Polymorphic Inter-simple Sequence Repeat Research Articles

Molecular Characterisation of Post-Fire Naturally Regenerated Populations of Maritime Pine (Pinus pinaster Ait.) in the North of Portugal

Wildfires act as a selection force threatening the sustainability and diversity of forest genetic resources. Few studies have investigated the genetic effects of forest wildfires. Species with perennial canopy seed banks in serotinous cones and soil or with long-distance seed and pollen dispersion can preserve genetic diversity and population differentiation under normal fire regimes. To test this hypothesis, we characterised molecularly Pinus pinaster Aiton (maritime pine) seedlings produced from seeds sampled in post-fire, naturally regenerated populations that had been subject to different fire regimes in the North of Portugal using inter-simple sequence repeats (ISSRs). The sampled populations burned once (A), twice (B), or three (D) times or had no prior fire history (C, control). Given the globally low seed germination ability, only 104 plantlets regenerated and were described. These plantlets were grouped according to their origin population. Intra-group ISSR polymorphism ranged from 72.73% (B) to 89.41% (D), revealing genetic differentiation among groups originating from populations that had experienced different fire recurrence. Overall, the unaffected genetic diversity of the regenerated plantlets allowed us to accept the hypothesis. Our findings enhance our understanding of the species ability to withstand fire-induced challenges and their responses to wildfires, guiding conservation endeavours and forest management strategies to bolster ecosystem resilience.

Molecular and histological validation of modified in ovulo nucellus culture based high-competency direct somatic embryogenesis and amplitude true-to-the-type plantlet recovery in Kinnow mandarin.

Kinnow (Citrus nobilis Lour. × Citrus deliciosa Ten.) needs to be genetically improved for traits such as seedlessness using biotechnological tools. Indirect somatic embryogenesis (ISE) protocols have been reported for citrus improvement. However, its use is restricted due to frequent occurrences of somaclonal variation and low recovery of plantlets. Direct somatic embryogenesis (DSE) using nucellus culture has played a significant role in apomictic fruit crops. However, its application in citrus is limited due to the injury caused to tissues during isolation. Optimization of the explant developmental stage, explant preparation method, and modification in the in vitro culture techniques can play a vital role in overcoming the limitation. The present investigation deals with a modified in ovulo nucellus culture technique after the concurrent exclusion of preexisting embryos. The ovule developmental events were examined in immature fruits at different stages of fruit growth (stages I-VII). The ovules of stage III fruits (>21-25 mm in diameter) were found appropriate for in ovulo nucellus culture. Optimized ovule size induced somatic embryos at the micropylar cut end on induction medium containing Driver and Kuniyuki Walnut (DKW) basal medium with kinetin (KIN) 5.0 mg L-1 and malt extract (ME) 1,000 mg L-1. Simultaneously, the same medium supported the maturation of somatic embryos. The matured embryos from the above medium gave robust germination with bipolar conversion on Murashige and Tucker (MT) medium + gibberellic acid (GA3) 2.0 mg L-1 + ά-naphthaleneacetic acid (NAA) 0.5 mg L-1 + spermidine 100 mg L-1 + coconut water (CW) 10% (v/v). The bipolar germinated seedlings established well upon preconditioning in a plant bio regulator (PBR)-free liquid medium under the light. Consequently, a cent percent survival of emblings was achieved on a potting medium containing cocopeat:vermiculite:perlite (2:1:1). Histological studies confirmed the single nucellus cell origin of somatic embryos by undergoing normal developmental events. Eight polymorphic Inter Simple Sequence Repeats (ISSR) markers confirmed the genetic stability of acclimatized emblings. Since the protocol can induce rapid single-cell origin of genetically stable in vitro regenerants in high frequency, it has potential for the induction of solid mutants, besides crop improvement, mass multiplication, gene editing, and virus elimination in Kinnow mandarin.

