Genetic variation within and between two cultivars of red clover (Trifolium pratense L.): Comparisons of morphological,isozyme, and RAPD markers

Summary Morphological, isozyme and random amplified polymorphic DNA (RAPD) markers were used to estimate genetic variation within and between cultivars of red clover (Trifolium pratense L.), an important temperate forage legume. Two cultivars of red clover, Essi from Europe and Ottawa from Canada, were evaluated. Six monogenic morphological characters were observed for 80 plants from each of these two cultivars. All six morphological loci were polymorphic in the cultivar Essi whereas only four loci were polymorphic in the cultivar Ottawa. Forty plants from each cultivar were assayed for isozyme markers. A total of 21 enzyme-coding loci with 43 alleles was detected using twelve enzyme systems. Thirteen and nine of these loci were polymorphic in Essi and Ottawa, respectively. The mean number of alleles per locus was 1.81 in Essi and 1.67 in Ottawa. Seventeen random 10-mer primers were screened for RAPD markers. Nine primers which gave clear and consistent amplified products were used to assay 20 individuals from each cultivar. Each primer gave from 7 to 20 amplified bands with an average of 14.8 bands per primer. One hundred and eight of 116 putative loci were polymorphic in Essi and 90 of 98 loci were polymorphic in Ottawa. High within-cultivar variation was observed in both cultivars using both isozyme and RAPD markers. This high polymorphism makes these markers useful for germplasm characterization and genetic studies in red clover.     

Identification of rape seed cultivars (Brassica napus) by starch gel electrophoresis of enzymes

Summary Isozyme patterns were used to characterize ten commercial rape seed (Brassica napus) cultivars. Extracts of cotyledons were made 4–7 days after germination and separated by electrophoresis on starch gels. A sample of more than 100 plants of each variety was analysed and stained for 4 polymorphic enzyme loci (Lap, Gpi, Acon, and Sdh). Therewith, it was possible to distinguish all ten rape varieties qualitatively by at least one typical enzyme pattern. Further, it could be demonstrated that all pairs of varieties showed clear qualitative differences in isozyme patterns, when only tow loci were screened (Acon, Sdh). Using ² test of homogeneity all pairs of varieties differ significantly in their frequencies of isozyme patterns for Acon and in all but one for Sdh.     

利用EST同工酶谱鉴定黑稻品种纯度的研究

采用改进的聚丙烯酰胺凝胶电泳对5种黑稻主要栽培品种进行了EST同工酶谱分析.结果表明,此方法分辨率高.测试各品种的EST同工酶存在丰富的多态性,其电泳图谱具有鲜明的特征,并构成了各自的＂指纹＂.其中黑优粘和云黑酶谱条带数多达15条,a1和a3为5个品种的共有谱带.通过EST同工酶谱相似度指数分析,黑优粘与黑丰糯、乌贡1号相似度指数大于0.75,说明这3个品种间亲缘关系较近.     

应用过氧化物同工酶鉴定山西省马铃薯主栽品种的研究

为了快速、准确鉴定马铃薯品种,试验采用聚丙烯酰胺凝胶电泳分离马铃薯过氧化物同工酶(POD),并以酶带图谱作为鉴定依据,从而实现对马铃薯品种的鉴定。本文以14个不同品种的马铃薯的芽部、茎部、叶片为实验材料,分别提取其过氧化物同工酶进行聚丙烯酰胺凝胶电泳,分析比对同工酶的酶谱差异,进而确定不同品种的差异及其亲缘关系的远近。结果表明,马铃薯芽的过氧化物同工酶可以作为区分马铃薯品种的一个重要指标,14个品种中4、7号品种酶带相似,分别为晋薯11号和晋薯14号;6、9号品种酶带相似,分别为晋薯21号和晋薯18号;5、12号品种酶带相似,分别为同薯22号和同薯20号;13、14号品种酶带相似,品种分别为05-32-7和同薯23号;表明这些品种之间亲缘关系较近。     

Identification of broad bean cultivars based on isozyme patterns

Summary With the increasing rate of new cultivar production for different crop plants, there is great need for methods of identifying each cultivar discriminatingly. Starch gel electrophoresis was employed to study the differences between the esterase and cathodal peroxidase isozyme patterns of 40 broad bean (Vicia faba L.) cultivars. A total of 10 and 17 medium to darkly stained bands were obtained for esterase and peroxidase systems, respectively. Bands from each enzyme system could be gropuped into three zones. Bands belonging to zone 1 of esterase (E₁) and zones 2 and 3 of peroxidase (P₂ and P₃) were quite distinct, stained intensely, and were especially useful for identification purposes.The differences in banding patterns among cultivars of the same origin were as great as those of cultivars of unrelated origin. A large proportion of the cultivars could be completely differentiated using both of the isozyme systems. There were no bands present in either enzyme system which were common to all cultivars.     

