Inter-simple Sequence Repeat Molecular Markers Research Articles

Microsatellite marker-based analysis of the genetic diversity and population structure of three Arnebiae Radix in western China

Arnebiae Radix is an important medicinal and perennial herb found in Western China, particularly in the Xinjiang region. However, the assessment, utilization and conservation of Arnebiae Radix resources are still unexplored. In this study, we evaluated the genetic diversity of three Arnebiae Radix populations across 47 regions (Ae = 16, Ag = 16, Ad = 15) in Xinjiang, China, using inter-simple sequence repeat (ISSR) molecular markers. In total, 48 alleles were amplified by six pairs of primers screened with ISSR markers. The average number of effective alleles (Ne) was 1.5770. The percentage of interspecific genetic polymorphisms in A. guttata (Ag = 89.58 %) was greater than that in A. euchroma. and A. decumbens (Ae = Ad = 87.50 %). Intraspecific genetic polymorphisms, Bo Le (BL) population of A. euchroma exhibited the highest percentage of polymorphic bands (PPB% =58.33 %, Na = 1.313, Ne = 1.467, I = 0.0.366, H = 0.255), which indicated high genetic diversity. In contrast, the Tuo Li (TL) population of A. guttata had the lowest values for these parameters (PPB% =0.00 %, Na = 0.313, Ne = 1,000, I = 0.000, H = 0.000). The Arnebiae Radix germplasms were classified into two major groups (I and II) based on UPGMA cluster analysis (Fig. 8a) and principal coordinate analysis (PCOA). In addition, A. decumbens is placed in a separate category due to its high differentiation coefficient. The AMOVA and genetic differentiation coefficient results indicated that the genetic variation in Arnebiae Radix was predominantly due to intrapopulation differences (78 %). Additionally, the gene flow index (Nm) between populations was 2.4128, which further indicated that the genetic diversity of Arnebiae Radix was greater at the intrapopulation level. The destruction of the ecological environment leads to the continuous reduction and degradation of the genetic diversity of Arnebiae Radix germplasm resources. In this study, we used ISSR molecular markers to analyze the genetic diversity and relatedness of Arnebiae Radix, which revealed the genetic relationship of Arnebiae Radix germplasm resources at the molecular level and provided a scientific basis for future research on selecting and breeding good varieties, evaluating the quality of Arnebiae Radix, and conserving and utilizing its resources.

Morphological and molecular characterization of cubios (Tropaeolum tuberosum Ruiz & Pavón) collected in two municipalities in Boyaca - Colombia

In this study, we aimed to determine the extent of genetic variation among cubios (Tropaeolum tuberosum) in a diversity micro-center in the department of Boyacá, Colombia, with morphological and ISSR (Inter Simple Sequence Repeats) molecular markers. Twenty-five cubio morphotypes were collected across different farms within the municipalities of Belén and Ventaquemada in Boyacá and were sown for subsequent morphological and molecular characterizations. Tuber features were evaluated according to descriptors of the International Potato Center, namely predominant tuber surface color, secondary tuber surface color, secondary tuber surface color distribution, predominant tuber pulp color, secondary tuber pulp color, distribution of secondary tuber pulp color, tuber shape, and depth of tuber eyes. Molecular variation was assessed via eight ISSR primers. Morphological and molecular characterizations allowed us to identify 25 cubio morphotypes with significant genetic variation in the study area. This finding is likely attributed to mechanisms maintaining genetic variability, such as naturally occurring crosses between morphotypes modulated by environmental conditions and local agricultural practices, including seed exchange and a preference for given morphotypes.

