Reproducible Bands Research Articles

Evaluation of the Genetic Fidelity of <i>In-Vitro</i> Raised Plants of <i>Nardostachys jatamansi</i> (D.Don) dc. (Valerianaceae) using ISSR Markers

Genetic fidelity analysis of in vitro established N. jatamansi plants has been carried out. Inter Simple Sequence Repeats (ISSR) marker was used to assess the genetic fidelity of tissue culture products with the mother plant. Among 17 ISSR primers screened, nine produced clear, reproducible and scorable bands. A total of 26 distinct bands in the size range of 200 to 2900 bp were produced. Most of the banding profiles from micropropagated plants were monomorphic and similar to those of the control plant, however few samples produced polymorphic bands also. The percentage of polymorphic bands in the ISSR analysis was 11.53%. The genetic similarity was calculated on the basis of ISSR data among the selected plant materials which ranged from 0.93 to 1.00 (mean = 0.965). The similarity coefficient by ISSR analysis revealed that differences between tissue culture raised plants and mother plant was not remarkable, thus ascertaining the true nature of the in vitro raised plants. This study is likely to suggest an alternate method for large scale production carried forward with an in situ participatory approach which will thereby enhance the germplasm conservation. In future, with this approach N. jatamansi is likely to become a potent ethnomedicine in the pharma industry.

Plant parasitic nematodes occurrence and genetic diversity of banana cultivars grown in Tanzania

The genetic diversity of bananas (Musa spp.), one of the most economically important crops in Tanzania, is underestimated with scarce reports available. In addition, cultivation of banana is severely constrained by plant parasitic nematodes (PPN). We assessed genetic diversity and population structure of 159 banana varieties from four agro-ecological zones (Northern, Southern highlands, Lake and Zanzibar islands) of Tanzania using 20 SSR markers analysed with UPGMA method. We also, assessed the PPN occurrence through isolation and counting of PPN from each banana genotype where leaf samples were collected. SSR primer pairs were polymorphic; and generated 63 distinct reproducible bands. The polymorphic information content values of each SSR marker ranged from 0.50 to 0.75 indicating high level of genetic diversity among banana varieties. The 159 banana varieties were grouped into two clusters: A and B, based on UPGMA cluster and population structure analysis. A total of 128 independent and 31 overlapping genotypes were identified. Higher kinship among genotypes was observed in cluster A compared with cluster B. Some of the clusters in A and B genotypes showed high genetic distance. The most prevalent and abundant nematode species was Pratylenchus goodeyi with a mean count of 63%, followed by Radopholus similis (31%) and P. coffeae (6%). The results from this study provides a foundation for understanding genetic variability of bananas existing in Tanzania and PPN occurrence that will be valuable information for breeding disease and pest-resistant bananas with high yield traits.

Studies on Diversity of Sclerotium rolfsii Causing Collar Rot in Chickpea using Morphological and Molecular Markers

Sclerotium rolfsii is a soil borne fast spreading fungal pathogen that causes collar rot in chickpea. The variability among the pathogen isolates have made difficult to design efficient management practices and necessitates for a comprehensive study to know its diversity. The present study was planned to study the genetic diversity among the isolates of S. rolfsii collected from chickpea growing regions of Kurnool and Ananthapur districts of Andhra Pradesh at morphological and molecular level. The morphological characterization of isolates on PDA indicated that the isolates, CSR 14, CSR 18 and CSR 20, were fast growing and the overall growth i.e., sclerotia formation in terms of number, size and days to formation, was also faster. These isolates took only 4 days for production of sclerotial bodies. A total of 254 reproducible and scorable polymorphic bands ranging from 200 to 2000 bp were observed with twenty nine RAPD primers. The RAPD banding pattern reflected the presence of greater variability among the isolates and grouped the isolates into two clusters. The isolates, CSR 18 and 20, formed the cluster II revealing their distantness from other isolates at molecular level. The isolate, CSR 14, formed a separate sub cluster in the cluster I indicating its distant association with other isolates of this cluster.

