ISSR and DAMD markers revealed high genetic variability within Flavoparmelia caperata in Western Himalaya (India)

Flavoparmelia caperata (L.) Hale is medicinally very important and possesses antifungal and antibacterial activities. F. caperata is the only species found in India. Inter simple sequence repeat (ISSR) and Directed amplification of minisatellite DNA (DAMD) methods were used to analyze the genetic variability within F. caperata from the Western Himalayan region of India. Eleven ISSR and 10 DAMD primers produced 139 and 117 polymorphic bands, and detected 91.44 and 82.34 % polymorphisms, respectively. Cumulative band data generated for ISSR and DAMD markers resulted in 86.86 % polymorphism across all the accessions of F. caperata. The average Polymorphic information content (PIC) value obtained with ISSR, DAMD, and cumulative band data were 0.28, 0.27, and 0.27, respectively. The clustering of the F. caperata accessions in the UPGMA dendrogram showed that these accessions are intermingled with each other in different subclusters irrespective of their geographical affiliations. The pattern of genetic variations within F. caperata accessions could be due to free exchange of spores that might have taken place among these accessions in the wild. ISSR and DAMD markers efficiently and reliably resulted in discrete banding patterns and polymorphic profiles. These markers despite targeting different regions of genome, revealed almost similar levels of polymorphism across all the accessions. The wide range of genetic distance and high level of polymorphism detected by ISSR and DAMD reflected a high genetic variability among the different accessions of F. caperata.     

Genetic variability and population structure of Bergenia ciliata (Saxifragaceae) in the Western Himalaya inferred from DAMD and ISSR markers

Genetic variability and population structure of Bergenia ciliata (Haw.) Sternb., commonly known as “Pashanbheda” (Stone-breaker), collected from the Western Himalayan region of India were estimated using two DNA fingerprinting methods viz., directed amplification of minisatellite DNA (DAMD) and inter simple sequence repeats (ISSR). The cumulative data analysis of DAMD and ISSR markers for 74 accessions from eight populations showed 86.1% polymorphism. Analysis of molecular variance (AMOVA) showed highest percentage of variation within individuals of populations (73.6%) and 21.7% among populations. STRUCTURE and PCoA analyses on the hierarchical partitioning of genetic diversity showed strong admixture of individuals among the eight assumed geographical populations of B. ciliata. The data suggests that high genetic flow is one of the major factors responsible for low genetic differentiation. Preservation of genetic diversity of B. ciliata is important, both to promote adaptability of the populations to changing environment as well as to preserve a large gene pool for future prospection. The present study using DAMD and ISSR markers, therefore, provide the means of rapid characterization of accessions within the populations, and thus enable the selection of appropriate accessions for further utilization in conservation and prospection programmes.     

Isozyme analysis of genetic variability and population structure of Lactuca L. germplasm

Isozymes were used to investigate the genetic variability, population structure, and relationships of Lactuca germplasm. The isozyme systems revealed 16 putative loci of a total of 31 alleles. Out of these 16 loci, 11 were polymorphic. The average values of expected heterozygosity (H_e), observed heterozygosity (H_o), mean number of alleles per locus (A) and effective number of alleles per locus (A_e) were 0.2227, 0.266, 1.3005 and 1.369, respectively. The average fixation indices were lower than zero for most of the accessions studied, indicating an excess of heterozygotes. Genetic differentiation among accessions (F_ST) exhibited that 51.3% of the isozyme variation was recorded among accessions, and 48.7% of the genetic variation resided within accessions. The average values of total heterozygosity (H_T) and intra-accessional genetic diversity (H_S) were 0.352 and 0.171, respectively. Moreover, the inter-accessional genetic diversity (D_ST) ranged from 0 to 0.424 with an average of 0.18. Cluster analysis revealed that L. sativa cultivars were distributed throughout different Lactuca species. Thereby, isozymes results confirms the hypothesis of the polyphyletic origin of L. sativa. This high level of genetic variation proved that isozymes are efficient for polymorphism analysis of Lactuca germplasm.     