Genetic diversity of Guava varieties (Psidium guajava L.) based on morphology and ISSR molecular markers

Abstract. Hien PTT, Cham LTT, Hang VTT. 2024. Genetic diversity of Guava varieties (Psidium guajava L.) based on morphology and ISSR molecular markers. Biodiversitas 25: 1037-1045. Guava (Psidium guajava L.) is a fleshy-fruited representative belonging to the Myrtaceae family that has attracted attention due to its economic benefits. In this study, seventeen guava varieties in the Mekong Delta and Hanoi were collected and genetically validated by both morphology and DNA markers. The results revealed that fruit shape is a powerful indicator of guava discrimination. There are 12 out of 15 Inter Simple Sequence Repeats (ISSR) markers that show informative band patterns and 93 amplicons are generated with the size in length 200-2000 bp. The average polymorphic band and PIC value are 97.85% and 36%, respectively. Based on the dendrogram constructed by the Unweighted Pair Group Method using arithmetic Averages (UPGMA) method, samples were grouped into two main groups and the similarity level ranged from 55-98%. Group 2 contains Oi Tim (Purple guava), a special variety that showed high differences in terms of morphology and DNA profiles. Such information reflects the diversity of guava germplasm in Viet Nam, which is useful for conservation, and breeding strategies. The result revealed that 11 varieties expressed genetic diversity through both phenotypic traits and DNA profiles. Such information illustrates the genetic diversity among Psidium guajava, contributing to germplasm resources and plant breeding programs.

Genetic Assessment of Leucaena leucocephala (Lam.) de Wit Provenances by Using ISSR Markers

Inter Simple Sequence Repeats (ISSR) technique was deployed for the estimation of genetic diversity in18 Leucaena leucocephala provenances. Seeds of these provenances were collected from across the country and planted at a single location in South India. To estimate the genetic diversity/ polymorphism, DNA was extracted through Cetyl Trimethyl Ammonium Bromide (CTAB) method, visualized and analyzed the DNA bands on UV trans- illuminator gel doc (α-imager 3.2). Totally ten ISSR primers were used in this study. Among them, eight ISSR microsatellites showed a high degree of polymorphism in all the eighteen leucaena provenances used in our study. These primers successfully amplified a total of 645 bands, of which 577 bands (89 %) showed polymorphism. On average, 14.92 bands per accession were obtained and 68.28 % provenances were distinguished through these primers. The ISSR primer T(GT)9 was able to distinguish 100 % of the L. luecocephalla provenances. Cluster analysis based on the genotypic similarities assembled the 18 leucaena accessions in to three clusters. The dendrogram generated based on the 8 ISSR primers distinctively demarcated many of the L. leucocephalla accessions into different clusters indicating vast genetic diversity among the provenances. The high degree of genetic variability demonstrated by the ISSR molecular markers in L. leucocephalla can be used as a tool for assessing genetic diversity and in future leucaena genetic improvement programs.

Genotyping of Vanilla planifolia Jacks ex Andrews from the Totonacapan region using Inter-Simple Sequence Repeat markers

Vanilla is the only genus of plants in the Orchidaceae family used in the food industry, being the most widely used flavoring worldwide and the second most valuable economically. Cultivation is of great biocultural importance in the Totonacapan region, in the state of Veracruz, as this region is considered its center of origin and distribution. However, little is known regarding the number of genotypes present in this area, as well as the genetic diversity, which is vital for the improvement of this crop. Therefore, the objective of this study was to perform the genotyping of 60 Vanilla planifolia accessions from the Germplasm Bank of The National Institute of Forestry, Agriculture, and Livestock Research (Instituto Nacional de Investigaciones Forestales, Agrícolas y Pecuarias - INIFAP), Experimental Field Ixtacuaco, which were selected and collected in the Totonacapan region. For this purpose, the number of V. planifolia genotypes was determined based on ISSR (Inter-Simple Sequence Repeat) molecular markers. Three DNA extraction protocols from plants and fungi described in the literature were evaluated, and a fourth protocol was adapted with different procedures from the previous protocols, seeking to obtain a high concentration and good quality and integrity. Once quality DNA was obtained, fourteen ISSR primers were evaluated, of which three primers that showed the highest number of amplified bands were selected. Subsequently, the genotyping of the 60 accessions was carried out, resulting in nine genotypes with primer 808 and six genotypes with primers 812 and 856. These results allowed the genotyping of Vanilla planifolia accessions with ISSR markers, which will help evaluate the genetic diversity of this crop in the Totonacapan region in the future.