Genetic diversity and population structure of wild Persian grape hyacinths (Muscari neglectum Guss. ex Ten.) assessed by morphological and molecular markers

Due to the unique blue color and perennial growth habit, Muscari can be cultivated as a bedding plant, pot plant, or cut flower. Meanwhile, there is lack of information about genetic variations of Muscari neglectum Guss. ex Ten.; consequently, the diversity of nine populations (90 accessions) belonging to M. neglectum was evaluated by inter simple sequence repeat (ISSR) marker, start codon targeted (SCoT) marker and morphological marker. Ten ISSR primers used in this experiment amplified 142 reproducible bands with 137 (96.4%) polymorphism; and ten SCoT primers amplified 165 bands with 151 (91.12%) polymorphism. Both ISSR and SCoT analyses indicated a high level of genetic variation in the wild population of M. neglectum. Analysis of Molecular Variance (AMOVA) revealed a higher genetic diversity within the geographic group (0.69%) compared to the genetic variation among populations (0.31%). The gene flow between populations was low, while there was a greater gene flow between close areas. Morphological data divided nine populations into three clusters, while molecular data separated the populations into five groups, which were confirmed with structure analysis and discriminant analysis of principal component (DAPC) analysis. Our investigation showed that first, SCoT and ISSR markers are suitable for evaluation of genetic variation in Muscari genus, and there is appropriate genetic diversity among M. neglectum populations. Our results suggest some information to conserve the genetic resources and improve breeding program based on genetic data.

Synergistic effect of cytokinins and auxins enables mass clonal multiplication of drumstick tree (Moringa oleifera Lam.): a wonder

The synergistic effect of plant growth regulators on axillary bud proliferation for mass clonal multiplication of Moringa oleifera Lam. (vern. drumstick) has been assessed for the first time. Treatment of decoated seeds with 1% (w/v) Bavistin for 60 min, 0.33% (w/v) streptocycline for 30 min, and 0.1% (w/v) HgCl2 for 3.5 min resulted in complete removal of the surface contaminants. Maximum seed germination (89.13%) was obtained on quarter-strength Murashige & Skoog (MS) medium. Culture of nodal segments on MS + 6-benzyladenine (BA) at 3 mg L−1 resulted in multiple shoot proliferation with ~ 18 shoots per explant. All combinations of indole-3-acetic acid (IAA) + kinetin (Kn) resulted in elongated shoots, while only lower concentrations of BA (0.5 mg L−1), along with IAA (0.5 to 2 mg L−1), or Kn (0.5 to 5 mg L−1), showed significant synergy in the shoot morphogenesis. In addition, the maximum (100%) rooting efficiency was attained on half-strength MS medium supplemented with different concentrations of IAA and indole-3-butyric acid (IBA). The rooted plants were successfully established in the greenhouse for acclimatization. Clonality of the raised plants was assessed using 15 random primers of Operon® technologies (OPT and OPF series), and eight primers resulted in significant amplification with distinct, identical, and reproducible bands that confirmed clonality of the micropropagated plants. The present study provides a comprehensive analysis of the synergistic effect of plant growth regulators (PGRs) on in vitro shoot regeneration and proliferation for clonal mass multiplication disease-free plantlets, which can be utilized to maximize the yield of healthy and genetically identical plants of drumstick tree, which is considered to be a miracle multipurpose tree.

Gene-targeted molecular phylogeny, phytochemical profiling, and antioxidant activity of nine species belonging to family Cactaceae

Cactaceae plant family comprises over 130 genera and 2000 species of succulent flowering plants. The genera Mammillaria and Notocactus (Parodia), which have medicinal and nutritional applications as well as aesthetic appeal, are considered to be among the major genera of the family. Several species of both genera show morphological and chemical similarities and diversities according to environmental conditions and genotypes. Here, we assessed the genetic relationships of nine species belonging to two major genera Mammillaria and Notocactus under the family Cactaceae, using two modern gene-targeting marker techniques, the Start Codon Targeted (SCoT) Polymorphism and the Conserved DNA-Derived Polymorphism (CDDP). Besides, we screened the various phytochemicals and evaluated the antioxidant activities of the nine species of cacti. Five out of the 10 SCoT and eight CDDP primers used to screen genetic variations within the nine species yielded species-specific reproducible bands. The entire 156 loci were detected, of which 107 were polymorphic, 26 were monomorphic, and 23 were unique loci. The nine species were categorized into two groups based on the dendrogram and similarity matrix. Phytochemical profiling revealed that sterols, triterpenes, flavonoids, and tannins were found in all the tested species. Additionally, two Notocactus species (N. shlosserii and N. roseoluteus) and one Mammillaria species (M. spinosissima) revealed a considerable antioxidant activity. Our results demonstrated that gene-targeting marker techniques were highly powerful tools for the classification and characterization of the nine investigated species, despite displaying high similarities at both morphological and phytochemical levels.

