Application of RAPD markers for characterization of γ-ray-induced rose mutants and assessment of genetic diversity

Six parent and their 12 gamma ray-induced somatic flower colour mutants of garden rose were characterized to discriminate the mutants from their respective parents and understanding the genetic diversity using Random amplification of polymorphic DNA (RAPD) markers. Out of 20 primers screened, 14 primers yielded completely identical fragments patterns. The other 7 primers gave highly polymorphic banding patterns among the radiomutants. All the cultivars were identified by using only 7 primers. Moreover, individual mutants were also distinguished by unique RAPD marker bands. Based on the presence or absence of the 48 polymorphic bands, the genetic variations within and among the 18 cultivars were measured. Genetic distance between all 18 cultivars varied from 0.40 to 0.91, as revealed by Jaccard’s coefficient matrix. A dendrogram was constructed based on the similarity matrix using the Neighbor Joining Tree method showed three main clusters. The present RAPD analysis can be used not only for estimating genetic diversity present in gamma ray-induced mutants but also for correct identification of mutant/new varieties for their legal protection under plant variety rights.     

基于主成分分析法的墨兰品种观赏价值评价

以福州地区栽培多年的30个墨兰品种为材料,对其植株高、花色、瓣型等20个观赏性状进行测定和观察,分析墨兰品种观赏性状的变异范围、性状间的相关性以及主成分因子,并利用主成分分析法对30个墨兰品种的观赏价值进行评价。结果表明,不同品种间的16个数量性状均存在不同程度的变异,花部形态存在较大差异,叶片形态和数量变异小;4个质量性状中,花色和叶艺的遗传多样性较高。叶部形态和花部形态的观赏性状间存在显著相关性。20个观赏性状指标可以分为7个主成分因子,分别为株高因子、瓣型因子、花色因子、叶片数因子、花径因子、叶宽因子和叶艺因子,7个主成分因子的方差贡献率累计达全部性状信息的87.35%。通过计算30个品种的重要主成分值并对其进行排序,筛选出4个观赏性状优良的品种,分别为‘白墨’、‘宝山爪’、‘龙梅’和‘金华山’。     

观赏南瓜及葫芦种质资源遗传多样性分子评价

利用RAPD和ISSR标记对28份观赏南瓜及葫芦种质资源进行遗传多样性分子评价。结果表明:12个RAPD引物和13个ISSR引物分别扩增出89条和93条清晰谱带,平均每个引物分别扩增出6.1条和6.2条多态性谱带,多态性比率分别为82%和86%。RAPD和ISSR标记检测供试材料的遗传相似性系数(GS)范围分别为0.31～0.99和0.33～0.99,ISSR(平均GS值0.68)检测多态性效果高于RAPD(平均GS值0.73)。利用UPGMA法基于RAPD与ISSR混合聚类,将28份观赏南瓜及葫芦种质分为3类,类群的划分与果实形状明显相关:第Ⅰ类群包括15份种质,为扁圆形、卵圆形、圆球形或圆筒状的早熟或晚熟果实;第Ⅱ类群包括11份种质,为汤匙形、梨形、扁球形或皇冠形的早中熟果实;第Ⅲ类群包括2份种质,为葫芦形的晚熟果实。     

Floral characteristics affect susceptibility of hybrid tea roses, Rosa x hybrida, to Japanese beetles (Coleoptera: Scarabaeidae)

