ISSR based analysis of genetic variability of plantlets culture of pineapple (Ananas comosus L.) from Sipahutar, North Sumatera, Indonesia

Sipahutar pineapple is very popular in North Sumatra, because of the distinctive sweet taste and normal water content. Furthermore, it is large in size and has a yellow fruit skin color with greenish tips. However, the problem with Sipahutar pineapple production is the limited amount of good quality seeds. The utilization of in vitro culture techniques on pineapples allows the emergence of somaclonal variations, especially in plantlets that have undergone the subculture stage. This somaclonal variation is one of the problems in commercial seedling production, therefore, it is necessary to conduct an initial examination using the inter simple sequence repeat (ISSR) molecular marker. This study aimed to determine the genetic and primary stability of ISSR, which can be used in pineapple plants native to Sipahutar. The methods used include sample preparation, DNA isolation, primer optimization, ISSR primer amplification by PCR method, and electrophoresis. Furthermore, a total of 15 samples were amplified with six ISSR primers, and the data were analyzed by cluster method using the NTSYS-PC software. The final result was visualized in a dendrogram and analysis of diversity was conducted using GenAlex. The results showed that the level of genetic variability of the Sipahutar pineapple, which has undergone in vitro culture using six ISSR molecular markers was 76-97%. Meanwhile, the genetic variability level of the native to Sipahutar pineapple can be influenced by the long culture period and the use of N6-benzyladenine. The primers can be used to observe the genetic variability, except for ISSR 25 with a PIC value of 0.000.

The utility of ISSRs for the identification of interspecific hybrids between pearl millet (Pennisetum glaucum [L.] R.Br.) × napier grass (Pennisetum purpureum Schumach)

Interspecific hybrids between pearl millet (Pennisetum glaucum) and napier grass (Pennisetum purpureum) give rise to perennial fodder crops characterized by high biomass, broad clumps and good palatability. These hybrids are triploid and developed by hand pollination of napier grass pollen on pearl millet panicles. The progeny shows a high percentage of pearl millet genotype due to self-pollination in the female parent. Identification of hybrids at a young stage based on morphological characters is difficult. DNA-based molecular markers have high discriminating power and were used to assess genetic differences between hybrids and their parents. Genetic diversity was studied in 18 pearl millet × napier grass hybrids along with their parents and two released national checks using inter simple sequence repeat (ISSR) markers. Eight ISSR primers gave rise to 125 bands, of which 120 bands were polymorphic. Polymorphic information content and ISSR primer index ranged from 0.40 to 0.49 and 8.88 to 11.14, respectively. The hybrids showed the presence of unique bands, besides those shared with male and female parents. Female (pearl millet) parents formed a separate group in the dendrogram constructed based on ISSR polymorphism. The male (napier grass) parents formed a separate group along with hybrids, indicating a higher similarity of hybrids with the male parents. Principal component analysis and STRUCTURE analyses showed a similar grouping. The close resemblance of hybrids to the male parents confirmed their interspecific origin. The study revealed that ISSR marker analysis could be a quick and reliable method to identify interspecific hybrids at an early stage of growth.

Genetic Diversity and Population Structure of Job’s Tears(Coix lacryma-jobi L.) Germplasm Based on ISSR Marker

Background: Job's tears（Coix lachryma-jobi L.）is a minor cereal and an important food item in some parts of Asia. It has also been used in the traditional Chinese medicine for relieving various ailments, therefore, it plays an important role in our lives. Lack of excellent new varieties hinders the development of coix as a sustainable crop, and it is urgent to provide new cultivars with excellent trait in Chinese Coix industry. Results: ISSR markers were used to assess the genetic diversity and population structure of 8 populations of Job’s tears in China. A genotyping analysis that utilized ten ISSR primer pairs resulted in the production of 116 bands, of which 98 were polymorphic. The Guizhou population (PPB = 81.90%, h = 0.3113, I = 0.4589) was the most genetically diverse, while the lowest was observed in the Hebei population (PPB = 46.55%, h = 0.1842, I = 0.2701). Genetic differentiation analyses including GST and AMOVA illustrated that genetic variation was most prevalent within populations while only minor variations were observed among populations. Genetic distance coefficients ranged from 0.0095 to 0.0948 for the 8 populations; the genetic relationship between the Guizhou and Chongqing populations was the closest, while the most distant genetic relationship occurred between the Hubei and Hunan populations. The results of an UPGMA cluster analysis that investigated genetic diversity among the populations were consistent with the genetic distance results. The results of a STRUCTURE analysis suggested that 94 Job’s tears accessions could be grouped into two subpopulations. Moreover, according to a cluster analysis based on the UPGMA for individuals of Job’s tears, accessions were divided into two major clusters. The results of the Bayesian cluster and UPGMA cluster analyses were largely consistent despite minor differences. There was no significant correlation between genetic distance and geographic distance (r = 0.055, p = 0.782). Conclusions: Our study was undertaken to systematically analyze genetic diversity and population structure in 94 Job’s tears accessions using ISSR markers. And this study provides us with valuable information pertaining to germplasm collection, genetic improvement, and systematic utilization of Job’s tears.

