中国桑树选育品种ISSR指纹图谱的构建及遗传多样性分析

利用ISSR标记构建了24个选育桑品种的指纹图谱,用3种独立的方法（特殊的标记;特异的谱带类型;不同引物提供的谱带类型组合）可以有效地鉴别桑树选育品种,证明ISSR标记在桑树品种的鉴别方面是一个有效的工具和方法。17个ISSR引物共扩增出80条带,40条带具有多态性,占50．0％。24份选育桑树品种间平均遗传相似系数、Nei’S基因多样性（gene diversity）和Shannon’S信息指数分别为0．8731,0．1210和0．1942。桑树选育品种间的遗传多样性较低,说明中国选育桑品种间遗传距离较小,亲缘关系较近,。遗传基础较狭窄。UPGMA法聚类和PCA分析都清楚地显示了24个桑树选育品种的亲缘关系,聚类结果与桑树品种的系谱基本一致。     

Construction of Fingerprinting and Genetic Diversity of Mulberry Cultivars in China by ISSR Markers

The ISSR fingerprintings of 24 mulberry cultivars were constructed. Totally 80 bands were produced using 17 primers selected from 20 primers. Of them, 40 bands showed polymorphism. From the bands amplified, there were three independent ways to identify the mulberry varieties, such as unique ISSR markers, unique band patterns and a combination of the band patterns provided by different primers. ISSRs were very effective in differentiating the mulberry varieties. The mean genetic similarity coefficient, the mean Nei's gene diversity (h), and the mean Shannon's Information index (I) of mulberry cultivars were 0.8731, 0.1210, and 0.1942, respectively. This suggests that the genetic diversity of mulberry cultivars was low and the genetic base was narrow. Both UPGMA cluster and PCA (Principal Coordinates Analysis) analysis showed clear genetic relationships among the 24 mulberry cultivars. The major clusters were related to known pedigree relationships.     

7种川产淫羊藿属植物遗传多样性及亲缘关系的ISSR分析

利用ISSR分子标记对7种12居群的川产淫羊藿属植物进行遗传多样性及亲缘关系分析。结果发现23个ISSR随机引物共扩增194条清晰条带,其中169条具多态性,平均多态性位点比率为87.11%,有效等位基因数Ne=1.534 2,Nei基因多样性指数H=0.314 4,Shannon多样性指数I=0.469 7,表明物种间遗传多样性丰富。7种川产淫羊藿属植物间的遗传相似系数为0.653 2～0.748 9,聚类分析和主成分分析直观的显示出12份供试材料间的亲缘关系,并将其分为两类。表明ISSR分子标记技术可以用于川产淫羊藿属植物的遗传多样性和亲缘关系分析。     

Applicability of inter-simple sequence repeat polymorphisms in wheat for use as DNA markers in comparison to RFLP and RAPD markers

 Inter-simple sequence repeat polymorphic DNA (ISSR) was evaluated for its applicability as a genetic marker system in wheat. PCR was carried out with primers that annealed to simple sequence repeats. The resultant products were subjected to agarose-gel electrophoresis, and the banding patterns were compared among six wheat accessions containing diploid, tetraploid, and hexaploid members. Out of 100 examined, 33 primers produced distinguishable as well as polymorphic bands in each of the six accessions. Although most of the primers that gave distinct bands (30 primers out of 33) contained dinucleotide repeats, each of the primers with tri-, tetra-, and penta-nucleotide motifs also yielded discrete bands. Primers based on (AC)_n repeats gave the most polymorphic bands. In total, 224 polymorphic bands were found in the comparison between Einkorn wheats whereas, on the average, 120 polymorphic bands were detected between common wheats. ISSR primers produced several times more information than RAPD markers. The extent of band polymorphism was similar to that of RFLP markers, and greater than that of RAPDs. The genetic relationships of wheat accessions estimated by the polymorphism of ISSR markers were identical with those inferred by RFLP and RAPD markers, indicating the reliability of ISSR markers for estimation of genotypes. These polymorphic bands are potential candidates as novel markers for use in linkage-map construction in wheat. The characteristic features of ISSR markers, i.e. polymorphism, generation of information and ease of handling, suggest their applicability to the analysis of genotypes as well as to the construction of PCR-based genome maps of wheats. Received: 15 September 1996 / Accepted: 25 October 1996     