The Japanese beetle, Popillia japonica Newman, feeds on the flowers and foliage of roses. Rosa x hybrida. Beetles attracted to roses land almost exclusively on the flowers. This study evaluated characteristics of rose flowers including color, size, petal count and fragrance, as well as height of plants and blooms within plant as factors in attractiveness to Japanese beetles. Artificial flowers that had been painted to match the spectral reflectance of real blooms were attached to potted nonflowering rose plants in the field and the number of beetles that landed on each model was recorded. More beetles landed on the yellow- and white-colored flower models than on the five other bloom colors that were tested. Large (15 cm diameter) yellow flower models attracted more beetles than did smaller (8 cm diameter) yellow models. There was no difference in beetle response to yellow flower models of the same size that differed in bloom complexity (i.e., number of petals). Experiments in which blooming rose plants were elevated above controls, or in which flower models were placed at different heights within plant canopies, failed to support the hypothesis that height per se accounts for beetles' attraction to flowers over leaves. Attractiveness of selected rose cultivars that varied in fragrance and flower color also was evaluated in the field. Yellow-flowered cultivars were more susceptible than those with red flowers, regardless of fragrance intensity as rated by breeders. Growing cultivars of roses that have relatively dark and small-sized blooms may have some benefit in reducing Japanese beetles' attraction to roses.     

Assessment of genetic variation withinBrassica campestris cultivars using amplified fragment length polymorphism and random amplification of polymorphic DNA markers

Genetic relationships were evaluated among nine cultivars ofBrassica campestris by employing random amplification of polymorphic DNA (RAPD) and amplified fragment length polymorphism (AFLP) markers. RAPDs generated a total of 125 bands using 13 decamer primers (an average of 9.6 bands per assay) of which nearly 80% were polymorphic. The per cent polymorphism ranged from 60–100%. AFLP, on the other hand generated a total of 319 markers, an average of 64 bands per assay. Of these, 213 were polymorphic in nature (66.8%). AFLP methodology detected polymorphism more efficiently than RAPD approach due to a greater number of loci assayed per reaction. Cultivar-specific bands were identified, for some cultivars using RAPD, and for most cultivars with AFLP. Genetic similarity matrix, based on Jaccard’s index detected coefficients ranging from 0.42 to 0.73 for RAPD, and from 0.48 to 0.925 for AFLPs indicating a wide genetic base. Cluster analyses using data generated by both RAPD and AFLP markers, clearly separated the yellow seeded, self-compatible cultivars from the brown seeded, self-incompatible cultivars although AFLP markers were able to group the cultivars more accurately. The higher genetic variation detected by AFLP in comparison to RAPD was also reflected in the topography of the phenetic dendrograms obtained. These results have been discussed in light of other studies and the relative efficiency of the marker systems for germplasm evaluation.     

Molecular mapping of quantitative trait loci in japonica rice.

Rice (Oryza sativa L.) molecular maps have previously been constructed using interspecific crosses or crosses between the two major subspecies: indica and japonica. For japonica breeding programs, however, it would be more suitable to use intrasubspecific crosses. A linkage map of 129 random amplified polymorphic DNA (RAPD) and 18 restriction fragment length polymorphism (RFLP) markers was developed using 118 F2 plants derived from a cross between two japonica cultivars with high and low seedling vigor, Italica Livorno (IL) and Labelle (LBL), respectively. The map spanned 980.5 cM (Kosambi function) with markers on all 12 rice chromosomes and an average distance of 7.6 cM between markers. Codominant (RFLP) and coupling phase linkages (among RAPDs) accounted for 79% of total map length and 71% of all intervals. This map contained a greater percentage of markers on chromosome 10, the least marked of the 12 rice chromosomes, than other rice molecular maps, but had relatively fewer markers on chromosomes 1 and 2. We used this map to detect quantitative trait loci (QTL) for four seedling vigor related traits scored on 113 F3 families in a growth chamber slantboard test at 18 degrees C. Two coleoptile, five root, and five mesocotyl length QTLs, each accounting for 9-50% of the phenotypic variation, were identified by interval analysis. Single-point analysis confirmed interval mapping results and detected additional markers significantly influencing each trait. About two-thirds of alleles positive for the putative QTLs were from the high-vigor parent, IL. One RAPD marker (OPAD13720) was associated with a IL allele that accounted for 18.5% of the phenotypic variation for shoot length, the most important determinant of seedling vigor in water-seeded rice. Results indicate that RAPDs are useful for map development and QTL mapping in rice populations with narrow genetic base, such as those derived from crosses among japonica cultivars. Other potential uses of the map are discussed. Key words : QTL mapping, RAPD, RFLP, seedling vigor, japonica, Oryza sativa.     