ISSR primer screening for analysis of genetic diversity among Scutellaria tuvensis (Lamiaceae) populations

Using the Diamond DNA kit, high quality nuclear DNA was isolated from dry leaves of the endemic species Scutellaria tuvensis. The selection of primers for ISSR analysis of genetic polymorphism of natural populations is described. During the experiment, 22 primers were tested, their effectiveness was assessed on a point scale. When assessing the primers, the number of reproducible amplified DNA fragments, the clarity and brightness of the obtained fragments, and only distinctive bands were taken into account. As a result, 10 ISSR markers were selected that are the most informative for assessing the population diversity of the species.

Genetic diversity of the Andean blackberry (Rubus glaucus Benth.) in Ecuador assessed by AFLP markers

Andean blackberry (Rubus glaucus Benth.) is an emerging fruit crop with significant commercial potential. Despite its growing popularity, basic research about its genetic resources and breeding remains insufficient. The aim of this study was to assess the genetic diversity of Andean blackberry cultivars and related berries species from the main production areas in Ecuador. We analysed a total of 106 samples and performed DNA screening with different molecular markers: random-amplified polymorphic DNAs (RAPDs), inter-simple sequence repeats (ISSRs) and a set of representative samples with amplified fragment length polymorphisms (AFLPs). The tested RAPD primers did not reveal any differentiation among accessions identified as R. glaucus, however one ISSR primer was useful to find polymorphisms allowing the selection of 29 accessions for the analysis with AFLP markers. AFLP-M13 technology was used for screen genetic variations among these accessions and eight wild Rubus accessions. We scored 203 bands using five primer combinations; out of these 152 were informative in R. glaucus. AFLP markers clearly distinguish R. glaucus from the screened wild Rubus species, also an unexpected genetic structure was revealed among R. glaucus cultivars. This genetic differentiation and detection of admixed genotypes suggest a possible introgression of wild Rubus species in R. glaucus. Our findings are relevant for blackberry genetic breeding and use of these genetic resources.

Genetic Diversity Corylus avellana L. in Lesser Caucasus of Azerbaijan

The article is devoted to the results of a study of the genetic polymorphism of hazel (Corylus avellana (L.) H. Karst.), growing in the Lesser Caucasus within Azerbaijan. For the analyzes used nuclear DNA extracted from sheet material. DNA extraction, PCR and ISSR analyzes were carried out according to standard methods (CTAB, PCR, ISSR protocols). According to the results of analyzes using 4 ISSR primers, the number of identified fragments was 42, which corresponds to 9–12 loci per primer (~10.8). Of the 42 fragments identified, 34 (80.95%) are polymorphic, and 8 (19.05%) are monomorphic. The number of polymorphic loci varies in the range of 7–10. With the smallest number of amplified loci in the UBC811 primer, the largest number of them occurs in the UBC827 primer. Depending on the primer, the number of amplified polymorphic loci varies within 63–90%. The level of ISSR primer polymorphism is on average 86% (75–96%). The average value of the actual heterozygosis (H0) is 0.359, and the expected heterozygosis (HE) is 0.414. According to the results of the cluster analysis, 70 hazel genotypes are combined in 9 clusters. Despite the fact that the populations are remotely and orographically sufficiently isolated, which excludes the flow of genetic information between them, the results of the cluster analysis show that genotypes from different populations are combined into a common sub-cluster in terms of genetic similarity. This is due to the common origin of hazel in populations. In the distant past, this species was represented by an extensive common range, which was fragmented as a result of geological processes. The homogeneous disjunction of the continuous range occurred.

