Cryopreservation-Based Strategy for the Long-Term Conservation of European Pear (Pyrus communis) Germplasm

In order to assess genetic variations among Rhodendron trifolium populations sampled from Tibet and determine the correlation among genetic variations, the geographic location of a population, and factors that influence high-level genetic diversity, in total of 107 R. triflorum samples using inter simple sequence repeats (ISSR) and amplified fragment length polymorphisms (AFLP). All genotypes were collected from six different areas. Eleven ISSR primers and five AFLP primer pairs were screened, and 118 and 169 amplification products were produced, of which, 96.61% in ISSR and 95.27% in AFLP were polymorphic. High genetic diversity was observed at the species level: Nei's genetic diversity (H) was 0.3382 and 0.306, and Shannon's information index (I) was 0.5085 and 0.4642 in ISSR and AFLP, respectively. Both the coefficient of gene differentiation (GST 0.3752 in ISSR, 0.31 in AFLP) and AMOVA analysis (75% in ISSR, 71% in AFLP) indicated that most genetic diversity was distributed within populations. Gene flow (Nm) was 0.8326 in ISSR and 1.1127 in AFLP. The analyze of unbiased genetic distances determined by an unweighted pair group method using arithmetic mean (UPGMA) phonograms indicated that there was a certain degree correlation between the genetic distance and the geographic distance, which was confirmed by a principal coordinate analysis. The results maybe indicated that difference of geographical environment and variation of habitat types caused the genetic differentiation of R. triflorum. At last, some conservation strategies for R. triflorum germplasm were put forward according to these results.

The power of microsatellite markers and AFLPs in revealing the genetic diversity of Hashemi aromatic rice from Iran

In order to get an overview on the genetic relatedness of sorghum (Sorghum bicolor) landraces and cultivars grown in low-input conditions of small-scale farming systems, 46 sorghum accessions derived from Southern Africa were evaluated on the basis of amplified fragment length polymorphism (AFLPs), random amplified polymorphic DNAs (RAPDs) and simple sequence repeats (SSRs). By this approach all sorghum accessions were uniquely fingerprinted by all marker systems. Mean genetic similarity was estimated at 0.88 based on RAPDs, 0.85 using AFLPs and 0.31 based on SSRs. In addition to this, genetic distance based on SSR data was estimated at 57 according to a stepwise mutation model (Deltamu-SSR). All UPGMA-clusters showed a good fit to the similarity estimates (AFLPs: r = 0.92; RAPDs: r = 0.88; SSRs: r = 0.87; Deltamu-SSRs: r = 0.85). By UPGMA-clustering two main clusters were built on all marker systems comprising landraces on the one hand and newly developed varieties on the other hand. Further sub-groupings were not unequivocal. Genetic diversity (H, DI) was estimated on a similar level within landraces and breeding varieties. Comparing the three approaches to each other, RAPD and AFLP similarity indices were highly correlated (r = 0.81), while the Spearman's rank correlation coefficient between SSRs and AFLPs was r = 0.57 and r = 0.51 between RAPDs and SSRs. Applying a stepwise mutation model on the SSR data resulted in an intermediate correlation coefficient between Deltamu-SSRs and AFLPs (r = 0.66) and RAPDs ( r = 0.67), respectively, while SSRs and Deltamu-SSRs showed a lower correlation coefficient (r = 0.52). The highest bootstrap probabilities were found using AFLPs (56% on average) while SSR, Deltamu-SSR and RAPD-based similarity estimates had low mean bootstrap probabilities (24%, 27%, 30%, respectively). The coefficient of variation (CV) of the estimated genetic similarity decreased with an increasing number of bands and was lowest using AFLPs.