Evaluation of common bean cultivar relationships by means of isozyme electrophoretic patterns

Summary Electrophoretic isozyme technique was applied on primary leaf, stem, and root tissues from seedlings of 34 U.S. major common bean (Phaseolus vulgaris L.) cultivars belonging to 19 commercial classes (Great Northern, Kidney, Navy, Pinto, Red Mexican, Tropical Black, California Small White, Idaho Flat Small White, Pink, and Cranberry). Among the isozyme systems studied, peroxidase (PER) and esterase (EST) were found to be suitable for cultivar identification within most commercial classes and for estimating the genetic relationships among the cultivars of the same class or among the classes. Acid phosphatase (PHOS), due to high proportions of monomorphic bands, could not be considered a good system for such purposes. Within each isozyme system, no pattern was found to be exclusive to any particular commercial class.Based on the number of polymorphic bands in common between each cultivar pair, a banding-similarity index was calculated. The indices were found to be highly significantly correlated with genetic distances obtained by Principal Component Analysis (PCA). In those comparisons where a pedigree relationship could be calculated, a non-significant correlation with similarity indices was obtained. Certain cultivar pair relationships, a minority of the whole, were incorrectly predicted by the isozyme technique. Caution is indicated when this technique is the only basis of assigning relationship. In a few cases, the similarity indices pointed either to close genetic relationships or the lack of such relationships, whereas the reverse is known from pedigree or PCA distance estimates. The reason for such discrepancies is discussed.Some isozymes were unique to a certain tissue, while others were present in more than one. Upon the compilation of bands from all the cultivars, for the leaf, stem, and root tissues respectively. 6, 4, and 0 EST, 9, 10, and 8 PHOS, and 7, 6, and 7 PER bands were obtained.     

Analyses of genetic diversity in Cuban rice varieties using isozyme, RAPD and AFLP markers

A survey of the genetic diversity among the major cuban rice cultivars was conducted using isozyme, RAPD and AFLP markers. Polymorphisms were detected for esterases, peroxidases, alcohol dehydrogenases and polyphenoloxidases systems; 21 RAPD primers and four AFLP primer combinations. Heterozygosity arithmetic mean value (H_av(p)), the effective multiplex ratio (EMR) and the marker index (MI), were calculated for isozyme, RAPD and AFLP markers. The mean value of genetic similarity among the different varieties was 0.92 for isozyme, 0.73 for RAPD and 0.58 for AFLP analyses. Thus, AFLP were able to detect polymorphisms with higher efficiency than RAPD (+15%) and isozyme (+34%). Data from the isozyme, RAPD and AFLP analyses were used to compute matrices of genetic similarities. The efficiency of the UPGMA for the estimation of genetic relatedness among varieties was supported by cophenetic correlation coefficients. The resulting values indicated that the distortion level for the estimated similarities was minimal. The correlation coefficients obtained by the Mantel matrix correspondence test, which was used to compare the cophenetic matrices for the different markers, showed that estimated values of genetic relationship given for isozyme and RAPD markers (r = 0.89), as well as for AFLP and RAPD markers (r = 0.82) were properly related. However, AFLP and isozyme data showed only moderate correlation (r = 0.63). Although the genetic variability found among the different cultivars was low, both RAPD and AFLP markers proved to be efficient tools in assessing the genetic diversity of rice genotypes. This revised version was published online in July 2006 with corrections to the Cover Date.     

An electrophoretic study of esterase and peroxidase isozymes in Brassicoraphanus

Summary A comparative electrophoretic study of esterase and peroxidase isozymes from the leaves of Brassicoraphanus and its parental species (Brassica japonica and Raphanus sativus) was carried out by means of the polyacrylamide gel isoelectrofocusing technique. The isozyme bands of Brassicoraphanus could be regarded as a summation of parent-derived bands, some of which were missing, in addition to some new bands. The qualitative and quantitative variation of isozyme patterns among individual plants was found within each strain of Brassicoraphanus as well as each parental species. The range of the enzymatic variation of a certain strain seemed to reflect the genetic homogeneity of the strain in question. Every strain of Brassicoraphanus was less variable in esterase patterns than the parental species, but in peroxidase patterns the variations of Brassicoraphanus were intermediate between those of both parents. Some strains of Brassicoraphanus were uniform in isozyme patterns, whereas others were variable. The difference in enzymatic variation among strains was perhaps due to the difference in the source of the strains and the selection which they received.     