Genetic Diversity as Revealed by Intersimple Sequence Repeat Polymorphism in Narcissus Accessions to Identify the Tolerant Genotypes for Deficit Irrigation

There are many species of Narcissus in diverse areas of the world in natural or cultured form and there is no complete information about their genetic status, especially the relatedness within a species. Thus, the current study applied intersimple sequence repeat (ISSR) markers to estimate the genetic diversity of 31 accessions, including 30 accessions of Narcissus tazetta, collected from 16 regions of Iran and one known exotic narcissus species that is being cultivated in Iran, and identification of tolerant genotypes for deficit irrigation by evaluation of their morpho-physiological characteristics. Seventeen anchored ISSR primers from a total of 19 tested ISSR primer pairs were used and produced 206 bands of different sizes. The average percentage of polymorphic bands was 96.02%. The maximum resolving power (8.32), polymorphic information content average (0.44), and marker index values (5.61) were observed for the primers of 811, 828, and 811, respectively. The unweighted pair group method with arithmetic mean based on Jaccard’s coefficients was used to assign the genotypes to one of two major clusters. Both clusters were divided into two subclusters, with single and double flowers separating into subgroups. The results showed that ISSR markers can be used as a diagnostic tool to evaluate genetic variation in Narcissus genotypes and reveal their relationships. The results of screening study identified drought-tolerant accessions. They were clustered into two major groups: drought-tolerant accessions with single flowers and drought-sensitive accessions having double and semidouble flowers. The findings presented can be used in breeding programs for different Narcissus genotypes.

KERAGAMAN GENETIK ALANG-ALANG (Imperata cylindrica (L.) Beauv.) BERDASARKAN MARKA INTER-SIMPLE SEQUENCE REPEATS (ISSR)

Alang-alang (Imperata cylindrica (L.) Beauv.) has been widely used as a medicinal plant to treat some diseases, such as fever, headache, and diuretic. Nowadays, there is no information of genetic diversity of this plant used in herbal formula by ethnic groups in Indonesia. The main objective of this study was to asses genetic diversity of alang-alang from 18 selected ethnic groups in Indonesia based on Inter Simple Sequence Repeats (ISSR). Location of sample collection was identified by using data on Research on Medicinal Plant (Ristoja) 2012. Total DNA genome was isolated and ISSR primer screening were done on collected samples. Ten selected ISSR primers produced 74 amplified DNA fragments 58, fragments (78.4 %) were polymorphic. Dice index similarity was used to construct UPGMA dendrogram. The genetic similarity indexing which among accessions was ranged from 70.5–90.5% thereby indicating a low level of genetic diversity occurred in alang-alang. The results of this study also showed that ISSR markers were able to genetically differentiate alang-alang accessions by which this information can be useful for further researchs such as for standardization of medicinal plants.

INTRA SPECIFIC ANALYSIS OF GLORIOSA SUPERBA (L) THROUGH ISSR FINGER PRINTING AND DNA SEQUENCING OF ECOTYPES COLLECTED FROM DIFFERENT ACCESSIONS OF TAMIL NADU STATE, INDIA

Diversity within and among the population of Gloriosa superba collected from five different location of Tamil Nadu State, India, were explored by using Inter Simple Sequence Repeat (ISSR) DNA sequencing method. A total of 86.27% of polymorphic bands were seen in 94 reproducible bands generated from 12 number of ISSR primer ranging between 200bp to1000bp of band width. ISSR primer produces the bands with an average polymorphism of more than ninety nine percent among all the ecotypes. The dendrograms drawn for the analysis of genetic similarity in all the ecotypes. Our studies revealed that, genetic variations among different accessions of Gloriosa superba (L) was very well identified with the help of ISSR finger printing technique.

Largely invariant trnL-F/nrITS sequences and ISSRs do not resolve morphospecies of Warburgia (Canellaceae)

The aim of this study was to provide a molecular perspective on the taxonomy of Warburgia across most of its range, including South Africa, Zimbabwe, Mozambique, Tanzania, Kenya and Uganda. An accurate taxonomic treatment of Warburgia is important for the construction of sound conservation strategy, as a number of the proposed species and subspecies are regarded as endangered or critically endangered. Analyses based on 702 nucleotides of the plastid trnL-F region, 532 nucleotides of the nuclear ribosomal (nr) ITS region and eight polymorphic ISSR primer pairs yielded congruent results. Molecular clock analyses yielded a crown age of 45.5 (31.2–64.9) MY for Canellaceae. The crown age of Warburgia was dated to the early Miocene, 19.6 (11.4–29.1) MYA, with diversification into moderately supported lineages in the later Miocene, 11.6 (6.0–19.0) and 7.2 (3.2–12.7) MYA. Warburgia samples formed three major clades: a widespread clade covering most of the range, a northeast clade from parts of Kenya and Tanzania, and a stuhlmannii clade formed by GenBank samples accessioned as W. stuhlmannii. There was some ambiguity in the association between these clades and morphologically defined Warburgia species. We found no evidence to support W. elongata as a genetically distinct species. Genetic diversity in Warburgia was low in both DNA markers. We propose inclusion of these haplotypes in conservation strategies. Further molecular analyses based on a greater number of more variable DNA regions and an expanded sample set with more extensive geographical coverage are required to clarify whether the above groupings warrant status as genetic or phylogenetic species and to refine conservation strategies so as to include greater genetic variability.