长江、黄河流域两棉区陆地棉品种的遗传多样性比较研究

从300个随机引物中筛选到带型清晰且重复性强的41个多态性引物,用于长江、黄河流域两棉区的91陆地棉品种的RAPD标记分析。分析采用Jaccard‘s相似系数,使用NTSYS－pcl.80数据分析软件,非加权组平均法（UPGMA）聚类。来自长江流域棉区的23个陆地棉品种产生了84个多态性位点,品种之间的平均相似系数为0．631。而来自黄河流域棉区的68个陆地棉品种产生了96个多态性位点,品种之间的平均相似系数为0．632.两棉区品种的相似系数矩阵比较及成对相似系数分布的比较均表明,长江流域和黄河流域棉区陆地棉品种的遗传多样性水平相当。共同的基础种质资源、相同的育种目标及相近的育种方法和策略可能是造成两大棉区品种遗传多样性水平相当的重要因素。     

利用RAPD和ISSR分子标记分析怀地黄种质遗传多样性

用RAPD与ISSR技术对怀地黄的8个品种和2个脱毒品系进行了种质遗传多样性分析。分别从80条RAPD引物和44条ISSR引物中筛选出适合怀地黄种质分析的17条RAPD引物和10条ISSR引物,用于RAPD和ISSR分析。17条RAPD引物共扩增出177条带, 多态性位点数为109; 多态性位点比率为61.58%;平均多样性指数(I)为0.3135;每个位点的有效等位基因数（Ne）是1.3641; 10条ISSR引物共扩增出110条带. 多态性位点数为79; 多态性位点比率为71.58%;平均多样性指数(I)为0.3577;每个位点的有效等位基因数（Ne）是1.4037。 基于扩增条带数据库建立了各自的Jaccard遗传相关系数矩阵,构建了相似的分子树状图,将10个供试材料分为2类:一类群含组培85.5、大田85.5、组培9302、大田9302、金状元和金白6个材料;另一类群含北京1号、大红袍、地黄9104和野生地黄4个材料。两种分子标记的分析结果呈极显著正相关(r＝0.649)。结果表明,RAPD与ISSR标记适合于怀地黄种质遗传多样性分析,ISSR标记技术是一种多态性和重复性优于RAPD技术的实用技术。     

Comparison of RAPD and ISSR markers for genetic diversity analysis among different endangered Mangifera indica genotypes of Indian Gir forest region

The genetic variability and relationships among 20 Mangifera indica genotypes representing 15 endangered and 5 cultivars, obtained from Indian Gir forest region, were analyzed using 10 random amplified polymorphic DNA (RAPD) and 21 inter simple sequence repeat (ISSR) markers. RAPD markers were more efficient than the ISSR assay with regards to polymorphism detection. Also, the average numbers of polymorphic loci per primer, average polymorphic information content (PIC) and primer index (PI) values were more for RAPD than for ISSR. But, total number of genotype specific marker loci, Nei’s genetic diversity (h), Shannon’s information index (I), total heterozygosity (Ht), average heterozygosity (Hs) and mean coefficient of gene differentiation (Gst) were more for ISSR as compared to RAPD markers. The regression test between the two Nei’s genetic diversity indexes showed low regression between RAPD and ISSR based similarities but maximum for RAPD and RAPD + ISSR based similarities. The pattern of clustering of genotypes within groups was not similar when RAPD and ISSR derived dendrogram were compared. Thus, both the markers were equally important for genetic diversity analysis in M. indica.     