Dimeric phenylphenalenones from Musa acuminata and various Haemodoraceae species. Crystal structure of anigorootin

Three fused octacyclic phenylphenalenone dimers were isolated from Musa acuminata: Anigorootin, which was first isolated from Anigozanthos flavidus and hitherto represented the only compound of that type, the new 4'-hydroxy-anigorootin, and 4',4"-di-hydroxy-anigorootin, which is a revised structure. The crystal structure of anigorootin was determined by X-ray crystallography. 3,3'-Bis-hydroxyanigorufone, a dimer of the conventional type known from Anigozanthos preissii, was also found in Musa acuminata. Phytochemical analysis of several Haemodoraceae species revealed the occurrence of anigorootin, 3,3'-bis-hydroxyanigorufone, and the novel metabolite 3,3'-bis-anigorufone. The occurrence of the same compounds in Musaceae and Haemodoraceae indicates the close chemotaxonomic relationship of both plant families.     

Purification and characterization of pectate lyase from banana (Musa acuminata) fruits

Pectate lyase (PEL) has been purified by hydrophobic, cation exchange and size exclusion column chromatographies from ripe banana fruit. The purified enzyme has specific activity of 680 +/- 50 pkat mg protein(-1). The molecular mass of the enzyme is 43 kDa by SDS-PAGE. The pI of the enzyme is 8 with optimum activity at pH 8.5. Analysis of the reaction products by paper and anion exchange chromatographies reveal that the enzyme releases several oligomers of unsaturated galacturonane from polygalacturonate. The K(m) values of the enzyme for polygalacturonate and citrus pectin (7.2% methylation) are 0.40 +/- 0.04 and 0.77 +/- 0.08 g l(-1), respectively. PEL is sensitive to inhibition by different phenolic compounds, thiols, reducing agents, iodoacetate and N-bromosuccinimide. The enzyme has a requirement for Ca(2+) ions. However, Mg(2+) and Mn(2+) can substitute equally well. Additive effect on the enzyme activity was observed when any two metal ions (out of Mg(2+), Ca(2+) and Mn(2+)) are present together. The banana PEL is a enzyme requiring Mg(2+), in addition to Ca(2+), for exhibiting maximum activity.     

Endophytic fungi from Musa acuminata leaves and roots in South China

One hundred and sixty-three endophytic fungal cultures were isolated from 200 leaf samples of Musa acuminata trees, which were soaked in 36% formaldehyde solution for surface sterilization. They belonged to the genera of Gloeosporium musae (45%), Myxosporium spp. (11%), Deightoniella torulosa (8.5%), Alternaria tenuis (7.9%), Sphaceloma spp. (7.4%), Aureobasidium spp. (4.3%), Melida spp. (1.8%), Uncinula spp. (1.8%), Penicillium spp. (1.8%), Aspergillus spp. (1.2%), Sarcinella spp. (1.2%), Cladosporium sp. (0.6%), Cephalosporium sp. (0.6%) and sterile mycelium (6.7%). Sixty-eight endophytic fungal cultures were isolated from 100 root samples. They respectively belonged to the genera of Aspergillus spp. (31%), Paecilomyces spp. (16%), Penicillium spp. (15%), Fusarium spp. (10%), Gloeosporium musae (6%), yeast (3%), Deightoniella torulosa (3%), Spicaria sp. (1.4%), Cephalosporrium sp. (1.4%), Meliola sp. (1.4%) and sterile mycelium (10%). Water agar (containing 50 g chloramphenicol ml^–1 and 50 g streptomycin ml^–1) seemed to be a better medium for isolation of endophytic fungi than potato-dextrose agar (PDA, containing 50 g chloramphenicol ml^–1 and 50 g streptomycin ml^–1).     

