核桃早实性状相关联的RAPD标记

运用RAPD技术进行核桃早实性状相关的分子标记研究,用180个随机引物,共扩增出1600条DNA带,选出5个多态性引物,进一步用这5个引物进行单株确认,发现只有引物OPG15在早实类群的8个个体中扩增出一条710bp的特异带,而晚实类群的8个个体中无此特异带。初步分析认为,OPG15710可能是与核桃早实性状相关的分子标记,该标记需进一步确定。     

核桃早实性相关性状的SCAR标记

用RAPD技术对新疆核桃早实特性的分子标记进行了研究。用180个10-mer随机引物分别扩增早实和晚实近等基因池DNA筛选出5个多态性引物,结果只有引物OPG15(5′-ACTGGGACTC-3′)扩增得到1条约710bp的早实核桃特异片段。对该片段进行克隆和序列分析,并根据序列分析结果将该RAPD标记转化为重复性和特异性更好的SCAR标记。研究设计出了1对早实核桃特异SCAR引物P1(5'-ACTGGGACTCCAATTGTATC-3')和P2(5'-ACTGGGACTCTCAACTAT-3'),用这对特异引物对14份材料进行PCR扩增,结果所有晚实核桃材料无任何扩增,但早实材料均扩增出了预期大小759bp的特异带。     

黄瓜全雌性基因连锁的AFLP和SCAR分子标记

 本研究以全雌品种‘戴多星’自交系和弱雌品种‘北京截头’自交系为双亲杂交获得F₁ ,然后得到F₂ 性型分离群体, 利用分离群体分组分析法(Bulked Segregant Analysis, BSA) 构建全雌和弱雌两个基因池, 筛选了64对AFLP选择性引物EcoR I-NN +Mse I-NNN组合, 发现EcoR I-TG +Mse I-CAC引物组合在全雌基因池中扩增出一条分子量为234 bp的特异带。经F₂ 代单株验证, 该特异条带能在全雌单株中稳定出现。以MAP MAKER (Version 310) 软件分析, 该标记与全雌性位点的连锁距离在617 cM。命名该连锁标记为TG/CAC₂₃₄。将该特异条带回收、克隆、测序, 设计特异SCAR引物, 再对F₂ 代单株基因组DNA进行扩增, 仅在全雌单株中扩增出1条分子量为166 bp 的特异带, 表明已成功地将与黄瓜全雌性连锁的AFLP标记转化为操作简便、表现稳定的SCAR标记, 该标记命名为SA₁₆₆。     

白菜抗霜霉病基因的RAPD标记

研究了与抗霜霉病基因紧密连锁的RAPD分子标记, 利用白菜抗病品种014和感病品种010杂交的F₂群体144个单株为材料, 通过人工接种鉴定, 采用高抗单株和高感单株分别构建抗、感病池, 利用BSA法筛选了560条RAPD引物, 其中引物AY12在抗感病池中扩增出多态性片段AY12₁₂₃₈ , 通过F₂单株验证后证明AY12₁₂₃₈与抗霜霉病基因紧密连锁, 其遗传距离为6.7 cM。     

茄子抗青枯病基因的RAPD标记研究

 为探明茄子抗青枯病的遗传机制, 以高感青枯病品种北京六叶茄064, 高抗青枯病半栽培种马来西亚S3及其F₂ 代为材料, 用RAPD技术对供试材料的抗青枯病基因进行了研究。结果表明: 通过300个随机引物的PCR扩增分析, 发现15.6%的引物在双亲间表现出多态性, 找到了一个与茄子抗青枯病亲本S3中的抗病基因紧密连锁的分子标记S264₇₈₀ (该引物的碱基序列为CAGAGCGGA) , 该标记与S3的抗病基因的交换值为4.32% , 遗传距离为4.33 cM。     

番茄抗晚疫病基因Ph-3的RAPD标记

 以番茄抗晚疫病材料CLN2037和感病材料T2-03杂交的F₂分离群体的147个单株为试材, 通过抗病性鉴定, 选择高抗单株和高感单株分别建抗、感病池。采用BSA法筛选了230条RAPD引物, 其中引物CCPB272-03 (序列为GGTCGATCTG) 在抗、感病池扩增出多态性片段CCPB272-03₇₄₀ , 通过F₂单株验证后证明CCPB272-03₇₄₀与抗晚疫病基因Ph-3紧密连锁, 遗传距离为518 cM。     

