Combined Inter-simple Sequence Repeat Research Articles

Understanding the genetic diversity and population structure of Dendrobium chrysotoxum Lindl.-An endangered medicinal orchid and implication for its conservation

Genetic variation study in 11 populations of D. chrysotoxum was conducted using 20 different inter-simple sequence repeats (ISSR) and 9 start codon targeted (SCoT) primers. Out of 199 bands produced by ISSR markers, 196 bands were polymorphic, resulting in high polymorphism (98.49%). SCoT markers, however, produced 111 polymorphic fragments generating 100% polymorphism. The Nei's genetic diversity at the species level (H = 0.184±0.11 for ISSR; H = 0.236±0.12 for SCoT) was higher than the average value at the population level (H = 0.160±0.15 for ISSR; H = 0.174±0.15 for SCoT). Low genetic differentiation among the populations (Gst = 0.1566 for ISSR; Gst = 0.2677 for SCoT) was observed due to high estimate of gene flow (Nm = 2.6938 for ISSR; Nm = 1.3678 for SCoT). Mantel test revealed a non-significant correlation between the genetic and geographical distance (r = -0.62; p = 0.86 for ISSR; r = -0.68; p = 0.82 for SCoT). Two-dimensional principal coordinate analysis (PCoA) determined the contribution of the first two principal components to the total genetic variation for combined ISSR and SCoT data at 13.71%. The distribution of individuals in the PCoA plot was in accordance with the unweighted pair group method with arithmetic averages (UPGMA) cluster analysis. Bayesian model-based STRUCTURE determined three (K = 3) gene pools for both ISSR and SCoT markers with a high degree of admixture in the populations. GENELAND further assigned the 11 different populations into 7 genetic clusters. Based on the genetic information obtained from the present investigation, we recommended different conservation strategies suited for effectively managing the endangered orchid. To the best of our knowledge, this is the first report of estimating the genetic diversity and population genetic structure of D. chrysotoxum in Manipur, a north-eastern state of India bordering Myanmar.

Farklı konukçulardan elde edilen Botrytis cinerea populasyon yapısının moleküler tanılanması

Botrytis cinerea (teleomorph: Botryotinia fuckeliana) causes gray mold disease on vegetable crops in greenhouses. Profound knowledge on pathogen diversity is necessary for efficiently disease management. In this study, forty-two B. cinerea isolates collected from 36 different greenhouses in Antalya province of Turkey were investigated. Twelve SRAP (sequence-related amplified polymorphism) and 18 ISSR (inter simple sequence repeat) primers producing high polymorphic fragments were used to genetic diversity of B. cinerea isolates infecting dill, basil, lettuce, bean, cucumber, tomato, pepper and eggplant. The unweighted pair-group method with arithmetic average analysis (UPGMA) was used to evaluate of combined ISSR and SRAP data showing a similarity range 0.15-0.90 among the isolates. Cophenetic correlation of the tree was high level (r=0.93). Interestingly, cluster analysis showed a divergent group consisting of lettuce isolates which were genetically different from the other isolates. On the other hand, transposable elements (Flipper and Boty) were detected among isolates from all the hosts. Isolates containing only the Fliper element were detected. The results showed that genetically characterized B. cinerea populations by a high level of genetic diversity were associated with genotype flow and the evolutionary potential of B. cinerea. In further studies, the newly tested molecular markers are useful and can be suggested for analyzing of genetic diversity and population structure of this pathogen on different hosts.

ASSESSMENT OF GENETIC COMPONENTS AND GENETIC DIVERSITY OF SIX EGYPTIAN CLOVER (Trifolium alexandrinum L.) GENOTYPES USING ISSR AND URP MARKERS

Six Egyptian clover genotypes, i.e., Helaly, Sakha 4, Gemmeiza 1, Serw1, Giza6 and Sakha composite were used in this study. Data was calculated on four cuts and seasonal yield for fresh and dry forage yield. The results indicated that the mean square values were highly significant for genotypes for all studied traits. Helaly cultivar gave the highest values in four cuts and seasonal yield for fresh forage yield. High heritability values were recorded for all studied traits except 1st fresh yield which recorded moderate value. Phenotypic coefficient of variability (PCV) was higher than genotypic coefficient of variability (GCV) in all cuts and seasonal yields. Genetic variations among the six Egyptian clover cultivars were evaluated using universal rice primers (URP) and inter simple sequence repeat (ISSR) markers. URP fingerprinting detected more polymorphic loci (90%) than the ISSR fingerprinting (77.55%). Mean PIC (polymorphism information content) and band informativness (Ib) for each of these marker systems were (0.21, 0.26 for ISSR and 0.32, 0.46 for URP) respectively, suggested that URP marker systems were effective than ISSR in determining polymorphisms. Pairwise similarity index values ranged from 0.37 to 0.72 for ISSR, 0.23 to 0.62 for URP and 0.29 to 0.65 for combined ISSRs and URP indicated the genetic distinctness among the studied genotypes.