Micropropagtion of Terminalia bellerica from nodal explants of mature tree and assessment of genetic fidelity using ISSR and RAPD markers

The present study reports an efficient in vitro micropropagation protocol for a medicinally important tree, Terminalia bellerica Roxb. from nodal segments of a 30 years old tree. Nodal segments taken from the mature tree in March-April and cultured on half strength MS medium gave the best shoot bud proliferation response. Combinations of serial transfer technique (ST) and incorporation of antioxidants (AO) [polyvinylpyrrolidone, PVP (50 mg l⁻¹) + ascorbic acid (100 mg l⁻¹) + citric acid (10 mg l⁻¹)] in the culture medium aided to minimize browning and improve explant survival during shoot bud induction. Highest multiplication of shoots was achieved on medium supplemented with 6-benzyladenine (BA, 8.8 μM) and α-naphthalene acetic acid (NAA, 2.6 μM) in addition to antioxidants. Shoot elongation was obtained on MS medium containing BA (4.4 μM) + phloroglucinol (PG, 3.9 μM). Elongated shoots were transferred to half strength MS medium containing indole-3-butyric acid (IBA, 2.5 μM) for root development. The acclimatization of plantlets was carried out under greenhouse conditions. The genetic fidelity of the regenerated plants was checked using inter simple sequence repeats (ISSR) and randomly amplified polymorphic DNA (RAPD) analysis. Comparison of the bands among the regenerants and mother plant confirmed true-to-type clonal plants.     

Foundation characteristics of edible Musa triploids revealed from allelic distribution of SSR markers

Background and Aims

The production of triploid banana and plantain (Musa spp.) cultivars with improved characteristics (e.g. greater disease resistance or higher yield), while still preserving the main features of current popular cultivars (e.g. taste and cooking quality), remains a major challenge for Musa breeders. In this regard, breeders require a sound knowledge of the lineage of the current sterile triploid cultivars, to select diploid parents that are able to transmit desirable traits, together with a breeding strategy ensuring final triploidization and sterility. Highly polymorphic single sequence repeats (SSRs) are valuable markers for investigating phylogenetic relationships.

Methods

Here, the allelic distribution of each of 22 SSR loci across 561 Musa accessions is analysed.

Key Results and Conclusions

We determine the closest diploid progenitors of the triploid ‘Cavendish’ and ‘Gros Michel’ subgroups, valuable information for breeding programmes. Nevertheless, in establishing the likely monoclonal origin of the main edible triploid banana subgroups (i.e. ‘Cavendish’, ‘Plantain’ and ‘Mutika-Lujugira’), we postulated that the huge phenotypic diversity observed within these subgroups did not result from gamete recombination, but rather from epigenetic regulations. This emphasizes the need to investigate the regulatory mechanisms of genome expression on a unique model in the plant kingdom. We also propose experimental standards to compare additional and independent genotyping data for reference.     

Genetic diversity and genetic relationships in Hyacinthaceae in India using RAPD and SRAP markers

Genetic diversity and relationship among three genera namely Drimia, Dipcadi and Ledebouria of Hyacinthaceae in India was studied using RAPD and SRAP markers. Twenty one RAPD primers and nine SRAP were used for analyzing 41 accessions. RAPD gave an average 12.6 markers per primer, while SRAP generated 10.1 markers per primer pair. The family emerged very diverged with high polymorphism. The study resolved the three genera into monophyletic groups corresponding to three subfamilies; Urginoideae, Hyacinthoideae and Ornithogaloideae. Drimia wightii emerged a very distinct species and species specific markers were obtained with both marker systems. AMOVA analysis also revealed the genera to be quite well diverged. The two markers showed high correlation (r = 0.932) in Mantel matrix crresspondance test. The combined data also showed a very good correlation with the respective markers individually.     

Soluble and wall-bound phenolics and phenolic polymers in Musa acuminata roots exposed to elicitors from Fusarium oxysporum f.sp. cubense