Possibility of utilizing Inter Simple Sequence Repeat regions, bark powder morphology and floral morphometry to characterize the <em>Cinnamomum</em> species in Sri Lanka

The genus Cinnamomum of the family Lauraceae is an economically important crop. The Ceylon cinnamon (Cinnamomum verum syn. C. zeylanicum) has an exceptional position in the global cinnamon market. In addition to the cultivated species, Sri Lanka is home to seven endemic wild Cinnamomum species, C. capparu-coronde, C. citriodorum, C. dubium, C. litseifolium, C. ovalifolium, C. rivulorum, and C. sinharajaense. Nevertheless, the species delimitation has not been successful with some morphometric and molecular traits. Therefore, this study focused on molecular characterization with an Inter Simple Sequence Repeats (ISSR) nuclear marker, floral morphometric characteristics, and microscopy of the powered bark of Cinnamomum species reported in Sri Lanka. According to the results four polymorphic ISSR regions resulted in an average of 83.7% polymorphism among all collected species, suggesting considerable polymorphism. The bark fiber size of the cultivated species is different from the studied wild species and could be used as a key to identify adulterants during export. The cultivated cinnamon, C. verum and two wild species (C. sinharajaense, and C. capparu-coronde) have considerably larger flowers compared to other species. Moreover, floral traits such as flower colour and shape could differentiate species C. sinharajaense and C. capparu-coronde. The Scanning Electron Microscopy (SCM) of pollen showed that the pollen size, spine length, interspinal distance, and spine ornamentation significantly contribute to species variation, and such variations need to be studied comprehensively. Nevertheless, the phylogeny of Cinnamomum species could not be completely resolved using ISSR regions, bark powder morphology, and floral morphological traits assessed and suggested for further studies.

Genetic variability and pathogenicity of Fusarium verticillioides isolates from the summer‐sown maize regions in China

AbstractMaize ear rot, one of the most destructive diseases in summer‐sown maize areas of China, is caused by several Fusarium spp. Not only does this pathogen lower maize yields, but also negatively impacts both animal and human health through the production of fumonisin mycotoxins. In this study, we collected 472 unique isolates of F. verticillioides from 480 samples of maize ear rots across eight Chinese summer‐sown maize regions and evaluated their genetic variability and aggressiveness. Analysis of polymorphic intersimple sequence repeat (ISSR) loci revealed high levels of genetic variation, particularly in Henan and Shandong provinces. The STRUCTURE result showed that all F. verticillioides isolates clustered into three populations, rather than eight distinct geographical provinces, and there was no significant positive correlation between genetic distance and geographical distance. Most of the variation resulted from genetic differences within rather than between populations. Pathogenicity varied among the evaluated F. verticillioides isolates, and most isolates examined, caused a moderate level of disease; there was no strong correlation between pathogenicity and genetic diversity. This is the first comprehensive report of both the genetic variability and pathogenicity of F. verticillioides across the whole summer‐sown maize region in China. These results provide important information that can inform efforts to control maize ear rot and fumonisin contamination caused by F. verticillioides.

Inter Simple Sequence Repeat-Based Genetic Divergence and Varietal Identification of Banana in Pakistan

Banana is one of the major cash and fruit crops of Pakistan. The lack of information concerning genetic diversity and purity within locally cultivated banana varieties is a major bottleneck in improving its genetics. Due to the existence of a narrow genetic background, it’s quite important to find genomic variations in banana varieties. DNA marker-based techniques have been used to effectively characterize banana varieties. In the current study, Inter Simple Sequence Repeat (ISSR) markers were used to characterize banana cultivars and to assess the genetic diversity of 14 local banana varieties grown in Pakistan. Out of the 45 primers used, 40 primers revealed reproducible results and produced 121 polymorphic bands, which contributed a ratio of 47.87 polymorphism. The ISSR UBC-835 and UBC-834 possessed the highest PIC ranged between (86–88%) in banana varieties, while the lowest PIC (46%) was detected in the case of UBC−857 marker with (100–1500 bp) PCR product size. Pairwise Jaccard’s similarity coefficient values were also calculated, and these were ranged from 0.56–0.88. Multivariate analysis divided 14 banana varieties into two distinct groups—A and B respectively—and furthermore into subgroups, clusters, and sub−clusters. Our results indicated that at the molecular level, the banana varieties in group—A were found to be 66% similar whereas in group B were 88% similar. Nei’s genetic diversity, PCA analysis, and a minimum spanning tree depicted Fenjiao, Dajiao, and NIGAB-2 as the most diverse members as compared to all other varieties of the three populations. Out of 14 varieties used, 11 varieties were uniquely identified by 54 polymorphic ISSR bands of different sizes. Some varieties like NIGAB-2 and NIGAB-3 were uniquely identified only with one band while others were tagged by multiple unique bands. In future, this study will be utilized to establish a molecular-based protocol for the identification of banana varieties.

