Analysis of genetic diversity of Tunisian pistachio (Pistacia vera L.) using sequence-related amplified polymorphism (SRAP) markers.

Sequence-related amplified polymorphism (SRAP) markers preferentially amplify open reading frames and were used to study the genetic diversity of Tunisian pistachio. In the present study, 43 Pistacia vera accessions were screened using seven SRAP primer pairs. A total of 78 markers was revealed (95.12%) with an average polymorphic information content of 0.850. The results suggest that there is strong genetic differentiation, which characterizes the local resources (GST = 0.307). High gene flow (Nm = 1.127) among groups was explained by the exchange of plant material among regions. Analysis of molecular variance revealed significant differences within groups and showed that 73.88% of the total genetic diversity occurred within groups, whereas the remaining 26.12% occurred among groups. Bayesian clustering and principal component analysis identified three pools, El Guettar, Pollenizers, and the rest of the pistachios belonging to the Gabès, Kasserine, and Sfax localities. Bayesian analysis revealed that El Guettar and male genotypes were assigned with more than 80% probability. The BayeScan method proposed that locus 59 (F13-R9) could be used in the development of sex-linked SCAR markers from SRAP since it is a commonly detected locus in comparisons involving the Pollenizers group. This is the first application of SRAP markers for the assessment of genetic diversity in Tunisian germplasm of P. vera. Such information will be useful to define conservation strategies and improvement programs for this species.

Assessment of genetic diversity of Tunisian orange, Citrus sinensis (L.) Osbeck using microsatellite (SSR) markers.

Citrus are one of the most cultivated crops in the world. Economically, they are very important fruit trees in Tunisia. Little is known about the genetic diversity of the Tunisian Citrus germplasm. Exploring this diversity is a prerequisite for the identification and characterization of the local germplasm to circumvent and controlling genetic erosion caused by biotic and abiotic stress to aid its conservation and use. In the present study, we explored the genetic diversity of 20 Tunisian orange cultivars [Citrus sinensis (L.) Osbeck] and established their relationships by using seven simple sequence repeat (SSR) loci. In total, 37 alleles and 44 genotypes were scored. The sizes of alleles ranged from 90 to 280 bp. The number of alleles per locus was from 4 to 7, with an average of 5.28. Polymorphic information content value changed from 0.599 to 0.769 with an average of 0.675. Analysis of the genotypes revealed a heterozygote deficiency across all the genotypes. The observed heterozygosity varied from 0 to 1 (average of 0.671). Cluster analysis showed that three groups could be distinguished and the polymorphism occurred independently of the geographical origin of the studied orange cultivars. The detected SSR genotypes allowed the establishment of an identification key with a discriminating power of 100%. Multivariate analysis and the neighbor-joining phylogenetic tree indicated a narrow genetic base for the orange cultivars. The usefulness of SSR markers for orange fingerprinting and evaluation of the genetic diversity in the Tunisian germplasm are discussed in this paper.

tRNALeu intron (UAA) of Ficus carica L.: genetic diversity and evolutionary patterns.

Cytoplasmic chloroplast DNA was explored to establish genetic relationships among Ficus carica cultivars and elucidate the molecular evolution of the species. The results suggest the occurrence of haplotype and nucleotide diversity. Conserved group I intron sequence motifs were detected and showed a common secondary structure, despite the presence of some mutations on their sequences. The neighbor-joining dendrogram showed a continuous diversity that characterizes local resources. The maximum parsimony tree, with an RI index of 0.507, indicated minimal homoplasy within the data set. Furthermore, our results demonstrate that the trnL intron is the seat of numerous substitutions. Herein, new insight on the mechanism involved in the evolution of the trnL intron in the fig is presented. From the study, it appears that there is an explicit rejection of the null hypothesis in F. carica. A scenario of positive selection and recent expansion of F. carica genotypes across Tunisia seems to be retained.

Cytoplasmic polymorphism and evolutionary history of plum cultivars: Insights from chloroplast DNA sequence variation of trnL-trnF spacer and aggregated trnL intron & trnL-trnF spacer.

We screened for polymorphisms of the non-coding region of plastid DNA in plum trees. Sequencing data from the trnL-trnF chloroplast region were used to reveal a pattern of diversity, establish phylogenetic relationships, and test the selection pressure or evolutionary demography scenario for plastome DNA. The size of the non-coding regions varied from 398 to 563 and 865 to 1084 bases pairs for the trnL-trnF spacer and combined sequences, respectively. The average GC contents were 33.8 and 34.4% in the spacer and pooled sequences, respectively. Genetic distances calculated within the plums were 0.077 and 0.254, on average, for the trnL spacer and combined sequences, respectively. The neighbor-joining trees showed clustering relationships among cultivars that were independent of their geographic origins and designations. The neutrality tests and site-frequency spectra indicated that spacer and pooled sequences fit the neutral theory model at equilibrium between mutation and genetic drift and reject the hypothesis of a recent demographic expansion. The mismatch distribution shows variation patterns, thus providing evidence of an important genetic diversity explained by an excess of intermediate variants that occurred in the sequences analyzed. Further implications of the findings with regard to plum germplasm management and its utilization in breeding programs are also discussed.

