Self-(in)compatibility in Tunisian apple accessions [Malus domestica. Borkh]: S-genotypes identification and pollen tube growth analysis.

MAIN CONCLUSION: Self-incompatibility studies have revealed a potential use of Tunisian apple resources for crop improvement and modern breeding programs and a likely correlation between the pollen tube growth and flowering period. Apples [Malus domestica. Borkh] exhibit an S-RNase-based gametophytic self-incompatibility (GSI) system. Four primer combinations were used to S-genotype eighteen Tunisian local apple accessions and twelve introduced accessions that served as references. Within the Tunisian local accessions, S2, S3, S7, and S28 S-alleles were the most frequent and were assigned to 14 S-genotypes; among them, S7S28, S3S7, S2S5, and S2S3 were the most abundant. PCA plot showed that population structuring was affected by the S-alleles frequencies and revealed a modern origin of the Tunisian varieties rather than being ancient ones. Nonetheless, the results obtained with 17 SSR markers showed a separate grouping of local Tunisian accessions that calls into question the hypothesis discussed. Pollination experiments showed that the pollen started to germinate within 24 h of pollination but 48 h after pollination in the "El Fessi" accession. The first pollen tubes arrived in the styles within 36 h of pollination in two early flowering accessions known as "Arbi" and "Bokri", and after 72 h of pollination in late flowering "El Fessi" and 48 h after pollination in remaining accessions. The first pollen tube arrests were observed in accessions "Arbi" and "Bokri" within 84 h of pollination, within 108 h of pollination in "El Fessi" and within 108 h of pollination in remaining accessions. In the apple accession called "Boutabgaya," the pollen tubes reached the base of the style within 120 h of pollination without being aborted. Nevertheless, the self-compatible nature of "Boutabgaya" needs more studies to be confirmed. However, our results revealed the malfunction of the female component of the GSI in this accession. To conclude, this work paved the path for further studies to enhance the insight (i) into the relation between the flowering period and the pollen tube growth, (ii) self-compatible nature of "Boutabgaya", and (iii) the origin of the Tunisian apple.

Vitamin D receptor gene BsmI (rs1544410) polymorphism: role in multiple sclerosis and genotype-phenotype correlations.

BACKGROUND: Multiple sclerosis (MS) has a complex pathophysiology which depends on many endogenous and exogenous factors. Vitamin D involvement has been largely studied in MS. The large distribution of the vitamin D receptor (VDR) in different immune cells is suggestive of an immunomodulatory role. The VDR gene polymorphisms have been proposed as potential risk factors for MS development or evolution with non-conclusive results. METHODS AND RESULTS: We conducted a cross-sectional study including patients ≥ 18 years, with a diagnosis of relapsing remitting MS according to the McDonald Criteria and having a minimum follow-up period of one year after starting a disease modifying therapy. Two study groups were compared based on the Multiple Sclerosis Severity Scale or MSSS: "a slow progressor" group for an MSSS ≤ 5, and a "fast progressor" group for an MSSS > 5. The rs1544410 VDR gene polymorphism was studied for all patients. Eighty patients were included. The fast progressor groups had a higher EDSS at onset, a higher total number of relapses, more frequent and shorter time to secondary progression. The progression profile was not statistically different between genotypes and alleles of the VDR gene polymorphism rs1544410. The CC genotype and wild-type allele exhibited a more aggressive disease phenotype with a higher number of relapses the first year, shorter time to secondary progression and cerebral atrophy on assessment. CONCLUSIONS: Our results suggest potential genotype-phenotype correlations for the rs1544410 VDR gene polymorphism in the disease course of MS. Future research on a larger scale is needed to confirm these findings.

Molecular and Evolutionary Characterization of Pollen S Determinant (SFB Alleles) in Four Diploid and Hexaploid Plum Species (Prunus spp.).

