利用RAPD分析鉴定花椰菜杂种纯度

应用随机扩增多态性DNA(RAPD)分析方法,鉴定花椰菜F1代杂种纯度。筛选出20个10bp随机引物对杂种F1代和父母本基因组DNA进行RAPD分析,共获得扩增片段124条,分子量在0.3-3kb之间,其中2个引物S120和S174可用来鉴定杂种纯度。RAPD分析结果与田间形态鉴定结果基本一致,表明RAPD分析方法适用于花椰菜杂种的纯度鉴定。     

RAPD法检测番茄种子纯度的研究

以番茄F1代杂交品种“强选1号”的干种子为材料,用RAPD方法对种子的纯度进行了初步的鉴定。所用RAPD引物是从110个RAPD随机引物中筛选出的BA1072号引物。使用这种方法不需进行田间种植即可完成F1代要种的纯度鉴定工作。     

利用RAPD分子标记对番茄杂交种纯度的鉴定研究

应用RAPD(RandomlyamplifiedpolymorphicDNA)分子标记对番茄京丹1号和毛粉802的F1代杂交种纯度进行鉴定的实验研究。该项研究使用了10个碱基的单随机引物和10个碱基的双随机引物进行扩增。在60个单引物扩增反应中获得7个京丹1号父本特有的核酸标记片段。但在14个双随机引物对京丹1号和毛粉802杂交组合的扩增反应中获得了7个京丹1号F1代杂交种特有的核酸标记片段和5个毛粉802父本特有的标记带。实验结果显示,双引物的扩增反应对鉴定双亲亲缘关系极近的杂交种纯度较单引物扩增反应更有效。其中,京丹1号的14个标记片段在北京蔬菜研究中心,种子纯度检测室又进行了重复扩增实验。实验结果为87%的RAPD标记可以在使用不同的PCR仪和不同来源的Taq酶的实验条件下得到。RAPD分子标记技术对鉴定双亲亲缘关系极近的杂交种纯度是真实可靠的。     

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.     

Assessment of RAPD markers for barley doubled haploid lines resistant and susceptible to Fusarium culmorum at seedling and adult plant growth stages

Thirty doubled haploid (DH) lines of barley derived from F(1) of a cross between the six-rowed cultivar Pomo and two-rowed cultivar Maresi were examined for susceptibility to Fusarium seedling blight (SB) and head blight (FHB), measured by mycotoxin (nivalenol) content of kernels. RAPD (random amplified polymorphic DNA) polymorphism was analysed by using 53 decamer primers. Amplification products (APs) were 200 bp up to 2000 bp in size on average 5.7 per primer and the total number of APs was 284, 51.06% of which were polymorphic. Only 32 APs differentiated the examined DH lines - 19 APs for nivalenol content of kernels and 13 for seedling resistance. DH lines segregated with continuous distribution of resistance to FHB and SB. At the seedling stage all DH lines exhibited lower susceptibility than parental cultivars, but in the adult stage only two lines (MP 2 and MP 7) appeared to be more resistant to FHB, i.e. accumulated in kernels a lower amount of mycotoxin than cultivars Maresi and Pomo.     

红莲型杂交稻(红莲2号)及其骨干亲本的RAPD分析与鉴定

利用RAPD技术,从248个随机寡核苷酸引物（10－mer）中筛出18个引物对红莲型杂交稻组合红莲2号及其亲本（T－07A、T－07B、YD6－05）,另6个红莲型胞质不育系的骨干恢复和汕优63及其亲本共14份水稻材料进行分析。共检测到173个多态性标记。聚类分析结果表明：不育系与保持系间因核背景相似,遗传差异很小;杂种（F1）的基因型更倾向于恢复系;恢复系与保持系间遗传距离的相对较大,但各恢复系之间的遗传距离较小。利用这些标记能有效地地区交组合中不育系,保持系、恢复系和杂种（F1）。     