Species delimitation in the genus Tamarix: Morphological and molecular data<br />

Tamarix (Tamaricaceae) is an ancient genus in Asia that contains about 54 to 90 species with unresolved taxonomy. Tamarix has major center of speciation in the Pakistan-Afghanistan-Iran-Turkmenistan-S-Kazakhstan-W-China area and in the eastern Mediterranean area. Tamarix species are highly plastic and tolerant to adversity being able to occupy different habitats, thus showing a wide range of ecological variations. Tamarix species have commercial value in the landscape and horticultural trade and are planted for erosion control and as wind breaks. In the genus Tamarix, many species are morphologically very similar probably due, in part, inter-specific hybridization. Tamarix species live in sympatry in Sistan and Baloochestan province of Iran and we have no report on the number of species and their genetic structure. Therefore, we performed morphometric study to delimit the species growing in these two provinces and also used combined ISSR (Inter simple sequence repeat) and RAPD (Random amplified polymorphic DNA) molecular markers to study their genetic structure. Morphological analyses separated plants of 6 studied species in different groups, indicating that morphological characters used can delimit Tamarix species. We also encountered some plants having intermediate morphological characters. AMOVA and Hickory test showed significant genetic differences of the studied species and Mantel test showed significant correlation between genetic distance and geographical distance. STRUCTURE analysis and population assignment test revealed some degree of genetic admixture among the studied species. Assignment tests showed that gene flow mainly occurred among the species that were growing in neighborhood regions. This genetic admixture may be the reason for intermediate morphological characters observed in some the plants.

Natural population structure and genetic differentiation for heterodicogamous plant: Cyclocarya paliurus (Batal.) Iljinskaja (Juglandaceae)

Cyclocarya paliurus, a Chinese monotypic genus belonging to the Juglandaceae, exhibits a widespread distribution in southern China. To provide resource support for its future breeding and medicinal utilization, much information involving natural population succession and genetic diversity of C. paliurus should be obtained. In this study, the structures of 30 natural populations throughout its distribution were analyzed based on the investigation, showing the ratio of individuals with DBH < 40 cm accounting for 71.7%. Inter-simple sequence repeat (ISSR) and simple sequence repeat (SSR) were used for evaluation of 244 individuals from 29 natural populations. High-level polymorphism (100%) indicates high efficiency of two markers. Analysis demonstrated that genetic diversity (value of 0.18 for ISSR and 0.09 for SSR) was low among populations. While gene flow among populations reached 1.182 and 1.335 for ISSR and SSR, respectively, indicating genetic variations mainly existing within population. Twenty-six populations (size ≥5 individuals) were grouped into five clusters based on the combined ISSR and SSR loci. The value of Nei’s coefficient varying from 0.87 to 0.96 indicates small differentiation among groups. Furthermore, we speculate that C. paliurus is an expanding species in subtropical China (most of populations are increasing type (IP)) and discuss the strategy of germplasm conservation.

Analysis of Genetic Diversity in Chrysopogon aciculatus Using Intersimple Sequence Repeat and Sequence-related Amplified Polymorphism Markers

Molecular genetic diversity and relationships among 86 Chrysopogon aciculatus (Retz.) Trin. accessions were assessed using intersimple sequence repeat (ISSR) and sequence-related amplified polymorphism (SRAP) markers. Twenty-five ISSR markers generated 283 amplification bands, of which 266 were polymorphic. In addition, 576 polymorphic bands were detected from 627 bands amplified using 30 SRAP primers. Both marker types revealed a high level of genetic diversity, with ISSR markers showing a higher proportion of polymorphic loci (PPL; 94%) than SRAP markers (91.87%). The ISSR and SRAP data were significantly correlated (r = 0.8023). Cluster analysis of the separate ISSR and SRAP data sets clustered the accessions into three groups, which generally were consistent with geographic provenance. Cluster analysis of the combined ISSR and SRAP data set revealed four major groups similar to those based solely on ISSR or SRAP markers. The findings demonstrate that ISSR and SRAP markers are reliable and effective tools for analysis of genetic diversity in C. aciculatus.

