Genome variability of domestic tomato varieties: data from AFLP analysis.

Tomato Solanum lycopersicum L. is one of the main vegetable crops, accessions and cultivars of which are characterized by a low level of genomic polymorphism. Introgressive tomato breeding uses related wild Solanum species to improve cultivars for stress tolerance and fruit quality traits. The aim of this work was to evaluate the genome variability of 59 cultivars and perspective breeding lines of S. lycopersicum and 11 wild tomato species using the AFLP method. According to the AFLP analysis, four combinations of primers E32/M59, E32/M57, E38/M57, and E41/M59, which had the highest PIC (polymorphism information content) values, were selected. In the process of genotyping a collection of 59 cultivars/lines of S. lycopersicum and 11 wild tomato accessions, the selected primers revealed 391 fragments ranging in size from 80 to 450 bp, of which 114 fragments turned out to be polymorphic and 25 were unique. Analysis of the amplif ication spectra placed wild tomato accessions into separate clades. Sister clades included cultivars of FSCV breeding resistant to drought and/or cold and, in part, to late blight, Alternaria, Septoria, tobacco mosaic virus and blossom end rot, as well as tomato accessions not characterized according to these traits, which suggests that they have resistance to stress factors. In accessions of distant clades, there was clustering on the basis of resistance to Verticillium, cladosporiosis, Fusarium, tobacco mosaic virus, gray rot, and blossom end rot. The combination of ac cessions according to their origin from the originating organization was shown. The primer combinations E32/M59, E32/M57, E38/M57 and E41/M59 were shown to be perspective for genotyping tomato cultivars to select donors of resistance to various stress factors. The clade-specif ic fragments identif ied in this work can become the basis for the development of AFLP markers for traits of resistance to stress factors.

Analysis of the structure and function of the tomato Solanum lycopersicum L. MADS-box gene SlMADS5.

At all stages of f lowering, a decisive role is played by the family of MADS-domain transcription factors, the combinatorial action of which is described by the ABCDE-model of f lower development. The current volume of data suggests a high conservatism of ABCDE genes in angiosperms. The E-proteins SEPALLATA are the central hub of the MADS-complexes, which determine the identity of the f loral organs. The only representative of the SEPALLATA3 clade in tomato Solanum lycopersicum L., SlMADS5, is involved in determining the identity of petals, stamens, and carpels; however, data on the functions of the gene are limited. The study was focused on the SlMADS5 functional characterization. Structural and phylogenetic analyses of SlMADS5 conf irmed its belonging to the SEP3 clade. An in silico expression analysis revealed the absence of gene transcripts in roots, leaves, and shoot apical meristem, and their presence in f lowers, fruits, and seeds at different stages of development. Two-hybrid analysis showed the ability of SlMADS5 to activate transcription of the target gene and interact with TAGL1. Transgenic plants Nicotiana tabacum L. with constitutive overexpression of SlMADS5 cDNA f lowered 2.2 times later than the control; plants formed thickened leaves, 2.5-3.0 times thicker stems, 1.5-2.7 times shortened internodes, and 1.9 times fewer f lowers and capsules than non-transgenic plants. The f lower structure did not differ from the control; however, the corolla petals changed color from light pink to magenta. Analysis of the expression of SlMADS5 and the tobacco genes NtLFY, NtAP1, NtWUS, NtAG, NtPLE, NtSEP1, NtSEP2, and NtSEP3 in leaves and apexes of transgenic and control plants showed that SlMADS5 mRNA is present only in tissues of transgenic lines. The other genes analyzed were highly expressed in the reproductive meristem of control plants. Gene transcripts were absent or were imperceptibly present in the leaves and vegetative apex of the control, as well as in the leaves and apexes of transgenic lines. The results obtained indicate the possible involvement of SlMADS5 in the regulation of f lower meristem development and the pathway of anthocyanin biosynthesis in petals.

Variability and Phylogeny of the Pepper Phytoene Synthase Paralogs PSY1 and PSY2 in Species of Various Capsicum Complexes.