猕猴桃观花品种‘江山娇’与中华猕猴桃回交后代的性别分离与开花性状变异

以猕猴桃种间杂种品种‘江山娇’（Actinidia chinensis Planch×A.eriantha Benth）与中华猕猴桃（A.chinensis Planch）雄株杂交得到的杂交后代群体为实验材料,于2012、2013和2016年分别对该群体的雌雄性别比及其开花性状进行了调查。结果表明,杂交群体的雌雄性别比例小于1：1,即雄株偏多。杂交后代的花瓣颜色以红色为基础,但红色的分布区域、深浅及类型出现明显分离。聚类分析显示,这些杂交后代可通过花瓣CMYK色卡取值分为4个类群,其中猩红色与紫罗兰红色2个变异类型与观察表型完全一致。杂交后代群体的始花期、开花天数、花朵大小、开花量及单花花瓣数等性状均出现广泛分离,且因不同年份而出现变化。群体中杂交个体进入始花期的平均时间跨度为14 d,群体的始花期进入高峰时个体平均比例仅占群体的25.5%。2016年群体开花时间最长,最多有47.4%的个体开放10~13 d;2013年杂交群体的开放时间最短,有55.2%的后代开花3~5 d。本研究筛选出一批花瓣数多、花朵较大或单花序花朵较多的优良单株,并在后代群体中共发现21个含不同花数的花序组合类型。     

中国茶树初选核心种质遗传多样性的RAPD分析

以中国茶树初选核心种质中的69份种质为实验材料,采用改良的SDS法提取它们的基因组DNA,并运用优化的RAPD 分析体系对基因组DNA进行分子标记遗传差异研究。从50个随机引物中筛选出32个扩增效果好的引物,对全部试验材料进 行了RAPD扩增共得到348条有效带,其中多态性带为328条(占94.3%),它们之间的遗传距离为0.223～0.723。研究结果表 明中国茶树初选核心种质的遗传结构、遗传多样性和遗传距离基本上能较好的代表中国的茶树种质资源。同时,指出结合形 态标记和DNA分子标记是构建茶树核心种质较好的选择。     

Genetic linkage mapping in peach using morphological,RFLP and RAPD markers

We have constructed a genetic linkage map of peach [Prunus persica (L.) Batsch] consisting of RFLP, RAPD and morphological markers, based on 71 F₂ individuals derived from the self-fertilization of four F₁ individuals of a cross between New Jersey Pillar and KV 77119. This progeny, designated as the West Virginia (WV) family, segregates for genes controlling canopy shape, fruit flesh color, and flower petal color, size and number. The segregation of 65 markers, comprising 46 RFLP loci, 12 RAPD loci and seven morphological loci, was analyzed. Low-copy genomic and cDNA probes were used in the RFLP analysis. The current genetic map for the WV family contains 47 markers assigned to eight linkage groups covering 332 centi Morgans (cM) of the peach nuclear genome. The average distance between two adjacent markers is 8 cM. Linkage was detected between Pillar (Pi) and double flowers (Dl) RFLP markers linked to Pi and flesh color () loci were also found. Eighteen markers remain unassigned. The individuals analyzed for linkage were not a random sample of all F₂ trees, as an excess of pillar trees were chosen for analysis. Because of this, Pi and eight other markers that deviated significantly from the expected Mendelian ratios (e.g., 121 or 31) were not eliminated from the linkage analysis. Genomic clones that detect RFLPs in the WV family also detect significant levels of polymorphism among the 34 peach cultivars examined. Unique fingerprint patterns were created for all the cultivars using only six clones detecting nine RFLP fragments. This suggests that RFLP markers from the WV family have a high probability of being polymorphic in crosses generated with other peach cultivars, making them ideal for anchor loci. This possibility was examined by testing RFLP markers developed with the WV family in three other unrelated peach families. In each of these three peach families respectively 43%, 54% and 36% of RFLP loci detected in the WV family were also polymorphic. This finding supports the possibility that these RFLP markers may serve as anchor loci in many other peach crosses.     