Interspecific Hybridization between Ganoderma lingzhi and G. resinaceum by PEG-Induced Double-Inactivated Protoplast Fusion

Ganoderma lingzhi is an important medicinal fungus, and it is particularly important to select strains with high yields and active substance contents. In this study, protoplasts of G. lingzhi were thermally inactivated to destroy intracellular enzyme proteins and preserve DNA. The DNA of G. resinaceum was damaged by ultraviolet (UV) radiation, and other components of the protoplasm except DNA were preserved. Then, the protoplast was induced using polyethylene glycol (PEG) for fusion. The results showed that the optimal thermal inactivation conditions for G. lingzhi were 30 min in a 45 °C water bath, and the optimal UV inactivation conditions for G. resinaceum were 70 s of irradiation using a 20 W UV lamp at a vertical distance of 15 cm. Antagonistic tests, internal transcribed space (ITS) and mitochondrial DNA identification, intersimple sequence repeat (ISSR) molecular markers and morphology were used to distinguish the parents from the fusants. Four true fusants were obtained, and the yield was 2.5%. The fruiting body yield of the fusants was significantly higher than that of G. lingzhi, and the polysaccharide and triterpene contents of the RAD-64 fusant were significantly higher than those of G. lingzhi. The results presented in this paper show that protoplast fusion technology can effectively improve G. lingzhi varieties and support the breeding of new varieties.

Evaluation of genetic diversity and production adaptability in different germplasms of Paspalum dilatatum

AbstractPaspalum dilatatum is an excellent warm‐season forage grass and soil conservation plant in subtropical regions. However, information on P. dilatatum cultivation in southwestern China is limited. A preliminary evaluation of the genetic diversity and adaptability of six P. dilatatum germplasms was conducted in Guizhou based on inter simple sequence repeat (ISSR) markers and field trials. Fifteen polymorphic primers were screened from 50 ISSR primers. A total of 141 bands were generated, 124 of which were polymorphic, yielding 88.63% polymorphic bands. The number of polymorphic loci detected for the different germplasms ranged from 23 to 61, and the percentage of polymorphic loci was 40.67%. The mean number of alleles was 1.23, and the effective allele number was 1.15. Nei's genetic diversity index was 0.08. The Shannon information index was 0.12, and the genetic similarity coefficient (GSC) ranged from 0.6002 to 0.8955, indicating a relatively rich genetic base among the germplasms. The unweighted pair group method with arithmetic mean approach was used to construct the clustering map. Using a GSC of 0.83 as the threshold value, the six germplasm materials were divided into four clusters, indicating that ISSR molecular markers can be effectively applied to analyze the genetic diversity of P. dilatatum germplasms. Cluster analysis of 12 growth characteristics of the germplasms of P. dilatatum showed that germplasm 4 exhibited strong tillering ability and a high crude protein content. Germplasm 3 showed high plant height, fresh yield and hay yield. Germplasm 1 showed the lowest forage yield and crude protein content. Germplasms 2, 5 and 6 showed excellent crude protein and crude fat contents; the remaining indicators showed intermediate performance. This study provides important references for the utilization and variety selection of introduced P. dilatatum resources in the karst areas of Guizhou.

The efficiency of SCoT, ISSR, and SRAP markers for detecting genetic polymorphism among Egyptian barley genotypes

The results of the present study support the idea that, the tested Egyptian barley cultivars could make a useful case study for genetic diversity research. When those cultivars were examined using three distinct markers (codon targeted (SCoT), sequence-related amplified polymorphism (SRAP) and inter-simple sequence repeats (ISSR)). According to SRAP, ISSR, and SCoT analyses, respectively 85, 76, and 66 polymorphism percentages among the seven tested barley cultivars were detected. The genotype specific markers for each genotype used were determined. The data revealed that Giza127 had the highest number of unique markers, showing six specific SCoT markers, three specific SRAP markers, and one specific ISSR marker. The genetic relationships between the seven Egyptian barley cultivars are explained by the phylogenetic tree built using information gathered from various molecular markers. These gene-based molecular markers demonstrated the reliability of SCoT, ISSR, and SRAP molecular markers for identifying DNA polymorphism levels and genetic relationships in barley.

