Molecular characterization and genetic relationship among almond cultivars assessed by RAPD and SSR markers

RAPDs and SSRs were used to study the genetic diversity of Iranian almond cultivars and their relationship to important foreign cultivars and three related species. Eight unidentified almond Shahrodi cultivars and three wild almonds (Prunus communis, Prunus orientalis and Prunus scoparia) were also included. Of the primers tested, 42 (out of 80) RAPD and 18 (out of 26) SSR primers were selected for their reproducibility and high polymorphism. A total of 664 polymorphic RAPD bands were detected out of 729 bands. The number of presumed alleles revealed by the SSR analysis ranged from 3 to 10 alleles per locus with a mean value of 6.64 alleles per locus. Both techniques discriminated the genotypes very effectively, but only RAPDs were able to discriminate the cultivars Monagha and Sefied. Results demonstrated an extensive genetic variability within the tested cultivars as well as the value of SSR markers developed in peach for characterization of almond and related species of Prunus. Dice similarity coefficient was calculated for all pair wise comparisons and was used to construct a UPGMA dendrogram. For both markers a high similarity in dendrogram topologies was obtained although some differences were observed. All dendrograms, including that obtained by the combined use of both the marker data, depicted the phenetic relationships among the cultivars and species, depending upon their geographic region and/or pedigree information. Almond cultivars clustered with accession of P. communis showing their close relationship. P. orientalis and P. scoparia were clustered out of the rest of P. dulcis.     

The genetic structure of Mytilus chilensis (Hupé 1854) populations along the Chilean coast based on RAPDs analysis

The Chilean blue mussel Mytilus chilensis is an important commercial species. However, little has been published on the population genetics of this species, despite the need to implement management and conservation policies. Randomly amplified polymorphic DNA‐polymerase chain reaction analysis was used to estimate genetic variation within and between eight natural populations along the whole range of its Chilean natural distribution (ca. 1900 km from Arauco (VIII Region) to Punta Arenas (XII Region)). The values of Nei's unbiased genetic distance, D (0.030–0.107), among populations were small, despite the large geographic separation. A mantel test using 50 000 randomizations showed evidence for a significant correlation (r=0.74, P<0.05) between genetic and geographic (coastal) distance. Punta Arenas population was the most genetically differentiated from the others, although the scale of differentiation was not large (D=0.076–0.107). The levels of gene flow (Nm=1.55) found in this study prevent differentiation among populations by genetic drift. This is the result of the long‐lived planktotrophic larvae of M. chilensis, which provides this species with considerable dispersal ability throughout its range, which is favoured by the ocean currents along the Chilean coast. A restricted larval dispersal towards the north due to the Cape Horn Current derived from the West Wind Drift could be the cause of the higher genetic differentiation of Punta Arenas population from the northern populations. For management purposes of the M. chilensis fishery, the results provide no evidence for discrete stocks, with the possible exception of the Punta Arenas population. The present study provides the baseline data in order to continue further characterization of these mussel populations, considering the great increase in aquaculture of this species.     

Genetic characterization of populations of the ectoparasitic caligid, Lepeophtheirus salmonis (Krøyer 1837) using randomly amplified polymorphic DNA

Genetic variability within salmon louse, Lepeophtheirus salmonis (Caligidae: Copepoda), populations parasitizing farmed and wild Scottish Atlantic salmon (Salmo salar) was investigated using analysis of randomly amplified polymorphic DNA (RAPD) fragments. Seven individual decamer primers were used to analyse samples of salmon lice collected from 15 different locations in Scotland. The polymerase chain reaction products were separated using agarose gel electrophoresis and the resulting band patterns were analysed using a semi‐automated analytical scoring system. Dendrograms were produced using the unweighted pair‐group average (UPGMA) method using Dice similarity values. The summary dendrogram of the analysis of all RAPD bands showed two separate clusters of salmon lice, the larger being sub‐divided into a further two sections. The collections of lice occupying each of these sub‐divisions, however, were a mix of sites, which did not exhibit a structured geographical pattern.     

