DNA extraction and RAPD markers to assess the genetic similarity among Gelidium sesquipedale (Rhodophyta) populations

A simple method developed for genomic DNA isolation from fungus was tested on the red alga, Gelidium sesquipedale (Clem.) Born et Thur., which is commercially exploited for its high sulfated polysaccharide (agar) content. This method is faster, cheaper, and less toxic than conventional phenol/chloroform methods. Random amplified polymorphic DNA (RAPD) amplifications were performed successfully without the necessity of purifying the DNA. RAPD markers were used to investigate the genetic similarity among three natural populations of G. sesquipedale from southern Portugal. Bulked-genomic DNA samples of 15 different individuals were made in each population. These can be conceived of as a sample of the population DNA. Of the 62 primers screened, 41 produced bands and 22 revealed polymorphisms. Genetic similarities among populations were high. Populations that are further away from each other have the lowest similarity coefficients, whereas the intermediate Ingrina population, located on the south coast, showed higher genetic similarity with the Odeceixe population located on the southwest coast, than with the Sao Rafael southern population. This suggests a higher genetic flow between Odeceixe and Ingrina or the result may be a founder effect in the sense that the species has propagated from the east coast to the south coast of Portugal. We conclude that the use of this isolation method with RAPD analysis is appropriate to characterize the genetic variability of this commercial species along its geographical distribution. Large sample sizes can be screened at a relatively low cost. Finding genetic markers for commercial populations of C. sesquipedale may be of industrial interest.

Genetic considerations on the introduction of farmed fish in marine protected areas: The case of study of white seabream restocking in the Gulf of Castellammare (Southern Tyrrhenian Sea)

Human exploitation has drastically reduced the abundance and distribution of several marine fish and invertebrate populations through overfishing and habitat destruction. Restocking can potentially mitigate these impacts and help to reconstitute depleted stocks but genetic repercussions must be considered. In the present study, the degree of genetic similarity between white seabream (Diplodus sargus Linnaeus 1758) individuals reared for restocking purposes and the receiving population in the Gulf of Castellammare fishery reserve (Sicily, Italy) was assessed using microsatellites. We also inferred the spatial pattern of the genetic structure of D. sargus and connectivity along Sicilian coasts. The farmed population showed significant heterozygosity deficiency in 6 loci and an important reduction in the number of alleles, which could indicate an incipient inbreeding. Both the farmed population and the target one for restocking (Castellammare fishery reserve), showed high and significant values of genetic differentiation due to different allele frequencies, number of privative alleles and total number of alleles. These findings indicate a low degree of genetic similarity between both populations, therefore this restocking initiative is not advisable. The genetic connectivity pattern, highly consistent with oceanographic currents, identified two distinct metapopulations of white seabream around Sicily. Thus it is recommended to utilize broods from the same metapopulation for restocking purposes to provide a better genetic match to the wild populations.

Correction of the misclassification of species in the Portuguese collection of Cucurbita pepo L. using DNA markers

In this study, the genetic variability among 130 accessions of the Portuguese germplasm collection of Cucurbita pepo L. maintained at the Banco Portugues de Germoplasma Vegetal was assessed using AFLP (amplified fragment length polymorphism) and RAPD (random amplified polymorphic DNA) techniques for the identification of a genetically diverse core group of accessions for field phenotypic analysis. The surprisingly completely different molecular patterns exhibited by multiple accessions was later confirmed in the distribution of the putative C. pepo plants into two clusters drastically separated at a very low level of genetic similarity (DICE coefficient = 0.37). Additional analyses with RAPD and ISSR (inter single sequence repeat) markers and the introduction of standard genotypes of C. maxima L. and C. moschata L. into the analyses allowed the identification of multiple accessions of the last species wrongly included in the C. pepo collection. This study is a good example of the usefulness of DNA markers in the establishment and management of plant germplasm collections.

Genetic diversity assessment of the almond (Prunus dulcis (Mill.) DA Webb) traditional germplasm of Algarve, Portugal, using molecular markers

In this study, 123 almond (Prunus dulcis (Mill.) D. A. Webb) trees identified among traditional orchards in the Algarve region and 53 trees of the local field collection managed by the regional office of the Portuguese Ministry of Agriculture (DRAALG) were assessed using isozyme, inter- single sequence repeat and simple sequence repeat or microsatellite techniques for the evaluation of genetic diversity and genetic relatedness and identification of new accessions for the field collection. The isozyme analysis allowed the distribution of the 176 plants into 13 different classes of enzyme similarity, while the use of DNA markers increased the distribution of the analysed trees among 140 discriminating DNA patterns. Multiple cases of homonymy and synonymy were identified in the local germplasm. Some traditional varieties, such as Lourencinha, appeared to be relatively homogeneous, while other local denominations, e.g. Galamba, included diverse genotypes. Of the 13 commercial varieties analysed in this study, 11 assembled in one major cluster clearly differentiated from the majority of the local genotypes. These results reinforced the perception that the Algarve traditional germplasm constitutes an important repository of genetic diversity, eventually carrying alleles of high agricultural interest such as the recently identified Phomopsis resistance in the traditional variety Barrinho Grado.