‘石硖’龙眼大果型桂花味芽变系的RAPD分析鉴定

 用160个10 bp单个和混和随机引物对‘石硖’龙眼大果型桂花味芽变母树芽变枝及普通枝的基因组DNA进行RAPD扩增分析, 从中筛选出6个单引物( S71、S161、S303、S304、S341、S1212) 和4个双引物( Z7、Z59、Z60、Z70) 能在二者之间扩增出稳定的多态性片段。10个引物共扩增出99条片段, 其中多态性片段有S71-560、S161-813、Z70-800等13条, 占13.1% , 表明芽变枝果实大小及风味的变化与其遗传物质的改变密切相关。用两个特异引物S71、Z70对来源于同一芽变枝不同树龄的芽变嫁接单株及对照进行RAPD扩增, 所得特异片段与引物筛选中完全一致, 进一步表明芽变引起的遗传物质的改变可通过无性繁殖的方式稳定存在, 利用特异引物可对芽变嫁接单株的结果性状做早期预测。     

山楂（Crataegus pinnatifida Bge.)遗传多样性的RAPD和ISSR标记分析

 利用RAPD和ISSR标记对35份山楂（Crataegus pinnatifida Bge.）资源进行了DNA多态性分析。12个RAPD引物共扩增出110条清晰的谱带,其中89条显示多态性,平均每个引物扩增出7.4条多态性谱带。13个ISSR引物共扩增出110条清晰的谱带,其中94条显示多态性,平均每个引物扩增出7.2条多态性谱带。基于RAPD和ISSR标记,利用UPGMA分别构建了35份山楂资源的聚类树状图。距离系数分别为0～0.62（RAPD）和0～0.64（ISSR）,表明山楂具有较高的遗传多样性。     

中国工厂化栽培杏鲍菇菌株的RAPD和ISSR分析

为了研究国内工厂化栽培的杏鲍菇菌株的遗传多样性,对筛选的34个杏鲍菇菌株进行RAPD(Randomly Amplifiled Polyrnorphic DNA)和ISSR(Inter-Stmple sequence Repeat)分析。从31个RAPD引物中选取了23个引物对34株杏鲍菇工厂化生产菌株进行PCR扩增,共扩增出266条稳定清晰的DNA条带;从32个ISSR引物中选取了23个引物对34株杏鲍菇工厂化生产菌株进行PCR扩增,共扩增出211条稳定清晰的DNA条带。根据扩增结果,对34个杏鲍菇菌株进行了RAPD标记、ISSR标记及二者相结合的聚类分析。3种方法的分析结果相近,可分别将34个供试菌株分为4~5大类。研究为国内工厂化杏鲍菇栽培菌株的筛选、育种和栽培奠定基础。     

双孢蘑菇颜色基因分子标记的初步筛选

以4个白色和4个棕色双孢蘑菇(Agaricus bisporus)菌株,对其500个随机扩增多态性DNA(random amplified polymorphic DNA,RAPD)随机引物进行筛选,筛选出的S33、S438、S485 3个引物在棕色菌株组能扩增出4个特异条带(大小300~2000bp),将特异片段回收,克隆测序,将合成的4对特征序列扩增区域(sequence characterized amplified regions,SCAR)标记引物对48个菌株进行验证,结果表明:仅SCAR41200在24个棕色菌株的22个中能扩增出特异条带,而所有白色菌株中都没有此条带,表明该标记与棕色菌株的棕色基因相关。     

大丽菊杂合花色突变体的发现及RAPD鉴定

以新发现的大丽菊花色突变体为材料,选取10个具有10个碱基长度的随机引物,对5种突变个体的基因组DNA进行了RAPD分析.结果表明:10个随机引物共扩增出152条DNA条带,分子量大小在250～1 600 bp之间,其中有4个引物的扩增结果中检测到了差异性条带,在DNA水平上初步证实了5种花色突变体是由于遗传因素导致的稳定变异.     

