Chromosome-scale reference genome assembly of a diploid potato clone derived from an elite variety.

Potato (Solanum tuberosum L.) is one of the most important crops with a worldwide production of 370 million metric tons. The objectives of this study were (1) to create a high-quality consensus sequence across the two haplotypes of a diploid clone derived from a tetraploid elite variety and assess the sequence divergence from the available potato genome assemblies, as well as among the two haplotypes; (2) to evaluate the new assembly's usefulness for various genomic methods; and (3) to assess the performance of phasing in diploid and tetraploid clones, using linked-read sequencing technology. We used PacBio long reads coupled with 10x Genomics reads and proximity ligation scaffolding to create the dAg1_v1.0 reference genome sequence. With a final assembly size of 812 Mb, where 750 Mb are anchored to 12 chromosomes, our assembly is larger than other available potato reference sequences and high proportions of properly paired reads were observed for clones unrelated by pedigree to dAg1. Comparisons of the new dAg1_v1.0 sequence to other potato genome sequences point out the high divergence between the different potato varieties and illustrate the potential of using dAg1_v1.0 sequence in breeding applications.

Assessing the geographic scale of genetic population management with microsatellites and introns in the clam Ruditapes decussatus.

The clam Ruditapes decussatus is commercially important in southwestern Europe, suffering from population decline and hybridization with exotic Manila clam (R. philippinarum). Previous studies with intronic markers showed a genetic subdivision of the species in three races (Atlantic, West Mediterranean, and Adriatic-Aegean). However, detailed population genetic studies to help management of the main production areas in the southwest of Europe are missing. We have analyzed eight Atlantic and two Mediterranean populations from the Spanish coasts using 14 microsatellites and six intronic markers. Microsatellites confirmed the Atlantic and West Mediterranean races detected with introns and showed that genetic variability was higher in Mediterranean than in Atlantic populations. Both marker types showed that genetic differentiation of Atlantic populations was low and indicated that populations could be managed at the regional level in the case of Cantabrian and Gulf of Cadiz areas, but not in the case of Rias Baixas and the Mediterranean. This study shows the interest of including different types of markers in studies of genetic population structure of marine organisms.

Characterization of nineteen microsatellite markers and development of multiplex PCRs for the wedge clam Donax trunculus (Mollusca: Bivalvia).

The wedge clam Donax trunculus is an Atlantic-Mediterranean warm-temperate species found from Senegal to the northern coast of France, including the Mediterranean and Black Sea. It is commercially exploited in several European countries and constitutes an important fishing resource due to its high economical value. To contribute to its conservation and management, nineteen microsatellite markers were isolated from two enriched genomic libraries. These loci were characterized in 30 clams from a single population from northwest Spain. The number of alleles per locus ranged from 2 to 17 and observed and expected heterozygosity varied from zero to 0.714 and from 0.078 to 0.950, respectively. Linkage disequilibrium was not detected and nine loci were in agreement with Hardy-Weinberg equilibrium. Fifteen polymorphic markers were arranged into three multiplex PCR sets to reduce both time and cost of microsatellite genotyping. This is the first time that polymorphic microsatellite markers have been reported for D. trunculus. These new markers provide a valuable resource for future population genetics studies and management and culture of this species.

Development and multiplex PCR amplification of microsatellite markers in the commercial clam Venerupis rhomboides (Mollusca: Bivalvia).

Venerupis rhomboides is a commercial clam whose production could be enhanced through effective management of natural and hatchery stocks. This study provides the first panel of microsatellite markers for the exploitation of this species according to genetic criteria. A total of 22 polymorphic microsatellite loci were isolated and characterized from two genomic libraries enriched for different motifs. The number of alleles per locus ranged from 2 to 14 in a sample of 20 clams from Spain, and the observed and expected heterozygosity from 0 to 0.95 and 0.05-0.901, respectively. Sixteen loci were in agreement with Hardy-Weinberg equilibrium after sequential Bonferroni correction and linkage disequilibrium between loci pairs was not detected. To reduce the cost of the genotyping process, tri- and pentaplex PCRs, amplifying a total of 13 microsatellites loci were optimized. The microsatellites developed here represent the first nuclear markers described in V. rhomboides and will be useful tools for genetic studies involving assessment of genetic variation and population structure of natural and cultivated populations, assignment testing, construction of genetic linkage maps and dissection of production traits.

Identification, inheritance, and variation of microsatellite markers in the black scallop Mimachlamys varia.