Genetic relationships of Japanese and Indian mulberry (Morus spp.) genotypes revealed by DNA fingerprinting

Mulberry (Morus spp.), a deciduous tree, originated at the foothills of the Himalayas and is used in sericulture for its leaf to feed the silkworm Bombyx mori L. Species differentiation among the genotypes of the genus Morus has never been out of debate as inter-specific hybridization events are often fertile. In the present study attempts were made to elucidate the genetic relationships among 18 mulberry genotypes collected from India and Japan using 15 Inter Simple Sequence Repeat (ISSR) and 15 Random Amplified Polymorphic DNA (RAPD) primers. The ISSR primers generated 81.13% polymorphism while the RAPDs generated 71.78% polymorphism. The polymorphic index of the primers identified UBC-812, UBC-826, UBC827, UBC-881, OPA-01, OPA-02, OPA-04 and OPH-17 as informative primers in mulberry. The genetic similarity coefficients and the dendrograms showed considerable genetic similarity among the genotypes. However, using the DNA markers, these genotypes were discriminated into two major groups in accordance with their geographic origin and species status. Distribution of the genotypes on a two-dimensional figure on the basis of the ALSCAL algorithm using Euclidean distance further confirmed the genetic divergence between these two groups. From the study it can be concluded that though morphologically Japanese and Indian mulberry genotypes show little divergence, genetic analysis using DNA markers could unravel significant genetic variation between these two groups. Similarly, while the species status of Japanese mulberry genotypes agrees with the genetic analysis, the same does not apply to Indian genotypes, in agreement with many earlier reports. The information generated in this study is of much use for taxonomical grouping and also for utilization in breeding and conservation programs.     

Population structure and genetic diversity in bottle gourd [Lagenaria siceraria (Mol.) Standl.] germplasm from India assessed by ISSR markers

Genetic diversity analysis was undertaken in 42 geographically distant genotypes accessions of bottle gourd (Lagenaria siceraria) from India northeastern (14) and northern region (28) using inter-simple sequence repeat (ISSR) markers. A total of 209 amplified bands were obtained from 20 ISSR primers used in this study, of which 186 were polymorphic with 89.00 % band polymorphism. Various parameters namely, observed number of alleles, effective number of alleles, Nei’s gene diversity/heterozygosity, resolving power, Shannon’s information index and gene flow were estimated under experiment. Jaccard’s similarity coefficient matrix was generated for pairwise comparisons between individual ISSR profiles and UPGMA cluster analysis based on this matrix showed clustering into six groups. Jaccard’s coefficient of similarity values ranged from 0.409 to 0.847, with a mean of 0.628 revealing a moderate level of genetic diversity. The Bayesian model-based approach to infer hidden genetic population structures using the multilocus ISSR markers revealed two populations among the 42 genotypes. This is the first report on the assessment of genetic variation using ISSR markers in this medicinal vegetable plant, and this study of diversity analysis will be helpful in analyzing future hybrid breeding strategy and devising effective germplasm exploration and conservation strategy.     

Analysis of the genetic diversity of garlic (Allium sativum L.) by simple sequence repeat and inter simple sequence repeat analysis and agro-morphological traits

The genetic diversity of 39 garlic accessions was investigated using eight simple sequence repeat (SSR) primer combinations and 17 inter-simple sequence repeat (ISSR) primer combinations. A total of 109 polymorphic loci were detected among these accessions, with an average of 4.63 polymorphic loci per SSR primer combination and 4.29 polymorphic loci per ISSR primer combination. The mean effective number of alleles, the mean Nei's genetic diversity, and the mean Shannon's information index for SSR were 1.4799, 0.2870, and 0.4378, respectively; and those for ISSR were 1.4847, 0.2898 and 0.4415, respectively. Cluster analysis, using the unweighted pair-group method with arithmetic averages (UPGMA) based on the allele frequency data, classified the accessions into three groups. The results of principal component analysis (PCA) were consistent with those of the cluster analysis. PCA showed that each of these three groups exhibited significant variation in agro-morphological traits. These findings suggest that the eight SSR and 17 ISSR primers identified could define valuable markers for genetic diversity for use by plant breeders.     