月季''''金银岛''''的红花芽变品系的分析鉴定

芽变育种在月季新品种选育中至关重要.为了快速区分真正可遗传的芽变和环境因素引起的饰变,采用RAPD和ISSR对月季'金银岛'的疑似芽变株系'往日情怀'进行了标记分析,RAPD和ISSR引物都能扩增出差异多态性条带.结果表明:该芽变是由基因组变异引起的可遗传的稳定变异,是与花青素合成相关基因的突变.这为花色相关变异基因分离克隆以及'往日情怀'芽变新品种申报奠定了基础.     

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。     

石斛属植物遗传多样性RAPD分析

应用随机扩增多态性DNA(RAPD)技术,对石斛属植物16个种进行了基因组DNA多态性分析.从82个随机引物中筛选出20个多态性好的引物进行RAPD扩增,扩增DNA片段数据用UPGMA聚类法构建系统发育树.20条引物共扩增出142条带,多态性带118条,约占总数的83.1%.聚类结果显示RAPD分子标记构建的系统发育树与经典分类系统一致.RAPD标记可为石斛属植物的系统分类研究提供分子生物学依据.     

红掌组培变异株的特异DNA片段分析

利用ISSR引物对红掌亲本及其组织培养后代的形态变异株进行2次PCR扩增分析,回收特异DNA片段,测定其扩增产物的基因序列,并用BLAST方法进行同源性分析。结果表明:变异株的基因组特异DNA片段为680 bp,在核酸和蛋白质水平均没有发现同源性较高的基因或蛋白质,说明红掌在组织培养过程中可能产生新的遗传重组,出现新的基因型。     

西瓜隐性核雄性不育基因的RAPD标记

 应用RAPD分子标记技术, 采用BSA法对西瓜隐性核不育材料Se18的不育基因进行了分子标记研究。结果表明, 220个引物中, 只有引物S1167、S357在不育株DNA中扩增出了多态性特异片段, 在可育株DNA中未扩增出多态性片段。验证结果表明, 引物S1167在12个不育材料中的138个不育株全部扩增出特异性片段, 可以区分不育株与可育株。对特异性片段进行回收、克隆和测序, 其结果表明, S1167扩增特异片段有2 391个碱基对。     

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.     

与黄瓜矮生基因连锁的ISSR标记及其SCAR转换

以黄瓜矮生品系D8与蔓生品系JIN5杂交并自交得到的124株F₂代为试材,应用ISSR分子标记技术和BSA（混合群体分组分析法）法寻找与黄瓜矮生基因连锁的分子标记。从80条ISSR引物中筛选到一个黄瓜矮生性状特异的多态性引物UBC818,经F₂代单株验证,UBC818的扩增片段在蔓生黄瓜JIN5植株中稳定出现,而在矮生黄瓜D8植株中没有。连锁关系分析表明,标记与黄瓜矮生基因间的遗传距离为11.1 cM。根据对该片段的序列分析结果重新设计了1对引物,将ISSR标记成功转化成了SCAR标记, 并命名为UBC818-S。     

Identifying the markers and tagging a leucine-rich repeat receptor-like kinase gene for resistance to anthracnose disease in vegetable cowpea [Vigna unguiculata (L.) Walp.]

