resistance genes is discussed.     

Molecular mapping of the py-1 gene for resistance to corky root rot (Pyrenochaeta lycopersici) in tomato

 We report the molecular mapping of the py-1 gene for resistance to corky root rot [Pyrenochaeta lycopersici (Schneider and Gerlach)] in tomato using RAPD and RFLP marker analysis. DNA from near-isogenic lines (NILs) of tomato differing in corky root rot resistance was screened with 575 random oligonucleotide primers to detect polymorphic DNAs linked to py-1. Three primers (OPW-04, OPC-02, OPG-19) revealed polymorphisms between the NILs. Twelve resistant and eight susceptible DNA pools derived from segregating F₃ families were used to confirm that the RAPD markers were linked to the py-1 gene. Two of the linked amplified fragments, corresponding to OPW-04 and OPC-02, were subsequently cloned and mapped on the tomato molecular linkage map as RFLPs. These clones were located between TG40 and CT31 on the short arm of chromosome 3. Further analysis with selected RFLP markers showed that 7% (8.8 cM) of chromosome 3 of the resistant line ‘Moboglan’ was introgressed from the L. peruvianum donor parent. Three RFLP markers (TG40, TG324, and TG479) from the introgressed part of chromosome 3 were converted to cleaved amplified polymorphism (CAP) markers for use in a polymerase chain reaction (PCR) assay. These PCR markers will allow rapid large-scale screening of tomato populations for corky root rot resistance. Received: 2 January 1998 / Accepted: 12 January 1998     

DNA polymorphism among barley NILs of cv. Pallas, carrying genes for resistance to powdery mildew (Blumeria graminis f. sp. hordei)

Barley powdery mildew, caused by the pathogen Blumeria graminis f. sp. hordei is an important disease of barley (Hordeum vulgare L.). The random amplified polymorphic DNA (RAPD) method was used to detect DNA polymorphism among 7 Pallas near-isogenic lines (NILs) carrying Mla3, Mla12, Mlk, Mlp, Mlat, Mlg and MlLa genes for resistance to B. graminis f. sp. hordei. From among 500 random 10-mer primers tested, 3 were specific for NIL P2 (Mla3), 1 for P10 (Mla12), 6 for P17 (Mlk), 46 for P19 (Mlp), 4 for P20 (Mlat), 6 for P21 (Mlg), and 4 for P23 (MlLa). The results of this study demonstrated that the RAPD technique is a useful tool for detecting DNA polymorphism among Pallas NILs.