Somatic embryogenesis in Cymbopogon pendulus and evaluation of clonal fidelity of regenerants using ISSR marker

An efficient plant propagation system through somatic embryogenesis was established in Cymbopogon pendulus, an aromatic grass followed by analysis of genetic status of regenerants using ISSR markers. Optimum embryogenic callus induction was observed on MS basal medium supplemented with 13.57 μM 2,4-dicholorophenoxyacetic acid (2,4-D) with 8.88 μM N⁶-benzyladenine (BA). Subsequent culturing of embryogenic calli on MS medium containing 4.52 μM 2,4-D and 8.88–13.32 μM BA gave maximum number of somatic embryos. Addition of coconut water (CW) promoted induction, growth and differentiation of callus and somatic embryogenesis. Further development of embryos into plantlets was achieved on MS medium supplemented with lower concentration of biotin and calcium pantothenate (CaP) along with BA (4.44–13.32 μM) and kinetin (2.32–4.65 μM). The root meristems were established on half strength MS medium containing 2% sucrose and 2.46–9.84 μM Indole3-butyric acid (IBA) and successfully established in soil with 77.8% survival rate in field condition. Thirteen randomly selected regenerated clones were screened using six ISSR primers. Nine clones produced similar monomorphic amplification profiles while remaining clones showed minor variation with absence of certain parental bands and appearance of unique band. Majority of the regenerants maintained genetic fidelity with the generation of few variants as evidenced from similarity matrix estimates using Nei Li's coefficient of similarity data.     

Stability of micropropagated Musa acuminata cv. Grand Naine over clonal generations: A molecular assessment

The present work was undertaken to investigate clonal fidelity of banana (Musa acuminata cv. Grand Naine) regenerants from six different in vitro subculture generations and in the explant suckers by using ISSR and REMAP molecular markers. Both types of markers revealed high degree of monomorphism. Very low variation was observed up to the eighth subculture generation with polymorphic bands being low in both ISSR (0.96%) and REMAP (0.95%) markers. Epigenetic stability was studied by DNA methylation analysis of the eighth subculture generation samples. Single 570 bp methylation sensitive band was absent in two of the fifteen MspI predigested samples, while it was present in HpaII predigested and undigested samples. The results of the investigation confirmed that the micropropagation of banana up to the eighth subculture generation show low variation.     

15份葡萄种质亲缘关系的ISSR分析

利用ISSR标记对15份葡萄材料进行了基因组多态性分析,从60条引物中筛选出15条用于葡萄的ISSR扩增。共扩增出105条带,其中多态性条带91条,多态性百分率为86.7%。根据ISSR扩增结果,利用NTSYSpc2.10e软件进行Jaccard相似性系数分析,15份葡萄材料的遗传相似系数为0.27～0.75,平均遗传相似系数为0.471。通过UPGMA进行聚类分析,探索亲缘关系。在遗传相似系数0.39处,15份葡萄材料可分为2大类群。第1类包含7份山葡萄资源、2个山欧杂交品种,第2类包含3个欧亚种品种、2个欧美杂种品种和1个美洲杂种品种。由此可见,山葡萄与欧亚种、美洲杂种葡萄的亲缘关系较远,欧亚种与美洲杂种之间亲缘关系较近。     

Assessment of genetic diversity in cashew germplasm using RAPD and ISSR markers

Diversity and genetic relationship in 100 cashew germplasm accessions were analyzed by using RAPD and ISSR markers. Using 10 selected RAPD primers 60 bands were generated, of which 51 bands were polymorphic (85%), and with 10 selected ISSR primers 67 amplified bands were observed with 58 polymorphic bands (86.6%). Though both kinds of markers discriminated the accessions effectively, analysis of combined data of markers (RAPD + ISSR) resulted in better distinction of accessions. By combining markers, a total of 127 bands were detected, of which 109 bands (85.8%) were polymorphic and produced on an average of 5.45 polymorphic bands per primer. Primers with high polymorphic information content and marker index were identified for discriminating accessions. High percentage of polymorphism (>85%) observed with different markers indicated high level of genetic variation existing among the accessions. Genetic relationship estimated using similarity co-efficient (Jaccard’s) values between different pair of accessions varied from 0.43 to 0.94 in RAPD, 0.38 to 0.89 in ISSR and 0.43 to 0.87 with combined markers suggested a diversity (dissimilarity) ranging from 6 to 57%, 11 to 62% and 13 to 57% respectively and the diversity skewed around 50% indicated moderate diversity. The cluster analysis with UPGMA method separated the accessions broadly into 13 clusters and in that three into smaller clusters. Some correspondence between the molecular groupings and the morphological clusters were observed. Among the accessions, NRC-142 and NRC-12 were highly divergent and NRC-231 and NRC-232 were genetically similar.     