Geographical variation of β-amylase thermostability among varieties of barley (Hordeum vulgare) and β-amylase deficiency

Investigations onto the thermostability of β-amylase in 274 varieties of barley (Hordeum vulgare L.) indicated that all varieties except one were distributed into three types of high (type A), intermediate (type B), and low (type C) thermostability, respectively. One variety (TB29) from China showed no β-amylase activity. Geographical variation was observed in the thermostability of β-amylase. Type C varieties were not observed in East Asia (Japan, the Korean Peninsula, China and Nepal), although 36 out of 37 varieties in Ethiopia were type C. Most of the varieties were Type A in Japan, the Korean Peninsula and China, whereas the frequency of type A and type B were nearly equal in Nepal. Varieties in the other five areas (North America, North Africa, Southwest Asia, Turkey and Europe) consisted of types A, B and C. These results support the fact that East Asian cultivars are genetically different from those of the western regions, as previously reported.     

12种黑稻主要栽培种酯酶同工酶谱分析

对我国12种黑稻主要栽培种进行了酯酶同工酶谱分析.结果表明:它们的酯酶同工酶条带数在5～15之间,其中黑优粘和云黑酶谱条带数多达15条,黑粳R最少,为5条,a1和a3为12个品种的共有谱带.通过酯酶同工酶谱相似度指数分析,黑优粘与黑丰糯、乌贡1号相似度指数大于0.75,说明这3个品种间亲缘关系较近,泰稻2号与汉中黑糯、河大114、黑糯567、黑帅相似度指数大于0.86,表明这5个品种间亲缘关系很近.同时发现黑稻酯酶同工酶存在着器官的差异性.     

杂交稻部分不育系与恢复系的SSR分类

选用分布于水稻(Oryza sativa L.)12条染色体上的36对SSR(simple sequence repeats)引物，分析了5个光温敏核不育系、7个细胞质雄性不育系及54份来自不同生态类型恢复系或品种的遗传差异。在供试材料中共检测出300条多态性片段，平均每对SSR引物检测到8.33条。分析结果表明，(1) SSR标记明确地把供试的66份水稻材料中的65份区分开来，和已知系谱的亲缘关系多数吻合，与表型性状聚类结果也有一定的相似性。(2) 以GD=0.73为标准，可准确地将籼稻、粳稻、野生稻三大类区分开；以GD=0.63为标准，又可区分出籼稻大类中带有粳稻或野生稻血缘的品种。表明SSR标记对籼稻、粳稻、野生稻品种的分类灵敏度高、结果可靠；并筛选出可用于鉴别水稻籼粳类型的10对特异性引物。(3) 以GD=0.56为标准，将供试材料划分在8个生态类型中，为生产上杂优组合亲本的选配提供了一些有益的参考。同时，生产上常用的一些杂交稻亲本划分在不同的生态类型中，表明强优势组合亲本间的遗传差异一般较大。     

Allozyme variation in cultivars of Cucurbita pepo L.

Summary Cultivars of Cucurbita pepo were analyzed for their isozyme phenotypes by horizontal starch gel electrophoresis. Considerable allozymic variation was observed between cultivars, especially in the aspartate aminotransferase and malate dehydrogenase isozyme systems. Each of the five fruit types represented in the 21 cultivars tested (zucchini, pumpkin, spaghetti, acorn, scallop and yellow straightneck) could be distinguished by specific allozymes or combination of allozymes. Cultivars within a fruit type gave very similar allozyme phenotypes and often could not be distinguished on the basis of the 6 assays used. Despite the outcrossing nature of the species, allozyme polymorphism within most cultivars was low and did not seriously interfere with the analysis. Approximately half of the hybrid lines tested gave heterozygous phenotypes at one or more loci, indicating that such loci could be used to screen for the percentage of hybrid seed obtained from crosses.     