Genetic Divergence and Relationship Among Opisthopappus Species Identified by Development of EST-SSR Markers.

Opisthopappus Shih is an endemic and endangered genus restricted to the Taihang Mountains that has important ornamental and economic value. According to the Flora Reipublicae Popularis Sinicae (FRPS, Chinese version), this genus contains two species (Opisthopappus longilobus and Opisthopappus taihangensis), whereas in the Flora of China (English version) only one species O. taihangensis is present. The interspecific phylogenetic relationship remains unclear and undefined, which might primarily be due to the lack of specific molecular markers for phylogenetic analysis. For this study, 2644 expressed sequence tag-simple sequence repeats (EST-SSRs) from 33,974 unigenes using a de novo transcript assembly of Opisthopappus were identified with a distribution frequency of 7.78% total unigenes. Thereinto, mononucleotides (1200, 45.39%) were the dominant repeat motif, followed by trinucleotides (992, 37.52%), and dinucleotides (410, 15.51%). The most dominant trinucleotide repeat motif was ACC/GGT (207, 20.87%). Based on the identified EST-SSRs, 245 among 1444 designed EST-SSR primers were selected for the development of potential molecular markers. Among these markers, 63 pairs of primers (25.71%) generated clear and reproducible bands with expected sizes. Eventually, 11 primer pairs successfully amplified all individuals from the studied populations. Through the EST-SSR markers, a high level of genetic diversity was detected between Opisthopappus populations. A significant genetic differentiation between the O. longilobus and O. taihangensis populations was found. All studied populations were divided into two clusters by UPGMA, NJ, STRUCTURE, and PCoA. These results fully supported the view of the FRPS, namely, that O. longilobus and O. taihangensis should be regarded as two distinct species. Our study demonstrated that transcriptome sequences, as a valuable tool for the quick and cost-effective development of molecular markers, was helpful toward obtaining comprehensive EST-SSR markers that could contribute to an in-depth assessment of the genetic and phylogenetic relationships between Opisthopappus species.

Anatomical features and SCoT profiles provide new insight into phenotypic plasticity in the halophyte Suaeda maritima in Thailand

Abstract. Rittirongsakul K, Srinual A, Vanijajiva O. 2020. Anatomical features and SCoT profiles provide new insight into phenotypic plasticity in the halophyte Suaeda maritima in Thailand. Biodiversitas 21: 1082-1090. Phenotypic plasticity is the variation in phenotypic traits of organisms to survive with fluctuating environments. Halophytes are plants that can naturally tolerate high concentrations of salt in the soil, and their tolerance to salt stress, which reflects the ability of the plants to produce different phenotypic traits in response to changing environmental conditions. Suaeda maritima (L.) Dumort is a polymorphic halophyte and prominent species complex from tropical to temperate regions due to diverse stressful saline habitats. Consequently, the identification of S. maritima based on morphological data is challenging due to high phenotypic plasticity. In Thailand, S. maritima, known as Seablite or Cha Khram, has only been reported for the genus Suaeda which frequently found in coastal salt marshes. The peculiarities of the prevalence of three distinctive color traits, green, green-reddish and reddish, were commonly observed in Thailand. This preliminary study aims to examine the morpho-anatomical characters and molecular analysis of S. maritima and to determine how their morphological and genetic characterizations are related to phenotypic traits, particularly in color variation within the population at Bang Khun Tien shoreline in Bangkok, Thailand. This combined investigation of anatomical characters and start codon targeted (SCoT) fingerprinting profiles provide first evidence of phenotypic variations and genetic characterizations of halophytic S. maritima in Thailand. Anatomical characters of leaf structures confirmed that the species is austrobassioid (C3) types. Meanwhile, molecular investigation of thirty SCoT primers found that only two SCoT primers (SCoT10 and SCoT27) failed to amplify the S. maritima genomic DNA. Thoroughly, twenty-eight SCoT primers produced 162 unambiguous and reproducible banding profiles with 140-1200 bp product size. Despite, there was no genetic variation detectable within the population, SCoT technique was considered a suitable tool to produces adequate characterization for DNA fingerprinting of halophyte S. maritima. However, further studies involving additional samples of S. maritima are needed to obtain a more general view of the conspicuous variation and colonization history in Thailand.