莲雾种质资源遗传多样性的ISSR分析

 对12份莲雾资源和2个莲雾近缘种进行了ISSR分析, 18条引物共扩增出459个位点, 其中多态性位点435个(94.77% ) 。利用UPGMA聚类分析表明: 蒲桃、马六甲蒲桃与莲雾分属于3个不同的组; 12个莲雾品种和品系又分成4个亚组, 这与传统分类学上按照果实成熟期的划分结果基本一致。     

Genetic diversity of Chinese natural bermudagrass (Cynodon dactylon) germplasm using ISSR markers

Bermudagrass (Cynodon dactylon L.) is an important warm-season turfgrass. Although it is widely distributed in China, studies on the genetic variation and relationship among the large-scale indigenous bermudagrass were relatively insufficient, especially at molecular level. The purpose of this study was to assess the molecular variation and relationship among one commercial cultivar ‘Tift3’ and 95 wild bermudagrass accessions collected from 11 provinces in China by ISSR marker technique. The results indicated that 29 ISSR primers generated a total of 248 bands among which 242 (97.6%) were polymorphic bands. The genetic similarity coefficients among accessions ranged from 0.51 to 0.97 with an average of 0.74. All accessions could be clustered into 11 groups with UPGMA method. Accessions from the same or adjacent regions generally were clustered into the same group or subgroups. A few accessions, however, were greatly different from the majority of all accessions. The results suggested that ISSR marker is an effective tool for the study of genetic variation in Chinese natural bermudagrass.     

滇西北地区5种羊肚菌遗传多样性的ISSR分析

以滇西北地区11个羊肚茵居群56个羊肚菌样品为研究材料,应用ISSR分子标记方法,进行了遗传多样性与亲缘关系分析。结果表明,11个多态性ISSR引物对全部试验样品进行PCR扩增,共获得88条稳定的条带,其中多态性条带70务（占79．55％）。应用软件POPGENE32分析得出11个羊肚菌居群间遗传距离的变化范围在0．1715～0．4853之间,相似系数的变化范围在0．6155～0．8424之间。遗传分化分析表明,61．35％的变异存在于居群间,38．65％的变异存在于居群内。对56个羊肚菌样品进行分子系统聚类分析（UPGMA）将资源分为三大组,对11个羊肚菌居群进行亲缘关系聚类分析将居群分为两大类群,聚类结果与地理距离有明显的相关性。     

High multiplication frequency and genetic stability for commercialization of the three varieties of micropropagated tea plants (Camellia spp.)

RAPD and microsatellite markers were used to determine the genetic fidelity of micropropagated plants from the three varieties of tea plant derived from explants of field grown mother bush as well as in vitro germinated seedlings. Rate of shoot multiplication was better from nodal explants than from shoot tips. A maximum of 32–33 shoots was observed in cotyledonary node in 1/2 MS medium with BAP (6 mg/l), GA₃ (1 mg/l) and IBA (0.5 mg/l). 90% of the in vitro derived microshoots were micrografted into rootstocks. The micropropagated plantlets showed both cytological and genetical stability. SSR primers showed complete stability among the regenerants. The results convinced that plants derived from axillary as well as adventitious mode of propagation can be genetically true to type. This cost effective technique would help in fast clonal propagation at a commercial scale.     

Genetic diversity and relationships of lotus (Nelumbo) cultivars based on allozyme and ISSR markers

Genetic diversity and genetic relationships of lotus (Nelumbo Adanson) cultivars were evaluated using allozyme and ISSR markers. The samples used covered 11 accessions of possible hybrids between Nelumbo nucifera and Nelumbo lutea and 92 accessions of N. nucifera including 69 flower lotus, 13 rhizome lotus, 5 seed lotus and 5 wild lotus. For allozyme studies, a total of 31 alleles at 23 loci of 18 enzyme systems were detected of which 5 (21.7%) loci Aat, Idh, Mdh-2, Pgd, Sod were polymorphic. The loci of Aat and Idh included two alleles, Mdh-2, Pgd and Sod included three alleles. Eighteen genotypes were detected with the 13 alleles of the 5 polymorphic loci. The parameters of average allele number, observed heterozygosity, expected heterozygosity and Shannon information index of 92 N. nucifera samples were 1.35 ± 0.71, 0.06 ± 0.21, 0.05 ± 0.14, 0.10 ± 023, respectively. Thirteen ISSR primers generated 93 loci, of which 37.63% were polymorphic across all samples. The percentage of polymorphic loci, average allele number, expected heterozygosity and Shannon information index of 92 N. nucifera samples were 26.67%, 1.30 ± 0.46, 0.10 ± 0.18 and 0.15 ± 0.25, respectively for the ISSR data. The ‘Bottleneck effect’ and rapid propagation of clones after the ice ages may explain the low genetic diversity of lotus. The dendrograms based on ISSR and allozymes were not congruent. Based on the ISSR data, the 103 samples were divided into the N. nucifera group (Group I), and the group containing inter-specific hybrids between N. nucifera and N. lutea (Group II). The flower lotus, rhizome lotus, and seed lotus each has multiple sources of origin. Plant size, a criterion commonly used in the classification of cultivars of lotus, is not correlated with genetic variation. Flower color is correlated with the cultivar classification to some degree, but its variation is complex in the hybrids.     