RAPD及SRAP两种分子标记技术对苦瓜杂交种纯度的鉴定分析

以F1代苦瓜杂交种如玉11号及其亲本为材料,利用RAPD及SRAP两种分子标记技术对这3种苦瓜基因组DNA进行比较分析,以获得该杂交种及其亲本（或母本）差异目的基因片段。经过多次对该3种苦瓜叶片DNA提取,PCR扩增及其PCR产物的琼脂糖凝胶电泳分析,在供试的46个RAPD引物及121对SRAP引物中,筛选出1个RAPD引物及1对SRAP引物能区分该苦瓜杂交种及其母本种子,通过进一步验证分析,证明该两种分子标记的特异引物可作为如玉11号苦瓜杂交种子的纯度鉴定之用。     

Identification of the BrRHP1 locus that confers resistance to downy mildew in Chinese cabbage (Brassica rapa ssp. pekinensis) and development of linked molecular markers

Inheritance of resistance to downy mildew (Hyaloperonospora parasitica) in Chinese cabbage (Brassica rapa ssp. pekinensis) was studied using inbred parental lines RS1 and SS1 that display strong resistance and severe susceptibility, respectively. F(1), F(2), and BC(1)F(1) populations were evaluated for their responses to downy mildew infection. Resistance to downy mildew was conditioned by a single dominant locus designated BrRHP1. A random amplified polymorphic DNA (RAPD) marker linked to BrRHP1 was identified using bulked segregant analysis and two molecular markers designated BrPERK15A and BrPERK15B were developed. BrPERK15B was polymorphic between the parental lines used to construct the reference linkage map of B. rapa, allowing the mapping of the BrRHP1 locus to the A1 linkage group. Using bacterial artificial chromosome clone sequences anchored to the A1 linkage group, six simple polymerase chain reaction (PCR) markers were developed for use in marker-assisted breeding of downy mildew resistance in Chinese cabbage. Four simple PCR markers flanking the BrRHP1 locus were shown to be collinear with the long-arm region of Arabidopsis chromosome 3. The two closely linked flanking markers delimit the BrRHP1 locus within a 2.2-Mb interval of this Arabidopsis syntenic region.     

陕油8号种子纯度的RAPD鉴定研究

从杂交油菜“陕油8号”及其亲本中提取基因组DNA,用100个RAPD随机引物进行扩增,从中筛选出3个可将亲本和子代区分的引物BA208、BA1090、BA497。BA208产生亲本互补的特征带BA208-1050bp、BA2081250bp;BA1090产生母本特征带BA1090-700bp,BA497产生父本特征带BA497-870bp,上述谱带均在子代中出现。以BA208产生的特征谱带作为分子标记对杂交油菜种子纯度鉴定得到了一致的结果,并与大田纯度检测结果一致。BA497可将“陕油8号”与当地4个主栽品种有效区分。此外,还对双引物共同鉴定杂交种子纯度问题进行了初步探讨。     

同工酶差异位点分析在蔬菜杂交种纯度检测中的应用

用１０种同工酶和蛋白质和分析体系,分析了７种重要蔬菜的７１个杂交种与其亲本之间的差异位点以及这些差异位点用于杂交种纯度检测的可能性和存在的问题。试验表明,作物的不同种类,杂交种与亲本之间的亲缘关系以及作物种类的遗传多态性,都会影响同工酶差异位点产生的多寡,从而影响到这一技术是否能用于此种作物的纯度检测,分析了不同同工酶在不同蔬菜中的多态性以及在蔬菜种子纯度检测中的表现。     

大白菜杂交种''冠春''杂交率的RAPD分析

从春大白菜品种‘冠春’及其亲本中提取基因组DNA,用320个随机引物进行RAPD扩增,从中筛选出5 个可将亲本和子代区分的引物S4、S47、S73、S134和S194。S4产生父本特征带S4-370;S47和S134产生母本特征带S47-700 和S134-1200;S73和S494产生亲本互补的特征带S73-660、S73-730和S494-400、S494-1770,上述谱带均在子代中出现。以这5个引物产生的特征谱带建立杂交种‘冠春’及其亲本的RAPD特异指纹。通过对134个‘冠春’的种子进行纯度鉴定,结果表明2个父本和4个母本与大田检测结果完全一致。进一步验证了4种鉴定大白菜杂交种方法的可行性。     