In more than 60 families of angiosperms, the self- and cross-fertilization is avoided through a complex widespread genetic system called self-incompatibility (SI). One of the major puzzling issues concerning the SI is the evolution of this system in species with complex polyploid genomes. Among plums, one of the first fruits species to attract human interest, polyploid species represent enormous genetic potential, which can be exploited in breeding programs. However, molecular studies in these species are very scarce due to the complexity of their genome. In order to study the SFB gene [the male component of gametophytic self-incompatibility system (GSI)] in plum species, 36 plum accessions belonging to diploid and hexaploid species were used. A total of 19 different alleles were identified; 1 of them was revealed after analyzing sequences. Peptide sequence analysis allowed identifying the five domains features of the SFB gene. Polymorphism analysis showed a subtle difference between domesticated and open pollinated Tunisian accessions and suggested a probable influence of the ploidy level. Divergence analysis between studied sequences showed that a new specificity may appear after 5.3% of divergence at synonymous sites between pairs of sequences in Prunus insititia, 6% in Prunus cerasifera, 8% and 9% in Prunus domestica and Prunus salicina respectively. Furthermore, sites under positive selection, the ones more likely to be responsible for specificity determination, were identified. A positive and significant Pearson correlation was found between the divergence between sequences, divergence time, fixed substitutions (MK test), and PSS number. These results supported the model assuming that functionally distinct proteins have arisen not as a result of chance fixation of neutral variants, but rather as a result of positive Darwinian selection. Further, the role that plays recombination can not be ruled out, since a rate of 0.08 recombination event per polymorphic sites was identified.

Combination of Simple Sequence Repeat, S-Locus Polymorphism and Phenotypic Data for Identification of Tunisian Plum Species (Prunus spp.).

Plums (Prunus spp.) are among the first fruit tree species that attracted human interest. Artificial crosses between wild and domesticated species of plums are still paving the way for creation of new phenotypic variability. In Tunisia, despite a considerable varietal richness of plum as well as a high economic value, the plum sector is experiencing a significant regression. The main reason of this regression is the absence of a national program of plum conservation. Hence, this work was aimed to phenotypically and genetically characterize 23 Tunisian plum accessions to preserve this patrimony. Closely related Prunus species from the same subgenus may be differing at two characteristics: ploidy level and phenotypic traits. In this study, single sequence repeat (SSR) markers allowed distinguishing between eighteen diploid accessions and five polyploid accessions, but SSR data alone precluded unambiguous ploidy estimation due to homozygosity. In contrast, S-allele markers were useful to identify the ploidy level between polyploid species, but they did not distinguish species with the same ploidy level. Seven out of 12 phenotypic traits were shown to be discriminant traits for plum species identification. Molecular and phenotypic traits were significantly correlated and revealed a powerful tool to draw taxonomic and genotypic keys. The results obtained in this work are of great importance for local Tunisian plum germplasm management.

Conserved DNA-derived polymorphism, new markers for genetic diversity analysis of Tunisian Pistacia vera L.

Pistachio trees (Pistacia vera L.) have been cultivated in Tunisia for decades and the plantation was extended mostly in the center of the country contributing to the economic growth of marginalized areas. Herein we used conserved DNA derived polymorphism (CDDP) technique, which target specifically conserved sequences of plant functional genes, to assess the genetic diversity and construct genetic relationships among 65 Tunisian pistachio trees. A set of nine primers were used and 157 CDDP markers were revealed with an average of 17.44 showing a high degree of polymorphism (99.37%). The average of polymorphism information content of CDDP markers was of 0.86, which indicates the efficiency of CDDP primers in the estimation of genetic diversity between pistachios. UPGMA dendrogram and the principal component analysis showed four clusters of analyzed pistachios trees. Our results showed that the genetic structure depends on: (1) the gene exchanges between groups, (2) the geographical origin and (3) the sex of the tree. The same result was revealed by the Bayesian analysis implemented in STRUCTURE at K = 4, in which the pistachio genotypes of El Guettar, Kasserine and Sfax were assigned with more than 80% of probability. Our results prove polymorphism and efficiency of CDDP markers in the characterization and genetic diversity analysis of P. vera L. genotypes to define conservation strategy.

Towards a molecular taxonomic key of the Aurantioideae subfamily using chloroplastic SNP diagnostic markers of the main clades genotyped by competitive allele-specific PCR.