Genetic diversity and population structure of Stipa bungeana, an endemic species in Loess Plateau of China, revealed using combined ISSR and SRAP markers

Sequence-related amplified polymorphism (SRAP) and inter-simple sequence repeat (ISSR) markers were used to assess the genetic diversity within and among 15 natural populations of Stipa bungeana from the Loess Plateau of China. Using 15 SRAP primers, 504 (99.80%) polymorphic loci were detected, and 372 polymorphic loci (96.12%) were identified using 15 ISSR primers. At the species level, the S. bungeana populations showed relatively low levels of genetic diversity (HE = 0.2017 for SRAP; HE = 0.2066 for ISSR). The results of analysis of molecular variance indicated that genetic variation within populations (42.02% for SRAP and 38.40% for ISSR) is lower than that among populations (57.98% for SRAP and 61.60% for ISSR). The genetic distance was significantly correlated with geographical distance by the Mantel test (r = 0.3978, P = 0.002). Our results demonstrated that both SRAP and ISSR markers are effective and reliable for assessing the genetic diversity of S. bungeana. In addition, these data inform conservation and breeding strategies for S. bungeana.

Assessment of intrapathovar variability of Xanthomonas axonopodis pv. commiphorae through phenotypic and molecular markers

SummaryGummosis of guggal (Commiphora wightii) caused by Xanthomonas axonopodis pv. commiphorae (Xac) is one of the major reasons for drastic reduction in guggal population under natural plant stand. The pathogen spreads mainly through human activities (tapping). We isolated 43 Xac strains from 14 locations representing four different regions spread over three districts of Gujarat state, India. A polyphasic approach was followed to characterize these strains and to measure phenotypic and genetic variations among them. All strains were identical in colony morphology and were virulent on guggal. Some of the strains showed differential reactions towards certain biochemical tests viz., acid production from carbon sources. Whole cell protein profiles were apparently uniform for all strains with similarity coefficients ranging between 0.75 and 1.00. Clustering based on sodium dodecyl sulphate–polyacrylamide gel electrophoresis banding patterns showed heterogeneity among the isolates, but strains originating from same zone had similar protein profiles. Inter simple sequence repeats (ISSR)‐based and repetitive elements (rep)‐based PCR analyses revealed genetic heterogeneity among the strains. Both these methods were equally effective in deciphering variability among the strains and indicated similar types of variability with highly significant correlation (r = 0.91) on the similarity matrices. The variation matrix analysis on combined ISSR‐ and rep‐PCR data suggested 66.1% variability among Xac strains. The present study established that Xac strains from different geographical locations had profound genetic heterogeneity.

Isolation and identification of locally isolated bacterial strains effective against whitefly Bemisia tabaci

Potent bacterial strains effective against the whitefly, Bemisia tabaci, nymphs (second instar), were isolated from tomato cultivated fields at Fayoum governorate, Giza, Egypt. Of 72 isolates, 12 with the most morphologically distinct-looking bacterial colonies were selected and named A1, A2, A3, A6, A7, A9, A12, A13, A107, B37, B45 and B100. All isolates were preliminarily identified as members of the genus Bacillus based on morphological, physiological and biochemical characteristics. When tested for their pathogenicity against Bemisia tabaci, the 12 isolates revealed varying efficiencies with isolates A1 and A9 being superior, exhibiting maximum mortality of 92.2 and 90.8% on day 10, respectively. Isolate A7 recorded the lowest percentage at 18.3%. Further genetic characterization of the 12 isolates was performed using inter simple sequence repeat (ISSR), randomly amplified polymorphic DNA (RAPD) and 16S rDNA gene sequencing analysis. RAPD and ISSR results confirmed each other. The combined ISSR and RAPD phylogenetic tree showed two major clusters. With 16S rRNA gene analysis, isolate A1 and A12 sequences recorded 100% identity with Bacillus thuringiensis, while isolates A7 and B100 showed 95.7% and 95.6% identity with Bacillus cereus and Bacillus sphaericus, respectively.