Isozyme variation in taro (Colocasia esculenta (L.) Schott) from Asia and Oceania

Summary Isozyme variation was studied in 1,417 cultivars and wild forms of taro collected in Asia and Oceania. Seven polymorphic enzyme systems (MDH, IDH, PGI, 6-PGD, ME, SkDH, and ADH) revealed 143 isozyme phenotypes, or zymotypes, each uniquely characterized by the presence or absence of 56 electromorphs. Results showed greater isozyme variation in Asia than in Oceania, with Indonesia being the area of greatest diversity. No correlations were found between zymotypes and morphotypes or ploidy levels (as described by other investigators). Multivariate analyses of the isozyme data indicated that the majority of the Indonesian cultivars were different from the Philippine and Oceanian taro cultivars. Oceanian cultivars constituted a continuum of clusters and are thought to have originated from a narrow genetic base introduced from Indonesia. If taro breeding is to have any future in Oceania, it is important to exchange genotypes to broaden the base of existing breeding programmes.     

甜菜品种SSR指纹图谱的构建及遗传多样性分析

筛选出20对多态性高、带型清晰、重复性好的SSR标记引物对供试甜菜品种进行PCR扩增,根据扩增产物构建SSR指纹图谱,并分析其遗传多样性。结果显示,共检测出112个等位基因,平均每对引物5.6个,利用获得的等位基因计算遗传距离,107个品种的遗传距离变化范围在0.065~0.467之间,平均遗传距离0.298。Shannon’s多样性指数变异范围0.78~2.90;PIC值介于0.08~0.83;Nei’s指数介于0.39~1.87。利用类平均法（UPGMA）进行聚类分析,可将107个甜菜品种分为2个类群。类群Ⅰ29个品种,类群Ⅱ78个品种。类群Ⅰ和Ⅱ又可分为多个亚群。结果表明,每个甜菜品种有区别于其他品种唯一的数字指纹,说明用于试验的20对SSR标记适用于甜菜品种真实性的鉴定,同时甜菜品种指纹图谱库的构建也为甜菜品种鉴定提供技术基础。     

Isozyme electrophoregrams of sixteen enzymes in five tissues of cassava (Manihot esculenta Crantz) varieties

Summary Methodology, based on starch and polyacrylamide gel electrophoresis, was developed for determining isozyme electrophoregrams (patterns) of 16 enzymes of cassava (Manihot esculenta Grantz) varieties as potential genotype markers. Extracts of five different tissues (root, stem, leaf, petiole and bud) were examined. In general, the nodal portions of the shoots gave isozyme patterns with the largest number of bands. Petiole extracts gave similar results but bud extracts gave poor patterns. The limited number of varieties that were examined could be distinguished by sequential classification on the basis of the isozyme patterns of acid phosphatase, esterase, glutamate oxaloacetase transaminase and phosphoglucoisomerase.Joint publication of the Centro Internacional de Agricultura Tropical and the Department of Plant Science (No. 716), University of Manitoba. Presented in part at the 2nd International Symposium on Biochemical Approaches to Identification of Cultivars and Evaluation of Their Properties, 5–9 May, 1985, Braunschweig, West Germany.     

Assessment of genetic diversity within and among Basmati and non-Basmati rice varieties using AFLP,ISSR and SSR markers

Molecular markers provide novel tools to differentiate between the various grades of Basmati rice, maintain fair-trade practices and to determine its relationship with other rice groups in Oryza sativa. We have evaluated the genetic diversity and patterns of relationships among the 18 rice genotypes representative of the traditional Basmati, cross-bred Basmati and non-Basmati (indica and japonica) rice varieties using AFLP, ISSR and SSR markers. All the three marker systems generated higher levels of polymorphism and could distinguish between all the 18 rice cultivars. The minimum number of assay-units per system needed to distinguish between all the cultivars was one for AFLP, two for ISSR and five for SSR. A total of 171 (110 polymorphic), 240 (188 polymorphic) and 160 (159 polymorphic) bands were detected using five primer combinations of AFLP, 25 UBC ISSR primers and 30 well distributed, mapped SSR markers, respectively. The salient features of AFLP, ISSR and SSR marker data analyzed using clustering algorithms, principal component analysis, Mantel test and AMOVA analysis are as given below: (i) the two traditional Basmati rice varieties were genetically distinct from indica and japonica rice varieties and invariably formed a separate cluster, (ii) the six Basmati varieties developed from various indica × Basmati rice crosses and backcrosses were grouped variably depending upon the marker system employed; CSR30 and Super being more closer to traditional Basmati followed by HKR228, Kasturi, Pusa Basmati 1 and Sabarmati, (iii) AFLP, ISSR and SSR marker data-sets showed moderate levels of positive correlation (Mantel test, r = 0.42–0.50), and (iv) the partitioning of the variance among and within rice groups (traditional Basmati, cross-bred Basmati, indica and japonica) using AMOVA showed greater variation among than within groups using SSR data-set, while reverse was true for both ISSR and AFLP data-sets. The study emphasizes the need for using a combination of different marker systems for a comprehensive genetic analysis of Basmati rice germplasm. The high-level polymorphism generated by SSR, ISSR and AFLP assays described in this study shall provide novel markers to differentiate between traditional Basmati rice supplies from cheaper cross-bred Basmati and long-grain non-Basmati varieties at commercial level.The first two authors have equal contribution     