BACKGROUND: Chloroplast DNA is a primary source of molecular variations for phylogenetic analysis of photosynthetic eukaryotes. However, the sequencing and analysis of multiple chloroplastic regions is difficult to apply to large collections or large samples of natural populations. The objective of our work was to demonstrate that a molecular taxonomic key based on easy, scalable and low-cost genotyping method should be developed from a set of Single Nucleotide Polymorphisms (SNPs) diagnostic of well-established clades. It was applied to the Aurantioideae subfamily, the largest group of the Rutaceae family that includes the cultivated citrus species. RESULTS: The publicly available nucleotide sequences of eight plastid genomic regions were compared for 79 accessions of the Aurantioideae subfamily to search for SNPs revealing taxonomic differentiation at the inter-tribe, inter-subtribe, inter-genus and interspecific levels. Diagnostic SNPs (DSNPs) were found for 46 of the 54 clade levels analysed. Forty DSNPs were selected to develop KASPar markers and their taxonomic value was tested by genotyping 108 accessions of the Aurantioideae subfamily. Twenty-seven markers diagnostic of 24 clades were validated and they displayed a very high rate of transferability in the Aurantioideae subfamily (only 1.2 % of missing data on average). The UPGMA from the validated markers produced a cladistic organisation that was highly coherent with the previous phylogenetic analysis based on the sequence data of the eight plasmid regions. In particular, the monophyletic origin of the "true citrus" genera plus Oxanthera was validated. However, some clarification remains necessary regarding the organisation of the other wild species of the Citreae tribe. CONCLUSIONS: We validated the concept that with well-established clades, DSNPs can be selected and efficiently transformed into competitive allele-specific PCR markers (KASPar method) allowing cost-effective highly efficient cladistic analysis in large collections at subfamily level. The robustness of this genotyping method is an additional decisive advantage for network collaborative research. The availability of WGS data for the main "true citrus" species should soon make it possible to develop a set of DSNP markers allowing very fine resolution of this very important horticultural group.

Molecular signature of phylogenetic relationships and demographic history of Tunisian Mactra stultorum: Evidence from mitochondrial and nuclear DNA data.

The genetic structure of Mactra stultorum is inferred from partial sequence of a mitochondrial cox1gene and of the internal transcribed spacer region ITS1. The samples with two colors of shell (white and brown) were collected from three sites belonging to the Tunisian coasts: Kalaât El Andalous (KA) and Soliman (SM) and Gabes (GM)). The phylogenetic trees obtained from the 2 markers are similar and subdivided samples into 3 distinct clades; clade (1) regrouped GM, clade (2) regrouped KS (KS contains samples from SM and KAa) and clade (3) is formed by KAb. Using the external sequences from genbank, it can be suggested that M. stultorum from the three clades KS, GM and KAb are three subspecies. The two sympatric M. stultorum from KA (KAa and KAb) appear to be genetically isolated showing a high genetic distance and no common haplotypes where the shell color serves for segregating marker. A total of 29 and 18 haplotypes were detected in the examined cox1 and ITS1 regions, respectively. Our study revealed higher levels of genetic diversity for ITS1 compared to cox1. For both markers, significant clinal changes in haplotypes frequencies between the north and the south populations supported by the absence of common haplotypes were observed. The demographic history of M. stultorum populations has been assessed using neutral tests and mismatch distribution for cox1 marker. A unimodal curve of the Mismatch's distribution and negative significant neutral tests suggested a recent sudden demographic expansion for GM.

Development of molecular tools for characterization and genetic diversity analysis in Tunisian fig (Ficus carica) cultivars.

Fig, Ficus carica L., is a useful genetic resource for commercial cultivation. In this study, RAPD (60), ISSR (48), RAMPO (63), and SSR (34) markers were compared to detect polymorphism and to establish genetic relationships among Tunisian fig tree cultivars. The statistical procedures conducted on the combined data show considerable genetic diversity, and the tested markers discriminated all fig genotypes studied. The identification key established on the basis of SSR permitted the unambiguous discrimination of cultivars and confirmed the reliability of SSR for fingerprinting fig genotypes. The study findings are discussed in relation to the establishment of a national reference collection that will aid in the conservation of Tunisian fig resources.

Self-compatibility in peach [Prunus persica (L.) Batsch]: patterns of diversity surrounding the S-locus and analysis of SFB alleles.