Knowledge of genetic diversity within and among genotypes of any crop is fundamental for estimation of the potential genetic gain in a breeding programme and effective conservation of available genetic resources. Currently, different molecular marker techniques are being developed for measuring genetic diversity. Comparison among molecular marker techniques is important for effective marker selection. However such types of efforts are rare for arabica coffee. This study was conducted to compare the efficiency of simple sequence repeat (SSR) analysis for determining genetic relationships of 28 Coffea arabica L. genotypes collected from different parts of Ethiopia with work previously done using amplified fragment length polymorphism (AFLP) analysis. A total of 22 SSR fragments were amplified and compared with 712 previously amplified AFLP fragments. AFLP and SSR markers were positively and significantly correlated (0.217) in estimating genetic similarity among genotypes. The average genetic similarity coefficient calculated using SSR markers was much lower (0.560 with a range of 0.286–1.000) compared to AFLP markers (0.915 with a range of 0.860–0.982) indicating the higher information content of SSR markers. AFLP markers distinguished all genotypes, while SSR markers distinguished 64.3% of the genotypes. Results indicated that AFLP markers were more efficient compared to SSR markers for characterization of the evaluated coffee genotypes.

Six Egyptian pomegranate (Punica granatum L.) cultivars were characterized by fruit characteristics (physical and chemical) and two molecular markers; Inter simple sequence repeat (ISSR) and amplified fragment length polymorphism (AFLP). Genetic diversity of the pomegranate genotypes was evaluated. Physical fruit traits were determined (weight, volume and diameter), calyx [diameter, length (mm) and Carpels number], fruit firmness (Newton), peel as (weight and thickness), arils weight (g), volume of juice (ml), seeds [fresh and dry weight (g)], and color parameter of (fruit skin, internal peel, arils, juice and seeds). The chemical traits such as soluble solids contents (SSC), vitamin C content, anthocyanin content, pH, and titratable acidity (TA) were assessed and wide variations were observed in each of these traits among the studied cultivars. The genetic variability and relationships among six Egyptian pomegranate cultivars were tested using ISSR and AFLP analyses. The level of polymorphism across cultivars was 53 and 90.7% as revealed by ISSR and AFLP, respectively. ISSR and AFLP revealed different genetic similarities among the six pomegranate cultivars. Each analysis differs not only in its underlying principle, but also in their in-formativeness with regard to the type and amount of polymorphism detected. Genetic similarity matrices estimated from ISSR and AFLP data, showed similarity coefficients to range from 0.77 to 0.94 and 0.33 to 0.73, respectively. ISSR and AFLP characterized the six pomegranate cultivars by a large number of unique markers being 23 and 46 unique markers, respectively. The fruit weight ranged between 479.4 to 185 g of Ê»Nab El Gamalʼ and Ê»Assuityʼ, the firmness was 79.98 of Ê»Nab El Gamalʼ and 71.84 Newton of Ê»Manfaloutyʼ cv. The fruit peel thickness varied from 0.6 mm Ê»Arabyʼ, Ê»Hegazyʼ and Ê»Wardiʼ to 0.3 mm Ê»Assuityʼ. The arils weight ranged from 87.5 to 275 g of Ê»Assuityʼ and Ê»Nab El Gamalʼ cvs. The percentage net of arils weight/ fruit weight was the highest (59.34% of Ê»Manfaloutyʼ cv). The juice volume ranged from 62.41 to 71.81 ml/100 g arils for Ê» Wardiʼ and Ê»Nab El Gamalʼ cvs. The SSC content ranged between 16.01 Ê»Hegazyʼ and 12.55% Ê»Assuityʼ. V.C. content ranged from 3.21 to 14 mg. vitamin/100 ml juice of Ê»Nab El Gamalʼ and Ê»Assuityʼ. The anthocyanin content ranged from 1.47 to 10.03 for Ê»Arabyʼ and Ê»Hegazyʼ. The pH values varied from 3.3 (Wardi) to 2.9 (Araby). The Egyptain cultivars of pomegranate have a wide variation in the morphological and chemical characteristics for many uses of fresh fruit and of industry purpose.   Key words: Morphological and chemical fruits characterization, pomegranate, inter simple sequence repeat (ISSR), DNA markers, amplified fragment length polymorphism (AFLP).