利用SSR标记鉴定西瓜杂交种纯度的研究

以2个西瓜杂交品种(系)的种子黑公子和04-17及其亲本为材料,用SSR标记技术研究杂种与其双亲之间的扩增谱带多态性,以甄别真假杂种.结果发现,所试验的52对SSR引物中有13对引物分别在2个西瓜杂交种和其双亲之间存在扩增条带的多态性,表现为:多数SSR引物对自交系的扩增只出现1条带,但部分引物在某些自交系中扩增出2条带,杂交种条带均为父母本的互补型,很适合做杂交种纯度鉴定.用引物CMCT134b对黑公子和引物CMGA165对04-17进行了各100粒单种子SSR鉴定,所测纯度分别为96%和100%,与田间纯度95.6%和99.7%非常接近,表明SSR标记技术在西瓜杂交种子纯度室内快速检测中的应用前景.     

Hybrid rice (Oryza sativa L.): identification and parentage determination by RAPD fingerprinting

Summary DNA from three families of rice plants selected in Northern China (each comprising the male sterile, the restorer, the hybrid F1 and the maintainer lines) has been extracted and amplified by PCR with different random DNA primers (RAPD analysis). Then, DNA has been analysed by agarose gel electrophoresis and DNA bands scored as present or absent. The generated matrices are reproducible and amenable for identification of each single plant line. Thus, RAPD fingerprinting of the inbred parental lines and of the resulting hybrid is proposed as a convenient tool for the identification, protection and parentage determination of plant hybrids. Furthermore, by offering a molecular tool to verify the degree of dissimilarity between the parental lines, the RAPD analysis may also be used to search for new parental combinations.     

利用RAPD分子标记对三组三系杂交水稻及亲本的遗传分析和鉴定

利用RAPD技术,从248个随机寡聚核苷酸（10bp）中筛选出13个引物能在供试的三组三系杂交水稻及亲本间扩增出43条稳定性较好的多态性片段,其中6个引物能在供试材料间扩增出20个强的多态性标记。利用这些标记能有效地区分各组合中不育系、保持系、恢复系和F1,并能看出各组合中不育系与保持系、不育系与恢复系、F1与亲本间的遗传关系。 Abstract:A total of 248 arbitrary 10-mer oligonucleotide primers were screened using RAPD (random amplified polymorphic DNA) techniques with the genome DNA of three groups of three-line hybrid rice and their parents.Thirteen primers produced 43 polymorphism fragments.Six primers of them produced 20 obviously repeatable polymorphic markers among rice lines tested.Using this RAPD markers,the hybrid rice combinations (sterile-line,maintainer-line,restorer-line and F1)can be effectively identified,and the genetic relationship among them can be shown.     

Genetic Mapping of Soybean [Glycine max (L.) Merr.] Using Random Amplified Polymorphic DNA (RAPD)

Random amplified polymorphic DNA (RAPD) is based on DNA amplification by polymerase chain reaction (PCR) of random DNA segments using single arbitrary nucleotide sequences. We have adapted the assay to soybeans by using Stoffel Fragment DNA polymerase and by optimizing the reaction conditions. To increase the percentage of RAPD polymorphisms, the DNA template was digested with restriction enzymes before amplification. The combination of twenty-four primers and five DNA template treatments (Undigested, DraI, EcoRI, HindIII, and TaqI digested) revealed 94 polymorphic DNA fragments differing between soybean lines PI437654 and BSR101. Many polymorphic DNA bands were found unreliable or non-scoreable after re-screening of primers and verification of marker-allele segregation with 20 recombinant inbred lines (RILs). However, 28 RAPD markers were consistently polymorphic between the parental lines and followed Mendelian expectations. The use of DNA templates digested with DraI, EcoRI, HindIII or TaqI increased three times the number of RAPD markers compared to undigested DNA template alone. The 28 RAPD markers obtained were further screened with 72 RILs and placed on an existing RFLP map.     

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.     

