Genetic variability following selection for scrapie resistance in six autochthonous sheep breeds in the province of Bolzano (northern Italy)

Scrapie is an ovine transmissible spongiform encephalopathy, and its susceptibility is associated with polymorphisms in the prion protein gene (PRNP). Genetic selection is currently the most effective mean for eradication of the susceptible VRQ allele in favour of resistant ARR allele. Maintenance of genetic diversity should be one of the major objectives in breeding programmes, especially in endangered breeds, and genetic information are an excellent alternative to pedigree data where these information are missing. The aim of our study was to determine changes of genetic variability in six native sheep breeds from autonomous province of Bolzano, northern Italy, following simulation of scrapie selection scenarios. A total of 684 rams were investigated for PRNP polymorphisms and for 10 microsatellite loci to estimate genetic variability. Across all loci, a total of 163 alleles were detected with a mean of 10.4 alleles per locus. Average observed (Ho) and unbiased expected (uHe) heterozygosity overall loci were 0.74 and 0.78, respectively, showing a statistically significant deviation from Hardy–Weinberg equilibrium (HWE) in all breeds. This heterozygosity deficit was confirmed by a positive fixation index (Fis), determining a moderate inbreeding in each breed. Simulating a soft selection, where only rams having at least a VRQ allele should be excluded from reproduction, Ho, uHe and Fis values remained almost unchanged, indicating that genetic variability should not be affected by the removal of these individuals. With a mild selection scenario, considering only rams with at least one ARR allele, we observed a decrease in the mean alleles per breed (8.9) and the maintenance of heterozygosity deficiency, except for two breeds, where it was any longer significant. These results showed that selection strategies allowing use of heterozygous as well homozygous ARR rams might be the right compromise to improve resistance to scrapie and to do not dramatically affect genetic variability of these breeds.     

Development of EST‐SSR markers for diversity and breeding studies in opium poppy

All publicly available opium poppy expressed sequence tag (EST) sequences, totalling 20 885, were assembled into unigenes and examined for simple sequence repeats (SSRs). Nearly 19% of the 14 957 unigenes contained SSRs with 4% harbouring more than one SSR. Average density of the SSRs was 1 SSR per 3.6 kb of non‐redundant EST sequence. Trinucleotide SSRs were most frequently identified (39%), and many of the most prevalent motifs were AT‐rich. Flanking primers were designed for 86% of the SSRs and 67 primer pairs were tested on 37 opium poppy accessions and seven related species. All markers were transferable to the related species. Polymorphism information content (PIC) values for the markers were intermediate for comparisons within opium poppy (average of 0.27) and slightly higher for comparisons across species (average of 0.29). The markers were found to be useful for diversity analysis as they successfully distinguished among Turkish opium poppy accessions and land races.     

A quick methodology to identify sexual seedlings in citrus breeding programs using SSR markers

In citrus breeding and genetics, it is very important to distinguish between zygotic and nucellar seedlings in order to eliminate unwanted genotypes. Usually, isozyme marker shave been employed to determine the genetic origin of young plants. In this work we propose the use of SSR markers as an alternative methodology and compare them with isozymes in this kind of screenings. Two different populations were analysed: one derives from an interspecific cross and the other from selfing. We conclude that, in most cases, microsatellites are more efficient than isozymic markers to identify the sexual origin of citrus seedlings, given their higher level of polymorphism and the scarce number of polymorphic isozymes in some populations. We describe a quick and efficient methodology for SSR analysis, including a fast DNA extraction in microcentrifuge tubes, and visualization through silver staining, which eliminates the need for a labelling step. This revised version was published online in July 2006 with corrections to the Cover Date.     

Heritability of juvenile growth traits in red drum (Sciaenops ocellatus L.)

Heritability of juvenile growth rate was estimated for red drum (Sciaenops ocellatus), an economically important sciaenid fish in the southern USA. Thirty‐eight families were generated via ‘natural’ spawning of multiple sets of five breeders (three dams × two sires) in individual brood tanks. Offspring were individually tagged with Passive Integrated Transponder (PIT) tags and mixed for grow‐out in replicate ‘common‐garden’ tanks. Juvenile growth was followed from 166.4±18.6 to 254.0±27.0 mm (total length). Offspring were assigned a posteriori to individual brood fish (dam and sire) based on genotypes at nuclear‐encoded microsatellites. Heritability (h²) of a thermal growth coefficient was estimated using an animal‐additive model and a restricted maximum‐likelihood algorithm. Estimates of h² were 0.33±0.08 and 0.31±0.08 for thermal growth coefficient based on length and weight respectively. These results indicate a significant genetic component in juvenile growth rate in red drum. Estimates of h² for condition coefficient (K) at various measurement dates averaged 0.38, suggesting a genetic component to shape in juvenile red drum.     