金针菇遗传多样性初步分析

应用ISSR分子标记技术和金针菇的子实体形态特征,对59个金针菇菌株进行遗传多样性初步分析。应用ISSR方法可以将供试的金针菇菌株划分为14类,而依据金针菇子实体形态特征可以将它们划分为11类,2种分析结果基本一致。     

巨大革耳遗传多样性的ISSR分析

为确定巨大革耳种质资源问的亲缘关系,本文应用ISSR分子标记技术,对来源不同地区的野生或栽培的9个巨大革耳菌株进行遗传多样性分析。从20个引物筛选获得4个ISSR多态性引物对巨大革耳菌株扩增,获得23条多态性条带,多态性比率为85．19％;对扩增结果进行聚类分析,当遗传距离为20％时,9个菌株聚为2类：I类包括C．m0002菌株;Ⅱ类包括其它的8个菌株。其中C．m0002菌株与其它8个菌株的遗传距离很远。经栽培出菇实验,结果表明,C．m0002菌株的子实体似多脂鳞伞（黄伞）,是同名异种;其它8个菌株均为巨大革耳。ISSR分析的结果与子实体形态特征一致。     

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.     

Use of ISSR markers to assess genetic diversity of African edible seeded Citrullus lanatus landraces

We used the twenty primers to evaluate the genetic variability of 80 individuals belonging to four accessions of edible seeded Citrullus lanatus originated from Côte d’Ivoire. Edible seeded C. lanatus, named “egusi” or “pistachio”, had a great importance in nutrition in West Africa. Nevertheless, due to its neglected status no study to our knowledge has been devoted to its genetic variability using DNA markers. The twenty ISSR primers generated 258 bands among which 252 were polymorphic (97.67%). On the whole, the bands generated revealed three types of profile sharply distinct from each other with minor differences within each type. One profile (P1) was most frequent with 65 individuals. Three accessions (NI084, NI127 and NI145) generated the three types of profile and had medium values of genetic diversity (GD = 0.246–0.275, respectively). On the opposite, the accession NI076 only contained individuals of the most represented type of profile (P1) and had the lowest genetic diversity (GD = 0.055 ± 0.017). The pairwise genetic distance between the 80 individuals varied from 0 to 0.61. The Factorial Component Analysis and the dendrogram clearly separated the 80 individuals into three clusters corresponding to the three types of profile. The results showed that clusters were well separated from each other whereas accessions were not. Our results suggest that high number of individuals should be taken into account for sampling missions and conservation strategies because accessions were not well differentiated from each other. Local agricultural practices consisting of frequent seeds exchanges between farmers and the conservation of harvested seeds for next year culture could be one explanation.     

Phylogenetic study and molecular identification of 31 Dendrobium species using inter-simple sequence repeat (ISSR) markers

The genetic diversity of the genus Dendrobium is not well known and the phylogenetic relationship of Dendrobium species are mainly determined by studies of the comparative vegetative anatomy and plant systematics. In the present study, we used the technique of inter-simple sequence repeats (ISSRs) to evaluate genetic diversity and phylogenetic relationship among 31 Dendrobium species from Yunnan region of China. In total, 2368 bands were amplified by 17 ISSR primers, resulting from 278 ISSR loci with 100% polymorphism at genus level. Thirty-one species were unequivocally distinguished based on ISSR fingerprinting. Species-specific ISSR markers were identified in nine of 31 tested Dendrobium species. Unweighted pair-group mean analysis (UPGMA) showed that 31 Dendrobium species were grouped into six clusters, indicating the genus was polyphyletic with several well-supported lineages. The high polymorphism and reliable amplification across species demonstrated the utility of ISSR marker for species diagnosis and genetic diversity study of the genus Dendrobium.     