30个枣树种质资源遗传多样性的ISSR分析

取30个枣树品种进行ISSR分子标记分析,其扩增条带分别进行琼脂糖和聚丙烯酰胺凝胶电泳检测,以期获得不同产地品种之间的遗传多态性。从100条选择扩增的ISSR引物中筛选出17条扩增清晰、重复性和稳定性好的引物,选取其中8条扩增条带多态性强的引物进行遗传聚类分析。结果表明：（1）1％琼脂糖凝胶电泳和5％聚丙烯酰胺凝胶电泳检测扩增总条带数分别为72和127条,其中多态性条带分别为51条和113条,多态性条带比率（PPB）分别为70．8％和88．9％。（2）基于UPGMA软件对30个品种的遗传差异性分析表明,8个ISSR引物可以将枣树品种之间遗传差异明显区分开来。两种电泳检测方法相比较,聚丙烯酰胺凝胶电泳检测可获得较为精细的枣树品种间遗传图谱。其遗传相似系数范围在0．56～1．00之间,以0．62为最低遗传相似系数,可将30个枣树品种分成3个大类,6个亚类,为进一步研究枣树品种间分类、起源进化关系和分子辅助育种奠定基础。     

Analysis of phylogenetic relationship among five mulberry (Morus) species using molecular markers.

Species identification in mulberry (Morus) continues to be a point of great debate among scientists despite the number of criteria such as floral characters, wood, and leaf anatomical and biochemical characters used to identify the species within this genus. However, no consensus system of classification has emerged. Hence, an investigation was undertaken with inter-simple sequence repeat (ISSR) and random amplified polymorphic DNA (RAPD) markers to find out the possibility of using these DNA markers to confirm the identity of genotypes in a particular species. Fifteen ISSR and 15 RAPD primers generated 86% and 78% polymorphism, respectively, among 19 mulberry genotypes. The polymorphism among the species varied from 50% to 57% in ISSR markers and 31% to 53% in RAPD markers. Similarity coefficients were higher among the genotypes of M. latifolia, M. bombycis and M. alba. Cluster analyses separated genotypes of M. laevigata and M. indica from those of the other species. Population structure analysis of these species further showed high genetic differentiation coefficients (GST), high heterozygosity between two species (DST), and total heterozygosity among populations (Ht) coupled with considerably low gene flow (Nm) when M. laevigata was paired with other species. Based on these parameters and the result of cluster analysis it is concluded that M. laevigata can be considered as a separate species of mulberry, whereas the other four species may be grouped together and treated as subspecies.     

Comparison of ISSR and RAPD markers to characterize three Chilean Nothofagus species

The present study is the first report of fingerprinting on three Chilean Nothofagus species using ISSR and RAPD markers; 61 Nothofagus nervosa, 32 Nothofagus obliqua and 32 Nothafagus dombeyi individual trees, sampled from collection and natural sites, were analyzed. Among 45 primers tested, the 6 ISSR and 6 RAPD primers selected for the analysis generated a total of 63 ISSR and 42 RAPD fragments. A high proportion of polymorphic bands, ranging from 97% and 80%, was found using both markers. A similar number of private and marker bands was generated by both markers in all the species examined and one discriminant ISSR fragment was obtained for N. dombeyi. Jaccard and Dice similarity indices were used to evaluate pairwise genetic divergence; cluster analysis of the similarity matrices was performed to estimate the intra- and inter-specific genetic diversity, and PCA analysis was employed to evaluate the resolving power of the markers to differentiate between the species. These analyses, carried out for both markers, allowed us to identify three main groups corresponding to the three Nothofagus species. The results of the present study can be seen as a starting point for future researches on the population and evolutionary genetics of these species.     