桃花粉可育基因连锁的RAPD 标记与SCAR标记的转化

 桃品种以重阳红(花粉不育) ×大久保(花粉可育) 的52株F₁代群体为试材, 应用RAPD技术, 结合集群分组分析法(Bulked Segregate Analysis, BSA) 构建花粉可育/不育基因池, 利用180个随机引物, 筛选在花粉可育基因池中稳定扩增的一个RAPD标记OPW03-900。经过重复性验证和群体单株验证, 该标记仅在花粉可育个体(重组型除去) 中出现, 与桃花粉可育/不育位点的连锁距离为5.80cM。将该特异性片段回收、克隆、测序, 并设计一对特异SCAR引物, 再对F1代个体进行PCR扩增, 发现该特异带的位点及重组型个体的数目与RAPD扩增结果一致, 片段大小为906 bp, 命名为SCW03-906,表明RAPD标记已成功转化为与桃花粉可育基因连锁的SCAR标记。该序列已被GenBank收录, 登录号为DQ659676。     

京白梨等品种S 基因型鉴定及新基因S28 和S30 的核苷酸序列分析

 根据梨S基因高度保守区C1和C3区, 设计1对引物P1和P2, 对梨品种的基因组DNA进行S基因特异扩增、克隆、测序, 并在GenBank中BLAST比较, 确定S 基因特异性片段, 对京白梨等6个供试自交不亲和品种的S基因型比对结果为: 白梨中的‘库尔勒香梨’为S₂₁ S₂₈ , ‘苹果梨’为S₁₇S₁₉ ; 砂梨中的‘台湾蜜梨’为S₁₁ S₂₂ ; 西洋梨中的‘葫芦梨’为S_a S_b; 秋子梨中的‘京白’为S₁₆ S₃₀ , ‘早梨18’为S₄ S₂₈。其中S₂₈和S₃₀为首次登录的新S 基因, 在GenBank的登录号分别为AY562394 (库尔勒香梨) 和AY876945 (京白) 。     

桃果实非酸/酸性状SCAR标记的转化与验证

 根据筛选到的与桃果实非酸/酸性状紧密连锁的3个AFLP标记特异片段序列信息, 设计特异引物BFPA1/A2、BFPB1/B2和BFPC1/C2。3个特异引物在‘京玉’桃、‘美味’油桃及其正反交F¹代69株群体中的PCR检测结果表明: 引物BFPB1/B2在果实性状表现为酸的后代个体中扩增出分子量大小为158 bp的特异条带, 且根据特异片段AT-CTA₁₉₉不同部位设计的引物都能稳定扩增, 只是片段大小有所差异;特异扩增检测F¹群体分离结果与原先AFLP_AT/CTA标记完全一致, 表明AFLP标记已成功转化为SCAR标记,该SCAR BFPB1/B2标记与D基因的连锁距离为2199 cM。利用SCAR BFPB1/B2标记对‘大久保’桃ב兴津’油桃的F²群体60株个体的果实非酸/酸性状进行检测, 基因型与表型性状符合率为96.7% , 并且表现为共显性标记。特异引物BFPA1/A2和BFPC1/C2的扩增多态性条带在亲本及后代中消失。     

与黄瓜抗炭疽病相关基因连锁的AFLP标记的筛选

以黄瓜抗炭疽病母本66和感炭疽病父本A18及其F₂代分离群体为试材, 采用BSA法和AFLP技术建立了对炭疽病的抗病组和感病组, AFLP引物组合E24M48在抗感组间表现多态性, 且呈共显性。经220个F₂单株分析, 在高抗单株和高感单株中分别仅扩增出251 bp和245 bp的特异片段, 而在中间类型个体中同时扩增出了该两个特异片段。连锁值测定结果表明, 该特异带与黄瓜炭疽病抗病相关基因紧密连锁,距离为21727 cM。测序结果显示, 该两个片段的大小分别为251 bp和245 bp, 并且两个片段的差异在于两个“TCT”重复序列(6个碱基) 的插入或缺失。     

番木瓜雄性性别的RAPD和SCAR标记

利用RAPD标记技术鉴定番木瓜雄性性状。在214条随机引物中,用引物Z18扩增得到1条雄性多态性片段,此片段存在于试验所用材料的所有雄株中而两性株和雌株没有,将其命名为Z18-1000。根据对该片段的序列分析结果重新设计了2对引物将RAPD标记成功转化成了SCAR标记,并命名为SD-1000。     

Identification of RAPD markers linked to nut weight and plant stature in cashew

Cashew is an important edible nut crop of tropics. Bulk segregant analysis (BSA) was carried out on DNA bulks constituted from F₂ population and germplasm in order to link or associate molecular markers with economic characters. In all 458 RAPD, 31 ISSR and 21 pairs of SSR primers were used and identified polymorphic markers between parents. Though screening F₂ bulks with these markers identified markers polymorphic between the bulks but none could be validated with the individuals of their bulks. Hence screening with germplasm bulks was carried out and could identify four RAPD markers polymorphic between the bulks for nut weight and plant stature and also between the individuals of their bulks. Of the four, three markers were associated with nut weight amplifying at 775, 475, 275, bp region in primers OPN 14, UBC 184 and UBC 185 respectively. Out of these three, two markers were specific to low nut weight and one marker was specific to high nut weight and their bands were present in greater frequency (50–77.8% and 75%) of individuals constituting the respective bulks. Similarly, the another marker UBC 185₂₇₅ was detected which was specific to low plant stature and was present in 66.7% and 10% individuals constituting short and tall bulks respectively. Markers identified with bulks and with the individuals of bulks were validated further with more individuals of F₂ and germplasm.     