Anthracnose caused by the fungus Colletotrichum lindemuthianum is the most destructive disease of cowpea. Field-type cowpeas show various levels of resistance, whereas pole-type vegetable cowpeas are highly susceptible. Transfer of resistance available in field types to vegetable types is a major breeding objective in cowpea. This paper details the development of an F₂ mapping population by crossing field-type cowpea variety Kanakamony (Vigna unguiculata ssp. cylindrica) with pole-type vegetable cowpea variety Sharika (Vigna unguiculata ssp. sesquipedalis), screening this population with artificial inoculation and Bulked Segregant Analysis (BSA) with random marker systems Random Amplified Polymorphic DNA (RAPD) and Inter-Simple Sequence Repeats (ISSR); the objective is to identify the markers linked with major resistance-contributing genes. RAPD primer OPA02 has yielded marker at 850 bp in susceptible genome, whereas ISSR primers UBC810 and UBC811 have yielded markers at 1.4 kb and 1.5 kb respectively in resistant genomes. These markers were reproducible and their linkage with resistance and suitability in marker assisted selection (MAS) were confirmed through co-segregation analysis in F₃ population. UBC811 marker was eluted, cloned on pGEM-T, and sequenced. The sequence had shown that this marker is anchored on LRR receptor-like serine/t\hreonine protein kinase gene which could be involved in the resistance mechanism.     

A comparative analysis of the genetic diversity between inbred lines of Zinnia elegans using morphological traits and RAPD and ISSR markers

Genetic diversity within Zinnia elegans is key to the genetic improvement of this important ornamental species. Here, morphological traits and RAPD and ISSR molecular markers were used to assess levels of polymorphism across 20 inbred lines. Thirty-four morphological traits were scored and also 147 RAPD marker-fragments, as amplified by 12 arbitrary primers, and 128 ISSR marker-fragments as generated by 9 primers. The number of polymorphic loci, the percentage of polymorphic loci, Shannon's Information index (I) and the effective number of alleles (Ne) were calculated from the RAPD data as 100, 68.03%, 0.3559 and 1.4169, respectively. From the ISSR data, these respective statistics were calculated as 97, 76.38%, 0.4013 and 1.4728. Thus, ISSR markers were considered slightly superior to RAPD markers for assessing genetic diversity between the accessions; however, Mantel's test indicated significant correlation (R = 0.733) of the RAPD and ISSR results. By contrast, the morphological matrix showed low correlation with both RAPD and ISSR data matrices (R = 0.3814 and 0.3765, respectively). Cluster analysis showed that groupings of the accessions according to all three methods correlated well with their geographic region of origin, but flower color was not strongly associated with the genetic classification of these inbred lines of Z. elegans.     

SRAP、ISSR和RAPD分子标记技术在银耳菌株鉴别上的应用

Three types of molecular markers (SRAP, ISSR and RAPD) were used to identify four Tremella fuciformis strains, T6 (white), T7 (white), T8 (yellow) and T9 (light yellow). Twelve SRAP primer pairs, ten ISSR primers and eight RAPD primers were screened, and identification data obtained using the three molecular markers were consistent in that the four T. fuciformis strains were divided into three groups with T7 and T9 clustered together in a single group. Each RAPD primer generated a higher average number of polymorphic bands than either the SRAP or ISSR primers, and the average similarity between the four strains was 81.34%. SRAP markers reflected more genetic information compared with the two other markers, and the average similarity was 68.98%. Genetic information reflected by ISSR markers was intermediate between SRAP and RAPD, and the average similarity was 77.48%.     

DNA fingerprinting and genetic diversity analysis of late-bolting radish cultivars with RAPD,ISSR and SRAP markers

Three molecular marker systems, RAPD (random amplified polymorphic DNA), ISSR (inter-simple sequence repeat) and SRAP (sequence-related amplified polymorphism), were employed for identification and genetic diversity analysis of 35 elite late-bolting radish cultivars. Detected by 35 RAPD primers, 22 ISSR primers and 17 SRAP primer combinations, the proportions of polymorphic bands were 85.44%, 85.2% and 85.41%, respectively, and the mean genetic similarity coefficients between pairs of genotypes were 0.781, 0.787 and 0.764, respectively. Each of the three molecular marker systems can identify all the cultivars. Five sets of three-RAPD primers, 3 sets of three-ISSR primers and 16 sets of three-SRAP primer combinations were able to distinguish all the cultivars. A linear relationship was observed between Resolving power (Rp) of a primer and its ability to distinguish genotypes. The 35 cultivars were clustered into three major groups based on the RAPD, ISSR and marker combination data with UPGMA, which are in high accordance with their own origins and main characteristics. The results demonstrated that these three marker systems could be useful for identification and genetic diversity analysis of radish cultivars.     