Determination of genetic diversity among Saccharina germplasm using ISSR and RAPD markers

Various species of genus Saccharina are economically important brown macroalgae cultivated in China. The genetic background of the conserved Saccharina germplasm was not clear. In this report, DNA-based molecular markers such as inter simple sequence repeats (ISSR) and random amplified polymorphic DNA (RAPD) were used to assess the genetic diversity and phylogenetic relationships among 48 Saccharina germplasms. A total of 50 ISSR and 50 RAPD primers were tested, of which only 33 polymorphic primers (17 ISSR and 16 RAPD) had an amplified clear and reproducible profile, and could be used. Seventeen ISSR primers yielded a total of 262 bands, of which 256 were polymorphic, and 15.06 polymorphic bands per primer were amplified from 48 kelp gametophytes. Sixteen RAPD primers produced 355 bands, of which 352 were polymorphic, and 22 polymorphic bands per primer were observed across 48 individuals. The simple matching coefficient of ISSR, RAPD and pooled ISSR and RAPD dendrograms ranged from 0.568 to 0.885, 0.670 to 0.873, and 0.667 to 0.862, revealing high genetic diversity. Based on the unweighted pair group method with the arithmetic averaging algorithm (UPGMA) cluster analysis and the principal components analysis (PCA) of ISSR data, the 48 gametophytes were divided into three main groups. The Mantel test revealed a similar polymorphism distribution pattern between ISSR and RAPD markers, the correlation coefficient r was 0.62, and the results indicated that both ISSR and RAPD markers were effective to assess the selected gametophytes, while matrix correlation of the ISSR marker system (r = 0.78) was better than that of the RAPD marker system (r = 0.64). Genetic analysis data from this study were helpful in understanding the genetic relationships among the selected 17 kelp varieties (or lines) and provided guidance for molecular-assisted selection for parental gametophytes of hybrid kelp breeding.     

Assessment of Genetic Diversity in Ziziphus mauritiana Using Inter-Simple Sequence Repeat Markers

Genetic diversity among 47 ber accessions belonging to cultivated species (Ziziphus mauritiana Lam) and one wild accession of Ziziphus nummularia (Burm F) Willed was investigated using Inter-Simple Sequence Repeat (ISSR) markers. A total of 167 amplification products were detected with 18 ISSR primers of which 152 (89.96%) were polymorphic. Most of the primers that produced distinct bands (14 primers out of 18) contained dinucleotide repeats. Primers based on (AC)n and (AG)_n repeats produced more polymorphic bands. Genetic similarity ranging from 43.07% to 90.30% suggested that the 48 Ziziphus genotypes used in the study were divergent. Cluster analysis based on UPGMA method and Bootstrap analysis separated all the 48 genotypes in four distinct clusters. The present study has successfully distinguished morphologically similar genotypes that emphasize the use of molecular markers to the taxonomists. Morphologically similar but genetically distinct genotypes, identified using ISSR markers could be potential sources for genotype identification and to resolve controversies over misnomination of ber genotypes. Present study is the first report on the exploitation of ISSR markers in ber for genetic diversity analysis.     

利用RAPD和ISSR标记分析烤烟品种间遗传关系

利用RAPD和ISSR标记对22份烤烟(Nicotiana tabacumL.)品种进行了遗传关系研究。在RAPD分析中筛选到13个引物,共扩增出167条带,其中多态性带50条,多态性比率为29.9%;在ISSR分析中筛选出7个引物,共扩增出96条带,其中多态性带44条,多态性比率为45.8%。两种标记相结合估算出的品种间遗传相似系数在0.881~0.979之间,平均为0.933。单独基于RAPD标记和ISSR标记的聚类结果有一定差异;两种标记结合起来的聚类分析结果与系谱信息吻合程度更高。定向选择可能对烤烟品种间遗传关系有较大影响;国外引进品种与国内育成品种并未完全分开,表明分子水平的遗传关系和地理来源间缺乏必然联系。     