The accumulation of soluble and wall-bound phenolics and phenolic polymers in Musa acuminata roots exposed to cell wall-derived elicitor from the pathogen, Fusarium oxysporum, f.sp. cubense, race four, was investigated. The root tissue from the banana cultivar "Goldfinger" was found to respond strongly and rapidly towards the elicitor through the increased synthesis of phenolic compounds. Following elicitation, the conjugated and non-conjugated phenolic metabolites in the induced root tissue were extracted and quantified. Induced phenolic synthesis was rapid and reached near maximum values after 16 h. High-performance liquid chromatography revealed both compositional and quantitative differences between induced phenolics (p-coumaric, ferulic, and sinapic acids) and those constitutively present (p-coumaric- and ferulic acid). In addition, vanillic acid was found in the ester-bound fraction and protocatechuic acid in the cell-wall bound fraction of elicited tissue. The deposition and accumulation kinetics of polymerized phenolic monomers as lignin and lignin-like polymers was quantified over a time period of 0-36 h and found to reach maximum values after 24 h. Ionization difference UV spectra of lignin indicated compositional differences in the newly synthesized lignin fraction and correlated with increased concentrations of ferulic acid and sinapic acids esters. The results show that the increased flux through the phenylpropanoid pathway resulted in the synthesis of cinnamic acid and benzoic acid derivatives that were esterified and incorporated into the cell wall fraction as part of the anti-microbial defenses activated in the root tissue.     

Micropropagation and assessment of genetic fidelity of Henckelia incana: an endemic and medicinal Gesneriad of South India

Physiology and Molecular Biology of Plants - Henckelia incana is an endemic medicinal plant used for the treatment of fever and skin allergy. In the present study shoot regeneration was evaluated...     

西双版纳野芭蕉先锋植物群落的结构特征及其演替动态

对西双版纳勐腊县麻木树地区的热带雨林刀耕火种撂荒后形成的不同演替阶段的野芭蕉先锋植物群落进行了植物种类组成、区系成分、结构特征及种群数量与年龄结构的动态变化分析。结果表明 :在 10 0 0m2 的样地上 ,随着群落的发展和演替的进行 ,群落的科、属、种组成日趋复杂 ,从侵入阶段的 17科 2 7属 30种 ,上升到定居阶段的5 0科 74属 98种 ,至扩散阶段已达 5 5科 87属 113种。 3个不同林龄阶段的群落区系组成可分为 9种类型 ,均以热带区系成分为主 ,占 90 %左右。随着群落的发展 ,泛热带分布、热带亚洲 (印度—马来西亚 )分布所占的比例随林龄逐渐增大 ,而旧世界热带分布及东亚至北美洲间断分布所占的比例在逐渐减少。群落的层次结构由简单趋于复杂 ,野芭蕉的种群数量急剧增长 ,各龄级的个体分布逐渐增多 ,群落正向着湿润性的热带森林方向发展。     

The extent of clonality and genetic diversity in the rare Caldesia grandis (Alismataceae): Comparative results for RAPD and ISSR markers

Genetic variation and clonal diversity of three natural populations of the rare, highly clonal marsh herb Caldesia grandis Samuelsson were investigated using random amplified polymorphic DNA (RAPD) and inter-simple sequence repeat (ISSR) markers. Both of the markers worked effectively in clone identification of C. grandis. RAPD markers detected more diversity than ISSR markers in the three populations examined. Of the 60 RAPD primers screened, seven produced highly reproducible bands. Using these primers, a total of 61 DNA fragments were generated with 52 (85.25%) being polymorphic indicating considerable genetic variation at the species level. Analysis of molecular variance (AMOVA) showed that a large proportion of genetic variation (81.5%) resided within populations, while only a small proportion (18.5%) resided among populations. With the use of 52 polymorphic RAPD markers, we were able to identify 127 genets among 342 samples from three populations. The proportion of distinguishable genets (PD: mean 0.37), Simpson's diversity index (D: mean 0.91), and evenness (E: mean 0.78) exhibited high levels of clonal diversity compared to other clonal plants. These results imply that sexual reproduction has played an important role at some time during the history of these populations. Nevertheless, the high level of diversity could have been also partially generated from somatic mutations, although this is unlikely to account for the high diversity generally found among C. grandis genets.     