Identification of RAPD marker linked to fruit skin color in Japanese pear (Pyrus pyrifolia Nakai)

In Japanese pear, fruit skin color is a very important trait for growers because the russet skin protects the fruit against external stress caused by disease, insects, bad weather, and shipping. This report describes the development of a randomly amplified polymorphic DNA (RAPD) marker linked to major genes controlling the fruit skin color in Japanese pear. Two F₁ progenies from the cross of ‘Kousui’ × ‘Kinchaku’ and ‘Niitaka’ × ‘Chikusui’ segregated by fruit skin color were used for bulked segregant analysis. Four kinds of bulked DNA were constructed and used for the polymerase chain reaction in RAPD analysis. After 200 random primers were screened against four bulks, a band named OPH-19₄₂₅ was selected in cooperation with green bulks. The recombination rate between OPH-19₄₂₅ and the green skin phenotype was 7.3%. The RAPD marker (OPH-19₄₂₅) could select green fruit with probability as high as approximately 92%. The marker was apparently useful for the selection of green-skinned individuals in a breeding program. This is the first report on developing a DNA marker closely linked to the fruit phenotype in Japanese pear.     

中国野生葡萄副梢长度的RAPD标记

为培育短副梢葡萄品种,对中国野生葡萄副梢长度性状进行调查,以葡萄短梢×长梢的葡萄种间杂交组合(燕山-1×河岸葡萄)F183个单株为试材,采用BSA法和RAPD技术,通过369个随机引物的筛选,获得了1个与中国野生葡萄副梢长度基因连锁的RAPD标记OPB07-2000,并在中国野生葡萄17个种46个株系中得到验证。这就为葡萄短副梢分子辅助育种提供了科学依据,进而从根本上解决葡萄生产上夏季副梢生长过旺的问题。     

桃早熟芽变品种‘大观一号’的RAPD分析及其特异片段的克隆

利用140个单个和混合随机引物对桃布日早生及其早熟芽变品种'大观一号'基因组DNA的多态性进行RAPD分析。其中有一个引物能检测出两者之间DNA的多态性，在1．1kb处'大观一号'多了一条特异性条带，将该片段克隆到pUC19载体，并作了酶谱分析。     

Combination of SCAR primers and Touchdown-PCR for sex identification in Pistacia vera L.

A combined method of sequence characterized amplified regions (SCAR) primers and Touchdown-PCR was used for the development of a female DNA marker in Pistacia vera L. The random decamer primer OPO-08 amplified a 905-bp fragment in all female trees, but also in several males. SCAR primers designed on the basis of the RAPD female molecular marker amplified a 905-bp female and a 909-bp male fragment. Sequencing these fragments revealed high homology, with several point mutations, four deletions in female and one in the male sequences. A second internal set of SCAR primers designed on the basis of a polymorphic locus, in combination with Touchdown-PCR technique, amplified a specific female 297-bp product. The diagnostic reliability of the new female specific marker was verified on 54 different genotypes. The method reported here offers a simple and reproducible way for early gender determination in P. vera.     

中国野生葡萄抗黑痘病基因的RAPD标记

应用随机扩增多态性DNA(RAPD)技术 ,对抗病×感病的葡萄种间杂交组合白河 35 1×佳利酿的亲本及其 2 3株F1代单株进行了抗黑痘病的遗传分析 ,通过对 196个随机引物的筛选获得了 1个与中国野生葡萄抗黑痘病基因连锁的RAPD标记OPS0 3 130 0 ,并在所有供试的中国野生葡萄 7个种的 2 0个株系和欧洲葡萄 8个品种中得到了验证。这一分子标记将为葡萄抗病育种和深入研究中国野生葡萄的抗黑痘病基因提供DNA依据     

桃果实离核性状的RAPD分子标记及克隆

为获得桃果实离核性状基因的分子标记,以桃(Prunus persica L.)品种京玉和美味的69株正反交F1代为试材,采用RAPD分析技术和BSA法,获得了控制桃果实离核性状基因的RAPD分子连锁标记OPI07-1000,该标记与目的基因的连锁遗传距离为2.5cM。对该片段回收、克隆后进行测序,得到全序列,长度为1054bp,该序列可作为进一步合成检测桃果实离核性状探针的基础,用于早期检测果实性状和分子标记辅助育种。