Improvement of Cercospora leaf spot and powdery mildew resistance of mungbean variety KING through marker-assisted selection

AbstractThe development of resistant mungbean varieties is one of the most efficient strategies to control major diseases such as Cercospora leaf spot (CLS) and powdery mildew (PM). The objectives of this study were to pyramid a CLS resistance gene and two PM resistance genes from the donor parent D2 into a susceptible variety KING through marker-assisted backcrossing (MABC) and to evaluate their agronomic traits and disease resistance under field conditions. Five markers linked to the resistance genes were used for foreground selection, while two marker sets [Set A containing 15 polymorphic simple sequence repeat (SSR) and expressed sequence tag-SSR (EST-SSR) markers and Set B containing 34 polymorphic inter-simple sequence repeat (ISSR) loci] were also used for background selection. Two pyramided backcross (BC) lines, namely H3 and H4, were homozygous at all five marker loci when confirmed in BC4F4 and BC4F5 generations. Their recurrent parent genome (RPG) recovery ranged from 96.4 to 100.0%, depending on the marker sets. During field evaluation, a moderate to high level of CLS and PM resistance was observed in both BC lines compared to the susceptible recurrent parent KING. One of these BC lines (H3) had all agronomic traits similar or superior to the recurrent parent KING at all environments, and had a higher yield than KING (18.0–32.0%) under CLS and PM outbreaks. This line can be developed into a new resistant mungbean variety in Thailand in the future. These results substantiate the usefulness of MABC for transferring multiple resistance genes into an elite variety.

Pathogenicity, anastomosis groups, host range, and genetic diversity of Rhizoctonia species isolated from soybean, pea, and other crops in Alberta and Manitoba, Canada

Root rot is a common disease in soybean (Glycine max) and field pea (Pisum sativum), which restrain increased production in Canada. Sixty-seven isolates of Rhizoctonia were recovered from various diseased plants in Alberta, Canada along with three isolates from diseased soybean plants in Manitoba, Canada. According to their anastomosis behavior, 23 (32.9%) of the isolates were identified as anastomosis group (AG) 4 (AG4), 7 (10.0%) were AG2-1, 10 (14.3%) were AG2-2, 7 (10.0%) were AG5, 3 (4.3%) were AG-E and the AGs of the remaining 20 (28.6%) isolates could not be determined. Isolates belonging to AG4 produced typical symptoms of stem rot and root rot on seedlings of soybean and pea and were more aggressive than the AG2-1, AG2-2, AG5 and AG-E isolates. Selected isolates of AG4, AG2-1, AG2-2, AG5 and AG-E were to some degree able to infect common crops in Alberta, which included barley, canola, corn, faba bean, flax, lupin, lentil, pea, potato, soybean, and wheat. The genetic variability among these isolates was evaluated using phylogenetic analysis based on the rDNA ITS sequences and inter-simple sequence repeat (ISSR) markers. For the ITS sequence analysis, a neighbour-joining tree was constructed using the PAUP program, which clustered the Rhizoctonia isolates into five groups (Groups I to V). However, no correlation was observed between AGs, locations, aggressiveness or host origins. For the ISSR analysis, 54 polymorphic ISSR patterns were identified, indicating a high level of diversity among the isolates.