Research on germplasm diversity of Amomum villosum. Lour in genuine producing area.

Genuine Chinese medicine is produced from medicinal plant cultivated in a specific region and is of better quality and efficacy, more consistently qualified and famous than that from the same medicinal plant cultivated in other regions. The cultivating region of genuine medicinal plant is known as the genuine producing area. Yangchun City, which is in Guangdong Province of China, is a genuine producing area for the famous Chinese medicine Amomi Fructus (also called Sharen). Amomi Fructus is the ripe and dry fruit of the Zingiberaceae plant A. villosum Lour.. A. villosum was introduced from the Persian Gulf region and has been cultivated in China for over 1000 years. Until now there are no reports on screening for good germplasm of A. villosum. The contents of volatile oil and bornyl acetate of Amomi Fructus from 14 populations were determined with GC method, and the relative contents of the main chemical components in the volatile oils were determined with GC-MS method. Evaluation and variance analysis of the comprehensive quality of the 14 samples were conducted by means of a multi-indicator entropy-weight TOPSIS model (Technique for Order Preference by Similarity to an Ideal Solution) combined with OPLS-DA (Orthogonal Partial Least Squares Discrimination Analysis) and HCA (Hierarchical Clustering Analysis). The ISSR (Inter-Simple Sequence Repeat) molecular marker technique and the UPGMA (unweighted pair-group method with arithmetic means) were employed to analyze the genetic relationship among A. villosum populations. The contents of volatile oil and bornyl acetate differed significantly among the different populations, but the main chemical component in the volatile oil was the same in all the samples, which was bornyl acetate. OPLS-DA results showed that 9 indicators were the main factors influencing the quality differences among the 14 populations. The entropy-weight TOPSIS results showed that there were significant differences in the comprehensive qualities of the 12 populations from the genuine producing area. The best quality of fruit was found in the genuine producing area of Chunwan Town; the qualities of 33% of genuine fruits were lower than that of non-genuine fruits. Twenty-three DNA fragments were obtained by ISSR-PCR amplification using four ISSR primers, eleven of which were polymorphic loci, which accounted for 47.8%. The similarity coefficients (GS) of different populations of A. villosum ranged from 0.6087 to 0.9565. There are significant differences among different populations of A. villosum in terms of the kinds of major chemical components and their contents, comprehensive quality and genetic diversity. The germplasm resources of A. villosum are rich in the genuine producing area. It means superior germplasm could be selected in the area. The comprehensive quality of the fruit of A. villosum from the non-genuine producing area is better than some of that from genuine producing area, proving that the non-genuine producing area can also produce Amomi Fructus with excellent quality.

Alginate encapsulation of micro-cuttings in endangered Satureja khuzistanica species: a promising method for obtaining genetically stable plants with high rosmarinic acid content