利用RAPD技术鉴定海南荔枝品种光明

应用RAPD标记对6份无性系材料进行检验,20个引物对6份材料均扩增出完全相同的125条带,不具多态性,证明6份材料遗传基础高度相似。利用30个引物对光明和其他42份材料的基因组进行多态性分析,多态性位点为212个,占总数的80、83％,显示多态性较高。光明和其他荔枝品种间的相似系数均较高,与大丁香的相似系数（0．884）最高。在相似系数0．78水平上,可将供试材料分成4大类,这些类型与成熟期有一定相关,光明属于第3类。光明不具特征谱带,但可用少数引物组合将其和很多品种区别开,如利用AP09、AM16和AK17即可将光明从其他荔枝材料中鉴别出来。     

辣椒种质遗传多样性的RAPD和ISSR及其表型数据分析

用RAPDI、SSR分子标记及28个表型性状数据对辣椒属5个栽培种的13份材料进行了分析,结果表明:23条RAPD引物共扩增出209条带,平均每个引物扩增出9.09条,多态性位点比率为83.73%;16条ISSR引物共扩增出94条带,平均每个引物扩增出5.88条,多态性位点比率为79.79%.与RAPD相比,ISSR标记检测到的有效等位基因数(Ne)及Shannon多样性指数(I)、遗传离散度(Ht)和遗传分化系数(Gst)等遗传多样性参数都较大,多态性位点比例在亲缘关系较近的一年生辣椒(Capsicum annuum)种内较高,说明ISSR有更高的多态性检测效率,并且适合亲缘关系较近的种群间遗传多样性分析.基于RAPDI、SSR的聚类与基于表型数据的聚类之间存在极显著正相关,且都能将C.annuum与其它栽培种区分开来.     

Comparison of RAPD and RFLP genetic markers in determining genetic similarity among Brassica oleracea L. genotypes

Genetic similarity among 45 Brassica Oleracea genotypes was compared using two molecular markers, random amplified polymorphic DNA (RAPD) and restriction fragment length polymorphisms (RFLPs). The genotypes included 37 broccolis (var. italica), five cauliflowers (var. botrytis) and three cabbages (var. capitata) which represented a wide range of commercially-available germplasm, and included open-pollinated cultivars, commercial hybrids, and inbred parents of hybrid cultivars. Fifty-six polymorphic RFLP bands and 181 polymorphic RAPD bands were generated using 15 random cDNA probes and 62 10-mer primers, respectively. The objectives were to compare RFLP and RAPD markers with regard to their (1) sampling variance, (2) rank correlations of genetic distance among sub-samples, and (3) inheritance. A bootstrap procedure was used to generate 200 random samples of size n (n=2,3,5,... 55) independently from the RAPD and RFLP data sets. The coefficient of variance (CV) was estimated for each sample. Pooled regressions of the coefficient of variance on bootstrap sample size indicated that the rate of decrease in CV with increasing sample size was the same for RFLPs and RAPDs. The rank correlation between the Nei-Li genetic similarity values for all pairs of genotypes (990) based on RFLP and RAPD data was 0.745. Differences were observed between the RFLP and RAPD dendrograms of the 45 genotypes. Overlap in the distributions of rank correlations between independent sub-samples from the RAPD data set, compared to correlations between RFLP and RAPD sub-samples, suggest that observed differences in estimation of genetic similarity between RAPDs and RFLPs is largely due to sampling error rather than due to DNA-based differences in how RAPDs and RFLPs reveal polymorphisms. A crossing algorithm was used to generate hypothetical banding patterns of hybrids based on the genotypes of the parents. The results of this study indicate that RAPDs provide a level of resolution equivalent to RFLPs for detemination of the genetic relationships among genotypes.     