Genetic Diversity in Diospyros Germplasm in the Western Caucasus Based on SSR and ISSR Polymorphism.

Simple SummaryPersimmon is an edible fruit consisting of several species in the genus Diospyros. The most widely cultivated and most commercially important species is the Oriental persimmon, Diospyros kaki. However, the inter- and intra-specific genetic diversity of the genus Diospyros remains largely unclear and is of great interest both for conservation and breeding purposes. This study describes the genetic diversity and genetic admixture of Diospyros germplasm in the Western Caucasus. The information can be used to support conservation measures and the breeding of persimmon.Persimmon germplasm in the Western Caucasus represent one of the most northerly collections. In our study, 51 commercial cultivars of D. kaki, 3 accessions of D. virginiana and 57 D. lotus accessions from six geographically distant populations were investigated using 19 microsatellite and 10 inter simple sequence repeat (ISSR) markers. After STRUCTURE analysis, the single accessions of Diospyros were allocated to three genetic clusters. Genetic admixtures in the important genotypes of D. kaki were revealed, whereas D. lotus accessions showed no admixture with other genetic clusters. The correspondence of genetic data and phenotypical traits was estimated in the D. kaki collection. The most frost tolerant genotypes of the collection, such as “Mountain Rogers”, “Nikitskaya Bordovaya”, “Rossiyanka”, “MVG Omarova”, “Meader”, “Costata”, “BBG”, and “Jiro”, showed a high percentage of genetic admixtures and were grouped close to D. virginiana. Some of these genotypes are known to be interspecific hybrids with D. virginiana. A low level of genetic diversity between the distant D. lotus populations was revealed and it can be speculated that D. lotus was introduced to the Western Caucasus from a single germplasm source. These results are an important basis for the implementation of conservation measures, developing breeding strategies, and improving breeding efficiency.

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

AbstractInterspecific 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.

Morphological variation, genetic diversity and phylogenetic relationships of Hypericum triquetrifolium Turra populations from Tunisia

Hypericum triquetrifolium Turra is an ecologically, medicinally and economically important species in Tunisia. Thirty-six Hypericum individuals sampled from 6 northern Tunisian locations were investigated for their diversity and relationships using 10 inter-simple sequence repeats (ISSR) markers and 10 morphological features at vegetative stage. The phylogenetic analysis, using 308 bp of sequenced ITS1 region, identified the Hypericum individuals as H. triquetrifolium that clustered with members of genus Hypericum section 9, 9a, 9b and 27, in agreement with the previous molecular classification of the genus. Among the 10 ISSR markers tested, 7 were scorable and yielded 91 loci with 94.5% of polymorphism. UBC848 and UBC836 were the most polymorphic ISSR markers. The level of genetic diversity (HT = 0.247) and gene flow between the six populations (N m = 1.169) were moderate. The structure analysis revealed three genetic subpopulations: individuals of Le Krib location formed a subpopulation divergent from two other subpopulations, probably due to its northwestern and high-altitude geographic barriers, and its sub-humid microclimate. Zaghouan, northeastern location in the lower semi-arid, with the highest genetic (I = 0.370) and morphological (I = 0.631) Shannon’s information indices and, regrouping two out of the three genetic subpopulations, is the most probable zone of origin for H. triquetrifolium. In addition, morphological data showed higher diversity than ISSR data; however, no evidence of correlation between genetic and morphologic traits could be suggested in this study. These results on the genetic diversity and phylogenetic analysis will contribute to the conservation of the gene pool of H. triquetrifolium in Tunisia.