Genes homologous to PSY1 and PSY2 that encode phytoene synthase isoforms in Capsicum species C. baccatum, C. chinense, C. frutescens, C. tovarii, C. eximium, and C. chacoense were identified. High conservatism of functionally significant sites of phytoene synthases of the analyzed accessions was revealed. It was found that only PSY1-based clustering of pepper species corresponds to the traditional Capsicum phylogeny; C. eximium was a part of the Purple corolla complex, and C. chacoense was equidistant from Annuum and Baccatum clades. The absence of significant differences between PSY1 and PSY2 of yellow-fruited C. chinense and red-fruited pepper accessions was shown. The yellow color of C. chinense fruit may be the result of both decreased PSY1 expression and increased PSY2 transcription. Thus, it was demonstrated that the acquired fruit pigmentation retains strict phylogenetic limitations, which, however, can be overcome using artificial selection for the activity of phytoene synthase PSY1.

Structural and Functional Diversification of SEPALLATA Genes TM5 and RIN in Tomato Species (Section Lycopersicon).

New TOMATO MADS 5 (TM5) homologous genes were identified in evolutionarily recent, red-fruited and more ancient, wild green-fruited tomato species. It was shown that the identified TM5 homologs belong to the SEPALLATA3 clade; thus, the SEP subfamily diversification was characterized. For the first time, the TM5 and RIN co-expression pattern was determined in flowers, immature green fruits, and ripe fruits of Solanum lycopersicum and in five wild tomato species. It was shown that, regardless of the species, the level of TM5 transcription in flowers was higher than that of RIN, whereas in fruits it was lower than the level of RIN transcription. The data obtained suggest that TM5, together with other transcription factors RIN and SlCMB1, is involved in the regulation of fruit development and ripening.

Structural and functional features of phytoene synthase isoforms PSY1 and PSY2 in pepper Capsicum annuum L. cultivars.

The fruits of various pepper cultivars are characterized by a different color, which is determined by the pigment ratio; carotenoids dominate in ripe fruits, while chlorophylls, in immature fruits. A key regulator of carotenoid biosynthesis is the phytoene synthase encoded by the PSY gene. The Capsicum annuum genome contains two isoforms of this enzyme, localized in leaf (PSY2) and fruit (PSY1) plastids. In this work, the complete PSY1 and PSY2 genes were identified in nine C. annuum cultivars, which differ in ripe fruit color. PSY1 and PSY2 sequence variability was 2.43 % (69 SNPs) and 1.21 % (36 SNPs). The most variable were PSY1 proteins of the cultivars 'Maria' (red-fruited) and 'Sladkij shokolad' (red-brown-fruited). All identified PSY1 and PSY2 homologs contained the phytoene synthase domain HH-IPPS and the transit peptide. In the PSY1 and PSY2 HH-IPPS domains, functionally significant sites were determined. For all accessions studied, the active sites (YAKTF and RAYV), aspartate-rich substrate-Mg2+-binding sites (DELVD and DVGED), and other functional residues were shown to be conserved. Transit peptides were more variable, and their similarity in the PSY1 and PSY2 proteins did not exceed 78.68 %. According to the biochemical data obtained, the largest amounts of chlorophylls and carotenoids across the cultivars studied were detected in immature and ripe fruits of the cv. 'Sladkij shokolad' and 'Shokoladnyj'. Also, ripe fruits of the cv. 'Nesozrevayuschij' (green-fruited) were marked by significant chlorophyll content, but a minimum of carotenoids. The PSY1 and PSY2 expression patterns were determined in the fruit pericarp at three ripening stages in 'Zheltyj buket', 'Sladkij shokolad', 'Karmin' and 'Nesozrevayuschij', which have different ripe fruit colors: yellow, red-brown, dark red and green, respectively. In the leaves of the cultivars studied, PSY1 expression levels varied significantly. All cultivars were characterized by increased PSY1 transcription as the fruit ripened; the maximum transcription level was found in the ripe fruit of 'Sladkij shokolad', and the lowest, in 'Nesozrevayuschij'. PSY2 transcripts were detected not only in the leaves and immature fruits, but also in ripe fruits. Assessment of a possible correlation of PSY1 and PSY2 transcription with carotenoid and chlorophyll content revealed a direct relationship between PSY1 expression level and carotenoid pigmentation during fruit ripening. It has been suggested that the absence of a typical pericarp pigmentation pattern in 'Nesozrevayuschij' may be associated with impaired chromoplast formation.