Genetic diversity of Ustilago scitaminea Syd. in Southern China revealed by combined ISSR and RAPD analysis

The polymorphism and similarity relationships among 35 mating-type isolates of Ustilago scitaminea collected from Southern China were determined with random amplified polymorphic DNA (RAPD) and inter-simple sequence repeat (ISSR) analyses. These fungal isolates were collected from 16 sugarcane cultivars including F134 that is resistant to the physiological race 1 but susceptible to the race 2 of U. scitaminea, and N: Co376 that is immune to both races 1 and 2. Unweighted pair group method with arithmetic mean (UPGMA) cluster analysis revealed that the U. scitaminea isolates could be divided into 2 groups with a coefficient of 0.74. The first group comprises two isolates collected from the sugarcane cultivar F134, while the remaining 33 isolates were clustered into the second group. The second group was further divided into two subgroups with most of the isolates from Guangdong Province which clustered in the same subgroup, and all the isolates from Guangxi and Yunnan Provinces were clustered in another subgroup. Given that the member of the second group could infect the cultivar N:Co376, which is immune to the races 1 and 2, our results suggest that majority of U. scitaminea in sugarcane-producing regions of Southern China may belong to or genetically similar to race 3. Key words: Ustilago scitaminea , sugarcane, inter-simple sequence repeat (ISSR), random amplified polymorphic deoxyribonucleic acid (DNA) (RAPD), genetic diversity.

Analysis of genetic diversity in Monilinia fructicola from the Ebro Valley in Spain using ISSR and RAPD markers

The genetic diversity of Spanish and French field populations of Monilinia fructicola, a quarantine fungal pathogen in Europe, was compared with that of Californian, Uruguayan, and New Zealand M. fructicola populations using inter simple sequence repeat (ISSR) and random amplified polymorphic DNA (RAPD) markers. Unweighted pair-group method with arithmetic average (UPGMA) cluster analysis and principal component analysis (PCA) of the ISSR data set revealed that the Spanish and French M. fructicola isolates were more closely related between themselves than to the non-European isolates. The levels of genetic diversity in the Spanish and French isolates are lower than those of the non-European isolates, indicating that M. fructicola is a recently introduced pathogen. UPGMA cluster analysis and PCA of the combined ISSR + RAPD data set of the European M. fructicola populations revealed that the Spanish isolates were more closely related among themselves than with the French isolates. Analysis of molecular variance partitioned the genetic variance to among the two regions (Spain and France) (20%), among the regional populations (35%), and within the populations in each region (45%) suggesting restricted gene flow between the three European populations. The observed index of association (IA) in each European M. fructicola populations indicates that the French and Spanish populations of M. fructicola are mainly asexually reproducing, with the Sudanell population potentially having a teleomorphic stage. The present finding of low genetic diversity in the Spanish and French M. fructicola populations is probably due to founder effects and genetic drift.

Assaying of diversity among soybean (Glycin max (L.) Merr.) and peanut (Arachis hypogaea L.) genotypes at DNA level

Developing successful strategies to ensure future increase in yield of soybean and peanut crops hinges in part, on improving the genetic basis of the cultivars. Knowledge of genetic relationships in crop breeding programs provides valuable information that can be used by plant breeders as a parental line selection tool. So far, a thorough analysis of genetic diversity among the soybean and peanut genotypes grown in Turkey had not been attempted at DNA level. In this study, inter-simple sequence repeats (ISSR) and sequence-related amplified polymorphism (SRAP) markers were used to evaluate the genetic diversity between 21 soybean and 32 peanut cultivars and breeding lines adapted to different regions of Turkey. The ISSR analysis, which was performed with 46 primers in soybean and 47 primers in peanut, yielded 31 and 26 polymorphic bands, respectively, while 26 and 17 polymorphic amplicons were amplified by 34 and 36 SRAP primer combinations in soybean and peanut, respectively. The unweighted pair group method with arithmetic means dendrograms (UPGMA), based on Jaccard similarities,were obtained from the combined ISSR + SRAP data for both soybean and peanut. In soybean, UPGMA dendrogram clustered all soybean cultivars into the same group except ´Yeşilsoy´, whereas, in peanut, it separated cultivars southwest runner and spantex from all the other cultivars, breeding lines, and plant introductions. In light of the narrow germplasm base of soybean and peanut genotypes grown in Turkey, a renewed emphasis should be placed on the introduction of new sources of germplasm into the breeding pool in order to enhance genetic variability to permit continued progress in developing high yielding cultivars and lead to greater gains for selections. The results obtained from this study will be helpful for soybean and peanut breeders in Turkey to gain information about genetic diversity and will enable them to make a future strategy for broadening the genetic basis of these crops.