BACKGROUND: Bitter gourd (Momordica charantia L.) is an important vegetable crop in tropical countries, including China and India. A wide range of genetic diversity exists in India with respect to fruit morphology such as colour, size and exocarp. A diversity assessment conducted using different DNA marker systems amplified fragment length polymorphism (AFLP), inter simple sequence repeat (ISSR) and random amplified polymorphic DNA (RAPD) markers) will be helpful in the establishment of a broad‐based description for improved germplasm curation and the identification of germplasm for genome mapping and breeding of bitter gourd.RESULTS: Genetic relationships between 38 bitter gourd accessions were determined with the aid of 29 RAPD, 15 ISSR and six AFLP markers. Greater polymorphism was detected by AFLPs when compared with RAPD and ISSR analyses using the same germplasm array (RAPD 36.5%, ISSR 74.5% and AFLP 78.5% polymorphism). The average marker index (MI) values derived from the three different marker systems differed dramatically, indicating that they vary in their discriminatory power (AFLP > ISSR > RAPD). The AFLP markers used were only weakly correlated with ISSR (r2 = 0.007) and RAPD (r2 = 0.04) marker analyses, whereas a comparatively high correlation (r2 = 0.77) was found between RAPD and ISSR marker systems.CONCLUSION: The studies using RAPD and ISSR markers were not able to uniquely discriminate all the bitter gourd accessions examined, whereas AFLP analysis was discriminatory and allowed for a more complete dissection of unique differences among accessions of bitter gourd within and between collection sites. Copyright © 2007 Society of Chemical Industry

Urtica dioica is an important medicinal plant which is widely distributed in Mazandaran province (North of Iran). In this study for the first time Amplified Fragment Length Polymorphism (AFLP) and Inter-simple Sequence Repeat (ISSR) markers were used for detection of genetic polymorphism in Mazandaran nettle. Ten AFLP primer combinations and seventeen ISSR markers were utilized. AFLP produced 830 scorable bands out of which 90.21% were polymorphic. ISSR primers amplified 234 bands, 181 being polymorphic (77.3%). Average heterozygosity for AFLP and ISSR markers were 0.25, 0.23 respectively. Marker Index obtains 22.25 for AFLP and 15.57 for ISSR. The number of cluster computed was same for both molecular makers but location of samples in branch were different. The total compare of these two marker systems shown AFLP marker was a useful tool for detection of U. dioica’sgenetic diversity. This plant is very variable and is genetically distinct in east, west and north of Mazandaran.

Applications of AFLP and ISSR techniques in detecting genetic diversity in the soybean brown stem rot pathogen Phialophora gregata

Zingiber moran, a rare ginger found only in a few parts of northeast India, faces the threat of extinction due to its exploitation as an important medicinal plant. In this study, the chromosome number of wild ecotypes of Z. moran was determined and the genetic analysis was carried out using two kinds of molecular markers: inter simple sequence repeats (ISSR) and amplified fragment length polymorphism (AFLP). A chromosome number of 2n = 22 was confirmed for all 10 ecotypes investigated. AFLP analysis revealed a greater amount of genetic polymorphism (95.06%), with a strong resolving power in detecting intraspecific variation, compared to ISSR analysis (34.61%). Genetic diversity parameters, such as number of observed alleles (na), mean number of effective alleles (ne), mean Nei's gene diversity index (h), and Shannon index (I) for ISSR and AFLP were in the range 1.34–1.95, 1.25–1.60, 1.44–0.33, and 0.20–0.49, respectively. Marker indices and polymorphic information content varied from 0.5–19.3 and 0.03–0.27 for ISSR, and 28.2–39 and 0.3–0.39 for AFLP, respectively. Dendrograms derived by unweighted pair group method of arithmetic averages cluster analysis based on molecular data classified the ecotypes into two major clusters. The Mantel test cophenetic correlation coefficient (r) for ISSR (0.85) and AFLP (0.91) showed a good degree of confidence in the association with 10 ecotypes. Clustering was further supported by principle component analysis which demarcated individuals of hilly topography from the ones of plain land.