Genetic Diversity in Various Accessions of Pineapple [<Emphasis Type="Italic">Ananas comosus</Emphasis> (L.) Merr.] Using ISSR and SSR Markers

Inter simple sequence repeat (ISSR) and simple sequence repeat (SSR) markers were used to assess the genetic diversity of 36 pineapple accessions that were introduced from 10 countries/regions. Thirteen ISSR primers amplified 96 bands, of which 91 (93.65%) were polymorphic, whereas 20 SSR primers amplified 73 bands, of which 70 (96.50%) were polymorphic. Nei’s gene diversity (h = 0.28), Shannon’s information index (I = 0.43), and polymorphism information content (PIC = 0.29) generated using the SSR primers were higher than that with ISSR primers (h =  0.23, I = 0.37, PIC = 0.24), thereby suggesting that the SSR system is more efficient than the ISSR system in assessing genetic diversity in various pineapple accessions. Mean genetic similarities were 0.74, 0.61, and 0.69, as determined using ISSR, SSR, and combined ISSR/SSR, respectively. These results suggest that the genetic diversity among pineapple accessions is very high. We clustered the 36 pineapple accessions into three or five groups on the basis of the phylogenetic trees constructed based on the results of ISSR, SSR, and combined ISSR/SSR analyses using the unweighted pair-group with arithmetic averaging (UPGMA) method. The results of principal components analysis (PCA) also supported the UPGMA clustering. These results will be useful not only for the scientific conservation and management of pineapple germplasm but also for the improvement of the current pineapple breeding strategies.     

Genetic diversity in a collection of Cucumis sativus L. assessed by RAPD and ISSR markers

The genetic diversity among 39 cucumber collections from Karnataka, India was assessed using 23 RAPD and 18 ISSR primers. A total of 309 bands were scored of which 147 (47.57 %) were polymorphic. The average number of bands per primer was 7.82 and an average number of polymorphic bands of 3.58 per primer. The primers UBC855 revealed the highest PIC (polymorphism information content) value of 0.49 followed by the primers UBC846, OPE13, OPC01 and OPR12 (0.48). The Jaccard’s similarity coefficients ranged from 0.36 to 0.84 and the first two principal components explained 53.33 % of the total variance. The UPGMA phenogram and the PCA (Principle component analysis) indicated that the populations formed five major clusters. CSC 83 (774 g per fruit) and CSC 71 (yellow skin) are considered to be the most important collections to be stressed for further breeding purpose. The available genetic resources of cucumber in Karnataka were characterized.     

Polymorphism analysis in advanced mutant population of oat (<Emphasis Type="Italic">Avena sativa</Emphasis> L.) using ISSR markers

Present investigation was carried out to evaluate genetic diversity among 38 M6 population of oat cv. JO-1. To validate the observed morpho-physiological variations, these lines were analyzed with 21 ISSR primers. A total of 132 loci were amplified by these 21 ISSR markers and 116 loci were found to be polymorphic (87.87 %). The genetic similarity coefficient values among 39 oat genotypes based on ISSR analysis ranged from 0.305 to 0.957. The cluster analysis divided the oat genotypes into two groups. Mutants JMO 81 and JMO 82 were found to be most divergent, hence can be used as parents in breeding program for the development of superior cultivars.     

Molecular characterization of Jatropha genetic resources through inter-simple sequence repeat (ISSR) markers