Assessment of Somaclonal Variations in Embryo-derived Axillary Shoots of Chickpea using Molecular Markers

Background: Somaclonal variation is considered as a source of genetic variation for crop improvement. It has been investigated using cytological, biochemical and molecular techniques.Methods: Genetic stability in the embryo-derived axillary shoots of 4 chickpea genotypes was assessed using eight inter-simple sequence repeat (ISSR) and 19 random amplified polymorphic DNA (RAPD) primers. Result: RAPD primers produced 94 and ISSR primers produced 38 distinct and scorable alleles, with an average of 4.9 alleles for RAPD and 4.75 for ISSR primers. The polymorphic information content (PIC) ranged from 0.36 to 0.90 for RAPD and from 0.50 to 0.87 for ISSR. ISSR recognized a 90%, but RAPD recognized 82% similarity value. No absolute similarity value was between the mother plant and the regenerated shoots for the overall genotypes. At a 90% similarity value, 15 out of the 20 regenerated shoots from ‘Giza 88’ group with their mother plant using ISSR markers; however, 11 regenerated shoots grouped with their mother plant in one central cluster for ‘Giza 4’ using RAPD markers. The observed variations in the total number of polymorphic RAPD and ISSR bands and the number of bands specific to the mother and regenerated shoots, detected intra-clonal variation and genetic instability seem to be genotype-dependent.

Short Communication: Genetic polymorphism of registered and popularly cultivated coffee (Coffea spp.) in the Philippines using inter-simple sequence repeats markers

Abstract. Panaligan AC, Baltazar MD, Alejandro GJD. 2020. Short Communication: Genetic polymorphism of registered and popularly cultivated coffee (Coffea spp.) in the Philippines using inter-simple sequence repeats markers. Biodiversitas 21: 4228-4233. Three Coffea species, namely Coffea arabica, Coffea canephora and Coffea liberica have been commercially cultivated in the Philippines. Genetic variability analysis of these species is important for the conservation of genetic resources and breeding programs. Hence, this study was carried out to identify polymorphic inter-simple sequence repeats (ISSR) markers and determine the genetic variation of these three commercially cultivated coffee species. Twenty-nine DNA samples from young coffee leaves were extracted and PCR amplified. Of the 29 primers used, seven produced clear and reproducible bands. In the 54 bands produced, 51 were polymorphic. The number of bands amplified by each primer varied from 5 to 12 with an average of 7.71 bands. Polymorphism percentage ranged from 80 to 100. This is the first time that ISSR markers were used to determine the genetic variation of coffee in the Philippines. The study demonstrated the efficiency of ISSR markers to assess genetic variation in cultivated coffee species. The ISSR markers were able to differentiate the coffee germplasm at the interspecific and intraspecific levels. These results suggest the potential of ISSR markers for genetic diversity analysis of commercial coffee and varietal identification of elite varieties using DNA fingerprinting.

Genetic diversity analysis in Plectranthus edulis (Vatke) Agnew populations collected from diverse geographic regions in Ethiopia using inter-simple sequence repeats (ISSRs) DNA marker system

BackgroundPlectranthus edulis (Vatke) Agnew (Lamiaceae), locally known as Ethiopian potato syno. Ethiopian dinich, is one of the native Ethiopian edible tuber crops that has been significantly contributing to household food security for millions of subsistence farmers. However, its current production is declining to the extent of total extinction from several administrative regions where it used to be widely cultivated. It is one of the less researched crops regardless of being indigenous and its contribution to food security during time of scarcity. Therefore, we intended to assess the level of genetic diversity in 67 accessions, representing nine populations that were collected from diverse agro-ecologies in the country, using ISSR markers and hence, to generate a baseline information that assists marker assisted breeding, conservation and germplasm management efforts.ResultsIn the present study, ten polymorphic ISSR markers were screened and optimized, that generated an average of 7.4 scorable bands per marker and revealed high overall percent polymorphism (95%), Nei’s gene diversity (h = 0.40) and Shannon index (I = 0.62) suggesting ISSR’s effectiveness in detecting high levels of genetic diversity. A considerably high overall populations gene diversity (Nei’s) (h = 0.32) and Shannon index (I = 0.47) were observed, revealing high potential of the populations for further breeding and conservation efforts particularly for population from Gurage administrative zone, which showed the highest values. Similarly, estimation of pairwise genetic distance revealed the importance of cross breeding population from Awi administrative zone to the rest populations. Analysis of hierarchical molecular variance (AMOVA) showed higher levels of genetic differentiation within populations (92%), and collection regions (94%) suggesting that either clonal mode of propagation in the crop or farmers selection pressure for important agronomic traits or both maintained the original heterozygosity in the crop. UPGMA phylogenetic analysis did not strictly group the populations based on their geographic region of origin, which could be attributed to the widely practiced tuber exchange and hence continuous human mediated exchange of genetic material and sharing of the same genetic base among the geographic regions.ConclusionsThe ISSR markers used in the present study were effective in revealing extent and patterns of genetic diversity in P. edulis populations. However, it is important to couple them with agro-morphological traits or codominant molecular markers to get more reliable information for use in breeding and conservation. Several of the potential administrative zones we covered are useful for P. edulis diversification and conservation. However, the crop is currently highly marginalized and this led to rapid decline in population size and loss of valuable agronomic traits. To address this challenge, there is an urgent need to take counteractive measures.