The genetic stability of in vitro propagated potato microtubers was assessed using random amplified polymorphic DNA (RAPD), inter simple sequence repeat (ISSR), simple sequence repeat (SSR) and amplified fragment length polymorphism (AFLP) markers. Microtubers were developed through in vitro from potato microplants using standardized protocols. The microtubers were conserved for 1year under three different culture media and consequently microplants were regenerated for the DNA analyses. During the study, a total of 38 (10 RAPD, 11 ISSR, 12 SSR and 5 AFLP) primers produced a total of 407 (58 RAPD, 56 ISSR, 96 SSR and 197 AFLP) clear, distinct and reproducible amplicons. Cluster analysis revealed 100% genetic similarity among the mother plant and its derivatives within the clusters by SSR, ISSR and RAPD analyses, whereas AFLP analysis revealed from 85 to 100% genetic similarity. Dendrogram analysis based on the Jaccard's coefficient classified the genotypes into five clusters (I-V), each cluster consisting of mother plant and its derivatives. Principal component analysis (PCA) also plotted mother plant and its genotypes of each cluster together. Based on our results, it is concluded that AFLP is the best method followed by SSR, ISSR and RAPD to detect genetic stability of in vitro conserved potato microtubers. The in vitro conservation medium (T2) is a safe method for conservation of potato microtubers to produce true-to-type plans.

Amplified fragment length polymorphism (AFLP) and inter-simple sequence repeat (ISSR) markers were used to study the DNA polymorphism in elite mungbean genotypes. A total of nine AFLP primer combination and 22 ISSR primers were used. Amplification of genomic DNA of the 30 genotypes, using AFLP analysis, yielded 300 fragments that could be scored, of which 192 were polymorphic, with an average of 21.3 polymorphic fragments per primer. Number of amplified fragments with AFLP primers ranged from 29 (E-AAC: M-CAG) to 10 (E-ACG: M-CAT). Percentage polymorphism ranged from 46.3% (E-AAC: M-CCA) to a maximum of 100% (E-AAC: M-CAC), with an average of 64%. The 22 ISSR primers used in the study produced 108 bands across 30 genotypes, of which 68 were polymorphic. The number of amplified bands varied from two UBC820) to ten URP 6F). The average numbers of bands per primer and polymorphic bands per primer were 4.9 and 3.1, respectively. Percentage polymorphism ranged from 25% (UBC844) to 85% (UBC846, UBC864, UBC895), with an average percentage polymorphism of 58.3% across all the genotypes. AFLP markers were more efficient than the ISSR assay, as they detected 64% polymorphic DNA markers in Vigna radiata as compared to 58.3% for ISSR markers. The Mantel test between the two Jaccard's similarity matrices gave r = 0.19, showing low correlation between AFLP- and ISSR-based similarities. Clustering of genotypes within groups was not similar when AFLP and ISSR derived dendrograms were compared. Key words: AFLP, ISSR, Vigna radiata (mung bean), marker index, unweighted pair-group method with arithmetic averages (UPGMA).

SummaryThe characterisation of sweet cherry (Prunus avium L.) genetic resources in Turkey may help to increase their use in breeding programmes worldwide, as Turkey is the centre of origin of sweet cherry. Amplified fragment length polymorphism (AFLP) and simple sequence repeat (SSR) markers were therefore used to analyse genetic diversity among a total of 78 local and introduced sweet cherry cultivars. Four AFLP primer combinations, and six SSR primer pairs for sweet cherry were used for genetic diversity analysis. A genetic similarity matrix was calculated using the combined data from AFLP and SSR analyses with simple matching coefficient. Genetic similarities among the sweet cherry genotypes studied were higher than 42%. No two accessions had an identical AFLP and SSR marker profile, indicating that all 78 genotypes were unique. An UPGMA dendrogram, based on the similarity matrix, revealed 18 separate Groups at or above the 70% similarity level. While some Groups consisted of both introduced and local genotypes, other Groups had only local genotypes. This result suggests that there was broad genetic diversity among the local Turkish sweet cherry genotypes, which was not present in the introduced sweet cherry accessions. The genetic variation present in local Turkish sweet cherry genotypes may be useful for future breeding programmes. We found that the use of both SSR and AFLP marker systems was effective for distinguishing between genetically-close sweet cherry genotypes. These marker systems can be used to complement pomological and morphological markers during the characterisation and identification of sweet cherry genotypes.