Genetic diversity and differentiation in Prunus species (Rosaceae) using chloroplast and mitochondrial DNA CAPS markers.

Chloroplast (cpDNA) and mitochondrial DNA (mtDNA) were analyzed to establish genetic relationships among Tunisian plum cultivars using the polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) technique. Two mtDNA regions (nad 1 b/c and nad 4 1/2) and a cpDNA region (trnL-trnF) were amplified and digested using restriction enzymes. Seventy and six polymorphic sites were revealed in cpDNA and mtDNA, respectively. As a consequence, cpDNA appears to be more polymorphic than mtDNA. The unweighted pair group method with arithmetic mean (UPGMA) dendrogram showed that accessions were distributed independently of their geographical origin, and introduced and local cultivars appear to be closely related. Both UPGMA and principal component analysis grouped Tunisian plum accessions into similar clusters. The analysis of the pooled sequences allowed the detection of 17 chlorotypes and 12 mitotypes. The unique haplotypes detected for cultivars are valuable for management and preservation of the plum local resources. From this study, PCR-RFLP analysis appears to be a useful approach to detect and identify cytoplasmic variation in plum trees. Our results also provide useful information for the management of genetic resources and to establish a program to improve the genetic resources available for plums.

Assessment of genetic diversity and relationships among wild and cultivated Tunisian plums (Prunus spp) using random amplified microsatellite polymorphism markers.

The usefulness of random amplified microsatellite polymorphism markers to study the genetic diversity and relationships among cultivars belonging to Prunus salicina and P. domestica and their wild relatives (P. insititia and P. spinosa) was investigated. A total of 226 of 234 bands were polymorphic (96.58%). The 226 random amplified microsatellite polymorphism markers were screened using 15 random amplified polymorphic DNA and inter-simple sequence repeat primers combinations for 54 Tunisian plum accessions. The percentage of polymorphic bands (96.58%), the resolving power of primers values (135.70), and the polymorphic information content demonstrated the efficiency of the primers used in this study. The genetic distances between accessions ranged from 0.18 to 0.79 with a mean of 0.24, suggesting a high level of genetic diversity at the intra- and interspecific levels. The unweighted pair group with arithmetic mean dendrogram and principal component analysis discriminated cultivars efficiently and illustrated relationships and divergence between spontaneous, locally cultivated, and introduced plum types. These procedures showed continuous variation that occurs independently of the status of the species and geographical origin of the plums. In this study, random amplified microsatellite polymorphism was found to be as a reliable molecular marker for fingerprinting and for examining the diversity study of the plum and its relatives.

Genetic diversity and phylogenetic relationship among Tunisian cactus species (Opuntia) as revealed by random amplified microsatellite polymorphism markers.

Opuntia ficus indica is one of the most economically important species in the Cactaceae family. Increased interest in this crop stems from its potential contribution to agricultural diversification, application in the exploitation of marginal lands, and utility as additional income sources for farmers. In Tunisia, O. ficus indica has been affected by drastic genetic erosion resulting from biotic and abiotic stresses. Thus, it is imperative to identify and preserve this germplasm. In this study, we focused on the use of random amplified microsatellite polymorphisms to assess genetic diversity among 25 representatives of Tunisian Opuntia species maintained in the collection of the National Institute of Agronomic Research of Tunisia. Seventy-two DNA markers were screened to discriminate accessions using 16 successful primer combinations. The high percentage of polymorphic band (100%), the resolving power value (5.68), the polymorphic information content (0.94), and the marker index (7.2) demonstrated the efficiency of the primers tested. Therefore, appropriate cluster analysis used in this study illustrated a divergence among the cultivars studied and exhibited continuous variation that occurred independently of geographic origin. O. ficus indica accessions did not cluster separately from the other cactus pear species, indicating that their current taxonomical classifications are not well aligned with their genetic variability or locality of origin.

DNA variation and polymorphism in Tunisian plum species (Prunus spp): contribution of flow cytometry and molecular markers.

Plums (Prunus spp) are among the most important stone fruit crops in the world. European (Prunus domestica) and Japanese (Prunus salicina) plums are characterized by different levels of ploidy. Because genetic variability is the prerequisite for any plant-breeding program, we aimed to establish the taxonomic status of Tunisian plums and study their genetic variability. The nuclear DNA content of 45 wild and cultivated Tunisian plums was determined by flow cytometry. Two arbitrary primers (AD10, AD17) were used to elaborate SCAR markers useful to identify plum species. Three wild trees, Zenou 1, Zenou 6, and Zenou 3, which had 2C nuclear DNA contents of 1.99, 2.05, and 2.13 pg, were shown to be hexaploid (2n = 6x = 48), whereas the others were diploid (2n = 2x = 16). These results suggest that the three hexaploid wild plums belong to Prunus insititia, and the others belong to Prunus salicina. No SCAR markers were revealed using the AD10 and AD17 RAPD primers in relation to the ploidy of plums. We note also that AD17 primer appears to be the most informative concerning the genetic diversity. Morphological and pomological traits revealed similarity between introduced and Tunisian plum cultivars. Despite the significant morphological differences found, all the cultivars studied belong to P. salicina. The information obtained in this analysis provided on local plum genetic resources will be helpful to establish a core collection, to evaluate genetic diversity, and to initiate an improvement and selection program.