Five polymorphic microsatellite loci were identified in the black scallop Mimachlamys varia after construction of a genomic library enriched for (GT)n. To examine the transmission pattern of microsatellite alleles, several families were created and genotypes scored for three loci. The expected Mendelian ratios were found in 12 of 14 segregations examined. Unexpected segregations may be explained by a genotyping error (allelic dropout), given that when a specific allele was treated as dominant, the phenotypic ratios conformed to Mendelian expectations. The five loci were also examined in two samples from the Spanish coast. The two localities displayed similar mean values for the number of alleles per locus (7.2-8.4), allelic richness (7.2-7.9), and observed (0.389-0.484) and expected heterozygosity (0.545-0.618). Significant Hardy-Weinberg deviations were observed at three loci, with heterozygote deficiency occurring in all cases. Global multilocus θ value and allele frequencies at one locus revealed significant differentiation between the two localities.

Evolutionary dynamics of the 5S rDNA gene family in the mussel Mytilus: mixed effects of birth-and-death and concerted evolution.

In higher eukaryotes, the gene family encoding the 5S ribosomal RNA (5S rRNA) has been used (together with histones) to showcase the archetypal example of a gene family subject to concerted evolution. However, recent studies have revealed conspicuous features challenging the predictions of this model, including heterogeneity of repeat units, the presence of functional 5S gene variants as well as the existence of 5S rDNA divergent pseudogenes lacking traces of homogenization. In the present work, we have broadened the scope in the evolutionary study of ribosomal gene families by studying the 5S rRNA family in mussels, a model organism which stands out among other animals due to the heterogeneity it displays regarding sequence and organization. To this end, 48 previously unknown 5S rDNA units (coding and spacer regions) were sequenced in five mussel species, leading to the characterization of two new types of units (referred to here as small-beta 5S rDNA and gamma-5S rDNA) coexisting in the genome with alpha and beta rDNA units. The intense genetic dynamics of this family is further supported by the first description of an association between gamma-5S rDNA units and tRNA genes. Molecular evolutionary and phylogenetic analyses revealed an extensive lack of homology among spacer sequences belonging to different rDNA types, suggesting the presence of independent evolutionary pathways leading to their differentiation. Overall, our results suggest that the long-term evolution of the 5S rRNA gene family in mussels is most likely mediated by a mixed mechanism involving the generation of genetic diversity through birth-and-death, followed by a process of local homogenization resulting from concerted evolution in order to maintain the genetic identities of the different 5S units, probably after their transposition to independent chromosomal locations.

Intron characterization and their potential as molecular markers for population studies in the scallops Aequipecten opercularis and Mimachlamys varia.

Exon-primed intron-crossing PCR was used on the European commercial scallops Aequipecten opercularis and Mimachlamys varia to characterize introns of four nuclear genes and to identify DNA markers useful for population studies. The primers used yielded the expected product, except those for the lysozyme gene that failed to work in M. varia and amplified a fragment of a proteasome subunit gene (APSM) in A. opercularis. According to the sequences characterized, A. opercularis has at least four calmodulin genes, one of arginine kinase and two of beta-tubulin, and M. varia five, one and one, respectively. Length polymorphism or/and restriction fragment length polymorphism was detected at two loci of A. opercularis (arginine kinase and APSM) and four of M. varia (calmodulin and beta-tubulin), distinguishing in each case two or three alleles. The polymorphic loci were not closely linked. The population survey included four localities from Spain and one from Northern Ireland for A. opercularis and two Spanish localities for M. varia. Observed heterozygosity (H(o)) per locus was 0.276 and 0.296 in A. opercularis. The Northern Ireland sample had the lowest H(o) value (0.200) and the Mediterranean Spanish sample the highest (0.350). In M. varia, H(o) per locus ranged from 0.172 to 0.391 and the two localities showed similar H(o) values (0.255 and 0.293). All population-locus combinations were in agreement with Hardy-Weinberg equilibrium, except two loci of M. varia that showed a strong heterozygote deficit in the two localities examined. Evidence for genetic differentiation among samples was not found.

Sequence characterization and phylogenetic analysis of the 5S ribosomal DNA in some scallops (Bivalvia: Pectinidae).