Application of Molecular Markers in Predicting Production Quality of Cultivated Cistanche deserticola

We developed a set of molecular markers in Cistanche deserticola Y. C. MA to evaluate the production quality of cultivated C. deserticola individuals. This application utilizes the inter-simple-sequence repeat (ISSR) polymerase chain reaction (PCR) and random amplified polymorphic DNA (RAPD) PCR as molecular markers to determine the echinacoside content in cultivated C. deserticola individuals. The unweighted pair-group method using arithmetic average clustering (UPGMA) confirmed that the combined ISSR and RAPD data could categorize all C. deserticola individuals into three groups according to their respective echinacoside content. The stepwise multiple regression analysis (SMRA) revealed six potential markers associated with echinacoside accumulation in C. deserticola and produced 18 echinacoside-marker prediction models, four of which were successfully used to predict the quality of C. deserticola from Neimenggu. Both clustering and SMRA showed a correlation between the echinacoside content and molecular markers in cultivated C. deserticola. The relative average deviation of prediction (RADP) of the prediction models could be improved by simplifying and adjusting the model. It was found that the RADP value could reach 2.6% after adjustment and the simplified prediction models could successfully predict the quality of cultivated C. deserticola individuals.

Genetic variation among lentil (Lens culinaris Medik) landraces from Southeast Turkey

AbstractThe molecular characterization of cultivated plant genepools is of foremost importance for germplasm utilization in plant breeding. However, no comprehensive genetic fingerprinting of Turkish lentil landraces existed so far. To overcome this gap, 38 lentil landraces from southeast Turkey, together with six commercial varieties, were molecularly characterized using inter simple sequence repeat (ISSR) and amplified fragment length polymorphism (AFLP) molecular markers. The ISSR analysis, performed with 14 primers, yielded 105 polymorphic bands and the AFLP analysis, carried out with six primer combinations, amplified 119 polymorphic fragments. Even though the AFLP produced more bands per primer combinations, the ISSR detected more polymorphisms. Unweighted pair‐group method with arithmetic means dendrograms based on Jaccard similarities obtained from three data sets: (i) ISSR, (ii) AFLP and (iii) combined ISSR and AFLP data, were similar and separated the landraces into two main groups. Turkish lentil landraces exhibited considerable genetic diversity. One landrace from Karacadag/Diyarbakir region was significantly different from the rest of the germplasm analysed. Jaccard distances highlighted sharp differences among landraces over short geographic distances. The knowledge of regional differentiation has practical utility in the management of germplasm and in breeding programmes.

Genetic diversity and relationships of sweetpotato and its wild relatives in Ipomoea series Batatas (Convolvulaceae) as revealed by inter-simple sequence repeat (ISSR) and restriction analysis of chloroplast DNA

Genetic diversity and relationships of 40 accessions of Ipomoea, representing ten species of series Batatas, were examined using ISSR markers and restriction-site variation in four non-coding regions of chloroplast DNA. A total of 2071 ISSR fragments were generated with 15 primers in these accessions and, on average, 52 bands per accession were amplified. Most of the primers contained dinucleotide repeats. The ISSR fragments were highly polymorphic (62.2%) among the 40 accessions studied. Restriction analysis of chloroplast (cp) DNA revealed 47 informative restriction-site and length mutations. Phylogenetic analyses of ISSR and cpDNA datasets generally revealed similar relationships at the interspecific level, but the high polymorphism of ISSRs resulted in a better separation of intraspecific accessions. However, the combined ISSR and cpDNA dataset appeared to be appropriate in resolving both intra- and interspecific relationships. Of the species examined, I. trifida was found to be the most closely related to cultivated sweetpotato, the hexaploid I.batatas, while I. ramosissima and I. umbraticola were the most distantly related to I. batatas within the series. Ipomoea triloba, hitherto considered to be one of the ancestors of sweetpotato, was only distantly related to sweetpotato based on ISSR similarity index.