DEVELOPMENT OF TERMITOMYCES PROTOCLONES AND CHARACTERIZATIONS THROUGH PCR BASED ISSR AND RFLP OF RRNA-ITS GENE

A total of five protoclones were successfully cultured on PDA medium out of regenerated twenty two colonies of Termitomyces protoplast and further studied. Liquid MYG grown mycelial tissue is used for protoplast isolation by enzymatic digestion in a mixture containing Lysing enzyme 2% and Cellulase R10 2% in 0.6 M mannitol. The incubation conditions like temperature, shaking and time were standardized at 24oC with shaking 60 rpm for 10 hours, respectively for healthy protoplasts liberation. The purified protoplasts showed 31.60±9.31% regeneration on specific medium and 77.12±2.72% viability by FDA test. Four ISSR primers were used in this study with a total of 27 reproducible bands with mean value 6.75 and they showed similar banding pattern in all with zero percent polymorphism ranged from 280 bp - 2700 bp. The amplified rRNA-ITS gene showed ~ 600 bp size in gel and found a single restriction site for enzyme HaeIII in all the protoclones and parent with similar fragment size in all.

Evaluation of Genetic Diversity in Antiaris toxicaria Lesch. from Sacred Groves of the Western Ghats, India

Antiaris toxicaria Lesch. is a deciduous tree belongs to the family Moraceae, is native to the Old World tropics. It is an important industrial forest plant for its veneer, plywood and medicinal values. In the present study, genetic diversity of different populations of A. toxicaria collected from sacred groves of the Western Ghats, India has been evaluated. Thirteen RAPD and ten ISSR primers produced a total of 175 and 143 reproducible bands, respectively. RPI 10 of RAPD primers used showed maximum (0.49) polymorphic information content (PIC), while UBC 809 (0.37) exhibits maximum PIC among ISSR primers. Nei’s genetic similarity showed that pairwise similarity coefficients ranged from 0.460 to 0.832 in RAPD analysis, whereas 0.550–0.842 in ISSR analysis. Understanding the genetic diversity among natural A. toxicaria populations will be useful for effective long-term conservation.

Genome Status of Lippia alba Polyploid Complex Long-term in vitro Cultivated

This study is the first report of a genetic stability analysis of a polyploid complex maintained in vitro for a long-time. Twenty-two accessions of Lippia alba, a medicinal species of economic importance, had been maintained under in vitro culture conditions for 7 years through sprouting of axillary buds. Four clones of each accession were analyzed, being three plants from in vitro bank and one cultivated in the field. We investigated the genetic stability of diploid, aneuploid, triploid, tetraploid, and hexaploid accessions. The investigation was carried out using flow cytometry, inter simple sequence repeat (ISSR) and simple sequence repeat (SSR) markers. No significant variation in nuclear DNA content was observed between the in vitro conserved plants and their respective field plant. Out of 23 ISSR primers screened, 8 primers were found to produce clear reproducible bands resulting in a total of 5456 bands. 86.36% of the analyzed plantlets (19 accessions) showed at least one polymorphic band. The polymorphic rate ranged from 1.61 to 33.87%. The SSR markers were used to confirm the absence or low occurrence of variation in accessions that showed no polymorphism or polymorphism for only one ISSR primer. The genetic instability detected in our study at the molecular level may be attributed to the natural instability of L. alba genome combined with the long-time in vitro maintenance.