Use of PCR-based methodologies for the determination of DNA diversity between Saccharum varieties

Summary In this study, two PCR-based methodologies were evaluated for potential use in the determination of DNA diversity between 20 commercial sugarcane hybrids and 6 outgroup varieties of S. spontaneum, S. officinarum and hybrids from early in the genealogy. The first method involved PCR amplification of sugarcane DNA in the presence of random, decamer primers (RAPDs), while the second protocol utilized specific microsatellite and telomere sequences as primers. A total of 41 RAPD primers (356 loci) were screened across the varieties of which 15 (160 loci) were used in the calculation of DNA diversity (expressed% similarity). This varied from 61 to 95%, with most of the commercial varieties showing more than 80% similarity in their DNA. The RAPD data indicated that there had been a gradual decline in DNA diversity (84% reduction) from the early inter-specific crosses to the commercial hybrids, probably as a result of backcrossing and in-breeding strategies used in the previous 5 to 6 generations of sugarcane breeding. The microsatellite and telomere data produced a much greater range in DNA similarity values (25–91%), probably due to the fact that these primers detect highly variable regions of the genome. It is suggested that these specific primers would not be suitable for determination of DNA diversity, but could be used more effectively in the development of a methodology for routine, rapid identification of sugarcane varieties.     

Random amplified polymorphic DNA (RAPDs) markers for genetic analysis in micropropagated plants of Robinia pseudoacacia L.

Four years' old micropropagated plants regenerated by enhanced axillary branching from shoot buds of a single genotype of Robinia pseudoacacia were characterized by RAPDs. Random amplified polymorphic DNA analysis was carried outusing 19 random 10-mer DNA primers and 286 RAPD bands were examined which showed 30% polymorphism. Similarity indices ranged from 0.86 to 0.96 among different plants based on RAPD data. The UPGMA dendrogram was constructed based on similarity indices which showed clustering of different plants into subgroups based on similarity values. Our results suggest that somaclonal DNA sequence variations are present even when organized cultures such as shoot buds were used as explant for micro-propagation. This revised version was published online in August 2006 with corrections to the Cover Date.     

Genetic variability measures among Bromus catharticus Vahl. populations and cultivars with RAPD and AFLP markers

The objectives of this study were to optimize RAPD and AFLP techniques in B. catharticus, and to determine the genetic variability of populations and commercial prairie grass cultivars with the aforementioned molecular markers. Two populations with contrasting morphological characteristics were evaluated from individual and bulked DNA samples using RAPD markers. Both analyses showed a similar information about inter population variability. Each accession was sampled by a single leaf bulk of 10 plants. Accession similarities were established with 276 RAPD and 714 AFLP bands using Jaccard similarity coefficient. The dendrogram of the accessions using RAPD markers showed that they shared high similarity values (>94%). A similar result was obtained with AFLP markers (similarity values >98%), revealing a narrow genetic basis in the analyzed accessions. Consequently, molecular characterization of germplasm should be considered in addition to morphological criteria, to choose the parental genotypes for breeding programs of this forage crop. The AFLP technique was more efficient to detect DNA polymorphism in our experiments and unique fingerprints were detected for all the accessions. RAPD is a simple and non expensive technique, suitable to estimate genetic similarity. This revised version was published online in August 2006 with corrections to the Cover Date.     