日本岛屿山茶种群遗传结构的ISSR分析

为查明日本岛屿山茶种群的生存状况及了解岛屿隔离对山茶种群遗传结构的影响,采用ISSR分子标记,利用筛选的20条引物对日本5个山茶（Camellia japonica）种群的遗传结构进行分析。结果表明：山茶种群的多态位点百分比（PPB）为70.29％,Nei’s基因多样性指数（H_E）为0.281 9,Shannon信息多态性指数（H）为0.409 5,与其它岛屿种群相比遗传多样性水平较高,表明山茶种群的生存状况较好。基因分化系数G_st=0.205 7,种群间具有较高的遗传分化;地理距离与遗传距离具有显著相关性（r=0.821 7,p<0.05）,UPGMA也将同岛种群聚在一起,表明岛屿隔离对山茶种群的遗传分化具有重要影响。借鉴日本岛屿山茶种群的保护经验,建议加强我国岛屿山茶种群的就地保护力度,同时建立山茶种质资源库,促进基因交流。     

40个黄皮品种的ISSR分析

采用ISSR-PCR分子标记技术对40个黄皮品种的遗传多样性进行分析。从96条ISSR引物中筛选出15条引物用于PCR扩增,共扩增出165条带,其中多态性条带100条,多态性比率为60.6%。应用SPSS软件计算各品种间的Jaccard相似系数介于0.714~1.000,UPGMA法将40个品种分成5组。     

Regression association analysis of fruit traits with molecular markers in cherries

The use of molecular markers supports the study of genetic marker–trait association of biological and agronomic interest in diverse genetic material. In this research, association between simple sequence repeat (SSR) and random amplified polymorphic DNA (RAPD) markers with fruit traits were investigated in two collections of cherries by applying multiple regression analysis (MRA). Thirty-eight SSR alleles and 135 RAPD fragments were found associated with 14 of affecting fruit traits. Some of SSR and RAPD markers were associated with more than one fruit trait in MRA. Such an association may arise due to pleiotropic effect of the linked quantitative trait locus on different traits. For example, some SSR and RAPD markers were associated with all four traits including fruit cracking, fruit firmness, total soluble solid (TSS) and fruit shape. Also, some markers had correlations with all four characters of TSS, anthocyanin, fruit skin color and fruit flesh color, indicating a significant correlation among these traits. Therefore, it is possible to use these markers along with morphological traits in cherry breeding programs for identification of suitable parents to produce mapping populations and hybrid cultivars. Also, these results could be useful in marker-assisted breeding programs when no other genetic information is available.     

Optimization of primer screening for evaluation of genetic relationship in rose cultivars

Optimization of primer screening for evaluation of genetic relationship in 34 cultivars of rose through random amplified polymorphic DNA (RAPD) markers was investigated. Four series of decamer primers were used for screening and optimization of RAPD analysis between which A and N series performed good amplification of fragments as compared with other series. The primers OPN-07 and OPN-15 produced maximum number of DNA fragments in Rosa hybrida cv. Anuraag. Some primer either did not produce amplification or produced very poor amplification. Further, ten selected primers were used for genetic analysis of 34 rose cultivars. The primer OPN-15 amplified 21 fragments in all cultivars tested. A total of 162 distinct DNA fragments (bands) ranging from 100 to 3400 base pairs were amplified by using 10 selected random primers. The cluster analysis indicated that these rose cultivars formed nine clusters.     

Development of microsatellite and amplicon length polymorphism markers for Camellia japonica L. from tea plant (Camellia sinensis) expressed sequence tags

Simple sequence repeats and amplicon length polymorphism markers for Camellia japonica were developed, based on Camellia sinensis sequences in the National Center for Biotechnology Information database. In total, 2495 gene sequences were used to design 216 primer pairs. To identify amplicon length polymorphism markers, 61 gene loci in 16 Camellia individuals were re-sequenced. In total, 10 markers (three expressed sequence tags-simple sequence repeats and seven amplicon length polymorphisms) yielded polymerase chain reaction products with clear polymorphic patterns and were used for genotyping 22 C. japonica individuals from a population. Numbers of alleles and expected heterozygosity ranged from two to 13 and from 0.28 to 0.90, respectively.