Genetic diversity of ‘Wan Chak Motluk’ (Curcuma comosa Roxb.) in Thailand using morphological characteristics and random amplification of polymorphic DNA (RAPD) markers

‘Wan Chak Motluk’ (Curcuma comosa Roxb.) is an herbaceous plant in the family Zingiberaceae. Its rhizomes and extracts are widely used as a traditional herbal remedy because of its powerful estrogen-like activity. The herbal product is commonly taken in capsules that contain a powdered form of the raw material. However, due to similarity in appearance and color, several related species can be confused. Another mistake in species identification could be caused by similar morphological traits among Curcuma sp. Hence, it is necessary to identify medicinal plants using an efficient method such as molecular markers. In this study, morphological characteristics and random amplification of polymorphic DNA (RAPD) markers were used to identify and assess the genetic diversity of ‘Wan Chak Motluk’ (Curcuma comosa Roxb.) and other plants belonging to Curcuma sp. collected from different regions of Thailand. The results showed that the Curcuma samples had a wide range of variation in morphological traits. A total of 183 reproducible bands were scored, of which 173 (94.54%) were polymorphic. The values of polymorphic information content (PIC) varied from 0.00 to 0.50 with an average of 0.30. Dendrogram constructed from unweighted pair group method with arithmetic average (UPGMA) showed that all samples could be classified into two major clusters; the ‘Wan Chak Motluk’ samples (I) and the other Curcuma sp. (II). Moreover, the ‘Wan Chak Motluk’ cluster could be divided into six sub-clusters. The results based on this RAPD information will be useful for collection, conservation and breeding programs in the future.

Genetic diversity analysis of Rhanterium eppaposum Oliv. by ISSRs reveals a weak population structure

Rhanterium eppaposum Oliv. (Arfaj) the national plant of Kuwait, is a perennial bushy shrub and the national flower of Kuwait. It is one of the most palatable forage plants and an excellent performer for desert rehabilitation. Due to several anthropogenic activities and climate change, this native plant has suffered severe degradation. In order to formulate conservation strategies, the genetic diversity of Rhanterium, was assessed by inter simple sequence repeat (ISSR) markers. A total of 93 wild landraces distributed in six remote locations in Kuwait were tested using 17 primers (PIC = 0.31; polymorphism = 92.8 %) that produced 195 unambiguous and reproducible bands. Overall, diversity indices were high enough (Nei’s gene diversity (h') - 0.286; Shannon’s diversity index - 0.414) for these populations. AMOVA test returned a fixation coefficient (FST) of 0.116 (p = 0.0) with higher genetic variations detected within (88 %) the populations as compared to among (12 %) the populations. Satisfactory levels of genetic differentiation (GST = 0.186; G’ST = 0.250; D’ = 0.295) were recorded for R. eppaposum with a gene flow (NM) maintained at 1.9. The maximum genetic distances were observed between Maqwa and Mina Abdulla (Ds- 0.158; FST- 0.228) whereas minimum was between Al Kabd and SANR (Ds - 0.06; FST- 0.05). A weak Mantel’s correlation (r2 - 0.139; P = 0.014) between the six populations was also recorded. A narrow population structure was observed through the PCoA (2 groups), STRUCTURE (2 groups, K = 2) and SPLIT decomposition (1 cluster). The high genetic diversity within the populations are encouraging, however the low genetic differentiation raises concerns. The population structure should be improved by planting genetically diverse genotypes in areas of very low diversity to broaden its genetic base.

Estimation of molecular genetic diversity in some indian sugarcane cultivars as revealed by fluorescent rapd markers

The purpose of this study was to estimate the genetic diversity in some cultivars of sugarcane at the DNA level with an efficient, better sensitivity and highly informative PCR based fluorescent random amplified polymorphic DNA (F-RAPD) and capillary electrophoresis techniques. Degenerate RAPD primers were used for yielding best reproducible banding patterns that were generated for 803 discernible and reproducible bands which included 645 polymorphic, 12 mono-morphic and 146 unique bands. The range of coefficient of similarity or genetic distance among sugarcane varieties was 0.25912-0.64111. Based on the genetic distance calculated from 381 alleles generated from 19 sugarcane cultivars, a dendrogram dividing the cultivars into six clusters was made using the UPGMA (unweighted pair group with arithmetic mean) method. This paper reports the results of a study on the genetic diversity of cultivated 19 sugarcane cultivars as revealed by fluorescent RAPD information which would help to conserve the cultivar-specific genetic diversity for the breeding program. The present study also revealed that molecular markers can be successfully utilized for inferring genetic diversity and genetic relationship of sugarcane cultivars more precisely.