菠萝蜜遗传多样性的ISSR分析

用ISSR标记方法对76份菠萝蜜(Artocarpus heterophyllus Lam.)种质资源DNA的遗传多样性进行了检测,24个引物共检测到477条带,其中427条具多态性(占89.52%),平均PIC为0.23,24个引物能把76份种质完全区分开来。遗传距离分析结果显示供试种质的遗传多样性较低,在DNA水平上的遗传相似系数为0.626～0.945,平均为0.775。聚类分析表明,76份菠萝蜜材料在遗传距离系数为0.752处可分为4大类,其中热带植物园的种质WJ2和GSYWJ1与其他种质的遗传距离相对较大,干胞和湿类型不能独立聚类。另外各地区的种质与雷州半岛种质明显分开聚类,而雷州半岛各地区的种质混杂聚类在一起,不能按地区单独聚类。     

Use of ISSR,SRAP, and RAPD markers to assess genetic diversity in Turkish melons

The genetic relationships among 63 melon (Cucumis melo L.) genotypes collected from various regions of Turkey were determined by comparing their molecular ISSR, SRAP, and RAPD markers with those of 19 foreign melon genotypes to investigate the taxonomic relationships and genetic variation of Turkish melon germplasm. Total 162 polymorphic markers (69, 18, and 75 obtained from ISSR, SRAP, and RAPD primers, respectively) were used to define the genetic similarity among the melon genotypes by dendrogram or two and three dimensional scalings. The average similarity (SM coefficient) between any two pairs of accessions examined as estimated by molecular variation was 0.73 ± 0.48. Within-group genetic similarities ranged between 0.46 and 0.96. Related genotypes or genotypes collected from similar regions were partitioned to similar clusters. Southeastern Anatolian genotypes were distinctly apart from group inodorus and group cantalupensis (sweet) genotypes. This reinforced the position of Turkey in the secondary genetic diversity center of melon. The genetic diversity among Turkish genotypes (H = 0.28 and I = 0.42) was only a little less than that of the world accessions (H = 0.30 and I = 0.45). On the other hand, the percentage of polymorphic loci among Turkish melon genotypes (90.7%) was even higher than that of the world accessions (87.6%).     

DNA分子标记在猕猴桃上的应用

猕猴桃为中国半特有属植物,由于雌雄异株,自然杂交明显,染色体倍性复杂等原因,用常规方法对其进行种类(品种)鉴定、系谱分析、遗传多样性研究等难度较大。DNA分子标记具有许多优点,它的发展和应用为猕猴桃研究提供了一条有效的途径。就DNA分子标记在猕猴桃系谱分析与品种(品系)鉴定、性别鉴定、染色体起源分析、胞质遗传与系统发育研究、遗传多样性研究和分子遗传图谱构建等方面的应用进行了详细阐述,同时指出了DNA分子标记在猕猴桃研究中存在的问题和今后研究工作的重点。     

Identification of Tremella fuciformis strains Using SRAP, ISSR and RAPD markers

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%.     

十一份不同地理居群野菊的ISSR分析

对11份野菊材料分别用ISSR分子标记和形态学特征进行了遗传分析。用13个引物共得到176个位点,其中166个为多态性位点,多态性比率(PPB)高达94.32%;结果表明:2种分类方法得到的结果不甚一致。说明了野菊是一个多型性的种,在形态上表现出体态、叶型、叶序、伞房花序式样以及茎叶毛被性等诸特征上的极大的多样性。从ISSR标记分析,湖北神农架野菊和安徽天堂寨野菊之间的遗传系数最大,为0.727;安徽天柱山野菊和南京野菊之间的遗传系数最小,为0.591。     

In vitro organogenesis of Abutilon indicum (L.) Sweet from leaf derived callus and assessment of genetic fidelity using ISSR markers

This study reports on in vitro regeneration of Abutilon indicum plantlets through callus mediated organogenesis. The leaf explants implanted on Murashige and Skoogs (MS) medium supplemented with 4.52 µM 2, 4-Dicholorophenoxy acetic acid (2,4-D) and 8.88 µM 6 Benzyladenine (BA) showed highest response (70.3%) for callus proliferation, but these callus did not showed any morphogenetic differentiation on the same medium even after 12 weeks. Whereas, subsequent sub-culture of this green proliferated callus on MS medium added with 2.68µM α-Napthalene acetic acid (NAA), 8.88µM BA and 543 µM Adenine sulphate showed the highest frequency (62.2%) of multiple shoot-buds production and also elongation of shoots. Well developed shoots were efficiently rooted in vitro on half strength MS medium supplemented with 7.38 µM Indole-3-butyric acid (IBA). Seventy per cent of in vitro regenerated plantlets were successfully established in garden and were morphologically alike to the donor plants. The genetic homogeneity of these in vitro regenerated plantlets was also affirmed by inter simple sequence repeat (ISSR) analysis using eight ISSR primers. This standardised in vitro organogenesis protocol supplements a good platform for the conservation of A. indicum germplasms and also caters for the needs of the herbal industry.