Evaluation of genetic diversity and uniformity of head cabbage DH lines by the use of RAPD markers

DH lines derived from cabbage cvs. Kamienna G?owa, S?awa z Enkhuizen and Langendijker, representing R1 generation, were analysed by the use of RAPD markers for their diversity and uniformity. For the evaluation of genetic diversity, eight primers yielding informative bands were used. Of the total of 83 RAPD bands scored in this study, 16.9% were polymorphic between a set of 13 DH lines. The similarity of the DH lines, estimated by Jaccard's coefficient, was depicted in the UPGMA dendrogram. Fourteen generated informative RAPD bands allowed the identification of DH lines developed from each cultivar. The evaluation of the uniformity for six closely related DH lines was possible by the use of three primers which generate one or two polymorphic bands. The lack of differences among ten plants of the five investigated DH lines manifested their uniformity. One line showed intraline polymorphism with two RAPD primers. The occurrence of the differences at the molecular level among ten plants indicated that their parental R0 plant was probably obtained from somatic cells, not by androgenesis.     

与小麦白粉病抗性基因Pm2紧密连锁RAPD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ＯＰＭ０８(１６００)、ＯＰＩ０４(１７００)、ＯＰＨ１９(１１００、ＯＰＥ０９(９００)及ＯＰＭ１６(８５０)。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ＯＰＩ０４(１７００)在１２个抗病材料中出现（另两个抗病材料中未检测到）,而在９个感病材料中均未出现。进一步用 ＯＰI(０４)对１０２株（Ｃｈａｎｃｅｌｌｏｒｘ Ｕｋａ／８＊Ｃｃ）Ｆ２分离群体进行分析,估算出标记ＯＰＩ０４(１７００)与Ｐｍ２基因间的遗传距离为１２．２±３．３ｃＭ。     

Molecular characterization of endophytic strains of Fusarium verticillioides (= Fusarium moniliforme) from maize (Zea mays. L)

The species Fusarium verticillioides (= F. moniliforme) is often found in maize seeds, constituting an important source of inoculum in the field. Fusarium spp., associated with symptomatic and asymptomatic plants, may be a primary causal agent of disease, a secondary invader or an endophyte. In the present work, endophytic fungi were isolated from two populations of Zea mays (BR-105 and BR-106) and their respective inbred lines. Within different inbred lines of maize, Fusarium was found at a frequency of 0 to 100% relative to the number of total isolated fungi. The frequency with which the genus occurred was practically the same in the two field sites (around 60%). Twenty-one F. verticillioides strains were analysed using the random amplified polymorphic DNA (RAPD) technique, employing 10 random primers. Variability analysis of endophytic isolates via RAPD showed genome polymorphism taxa of species around 60%. Endophytic isolates were clustered by their sites of origin. RAPD analysis clustered the endophytic isolates by their maize inbred lines hosts (Mil-01 to Mil-06), whereas at site A they clustered into two major groups related to the maize gene pool (BR-105 or BR-106 population). All strains isolated from seeds collected in Site A, except strains L9 and L10, were sub-grouped according to maize inbred lines. The analysis showed a discrete sub-grouping at site B. Results obtained here could be explained by a co-evolution process involving endophytic isolates of F. verticillioides and maize inbred lines.     

Stability of RAPD fingerprints in potato: Effect of source tissue and primers

Variations in random amplified polymorphic DNA (RAPD) profiles from leaf, stem, root, and tuber tissues were observed in case of two glasshouse grown potato cultivars using 40 decamer primers suggesting possible danger of cultivar misidentification. Genomic DNA extracted from the above four tissues of four in vitro grown potato cultivars, however, produced more uniform RAPD fingerprints. A significant effect of random primers on fingerprint uniformity was observed in case of both glasshouse and in vitro grown samples. A new concept of stability index for random primers based on homogeneity of RAPD profiles obtained from different tissues of a single plant have been introduced. It is concluded that RAPD analysis of genomic DNA extracted from any tissue of in vitro grown potato plants using 14 selected decamer primers could be used to develop RAPD fingerprints for identification of Indian potato cultivars.