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.

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.

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.

Identification of conserved genes linked to responses to abiotic stresses in leaves among different plant species.

As a consequence of global climate change, certain stress factors that have a negative impact on crop productivity such as heat, cold, drought and salinity are becoming increasingly prevalent. We conducted a meta-analysis to identify genes conserved across plant species involved in (1) general abiotic stress conditions, and (2) specific and unique abiotic stress factors (drought, salinity, extreme temperature) in leaf tissues. We collected raw data and re-analysed eight RNA-Seq studies using our previously published bioinformatic pipeline. A total of 68 samples were analysed. Gene set enrichment analysis was performed using MapMan and PageMan whereas DAVID (Database for Annotation, Visualisation and Integrated Discovery) was used for metabolic process enrichment analysis. We identified of a total of 5122 differentially expressed genes when considering all abiotic stresses (3895 were upregulated and 1227 were downregulated). Jasmonate-related genes were more commonly upregulated by drought, whereas gibberellin downregulation was a key signal for drought and heat. In contrast, cold stress clearly upregulated genes involved in ABA (abscisic acid), cytokinin and gibberellins. A gene (non-phototrophic hypocotyl) involved in IAA (indoleacetic acid) response was induced by heat. Regarding secondary metabolism, as expected, MVA pathway (mevalonate pathway), terpenoids and alkaloids were generally upregulated by all different stresses. However, flavonoids, lignin and lignans were more repressed by heat (cinnamoyl coA reductase 1 and isopentenyl pyrophosphatase). Cold stress drastically modulated genes involved in terpenoid and alkaloids. Relating to transcription factors, AP2-EREBP, MADS-box, WRKY22, MYB, homoebox genes members were significantly modulated by drought stress whereas cold stress enhanced AP2-EREBPs, bZIP members, MYB7, BELL 1 and one bHLH member. C2C2-CO-LIKE, MADS-box and a homeobox (HOMEOBOX3) were mostly repressed in response to heat. Gene set enrichment analysis showed that ubiquitin-mediated protein degradation was enhanced by heat, which unexpectedly repressed glutaredoxin genes. Cold stress mostly upregulated MAP kinases (mitogen-activated protein kinase). Findings of this work will allow the identification of new molecular markers conserved across crops linked to major genes involved in quantitative agronomic traits affected by different abiotic stress.

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.

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.

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.