Synthetic seed technology is a suitable approach for the rapid, and uniform mass production of Satureja khuzistanica Jamzad, a valuable medicinal plant of the Lamiaceae family. In this study, the encapsulation of micro-cuttings of S. khuzistanica using sodium alginate (SA) was investigated. In order to determine the best conditions for encapsulation, different concentrations of SA and CaCl2·2H2O, different types of explants and matrix culture media, different types and concentrations of plant growth regulators, and different cold storage conditions of synthetic seeds were tested. Also, the genetic stability of regenerated plants from synthetic seeds using the inter simple sequence repeat (ISSR) molecular marker as well as the content of rosmarinic acid in synthetic seed-derived plants were evaluated. The use of 3% SA and 100 mM CaCl2·2H2O was found to be optimal for gel complexation. Encapsulation of nodal segments with 1/2 Murashige and Skoog (MS) medium containing 2.5 µM 6-Benzylaminopurine resulted in the highest regrowth percentage (72.22%) and regrowth rate (0.173). Pre-culture of nodal segments in 1/2 MS medium containing 5 µM indole-3-butyric acid (IBA) and 0.2% activated charcoal for 10 days and the use of 2.5 µM IBA in the rooting medium resulted in the highest rooting percentage (88.88%) in synthetic seed-derived plants. The highest regrowth percentage (61.11%) and regrowth rate (0.173) of synthetic seeds were obtained using 2.0 µM thidiazuron (TDZ) in MS liquid medium. The placement of germinated seeds on the coco peat substrate resulted in the highest conversion rate (61.11%) of synthetic seeds. Also, storage of encapsulated nodal segments at 4 °C in MS culture medium, compared to cold storage without using MS medium, resulted in better regrowth of synthetic seeds. The highest regrowth percentage (44.44%) and regrowth rate (0.092) for cold-stored synthetic seeds occurred after 2 weeks. The genetic stability testing by ISSR molecular marker showed that synthetic seed-derived plantlets were genetically similar to their mother plants. Also, encapsulated nodal segments and shoot tip-derived plants significantly enhanced the rosmarinic acid content up to 7.77 times that of the natural seed-derived plants. Genetic restoration programs, short-term storage, and germplasm distribution in S. khuzistanica plants could all benefit from the encapsulation regeneration strategy reported here.

Gamma Radiation (60Co) Induces Mutation during In Vitro Multiplication of Vanilla (Vanilla planifolia Jacks. ex Andrews)

In vitro mutagenesis is an alternative to induce genetic variation in vanilla (Vanilla planifolia Jacks. ex Andrews), which is characterized by low genetic diversity. The objective of this study was to induce somaclonal variation in V. planifolia by gamma radiation and detect it using inter-simple sequence repeat (ISSR) molecular markers. Shoots previously established in vitro were multiplied in Murashige and Skoog culture medium supplemented with 2 mg·L−1 BAP (6-benzylaminopurine). Explants were irradiated with different doses (0, 20, 40, 60, 80 and 100 Gy) of 60Co gamma rays. Survival percentage, number of shoots per explant, shoot length, number of leaves per shoot, and lethal dose (LD50) were recorded after 60 d of culture. For molecular analysis, ten shoots were used for each dose and the donor plant as a control. Eight ISSR primers were selected, and 43 fragments were obtained. The percentage of polymorphism (% P) was estimated. A dendrogram based on Jaccard’s coefficient and the neighbor joining clustering method was obtained. Results showed a hormetic effect on the explants, promoting development at low dose (20 Gy) and inhibition and death at high doses (60–100 Gy). The LD50 was observed at the 60 Gy. Primers UBC-808, UBC-836 and UBC-840 showed the highest % P, with 42.6%, 34.7% and 28.7%, respectively. Genetic distance analysis showed that treatments without irradiation and with irradiation presented somaclonal variation. The use of gamma rays during in vitro culture is an alternative to broaden genetic diversity for vanilla breeding.

Genetic diversity and population structure of Tunisian wild Kermes oak (Quercus coccifera L.): Assessment by ISSR molecular markers and implication for conservation.