Genetic Diversity and Phylogenetic Relationship Among Eight Species of Order: Rodentia in Egypt Based on Random Amplified Polymorphic DNA (RAPD) Marker

Of all mammals the rodents have an importance and great diversity. The phylogenetic relationships between the different species of the order Rodentia has an interest and investigations for many researchers. The current study suggests the ability of Random Amplified Polymorphic DNA (RAPD)-PCR to estimate the genetic distance and reflect the genetic relations and variability among eight rodent species of order Rodentia in Egypt. After the use of five arbitrary short primers (OPA-4, OPT-7, OPG-2, OPN-4 and OPM-1) to generate polymorphic, reproducible and score able bands. The results explained that, the highest genetic distance 1 was pronounced between Rattus norvegicus and Mus musculus praetextus similarly between Mus musculus domesticus and Rattus norvegicus. This is followed by the genetic distance between Mesocricetus auratus and Mus musculus domesticus as well as between Cavia porcellus and Rattus norvegicus. Nonetheless, the lowest genetic distance 0.31 was noticeable between Mus musculus praetextus and Mus musculus musculus.

Molecular assessment of environmental risk of pollution in plants from cement factory

This study aimed at determining the genotoxic effects of different environmental pollutants in Bazian cement factory in Bazian district, Iraq. Three species of plants viz., Platycladus orientalis, Eucalyptus spp. and Melia azedarach were studied. These plants were collected from areas in and near the cement factory, while the control of this study was one collected from area far from the pollution sources. Atomic Absorption Spectrophotometry (AAS) was used for estimation of the pollution status of some heavy metal concentrations (Pb, Cd, Cr, Ni, Fe, Cu and Zn). Random Amplified Polymorphic DNA (RAPD) revealed new bands and/or disappearing of normal bands in comparison with control samples. In this study, the mean of heavy metal concentrations for all species of plants was below 100 mg/kg and resembled sample results of control. Statistical results for heavy metals in plant leaves indicated significant differences only among sites for most metals. The lowest was level of Genomic Template Stability (GTS) ratios which are related to CCR samples followed by Gate3 and Out. This is in agreement with distances from main stack, respectively, and might have led to a high level of genotoxic effect on three plants’ species (68.36, 69.38 and 89.79%; 79.2, 83.16 and 89.1% and 79.12, 87.91 and 99.94), respectively. Different primers (25) were examined. Twenty primers only resulted in reproducible and clear bands. The current study showed that the samples of the selected plants were suitable for the detection of genetic toxicity. The RAPD technique used was suitable for the study of genetic toxicity. The use of plants, which are indicated as biomarker indicator in the early detection of genetic toxicity factors, was highly sensitive in assessing the effect of air pollution.

Genetic diversity analysis in cashew (Anacardium occidentale L.) germplasm using RAPD marker

Anacardium occidentale L., commonly known as cashew nut, belongs to the family Anacardiaceae. It is regarded as a high valued fruit nut crop world-wide. Potential of this economically important nut is under-utilized in Bangladesh in spite of having all favorable agro-climatic conditions. The objective of the present investigation was to characterize six cashew accessions using Random Amplified Polymorphic DNA (RAPD) markers. Four random primers viz. OPE-02, OPE-18, OPK-03 and OPB-15 were used to amplify DNA segments. A total of 33 reproducible bands were obtained, out of which 11 were monomorphic and 22 were polymorphic. On average 74.12% polymorphism was observed. . Primers OPB-15 and OPK-03 yielded 100% polymorphism and OPE 02 and OPE 18 produced 33.33% and 63.16 % polymorphism, respectively. Cluster analysis revealed two main distinct groups, first group included GP-1 and the second consisted of five genotypes viz. GP-2, GP-3, GP-4, GP-5and GP-6 The major cluster- II was further subdivided into two minor clusters i.e. minor cluster- III and IV. Minor cluster- III contained only one genotype GP-4. Minor cluster- IV consists of four rest genotypes. The genetic distance between the groups was found low and varied from 0.002 to 0.0308. Maximum genetic distance was observed between GP-1 and GP-2cashew germplasm and minimum between GP-5 and GP-6. The low genetic distance which is unusual for this out crossing long-lived tree species, indicates the probability of having common ancestry among the germplasm or may be due to the use of a narrow range of populations for the investigation.
 