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.

Genetic diversity among melon ( Cucumis melo L.) accessions revealed by morphological traits and ISSR markers

The genetic relationships among 48 melon ( Cucumis melo L.) genotypes collected from various parts of Azerbaijan were determined by comparing their phenotypic and molecular traits. Eleven agromorphological traits and 10 polymorphic inter-simple sequence repeat (ISSR) primers were used to define the genetic diversity. Principal component analysis grouped the agromorphological traits into the first four axes, describing 78% of the total variations. The highest genetic variation coefficient was found for yield per hectare (20.32%) and for fruit length (17.35%). Calculated heritability for yield was 0.96. The analysis of morphological traits grouped the accessions into four clusters. The 10 ISSR primers yielded 35 polymorphic alleles, representing 85.4% of all the amplified loci. The average genetic diversity index determined was 0.70. The highest and the lowest similarity indexes were equal to 0.97 and 0.36, respectively. The 48 accessions were grouped into 10 clusters based on ISSR markers. Correlation between distance matrices based on agromorphological traits and ISSR markers was not statistically significant (r = 0.012).

Characterization of the Genetic Diversity of Acid Lime (Citrus aurantifolia (Christm.) Swingle) Cultivars of Eastern Nepal Using Inter-Simple Sequence Repeat Markers.

Acid lime (Citrus aurantifolia (Christm.) Swingle) is an important fruit crop, which has high commercial value and is cultivated in 60 out of the 77 districts representing all geographical landscapes of Nepal. A lack of improved high-yielding varieties, infestation with various diseases, and pests, as well as poor management practices might have contributed to its extremely reduced productivity, which necessitates a reliable understanding of genetic diversity in existing cultivars. Hereby, we aim to characterize the genetic diversity of acid lime cultivars cultivated at three different agro-ecological gradients of eastern Nepal, employing PCR-based inter-simple sequence repeat (ISSR) markers. Altogether, 21 polymorphic ISSR markers were used to assess the genetic diversity in 60 acid lime cultivars sampled from different geographical locations. Analysis of binary data matrix was performed on the basis of bands obtained, and principal coordinate analysis and phenogram construction were performed using different computer algorithms. ISSR profiling yielded 234 amplicons, of which 87.18% were polymorphic. The number of amplified fragments ranged from 7–18, with amplicon size ranging from ca. 250–3200 bp. The Numerical Taxonomy and Multivariate System (NTSYS)-based cluster analysis using the unweighted pair group method of arithmetic averages (UPGMA) algorithm and Dice similarity coefficient separated 60 cultivars into two major and three minor clusters. Genetic diversity analysis using Popgene ver. 1.32 revealed the highest percentage of polymorphic bands (PPB), Nei’s genetic diversity (H), and Shannon’s information index (I) for the Terai zone (PPB = 69.66%; H = 0.215; I = 0.325), and the lowest of all three for the high hill zone (PPB = 55.13%; H = 0.173; I = 0.262). Thus, our data indicate that the ISSR marker has been successfully employed for evaluating the genetic diversity of Nepalese acid lime cultivars and has furnished valuable information on intrinsic genetic diversity and the relationship between cultivars that might be useful in acid lime breeding and conservation programs in Nepal.