Molecular mapping and characterization of traits controlling fiber quality in cotton

Cotton (Gossypium spp) is the world's leading natural fiber crop. Genetic manipulation continues to play a key role in the improvement of fiber quality properties. By use of DNA-based molecular markers and a polymorphic mapping population derived from an inter specific cross between TM-1 (G. hirsutum) and 3-79 (G. barbadense), thirteen quantitative trait loci (QTLs) controlling fiber quality properties were identified in 3-79, an extra long staple (ELS) cotton. Four QTLs influenced bundle fiber strength, three influenced fiber length, and six influenced fiber fineness. These QTLs were located on different chromosomes or linkage groups and collectively explained 30% to 60%of the total phenotypic variance for each fiber quality property in the F₂ population. The effects and modes of action for the individual QTLs were characterized with 3-79 alleles in TM-1 genetic background. The results indicated more recessive than dominant, with much less additive effect in the gene mode. Transgressive segregation was observed for fiber fineness that could be beneficial to improvement of this trait. Molecular markers linked to fiber quality QTLs would be most effective in marker-assisted selection (MAS) of these recessive alleles in cotton breeding programs. This revised version was published online in August 2006 with corrections to the Cover Date.     

Stability of RAPD markers for determining cultivar specific DNA profiles in rye (Secale cereale L.)

Summary Cultivar specific DNA profiles in rye were revealed by polymerase chain reaction (PCR) using randomly amplified polymorphic DNA (RAPD) sequences. Ten base primers were used for the amplification of genomic DNA of rye cultivars by PCR. RAPD analysis was found to be reproducible among samples between PCR runs. When amplification profiles of different rye cultivars were compared using various primers, the overall profiles were cultivar specific. However, not all primers revealed polymorphisms. These primers appear to amplify conserved sequences in all rye cultivars. Intracultivar studies were conducted on two of the cultivars. In the cultivar Imperial, no polymorphisms were observed among ten plants analyzed with five primers. In the cultivar Balboa, polymorphisms were observed among fifty plants with four of the ten primers analyzed. Despite the small amount of intracultivar variability, RAPD analysis has the potential to be a rapid and reliable method of cultivar identification in this outcrossing species.     

Cell lines and plants obtained after protoplast fusions of Rosa+Rosa,Rosa+Prunus and Rosa+Rubus

Summary Protoplasts of three Rosa cultivars were fused with each other, with protoplasts of Prunus `Colt' and with protoplasts of Rubus laciniatus, using polyethylene glycol 4000 as a fusogen. Protoplasts of Prunus were incapable of cell division and those of Rosa and Rubus were disabled by treatments with metabolic inhibitors, either iodoacetate (IOA) or rhodamine 6-G (R6G). Parental protoplasts were then fused in combinations that required complementation for their survival. RAPD analysis of 41 fusion-derived cell lines showed that two lines resulting from fusions of Rosa + Rosa and one from a fusion of Rosa + Prunus, contained some DNA markers from both fusion partners. The others contained markers of only one fusion parent. This showed that after protoplast fusion, the heterokaryons did not develop into cell lines with stable hybrid nuclei. Plants regenerated from cell lines derived from Rosa + Prunus and Rosa + Rubus fusions contained DNA markers of only Rosa and their DNA amounts were no greater than that of the Rosa parent. However, they differed morphologically from the Rosa parent to a remarkable degree, possibly because they inherited undetected genes of Prunus or Rubus, or because they were somaclonal variants of the Rosa parent. Alternative strategies for the production of somatic hybrids are discussed.     

Analysis of genetic diversity in Piper nigrum L. using RAPD markers

RAPD analysis was conducted in 22 cultivars of P. nigrum(black pepper) from South India and one accession each of P. longum and P. colubrinum. Twenty-four primers generated 372 RAPD markers of which 367 were polymorphic. Jaccard's similarity between pairs of accessions ranged between 0.11 and 0.66 with a mean of 0.38. Among P. nigrum cultivars, the similarity ranged between 0.20 and 0.66 and the mean was 0.42. The existence of wide genetic diversity as revealed in the present study is supported by earlier reports of extensive inter- and intrapopulation morphological variability in pepper cultivars from South India. UPGMA dendrogram and PCO plot revealed P. colubrinum to be most distant of the three species. Genetic proximity among P. nigrum cultivars could be related to their phenotypic similarities or geographical distribution. Greater divergence was observed among landraces than among advanced cultivars. Landraces grown in southern parts of coastal India and those grown in more northern parts were grouped in separate clusters of the dendrogram.