Assessment of genetic diversity and population structure of Chinese wild almond, Amygdalus nana, using EST- and genomic SSRs

Amygdalus nana L., commonly known as wild almond, is an endangered wild relative of cultivated almond, which has great potential in almond crop breeding. In this study, we used microsatellite (SSR) loci derived from both expressed sequence tag (EST) and anonymous genomic sequence to explore the genetic diversity and population structure of A. nana in Xinjiang of China. Seven natural populations were collected across the whole distribution of A. nana in China, including populations from both inside (four populations) and outside (three populations) the established protected areas. A total of 22 and 19 alleles were detected from the seven pairs of EST and genomic SSR loci, respectively. Generally, the genomic SSRs showed lower levels of variation than EST-SSRs, which may partially due to the higher cross-species transferability in EST-SSRs than in genomic SSRs. The population-level genetic diversity (A = 1.84, P = 50.00%, H_o = 0.3491, H_E = 0.2271) was lower than cultivated almond and several wild fruit species with similar breeding system. Most of the genetic variation (82.16%) was partitioned within populations. In particular, the population collected from Tacheng County (outside the protected areas) had the highest levels of genetic diversity and had significantly different genetic constitution from other populations.     

High genetic differentiation and low genetic diversity in Incarvillea younghusbandii, an endemic plant of Qinghai-Tibetan Plateau,revealed by AFLP markers

Incarvillea younghusbandii Sprague (Bignoniaceae) is a perennial herbaceous plant endemic to Qinghai-Tibetan Plateau. As a species of medical and horticulture importance, I. younghusbandii is threatened by over exploitation and habitat fragmentation. In this study, we analyze the genetic diversity and population structure of I. younghusbandii using amplified fragment length polymorphism (AFLP) markers. Our data reveal very low levels of genetic diversity in seven natural populations across Tibet. Specifically, at population level, the average Nei's genetic diversity index (H_E) and Shannon's diversity index (I) were 0.063 and 0.096, respectively. In contrast, high genetic differentiation among populations (Gst = 0.6238, Φ_ST = 0.614) is detected. The results of Neighbor-joining cluster, PCO, and STRUCTURE assignment reveal consistent pattern, suggesting seven well-defined genetic groups that are concordant with their geographical origins. The possible mechanisms and implications of these findings for conservation are discussed.     

Lack of genetic variation of an invasive clonal plant Eichhornia crassipes in China revealed by RAPD and ISSR markers

Random amplified polymorphic DNAs (RAPDs) and inter-simple sequence repeats (ISSRs) markers were used to analyze genetic structure of six populations of invasive plant Eichhornia crassipes that were sampled from its introduced regions in Southern China. Using 25 RAPD primers and 18 ISSR primers, 172 RAPD bands and 145 ISSR bands were produced respectively. But no polymorphic band was detected either within population or among populations by both markers, indicating the genetic diversity of E. crassipes in Southern China is extremely low, and all populations most likely consist of the same genotype. This study suggested that some other adaptability related factors, other than the genetic diversity, are responsible to the E. crassipes rapid expansion in China.     

Evaluation of genetic variability in the collared peccary Pecari tajacu and the white-lipped peccary Tayassu pecari by microsatellite markers

In this study, the microsatellite technique was used to evaluate the genetic variability in populations of collared and white-lipped peccaries kept in captivity. Six primers developed for domestic pigs were used and amplified in both species. They revealed the presence of five polymorphic loci and one monomorphic locus. The polymorphic loci included 4 of the 16 alleles in collared peccaries, and 3 of the 10 alleles in the white-lipped peccaries. Polymorphic information content (PIC) in both species and all the loci was highly informative. The probability of paternity exclusion (PEC), if one of the parents is known, was almost as high in white-lipped peccaries (95.53%) as in the collared (99,48%). The Fst values for collared (0.042) and white-lipped (0.1387) peccaries showed that both populations are not structured. The Fis values for all loci, except ACTG2 in white-lipped peccaries (-0.0275) and in both species (0.1985 to 0.9284 in collared peccaries and 0.3621 to 0.4754 in the white-lipped), revealed a high level of homozygosis, probably caused by inbreeding. Data on heterologous amplification and genetic variability in collared and white-lipped peccaries are presented for the first time.     