The genetic diversity among eight Jatropha species and three Jatropha curcas accessions were analyzed using ISSR-PCR. Nine ISSR primers generated reproducible amplification banding pattern of 61 polymorphic bands out of 64 scored accounting for 98.14% polymorphism across the species. The ISSR primers viz., I1, I2, I3, I4, I5, I6, I7 and I10 generated 100% polymorphic patterns. Jaccard’s coefficient of similarity varied from 0.346 to 0.807, indicative of high level of genetic variation among the genotypes studied. The UPGMA cluster analysis indicated three distinct clusters, one comprising all accessions of J. curcas L (TNMJ1, TNMJ 22 and TNMJ 23), while second included four species viz., J. tanjorensis J. L. Ellis et Saroja., J. gossypifolia L., J. podagrica Hook and J. maheshwarii Subrum and M.P. Nayer and the third cluster included another four species viz., J. villosa Wight J. multifida L., J. integerrima Jacq and J. glandulifera Roxb. The overall grouping pattern of clustering corresponds well with principal component analysis (PCA) confirming patterns of genetic diversity observed among the species. So far, there are no reports on the molecular diversity of the Jatropha species through ISSR marker. This study provides valid guidelines for collection, conservation and characterization of Jatropha genetic resources and also for further breeding programme towards biodiesel production.     

Genetic diversity analysis of Cynodon dactylon (bermudagrass) accessions and cultivars from different countries based on ISSR and SSR markers

ISSR and SSR markers were used to evaluate genetic diversity among 33 Cynodon dactylon accessions and 22 cultivars from four different countries in order to provide information on how to improve the utilization of bermudagrass germplasms. Eighty eight bands were amplified by nine SSR primer combinations and 236 bands were observed from 23 ISSR primers. The results showed that 97.7% of the SSR primers and 86.9% of the ISSR primers were polymorphic. The genetic similarity coefficients (GSC), gene diversity (He) and Shannon index (I) were 0.58–0.97, 0.27 and 0.41, respectively, for ISSR and 0.52–0.97, 0.29, and 0.43 for SSR. The UPGMA analysis clustered the 55 accessions (cultivars) into three groups. The cluster results produced by the ISSR data were close to the SSR data results. Analysis based on the combined ISSR and SSR data was more closely related to the geographical distribution of the tested germplasm.     

基于ISSR标记的烤烟种质遗传多样性研究

利用ISSR标记分析了24份代表性烤烟种质的遗传多样性。从100个ISSR引物中筛选出10个引物,通过聚丙烯酰胺凝胶电泳可以检测到208条稳定的条带,片段大小介于200~2 400 bp之间,条带数在7~37条之间;扩增片段中多态性带141条,平均多态性比率(PPB)为67.79%。 通过UPGMA聚类分析,24个烤烟品种分为5类,最大一类有12个材料,主要衍生于Coker319。品种间遗传相似指数(GS)范围为0.66~0.85,表明其遗传多样性较低,需要拓宽烤烟种质的遗传基础。同时,利用2个多态性好的ISSR引物可以将这24份烤烟材料区分开,每个品种都有各自独特的指纹图谱,表明ISSR标记适于烟草品种鉴定和遗传多样性研究。     

Correspondence of ISSR and RAPD markers for comparative analysis of genetic diversity among different apricot genotypes from cold arid deserts of trans-Himalayas

The phylogenetic relationships of 36 locally grown Prunus armeniaca genotypes which are collected from nine sampling sites from two valleys viz. Nubra (9,600 ft) and Leh (11,500 ft) of trans-Himalayan region were analyzed using 31 PCR markers (20 RAPDs and 11 ISSRs). This is the first report of molecular genetic diversity studies in apricot from this region of the world. RAPD analysis yielded 139 fragments, of which 136 were polymorphic, with an average of 6.8 polymorphic fragments per primer. ISSR analysis produced 58 bands, of which 56 were polymorphic, with an average of 5.09 polymorphic fragments per primer. The primers based on (CT)n produced maximum number of bands (nine) while, (AT)n and many other motifs gave no amplification. RAPD markers were found more efficient with regards to polymorphism detection, as they detected 97.84 % as compared to 96.5 % for ISSR markers. Clustering of genotypes within groups was not similar when RAPD and ISSR derived dendrogram were compared, whereas the pattern of clustering of the genotypes remained more or less the same in RAPD and combined data of RAPD + ISSR. The results of PCA analysis were comparable to the cluster analysis. These analyses, allowed us to identify the groups corresponding to the two apricot collection sites.Key words: Prunus armeniaca, Apricot, Genetic Diversity, RAPD, ISSR, AMOVA