国家芝麻区域试验新品种(系)的DNA指纹分析

以我国近年参加国家芝麻区域试验的43个品种(系)为材料,利用12对SSR核心引物开展DNA指纹分析,初步构建参试品种的DNA指纹图谱,并根据指纹图谱分析了参试品种的特异性和一致性。聚类分析结果表明,12对引物检测到30个标记,可将43个品种完全区分开,来源于同一育种单位的品种遗传基础相近,而不同来源的品种其遗传背景相差较大;以遗传相似系数0.90为划分标准,95%的供试品种具备特异性;以引物位点的一致性为指标,80%的品种具有一致性;分配试验中,86%的单株都能正确地分配回到原来的品种,这些品种的一致性较好,个别品种的单株正确分配率较低,品种一致性差。表明大部分参加国家区试的品种都具有很好的特异性和一致性。     

Isozyme variation in Helianthus petiolaris and sunflower,H. annuus

Summary Helianthus petiolaris is a wild species used in genetic improvement of sunflower, as a donor of cytoplasmic male sterility and of genetic resistance to diseases. Isozyme variation for ADH, ACP, EST, GDH, LAP, PGI, PGD and SKDH in this species was studied using starch gel electrophoresis. The patterns thus obtained were compared with zymograms of inbred lines, hybrids and open pollinated varieties of H. annuus. The same alleles for EST and SKDH isozymes were found in both species, while ACP showed an allele that has not been found in sunflower. The rest of the isozyme systems showed both common alleles and characteristic ones for each species. ACP, GDH and PGD were monomorphic in H. petiolaris, while ADH and LAP were monomorphic in H. annuus. The isozyme markers obtained here could be useful in breeding programs involving interspecific crosses, and studies on introgression and on genetic variation in other populations of this wild species.     

Genetic analysis of forage grasses based on heterologous RFLP markers detected by rice cDNAs

Genetic polymorphism within and between three species of forage grasses, perennial ryegrass (Lolium perenne L), meadow fescue (Festuca pratensis Huds.) and tall fescue (Festuca arundinacea Schreb.), was analyzed using restriction fragment length polymorphism (RFLP) markers detected by rice cDNA probes developed at the Rice Genome Research Programme of Japan (RGP). One hundred and ninety‐seven rice cDNA clones were used for hybridization to genomic DNA of forage grasses. Many of the rice cDNA clones produced no visible band or only a smear with no discrete bands. Twenty‐three clones showed high efficiency cross‐hybridization to the genomic DNA of forage grasses. Genetic variation was evaluated for five varieties and one population of forage grasses using 12 polymorphic rice cDNA RFLP probes. Genetic variability within varieties as measured by Rogers’ genetic distance was considerably lower for the F. pratensis variety ‘Tomosakae’ than for the L. perenne and F. arundinacea varieties. To determine the genetic diversity between varieties of different species, cluster analysis was performed using data from the 12 RFLP probes. The two accessions of Lolium perenne were clustered more closely together than the three varieties of F. arundinacea. Two Japanese varieties of F. arundinacea were grouped in the same cluster. The variety‐specific RFLP markers were seen among six accessions of L. perenne, F. pratensis and F. arundinacea. Such variety‐specific RFLP markers would provide very useful tools for breeding programmes such as the intergeneric hybridization of Lolium and Festuca genera.     

过氧化物酶同工酶与棉花黄萎病抗性的相关研究

对20个来源及抗性不同的海岛棉和陆地棉品种和2个陆地棉(S)/海岛棉(R)杂交组合 的亲本、 F1、 F2世代的POX同工酶研究表明, 接种黄萎病菌前后, 棉花叶片中的 POX同工酶酶谱发生明显变化, 抗病品种与感病品种的阴性POX同工酶谱带均由原来的3 条 增 至6条, 但两者之间不存在明显差异; 阳性POX同工酶谱带接种前后均只有1条(