This study was designed to characterize the 5S ribosomal DNA repeat unit and to evaluate its phylogenetic informativeness in Mimachlamys varia, Hinnites distortus, Aequipecten opercularis and Pecten maximus, scallops of the bivalve family Pectinidae. The repeat unit was PCR amplified and several products cloned and sequenced. The deduced coding region covered 120 bp, showed 52-53% GC content and an identifiable internal promoter. The spacer region was 313-345 bp in length with 30-40% GC and the ends contained elements showing similarity with upstream and downstream sequences involved in the transcription. The sequences of P. maximus were identical and those of M. varia and H. distortus differed only at 2-3 positions. However, the sequences of A. opercularis showed a percentage of differences of 0.69-9.15%, distinguishing two types of units differing in sequence and length of the spacer region. All scallops displayed an identical gene sequence, except M. varia that differed by one nucleotide substitution, but a highly variable spacer. In the phylogenetic trees the sequences grouped by species with two well supported clades, one containing the sequences of M. varia and H. distortus and the other those of P. maximus and A. opercularis, the latter grouped according to the unit type. The topology obtained was in accordance with scallop evolutionary relationships inferred from previous phylogenetic studies.

Karyotype and chromosomal location of 18S-28S and 5S ribosomal DNA in the scallops Pecten maximus and Mimachlamys varia (Bivalvia: Pectinidae).

This work describes the karyotype and chromosomal location of the ribosomal DNA (rDNA) of Pecten maximus and Mimachlamys varia, two commercial scallop species from Europe. According to the chromosome centromeric index values found, the karyotype of P. maximus is composed of 1 metacentric, 2 metacentric-submetacentric, 1 telocentric-subtelocentric and 15 telocentric pairs, and that of M. varia of 4 metacentric, 2 subtelocentric-submetacentric, 9 subtelocentric, 3 subtelocentric-telocentric and 1 telocentric-subtelocentric pairs. In P. maximus, 18S-28S rDNA was located by FISH on a metacentric-submetacentric pair, and in M. varia on a subtelocentric-submetacentric pair using both silver staining and FISH. PCR amplification of the 5S rDNA unit yielded a single product of about 460 bp (P. maximus) and 450 bp (M. varia), that used as probe revealed a 5S rDNA site on a telocentric pair in P. maximus and a subtelocentric pair in M. varia. Two-color FISH or sequential silver staining of 5S rDNA-FISH-metaphases corroborated that the two gene families are located on different chromosomes in both species. A comparative analysis of the data allowed the inference of karyotypic relationships within scallops.

Cerastoderma glaucum 5S ribosomal DNA: characterization of the repeat unit, divergence with respect to Cerastoderma edule, and PCR-RFLPs for the identification of both cockles.

The 5S rDNA repeat unit of the cockle Cerastoderma glaucum from the Mediterranean and Baltic coasts was PCR amplified and sequenced. The length of the units was 539-568 bp, of which 120 bp were assigned to the 5S rRNA gene and 419-448 bp to the spacer region, and the G/C content was 46%-49%, 54%, and 44%-47%, respectively. Two types of units (A and B), differing in the spacer, were distinguished based on the percentage of differences and clustering in phylogenetic trees. A PCR assay with specific primers for each unit type indicated that the occurrence of both units is not restricted to the sequenced individuals. The 5S rDNA units of C. glaucum were compared with new and previously reported sequences of Cerastoderma edule. The degree of variation observed in C. edule was lower than that in C. glaucum and evidence for the existence of units A and B in C. edule was not found. The two cockles have the same coding region but displayed numerous fixed differences in the spacer region and group separately in the phylogenetic trees. Digestion of the 5S rDNA PCR product with the restriction enzymes HaeIII and EcoRV revealed two RFLPs useful for cockle identification.

Sequence analysis of the ribosomal DNA internal transcribed spacer region in some scallop species (Mollusca: Bivalvia: Pectinidae).

The internal transcribed spacer (ITS) region of the ribosomal DNA from the European scallops Aequipecten opercularis, Mimachlamys varia, Hinnites distortus, and Pecten maximus was PCR amplified and sequenced. For each species, three or five clones were examined. The size ranged between 636 and 713 bp (ITS1, 209-276 bp; 5.8S rRNA gene, 157 bp; ITS2, 270-294 bp) and GC content ranged between 47 and 50% (ITS1, 43-49%; 5.8S rRNA gene, 56-57%; ITS2, 44-49%). Variation within repeats was minimal; only clones from M. varia and P. maximus displayed a few variable sites in ITS2. Among scallops, including Chlamys farreri whose ITS sequence appears in databases, significant variation was observed in both ITS1 and ITS2. Phylogenetic analysis using ITS1, ITS2, or both spacer sequences always yielded trees with similar topology. Aequipecten opercularis and P. maximus grouped in one clade and the other three scallops (C. farreri, M. varia, and H. distortus) in another, where M. varia and H. distortus are the more closely related species. These results provide new insights into the evolutionary relationships of scallop species and corroborate the close evolutionary relationship between the tribes Aequipectinini and Pectinini previously deduced from 18S rDNA sequences.