Self-incompatibility (SI) to self-compatibility (SC) transition is one of the most frequent and prevalent evolutionary shifts in flowering plants. Prunus L. (Rosaceae) is a genus of over 200 species most of which exhibit a Gametophytic SI system. Peach [Prunus persica (L.) Batsch; 2n = 16] is one of the few exceptions in the genus known to be a fully self-compatible species. However, the evolutionary process of the complete and irreversible loss of SI in peach is not well understood and, in order to fill that gap, in this study 24 peach accessions were analyzed. Pollen tube growth was controlled in self-pollinated flowers to verify their self-compatible phenotypes. The linkage disequilibrium association between alleles at the S-locus and linked markers at the end of the sixth linkage group was not significant (P > 0.05), except with the closest markers suggesting the absence of a signature of negative frequency dependent selection at the S-locus. Analysis of SFB1 and SFB2 protein sequences allowed identifying the absence of some variable and hypervariable domains and the presence of additional α-helices at the C-termini. Molecular and evolutionary analysis of SFB nucleotide sequences showed a signature of purifying selection in SFB2, while the SFB1 seemed to evolve neutrally. Thus, our results show that the SFB2 allele diversified after P. persica and P. dulcis (almond) divergence, a period which is characterized by an important bottleneck, while SFB1 diversified at a transition time between the bottleneck and population expansion.

Analysis of Self-Incompatibility and Genetic Diversity in Diploid and Hexaploid Plum Genotypes.

During the last decade, S-genotyping has been extensively investigated in fruit tree crops such as those belonging to the Prunus genus, including plums. In plums, S-allele typing has been largely studied in diploid species but works are scarcer in polyploid species due to the complexity of the polyploid genome. This study was conducted in order to analyze the S-genotypes of 30 diploid P. salicina, 17 of them reported here for the first time, and 29 hexaploid plums (24 of P. domestica and 5 of P. insititia). PCR analysis allowed identifying nine S-alleles in the P. salicina samples allocating the 30 accessions in 16 incompatibility groups, two of them identified here for the first time. In addition, pollen tube growth was studied in self-pollinated flowers of 17 Tunisian P. salicina under the microscope. In 16 samples, including one carrying the Se allele, which has been correlated with self-compatibility, the pollen tubes were arrested in the style. Only in one cultivar ("Bedri"), the pollen tubes reached the base of the style. Twelve S-alleles were identified in the 24 P. domestica and 5 P. insititia accessions, assigning accessions in 16 S-genotypes. S-genotyping results were combined with nine SSR loci to analyze genetic diversity. Results showed a close genetic relationship between P. domestica and P. salicina and between P. domestica and P. insititia corroborating that S-locus genotyping is suitable for molecular fingerprinting in diploid and polyploid Prunus species.

Phylogeography analysis and molecular evolution patterns of the nematode parasite Heligmosomum mixtum based on mitochondrial DNA sequences.

Mitochondrial DNA was explored to study phylogeography of the nematode parasite Heligmosomum mixtum and elucidate molecular evolution pattern of cytochrome b gene. The size of cyt b gene ranged from 511 bp to 591 bp and the average of GC contents was 28.9%. The overall transition/transversion ratio R was 5.773 indicating that the transitions are more frequent than transversion. The aligned sequences allowed identifying 54 mtDNA haplotypes among the 119 examined individuals. The genetic divergence registered among the populations of H. mixtum was low (0.3% to 1.5%). Neighbor-joining and maximum Likelihood trees evidenced a huge polytomy and unstructured phylogeographic pattern among the studied populations. The demographic analyses tend to evidence a recent and rapid expansion of H. mixtum. Our results imply a positive selection and the genetic hitchhiking effect is unlikely. Parameters performed supported scenario of sweep selection and recent expansion of H.mixtum populations. Both positive selection and demographic histories have jointly contributed to the observed patterns of nucleotide diversity and haplotypes structure. The comparison of the phylogeographical pattern of H. mixtum with the one of its most common rodent host M. glareolus, confirmed a strong incongruence between the two species. These results strongly suggest that the parasite would not be specific to M. glareolus and that it would switch easily from one rodent species to another. The mitochondrial diversity seems to be unstructured with any biogeographic repartition of the variability and that the genetic structure of H. mixtum is probably associated with weak host specificity.