 J Bangladesh Agril Univ 17(4): 461–465, 2019

Evaluation of the genetic diversity of mango (Mangifera indica L.) seedling germplasm resources and their potential parents with start codon targeted (SCoT) markers

Seedling germplasm resources are important for mango breeding. Genetic diversity analyses based on molecular data can provide useful information for germplasm management and varietal characterization. Start codon targeted (SCoT) markers are efficient and highly polymorphic markers for phylogenetic relationship analyses of multiple species and were used in the present study to determine genetic relatedness and variability among 168 mango (Mangifera indica L.) germplasm resources. Forty-five SCoT primers generated unambiguous and reproducible bands and a total of 337 fragments, with a mean of 7.49 fragments per primer. Of these, 244 (72.4%) were polymorphic. The number of polymorphic bands varied from 1 (SCoT55) to 10 (SCoT4 and 52), with an average of 5.42 amplicons per primer. The average polymorphism information content, Nei’s genetic diversity, Shannon’s information index and marker index were 0.823, 0.426, 0.603 and 4.460, respectively. A dendrogram generated using the unweighted pair-group method with arithmetic mean delineated the 168 germplasm resources into two major clusters. Analysis of molecular variance revealed higher genetic diversity (89%) within each population than among the populations (11%). Most mango seedling germplasm resources grouped with their potential parents, and genetic differences were also detected. Thirty-four seedling germplasm resources were identified as potential parents or sister lines. These results demonstrate that SCoT markers are useful for cultivar identification and genetic diversity analyses of mango cultivars. This genetic information will support germplasm management and cultivar improvement.

Rapid plant regeneration in industrially important Curcuma zedoaria revealing genetic and biochemical fidelity of the regenerants.

The present investigation was carried out to establish an efficient and reproducible micropropagation protocol for the production of morphologically, genetically and chemically uniform plants of Curcuma zedoaria. Axillary bud explants of C. zedoaria were inoculated into MS basal medium supplemented with various combinations and concentrations of 6-benzyladenine (2.2-22.2µM, BA), kinetin (2.3-23.2µM, Kin), indole-3-acetic acid (2.9-11.4µM, IAA), α-naphthalene acetic acid (2.7-10.2µM, NAA) and adenine sulphate (33.9-203.6µM, Ads). Almost 95% of rhizome buds sprouted on MS medium supplemented with 13.3μM BA, 5.7μM IAA and 63.9μM Ads giving rise to an average of 12.89 ± 0.02 shoots within 6weeks. However, the maximum number of roots (25.8 ± 0.07 roots per explant) was obtained on half strength MS medium supplemented with 7.4µM of IBA after 4weeks of inoculation. Morphological characteristics were similar in both conventionally propagated and micropropagated plants. Additionally, genetic homogeneity of in vitro plants was further confirmed through ISSR and flow cytometry analysis. A total of 27 ISSR primers were screened, out of which 13 ISSR primers generated 58 monomorphic and reproducible bands thereby confirming the genetic uniformity of obtained plants. The mean 2C DNA content of the mother plant (2.96pg) was similar to that of in vitro derived plants (3.07pg). Gas chromatography-mass spectrometry (GC-MS) analysis showed similarity in the qualitative profile of chemical constituents of essential oil and high-performance liquid chromatography analysis revealed no significant differences in curcumin content in the tissue culture regenerants and mother plants of C. zedoaria. Therefore, the present micropropagation protocol could be effectively employed to generate true to type plantlets of C. zedoaria.

First study of Genetic diversity in watermelon (Citrullus lanatus) germplasm collected from Southern Tunisia using RAPD markers

The genetic diversity in local Tunisian watermelon (Citrullus lanatus) was studied using RAPD markers. Eight watermelon cultivars originating from south of Tunisia and belong to four populations were analysed and compared to two commercial varieties (Giza and Sugar-baby) widely produced in this area. Five of nine RAPD primers generated a total of 86 reproducible bands, 85 of which were polymorphic (98.4%). Cluster analysis of the accessions considered in this study employing RAPD data indicated that commercial varieties are significantly different of all the local cultivars. The relationships among the local cultivars (four populations) showed that Medenine population and the most genotypes of Benguerdane population were grouped together and significantly different from Kebili population. The AMOVA showed significant differentiation between populations (27%). In addition, the data showed clusters according to some fruit characteristics such as fruit shape and fruit weight. This proved that RAPD markers are useful for germplasm discrimination as well as for investigation of patterns of variation in watermelon.