豆瓣菜种质资源遗传多样性的RAPD和ISSR分析

以8个豆瓣菜的品种为试材，用筛选出的79个RAPD引物和34个ISSR引物对这8个品种的基因组DNA进行扩增，分别扩增出361条和179条谱带，每个引物扩增出的带在3～10条之间，平均每个引物扩增出约5条带。根据所得的条带进行聚类分析，两种标记产生的聚类图存在一些差异，但它们都可以较好地将8个品种按亲缘关系的远近划分为3个不同的类群。Mantel测试得出相关系数r=0．58155，表明RAPD和ISSR两种分子标记技术的相关度很低。     

Breaking the intergeneric hybridization barrier in Carica papaya and Vasconcellea cauliflora

The present investigation was undertaken to develop PRSV (Papaya ringspot virus) resistant hybrids through intergeneric hybridization. Intergeneric hybridization was done involving nine Carica papaya cultivars as female and Vasconcellea cauliflora as male. To break the intergeneric hybridization barrier, various nutrient combinations were used. Among the combinations used, sucrose 5%, sucrose 5% + boron 0.5% and sucrose 5% + CaCl₂ 0.5% improved the fruit set and seed set percentage. A total number of 1197 flowers were pollinated and 308 fruits were obtained. On extraction, 721 seeds were obtained from CO 7, Pusa Nanha and CP 50. Out of 721 F₀ seeds (crossed seeds) sown, 419 seeds germinated and artificial screening for PRSV was carried out 27 days after sap inoculation. Out of 29 F₁ hybrid plants from CO 7 x V. cauliflora cross, only six plants namely CO 7V1 to CO 7V6 were found free from PRSV symptoms. Similarly, out of 55 F₁ hybrids from cross involving Pusa Nanha x V. cauliflora only 23 plants namely PNV1 to PNV23 were found free from the symptoms and 70 plants namely CPV1 to CPV70 out of 335 plants of CP50 x V. cauliflora cross were found free from PRSV symptoms. Among the crosses, Pusa Nanha x V. cauliflora had higher yield under PRSV infected conditions, however, total soluble solids and total sugars were found lesser than the CO 7 x V. cauliflora cross. The hybridity of the progenies were confirmed by using ISSR (Inter Simple Sequence Repeats) primers by the amplification of DNA from progenies and their parents. ISSR primers UBC 856, UBC807 and ISSR primer combinations UBC 856-817, UBC 810-817, UBC 861-817, UBC 856-810, UBC 861-810 and UBC 856-817 clearly amplified specific bands of the male parent, which were present in F₁ progenies, but it was absent in female parents.     

Genetic diversity analysis in date palm (Phoenix dactylifera L.): a comparative assessment using ISSR and RAPD marker assays

SUMMARY

A comparative study was conducted to evaluate genetic diversity in 45 genotypes of date palm (Phoenix dactylifera L.), including both male and female plants, employing RAPD and ISSR marker systems. The data were analysed to calculate the total number of bands, the number of polymorphic bands, the percentage polymorphism, the average number of bands per primer, the effective multiplex ratio (EMR), the polymorphic information content (PIC), the marker index (MI), and genetic similarity coefficients. The 37 RAPD and 53 ISSR primers used generated 363 and 608 scorable amplified products, respectively, of which 95.0% and 90.9% were polymorphic. The ISSR markers produced more information than the RAPD markers due to their higher EMR and MI values. Jaccard similarity values among male plants, female plants, and between all male and all female plants varied between 0.72 – 0.80. The results indicate the effectiveness of these two marker systems for demonstrating genetic relationships among date palm genotypes.