Random amplified polymorphic DNA (RAPD), Inter simple sequence repeat (ISSR) and Amplified fragment length polymorphism (AFLP) markers were used to verify the segregation of the genus Cassia L. (sens. lat.) into three distinct genera namely, Chamaecrista Moench.,Senna P. Mill. and Cassia L. (sens.str.). Eighteen representatives of the three taxa were characterized using the molecular markers. 25 RAPD, six ISSR primers and six AFLP primer combinations resulted in the amplification of 612, 115 and 622 bands (loci), respectively. Most of the loci are found to be polymorphic, showing high degrees of genetic diversity among the different taxa studied. The dendrogram constructed on the basis of the RAPD, ISSR and AFLP data using the SHAN clustering, divided Cassia L. (sens. lat.) into three different clusters as Chamaecrista Moench., Senna P. Mill. and Cassia L. (sens.str.). High bootstrap value revealed that all the clusters were stable and robust. It was observed from the present investigation that these genera have their identity at molecular level, which supports the elevation of the genus Cassia L. sens. lat. to the level of sub tribe Cassiinae and segregationinto three distinct genera instead of intra-generic categories. In the present study taking the molecular markers into account the trifurcation of the sub tribe Cassiinae could be re-established. Key words: Cassia, molecular phylogeny, RAPD, ISSR, AFLP.

Limonium sinense is a halobiotic herb endemic to China that has been traditionally used for hundreds of years for its good restorative function. Genetic variation and population structure of this species were investigated by using amplified fragment length polymorphisms (AFLPs) and inter simple sequence repeats (ISSRs). A high level of genetic diversity was detected [AFLP: HE = 0.284, percentage of polymorphic loci (PPL) = 92.68 %; ISSR: HE = 0.257, PPL = 85.71 %] at the species level with POPGENE. Based on analysis of molecular variation (AMOVA), the among-population component accounted for 29.03 % (AFLP) and 28.81 % (ISSR) of the genetic variation, indicating that most of the genetic variation was between individuals within populations. The Shannon diversity index (I) was higher for AFLP (0.432) than for ISSR (0.395). Five main clusters were shown in the unweighted pair-group method with arithmetic mean (UPGMA) dendrogram created using TFPGA, consistent with the result of principal coordinate analysis using NTSYS. In situ conservation is advocated first. Keeping a stable environment for this halobiotic herb is necessary. For ex situ conservation, it is important to establish a germplasm bank. AFLP and ISSR markers were proved to be efficient tools in assessing the genetic variation among populations of L. sinense. The patterns of variation appeared to be consistent for these two marker systems, and they can be used for management of genetic structure, protection of the halobiotic plant, and conservation of germplasm.

Abies ziyuanensis is a highly endangered fir species endemic to South China. Unlike other Abies species that are distributed in areas with cold climates, A. ziyuanensis is restricted to several isolated island-like localities at subtropical mountains. In this study, we used dominant amplified fragment length polymorphism (AFLP) and co-dominant simple sequence repeats (SSR) markers to infer the genetic structure of A. ziyuanensis. Seven populations consisting of 139 individuals were sampled across their whole distribution. A. ziyuanenesis has a relatively low level of genetic variation, with a mean genetic diversity per population (He) of 0.136 (AFLP) and 0.337 (SSR), which is lower than that of other reported endemic species based on the same kind of marker. We observed high population differentiation, with Gst = 0.482 (AFLP) and Fst = 0.250 (SSR), among the seven populations. AMOVA also detected significant differentiation among populations (Phist (AFLP) = 0.550 and Phist (SSR) = 0.289) and among regions (Phict (AFLP) = 0.139 and Phict (SSR) = 0.135) in both marker types. Both ongoing evolutionary forces (e.g., genetic drift resulting from small population size) and historical events (e.g., population contraction and fragmentation during and after the Quaternary glacial cycles) may have contributed to the genetic structure in A. ziyuanensis.