In Tunisia, Kermes oak (Quercus coccifera L.) populations are severely destroyed due to deforestation. Nowadays, no preservation programs are attempted, yet, to conserve and promote the potential value of this resource. In this work, we assessed the genetic diversity of seven natural Tunisian populations of Q. coccifera from different bioclimates using Inter-Simple Sequence Repeats molecular markers. The distribution of the genetic diversity of Q. coccifera constitutes the pioneer step in the process of the conservation of the species. Nine selected ISSR markers were analyzed to characterize the genetic profiles of 70 different genotypes. The ISSR primers produced 64 loci ranging from 6 (UBC809 and UBC810) to 9 (UBC873) with an average of 7.11 at the species level. The average percentage of the polymorphic loci varied from 64.06% (Tabarka) to 76.56% (El Haouaria). The analyzed genotypes (70 individuals) revealed a high level of genetic diversity at species level (Na = 1.697; Ne = 1.517; He = 0.289; I = 0.418). The major proportion of the variation was attributable to individual differences within populations (76.07%). Analysis of molecular variance revealed also significant differentiation among all populations (ΦST = 0.245) and among populations within bioclimates (ΦSC = 0.233), even at a low scale space. The UPGMA and the PCoA analyses showed that most populations clustered independently to bioclimate or geographical distance indicating that genetic differentiation mainly occurs at local space scale due to genetic drift. The in-situ conservation of the species should be maintained on natural populations as a forest genetic resources. Moreover, ex-situ conservation should involve the selection of genotypes with extensive collection of seeds and cuttings from different populations of the target area.

Morphologic and Molecular Evaluation of Papaya Strains (Carica papaya L.) Under Mediterranean Conditions

Papaya is high in nutrients, delicious flavours, and potent antioxidants like lycopene; all of these bio constituents may reduce the risk of a variety of ailments, particularly those associated with ageing, such as heart disease and cancer. The papaya (Carica papaya L.) despite having a lengthy farming history and important germplasm, little is known about its genetic basis and variety. In response to environmental changes, the results of papaya strain diversity show variation in yield, vegetative traits, morphological characteristics of flowers, fruits, and leaves. To characterise the genetic assessment, morphological, agronomical traits, and an inter-simple sequence repeat (ISSR) molecular marker were studied across 9 papaya strains. For the past three decades, DNA Molecular markers have been used. The DNA contours provide data on the strains, display the entire genome, and showing difference in both the encrypting and non-encrypting regions, as well as polymorphism. A mix of morphologic, chemical, and molecular characteristics of papaya strains can be used to well measure the degree of variety and correlation among papaya strains. The primers UBC812 and UBC825 performed the best for identifying and estimating the diversity of papaya accessions, followed by UBC864, UBC809, UBC811 and HB-13. The 9 papaya strains were classified into two clusters using UPGMA cluster analysis and a similarity coefficient obtained from ISSR markers. Among the nine papaya strains, the pair S3 and S7 had the highest similarity (0.97). The morphological characteristics are similar to molecular analysis. This work provided a quick and steadfast method for estimating variability among different Papaya strains, which breeders could use to improve papaya.

High Genetic Diversity of 16 Indian lettuce (Lactuca indica L.) Accessions from Vietnam.

<b>Background and Objective:</b> Plant genetic resources provide the raw material for crop improvement and plant breeding program largely depends on it. Therefore, the evaluation of plant genetic resources plays a critical role in crop improvement and also in conserving valuable genetic resources for the future. In this study, the genetic diversity of 16 <i>Lactuca indica</i> L. accessions collected in Vietnam was investigated by using ISSR and RAPD markers. <b>Materials and Methods:</b> Genetic diversity of 16 <i>Lactuca sativa</i> L. genotypes collected in Vietnam were evaluated using Random Amplified Polymorphic DNA (RAPD) and Inter-Simple Sequence Repeat (ISSR) molecular markers. <b>Results:</b> In this study, 42 RAPD and ISSR primers were initially used, of which 12 and 9 primers, respectively were finally selected as they produced scorable patterns. RAPD markers produced a total of 113 loci, out of which 52 loci (45.96%) were polymorphic. The average percentage of the polymorphic band for RAPD primer is 45.96% and the genetic similarity based on simple matching coefficient ranged from 69.0-94.7%. ISSR analysis detected a total of 60 loci, out of which 22 loci (36.32%) were polymorphic and the genetic similarity ranged from 56.7-95.0%. In general, ISSR markers amplified fewer loci and showed lower variation in the percentage of polymorphism compares to the RAPD assay. <b>Conclusion:</b> These results indicate that the 16 collected Indian lettuce genotypes are genetically diverse. Because of these genetic diversities, the collected genotypes could be used for preserving or crossing programs to improve this precious medicinal plant in Vietnam.