Molecular authentication of Trichosanthes species traded as “Patola:” An ayurvedic drug resource

Background: Different interpretations of ayurvedic texts have recognized/recommended taxonomically distinct species/varieties of Trichosanthes as “Patola,” which is used extensively in the Indian traditional system of medicine for curing a variety of disorders. Sources of “Patola” also vary depending on region-specific availability and diverse traditions in different parts of India, which creates ambiguity in correct identity of “Patola.” Since “Patola” is one of the medicinal plant species traded in high volumes, its exact taxonomic identity and authentication is very essential. Objectives: Molecular profiling of different taxa recognized as “Patola” was carried out for correct identification and authentication of commercial crude drug samples. Materials and Methods: Molecular marker profiles of different taxa recognized as “Patola” were generated using inter-simple sequence repeat (ISSR) markers. Attempt was also made to authenticate commercial crude drug samples, using ISSR markers. Results: Selected 10 polymorphic ISSR primers generated distinct amplification profiles for the taxa traded as “Patola.” Moreover, two varieties of Trichosanthes cucumerina (var. cucumerina and var. anguina) could be clearly distinguished based on the specific bands produced by primers UBC 812, 822, 887, 888, and 889. Molecular profiling of market samples along with the genuine plant samples confirmed the trade of different Trichosanthes species as well as Momordica charantia under the name of “Patola.” Conclusion: To our knowledge, this is a first report on the application of ISSR markers for identification and authentication of “Patola.” Developed DNA profiles should be useful in investigations of adulteration in market samples and establishment of correct identity. Abbreviations used: AFLP: Amplified fragment length polymorphism; AHMA: Agharkar Herbarium at Maharashtra Association; DNA: Deoxyribonucleic acid; ISSR: Inter-simple sequence repeat; MC: Momordica charantia; PCR: Polymerase chain reaction; RAPD: Random amplified polymorphic DNA; TAE: Tris acetate-EDTA buffer; TC: Trichosanthes cucumerina; TCA: Trichosanthes cucumerina var. anguina; TCC: Trichosanthes cucumerina var. cucumerina; TD: Trichosanthes dioica; TT: Trichosanthes tricuspidata; TE: Tris-EDTA buffer; UBC: University of british Columbia, Vancouver, Canada.

Effects of fragmentation and anthropic pressure on the genetic structure of Canthon (Peltecanthon) staigi (Coleoptera: Scarabaeidae) populations in the Atlantic Forest domain

Canthon (Peltecanthon) staigi is an endemic dung beetle from the east coast of South America (Brazil, Paraguay and Argentina). It is a stenotopic species abundant in different habitats of the Atlantic Forest. In this study, we analyzed the influence of fragmentation and environmental changes on the population genetic structure of C. (P.) staigi using inter-simple sequence repeat polymorphisms, and the consequences for management and conservation. The expected heterozygosity (HE) ranged from 0.16 (restinga, TAM population) in the smallest and most disturbed forest patch to 0.24 (“brejo de altitude”, PES population) in the largest and most conserved area. Based on the low coefficient of genetic population differentiation (FST = 0.07) and large molecular variance within populations, we suggest historical gene flow among C. (P.) staigi populations. Additionally, the model ƒ = 0, which corresponds to a lack of or very low inbreeding rate, is another factor that indicates gene flow among populations. Two genetic traits (K = 2) were related with anthropic disturbance, suggesting that the six studied populations are genetically divided into two separate units, one related to preserved and another to disturbed areas. Based on these data, the recent habitat fragmentation may not have led to genetic differentiation, but rather the environmental modifications caused by anthropic pressure seem to be strongly related to the loss of genetic diversity in the species C. (P.) staigi.