Microsatellite variability in natural populations of the blackspot seabream Pagellus bogaraveo (Brünnick, 1768): a database to access parentage assignment in aquaculture

Eight microsatellite loci previously reported were assessed for their utility in parentage assignment in 96 individuals belonging to natural populations of the blackspot seabream Pagellus bogaraveo (Brünnick, 1768) from the Mediterranean and Northeast Atlantic regions. At the mtDNA level, no differentiation was found between these two regions but based on microsatellite data an overall discrete genetic differentiation is perceivable between the two regions separated by the Strait of Gibraltar. The number of alleles per locus ranged from 8 to 30. A database constructed with allele frequency data from six populations was used in a simulation parentage assignment test using the software cervus . The test showed that the number of markers used is enough to perform parentage assignments with real data. The polymorphic information content for each locus was very high (mean value of 0.849), with a total exclusionary power of 0.9995. In summary, seven of the eight microsatellites analysed proved to be sufficient and powerful tools for parentage assignment in hatcheries and the allele frequency data given here can be used to perform pedigree analysis against which real data may be tested.     

Factors influencing male mating success in bur oak,Quercus macrocarpa

Pollen donors of acorns and saplings in a stand of bur oak were identified by paternity exclusion using microsatellite genotype analysis. Here we examine the influence of several factors likely to affect reproductive success of males with wind-dispersed pollen, including distance of pollen donor from maternal tree, genetic relatedness of pollen donor to maternal tree, direction of pollen donor relative to maternal tree, and size of pollen donor (crown volume and trunk diameter). Surprisingly, none of these factors were strongly correlated with fertilization success, although weak but significant correlations with crown volume and distance were found. The slight influence of distance is actually overestimated here, because our analysis necessarily excluded a large proportion of pollinations that were effected by trees outside the stand. The pollen donors outside the stand were not identified, but were more than 150 meters away. Pollination patterns in this stand of bur oak are quite complicated. Seed parents were fertilized by multiple pollen donors within the stand from all directions and as far as 200 m away. These results caution against the use of models of pollination that are based on simple distance or directional parameters because they will not adequately predict gene flow or male reproductive success. Our results further emphasize the importance of genetically identifying successful pollen donors rather than relying on patterns of physical movement of pollen.     

The potential of microsatellites for high throughput genetic diversity assessment in wheat and barley

Microsatellite (SSR) profiles from 65 wheats and 135 barleys have been generated, involving 14 and 22 loci, respectively. The wheat and barley varieties were chosen to represent the bulk of the area sown to these crops in the UK over the past 70 years. The profiling identified genotypic mixtures in some seed samples. Null alleles were common in wheat, but rare in barley. We describe attempts to increase the efficiency of data acquisition. High resolution agarose gel electrophoresis was unable to satisfactorily resolve 1–2 repeat unit differences in the common size range for SSR loci, and was therefore unsuitable for mass screening of allelic variants. Multiplex PCR was very dependent on the choice of primer combinations and seldom produced amplifications as consistently as when primer pairs were used individually. Background (non-specific) amplification was common to many primer pairs, and this hindered the use of both multiplex PCR and multiple sample loading. Sequential sample loading was the most effective strategy, although this was the least time-efficient of the measures used.     

Low level of polymorphism detected by SSR probes in bread wheat

In-gel hybridization patterns were studied in a set of nine diverse bread wheat ( Triticum aestivum L. em. Thell) genotypes using 23 simple sequence repeat (SSR) probes in combination with 14 different restriction enzymes. Multilocus fingerprints due to SSR probes, shown earlier to be characteristic of a majority of plant genomes, were not obtained and only a very low level of polymorphism was detected when using as many as 142 probe-enzyme combinations. The hybridization of a prominent solitary high molecular weight fragment (> 23 kb) with a number of SSR probes suggested the presence of these SSRs (microsatellites) within the long stretches of repeated DNA sequences. This indicates that the genome of bread wheat differs from that of other plants in the organization and distribution of SSRs and that SSR probes detect very little polymorphism.     

Gene pool and connectivity patterns of Pinna nobilis in the Balearic Islands (Spain,Western Mediterranean Sea): Implications for its conservation through restocking

1. Pinna nobilis is an endemic bivalve of the Mediterranean Sea, and a vulnerable species registered as endangered and protected under the European Council Directive 92/43/EEC and Barcelona Convention.
2. In early autumn 2016, a mass mortality event impacted P. nobilis populations in the south‐western Mediterranean Sea, including the Balearic Islands. At the time of this study, P. nobilis still maintained high population densities along the Balearic coasts (Western Mediterranean).
3. This study evaluated the connectivity of P. nobilis post‐larvae and adults in seagrass habitats around the Balearic Islands and identified its source and sink populations. These objectives were reached through a multidisciplinary approach including population genetics (10 microsatellites) and hydrodynamic modelling.
4. High genetic diversity was found and significant genetic differentiation (inferred by fixation index F_ST) was detected between post‐larvae samples, but not between adult populations. Significant genic and genotypic differentiation was recorded for adults and post‐larvae.
5. This pattern was confirmed by correspondence analysis using allele frequencies. The genetic connectivity pattern was consistent with marine currents and dispersal models.
6. This work not only improves knowledge of the P. nobilis gene pool in south‐west Mediterranean populations and their connectivity patterns, but is also crucial to help evaluate the possibility of recovery from source populations and the possibility of restocking programmes, as well as provide a solid base to establish effective marine reserve networks.