Evaluation of genetic relationships in Dalbergia species using RAPD markers

Conservation of identified germplasm is an important component forefficient and effective management of plant genetic resources. Traditionally,species identification has relied on morphological characters like growth habit,floral morphology like flower colour, and agronomic characteristics of the plant.Dalbergia species are important wind-dispersed tropicaltimber trees which exhibit high intrafruit seed abortion because of intensesibling competition for maternal resources. Studies were undertaken foridentification and genetic relationships in five species ofDalbergia and to evaluate genetic diversity withinpopulations of Dalbergia sisso, D.latifolia, D. paniculata, D.assamica and D. spinosa by using randomamplified polymorphic DNAs (RAPD) markers. Analysis was started by using 30decamer primers that allowed to distinguish five species and to select a reducedset of primers. The selected primers were used for identification and forestablishing a profiling system to estimate genetic relationships and toevaluate the genetic variability among the individuals in a population ofDalbergia species. A total of 120 distinct DNA fragments(bands), ranging from 0.3 to 4.0 kb, were amplified byusing nine selected random decamer primers. The genetic similarity was evaluated onthe basis of presence or absence of bands, which revealed a wide range ofvariability within the species. The cluster analysis indicated that five speciesof Dalbergia formed two major clusters. The first clusterconsisted of D. spinosa, D. latifolia and D.sisso. The second cluster was represented by two species, i.e.D. paniculata and D. assamica.A maximum similarity of 60% was observed in D. paniculata andD. assamica and they formed a minor cluster.Dalbergia latifolia and D. sissoformed another minor cluster with more than 50% similarity. Dalbergiaspinosa shared up to 40% similarity with D.latifolia and D. sisso. All the species sharemore than 20% similarity among themselves. The closest genetic distance existedwithin populations of different Dalbergia species. Thus,these RAPD markers have the potential for conservation of identified clones andcharacterization of genetic relatedness among the species. This is also helpful intree breeding programs and provides an important input into conservation biology.     

Production of tropan alkaloids in the in vitro and callus cultures of Hyoscyamus aureus and their genetic stability assessment using ISSR markers

Green wild plants (dirctly before flowering) and seeds of Hyoscyamus aureus were collected from natural habitat at Al Qalamon region in Syria. Seeds were surface sterilized and cultured in vitro, after 21 days from germination stem-derived callus was induced on two different nutrient media. Tropane alkaloids were extracted from wild plants and 30 days old in vitro plants and callus, and then analyzed using GC-MS. Genetic variation was also studied between the wild and in vitro plants and the callus culture lines using twenty ISSR markers. The results showed that there were significant variations in tropane alkaloids contents between the wild plants, the in vitro plants and the callus culture lines. The highest content of hyoscyamine was in callus on line A medium, but the highest content of scopolamine was in the wild plants. However, the lowest content of tropane alkaloids was in callus on line B medium. Also the ISSR analyses showed that there was genetic variation between the wild and in vitro plants and the callus culture lines.     

Variation and genetic structure of Melipona quadrifasciata Lepeletier (Hymenoptera, Apidae) populations based on ISSR pattern

For a study of diversity and genetic structuring in Melipona quadrifasciata, 61 colonies were collected in eight locations in the state of Minas Gerais, Brazil. By means of PCR analysis, 119 ISSR bands were obtained, 80 (68%) being polymorphic. H_e and H _B were 0.20 and 0.16, respectively. Two large groups were obtained by the UPGMA method, one formed by individuals from Januária, Urucuia, Rio Vermelho and Caeté and the other by individuals from São João Del Rei, Barbacena, Ressaquinha and Cristiano Otoni. The Φst and θ^B values were 0.65 and 0.58, respectively, thereby indicating high population structuring. UPGMA grouping did not reveal genetic structuring of M. quadrifasciata in function of the tergite stripe pattern. The significant correlation between dissimilarity values and geographic distances (r = 0.3998; p < 0.05) implies possible geographic isolation. The genetic differentiation in population grouping was probably the result of an interruption in gene flow, brought about by geographic barriers between mutually close geographical locations. Our results also demonstrate the potential of ISSR markers in the study of Melipona quadrifasciata population structuring, possibly applicable to the studies of other bee species.