Evaluation of diversity in Bulgarian pepper cultivars by agronomical traits and ISSR markers

Information about the genetic variation among cultivars of vegetable crops is of vital importance for improvement of plant breeding programmes worldwide. The objectives of this study were to group 19 pepper (Capsicum annuum L.) cultivars from the collection of Maritsa Vegetable Crops Research Institute, Plovdiv, Bulgariainto clusters according to their distances as estimated by agronomic traits and 9 di-and tri -nucleotide inter simple sequence repeat polymorphism (ISSR) markers and to assess the relationships between them. The phenotypic characterization during 3 consecutive years revealed significant differences among Bulgarian cultivars for the studied 13 phenotypic traits. The biplot analysis of quantitative traits showed that the most strongly correlating traits with the first axis (55.6% of variance) were fruit width, fruit weight and pericarp thickness (in the negative direction of the axis), and plant height (PH) (in the positive direction). The most discriminative traits, considering the second axis (22.6% of variance) were fruit length (FL) and to a lesser extent the stem height (StH). The correspondence analysis of the qualitative traits showed that the intensity of the colour of the fruit (before and at maturity), fruit colourbefore maturity and fruit shape in longitudinal section were the most discriminative characteristics for the first two dimensions. The agronomic traits data and 7 dinucleotide ISSR primers were used to estimate the pairwise genetic distances. Higher mean phenotypic distance (0.414) in comparison to the genotypic ones (0.214) among the cultivars was observed, indicating higher phenotypic diversity among them. A highly signi?cant, positive correlation between the agronomic data and ISSR marker-based matrices (r=0.41, p=0.001)was detected. This indicates that ISSR distance tended to re?ect that of the agronomics ones. However, additional molecular studies and large collection of highly diverse genotypes are needed to reveal associations between the marker loci and specific QTLs. The research initiated is a base for more precise estimation of genetic distances between pepper genotypes from the available large collection of landraces, local and modern cultivars including large number of highly polymorphic markers.

Mutation Induction of Dendrobium ‘Earsakul’ Using Sodium Azide

Dendrobium ‘Earsakul’ is an important commercial orchid in Thailand. Breeding new Dendrobium varieties for improved quality and yield is crucial. The objectives of this research were to perform in vitro mutagenesis of Dendrobium ‘Earsakul’ protocorm-like bodies (PLBs) using sodium azide (NaN3) and to select and evaluate the putative mutants using morphological characters, molecular markers, and the cytological method. The percentages of mortality of PLBs increased as concentrations of NaN3 increased. At 2 weeks, the lethal dose 30 (LD30) and LD50 were obtained with 0.1 and 0.5 mm NaN3, respectively. These two NaN3 concentrations were used for in vitro mutagenesis with reverse osmosis water (ROW; control 1) and 0 mm NaN3 (control 2) as controls. After the plants were cultured for 6 months, morphological differentiation was observed in some putative mutants: reduced height, higher numbers of nodes, reduced node length, shorter and thicker leaves, and shorter and fewer roots, compared with controls. When genetic profiles of 24 putative mutants were compared with controls, altered DNA profiles were found in 20 of 24 putative mutants (83.33%). Sixty-three polymorphic bands were produced from a total of 181 bands (34.81%) amplified by 10 polymorphic intersimple sequence repeat (ISSR) primers. When genetic diversity and relatedness, which were evaluated by ISSR analysis, and morphological characters were compared, the two markers were found to be uncorrelated. ISSR had a higher mutant differentiation capability than the morphological characters, indicating its higher efficiency. The chromosome numbers were similar in putative mutants and controls (2n = 2x = 24), suggesting that neither of the concentrations of NaN3 had any effect on the chromosome numbers in this experiment. These results indicate that NaN3 can be used effectively to mutagenize Dendrobium ‘Earsakul’ PLBs, and ISSR is a powerful tool for the identification of mutants. Chemical name: sodium azide (NaN3); reverse osmosis water (ROW).