Evaluation of Genetic Diversity in Three Diospyros Species, Collected from Different Regions in Iran, Using ISSR and SCoT Molecular Markers

ABSTRACT The Diospyros genus contains approximately 400 to 500 species, among which D. kaki is cultivated as the edible persimmon. Knowledge about the genetic diversity of Diospyros genotypes is of high significance for breeding programs. In this research, the genetic diversity of 57 Diospyros genotypes collected from different regions of Iran was evaluated using inter simple sequence repeats (ISSR) and start codon targeted (SCoT) markers. Seven ISSR and 10 SCoT markers produced a total of 42 and 75 fragments with the averages of 6 and 7.5 fragments per primer, respectively. The average polymorphism information content (PIC) for ISSR markers was 0.3 and for SCoT markers was 0.36. The average marker index (MI) for ISSR and SCoT markers were 1.81 and 1.79, respectively. Cluster analysis based on Jaccard’s genetic similarity coefficient and UPGMA method classified 57 Diospyros genotypes into four main groups based on ISSR and SCoT markers. However, the clustering pattern based on the combination of ISSR and SCoT markers data separated the species genotypes better than each marker alone. Accordingly, groups 1 and 2 contained all D. kaki genotypes, both encompassing various subgroups, group 3 included D. lotus and group 4 included D. virginiana genotypes. Genotype distribution visualized with principal coordinate analysis (PCoA) was in accordance with the cluster analysis. Based on our results Iran is a rich source of Diospyrus germplasm, and a combination of ISSR and SCoT markers can be used as an accurate technique for evaluating genetic diversity among the Diospyros genotypes.

Efficient micropropagation of Thunbergia coccinea Wall. and genetic homogeneity assessment through RAPD and ISSR markers

Thunbergia coccinea Wall. ex D. Don being a rare, ornamental and medicinal plant of India, is needed to propagate for conserving the germplasm and analyzing its phytochemical compounds in the future. A reliable protocol for direct in vitro propagation using nodal shoot meristem of T. coccinea as explant was standardized. The highest number of shoots per explant (22.17 ± 0.54) with maximum shoot length (2.36 ± 0.28) in cm was obtained in Murashige and Skoog (MS) medium supplemented with 9.70 µM of 6-furfurylaminopurine (Kinetin) and 0.053 µM of α-naphthaleneacetic acid (NAA) combination, among all the different plant growth regulators (PGR’s) and concentrations tested. The aforesaid PGR’s combination was optimum for axillary shoot bud induction and multiplication in T. coccinea. The best rooting was observed on the half-strength MS medium fortified with 2.68 µM NAA with the highest number of roots per shoot (3.75 ± 0.12) and maximum length (5.22 ± 0.32) in cm. All the in vitro raised plantlets were acclimatized in sterile sand and soil mixture (1:1) with a survival rate of 70% on earthen pots under greenhouse conditions. PCR-based RAPD (Random Amplified Polymorphic DNA) and ISSR (Inter-Simple Sequence Repeat) molecular markers were employed to determine the genetic homogeneity amongst the plantlets. Twelve (12) RAPD and nine (9) ISSR primers developed a total of 104 and 91 scorable bands, respectively. The band profiles of micropropagated plantlets were monomorphic to the mother, donor in vivo plant, and similarity values varied from 0.9542–1.000. The dendrogram generated through UPGMA (unweighted pair group method with arithmetic mean) showed 99% similarities amongst all tested plants confirming the genetic uniformity of in vitro raised plants.

Diversidade genética por marcadores ISSR em populações naturais de Lippia origanoides Kunth.

ABSTRACT Lippia origanoides Kunth. is a medicinal plant that is widely available in the Northeast region of Brazil and is known as “alecrim-d’angola”. However, there is no information available on the genetic variability of this species in the region. Thus, the current study was aimed to analyze the genetic diversity and structuring of L. origanoides populations occurring in the states of Bahia and Pernambuco, Brazil, using Inter-Simple Sequence Repeat (ISSR) molecular markers. The evaluated Nei’s diversity index of the populations varied from 0.162 to 0.237, and the Shannon diversity index varied from 0.247 to 0.350. In molecular variance (AMOVA) analysis, a variation of 31% was observed among the populations, which denotes a high interpopulation structuring. The structure analysis and dendrogram indicated the possibility of classifying the 18 populations into four groups. As their genetic structure is extremely high, it is important to collect L. origanoides germplasm, including as many populations as possible. Since the region of Chapada Diamantina holds the most diverse populations of L. origanoides germplasm, it is a priority area to obtain the germplasm.

Genetic analysis of Lepidium spp. by SSR and ISSR molecular markers

Simple sequence repeat (SSR) and inter simple sequence repeat (ISSR) markers were employed to evaluate the genetic diversity and population structure of 22 accessions belonging to three Lepidium species. Results revealed a high level of genetic diversity in the Lepidium accessions. A total of 58 bands were amplified by 11 ISSR primers, of which all were polymorphic, while 11 SSR primers produced 30 bands, of which 27 (90.0%) were polymorphic. The average values of polymorphism information content (PIC), marker index (MI), effective multiplex ratio (EMR) and percentage of polymorphic loci (PPL) in the ISSR markers were higher than those in SSRs, whereas higher values of number of effective alleles (Ne) and Shannon's information index (I) were observed by SSR markers. Based on the number of observed alleles (Na), Ne, I, expected heterozygosity (He) and PPL, the highest genetic diversity was found in L.draba followed by L.sativum by both markers. The analysis of molecular variance showed that higher allelic diversity within species than among species. The 22 Lepidium accessions grouped into three main clusters based on neighbor-joining (NJ) clustering and principal coordinate analysis (PCoA), and three (ISSR) or four (SSR and pooled data) sub-populations based on STRUCTURE analysis independent of their taxonomy. This is the first report of genetic diversity and relationships among accessions of L.sativum, L.draba and L. latifolium using SSR and ISSR markers. Therefore, these results can be utilized to improve these Lepidium species for various desired traits through molecular and hybridization breeding techniques.

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.

Genetic diversity of improved genotypes of Tectona grandis in the state of Mato Grosso, Brazil

Teak (Tectona grandis) is a tree species native to South-east Asia and widely cultivated in Latin America. In Mato Grosso state, Brazil, teak is the second-most planted forest species due to its high productivity resulting from high adaptability to the soil and climate conditions of the region. However, there is little information about the genetic diversity of improved genotypes cultivated in the state. Thus, the objective of this study was to quantify, through ISSR (inter-simple sequence repeat) molecular markers, the genetic diversity of improved teak genotypes used in different plantations in Mato Grosso state. For this, 31 ISSR primers were tested on 113 genotypes separated into nine populations. Six primers were selected to perform the analyses, resulting in 55 amplified loci. All selected primers showed a high polymorphic information content. Considering all genotypes in the Mato Grosso population, the percentage of polymorphic loci (P) was 96.4%, the genetic diversity according to Nei՚s genetic diversity index (H) was 1.96, and according to Shannon՚s information/diversity index (I), it was 0.53. Considering each population individually, P varied from 50.9% to 90.9%, H from 0.21 to 0.33, and I from 0.30 to 0.49. Most of the genetic diversity observed was within populations (86%). Four genetic groups were identified, with a high level of admixture among them. The existence of a considerable genetic diversity within populations indicates the possibility of exploring this variation in genetic improvement programmes. However, the introduction of new genetic materials is recommended to increase the genetic base of this species in Brazil.