To develop tomato hybrids and varieties with a high fruit quality, we gradually solved the following tasks: development of DNAmarking methods for long shelf life genes; the genes modifying the biosynthesis of carotenoids and their composition; testing of the elaborated methods on the developed breeding material; the selection of samples with different allelic composition of fruit quality genes; the development of F 1 hybrids using the method of successive crosses and their study; the selection of tomato forms by DNA-typing methods with target genes in F 2 populations to develop valuable breeding samples; the study of carotenoids' accumulation peculiarities and their inheritance. We used DNA-identification methods for fruit quality genes:
Anthocyanins are high-value plant antioxidants; they also determine biotic and abiotic stress resistance. The aim of our research was to study the allelic polymorphism of Antocyanin 1 orthologs in the vegetable Solanaceae crops of C. annuum and S. melongena. The search revealed the following closest genes in C. annuum: Myb113-like1 TF and Myb113like2 transcription factors and Myb1 in S. melongena. Exon amplicons of those genes were obtained and then sequenced in the pepper and eggplant samples with contrasting anthocyanin fruit coloration. Primers to the identified polymorphisms were developed and their correlation with the anthocyanin accumulation in fruits was studied. A close correlation was found between a minimum accumulation or the complete absence of anthocyanin synthesis in fruits with a single nucleotide deletion (Myb113-like1), and in the pepper samples, 2 SNP (Myb113-like2) was detected using the CAPS marker Myb 113-AccI. In the eggplant samples, the deletions of 6 and 26 bp were detected using the SCAR marker MybMel and the CAPS marker MybmelPst1. The disturbance of anthocyanin synthesis in pepper forms with 1Indel in Myb113-like1 TF was determined by a shift in the reading frame and SNPs in Myb113-like2 TF lead to amino acid substitutions: Lys → Arg and Thr → Lys. In the eggplant, a deletion of 6 bp leads to the loss of ala and arg in the protein; a deletion of 26 bp causes disorder during the mRNA maturation. The developed markers allow identifying the Myb-like TF alleles under study, resulting in anthocyanin synthesis disturbance in fruits. C. annuum and S. melongena samples with different alleles were selected for a further study and new varieties in agriculture.
Efficiency in detecting of tomato forms with no chalcone-naringenin flavonoid in pink-fruited and yellow-fruited forms was evaluated using DNA markers for various polymorphisms of the SlMYB12 gene. The closest relationship between a phenotype with the transparent skin of fruits and a deletion in the promoter region of the SlMYB12 gene was shown. The highest efficiency in the detection of the recessive y allele of the regulatory SlMYB12 gene, leading to the chalcone-naringenin synthesis disruption and skin transparency, was established by a combination of markers MYB12-603delaF1/603del-aR6 (Myb-603del aF1/R6) and MYB12-603del-aF1/603del-aR5 (Myb12 aF1/R5). Fruit coloration peculiarities were shown depending on a combination of the structural alleles of a carotenoid biosynthesis pathway and SlMYB12 gene alleles. A combination of this y allele with the alleles of the gene of the lycopene-β-cyclase beta (b) and old gold crimson (ogc ) allows selecting pink and raspberry forms respectively. In tomato accessions with yellow and orange fruits, the y allele provides pale shades of the main coloration determined by carotenoid biosynthesis genes (yellow flesh (r), tangerine (t), Beta (B)). The presence of SNP T → C of the SlMYB12 gene (171476848 position of chromosome 1) was identified in 80 % of accessions with the transparent skin of fruits of the evaluated collection. The effect of the recessive y allele of the SlMYB12 gene on an increase in the lycopene concentration of tomato fruits in a combination with b, ogc alleles was shown. Using MAC methods by fruit quality genes, including the SlMYB12 gene, the cherry tomato variety Malinovyj koktel with a high lycopene accumulation was developed and included in the State Register
In order to use marker-assisted selection (MAS) methods in the development of hybrids and tomato varieties resistant to late blight, the efficiency of molecular markers to the alleles resistant to Phytophthora infestans known in the literature was assessed. High efficiency in the identification of homozygous and heterozygous forms using CAPS dTG63 (Hinf1) and SCAR NCLB-9-6678 markers to Ph2 and Ph3 alleles was established respectively. DNA extraction techniques, reaction mixture compositions, as well as amplification and visualization result modes were adapted. The presence of the Ph3 allele in F1-standard Adapt and Alamina hybrids, and F1 Azart, Agenchik, Dzivosny, and Black Brilliant hybrids included in the Register of Varieties of the Republic of Belarus, as well as in the breeding lines of the collections possessed by the Institute of Genetics and Cytology, NAS of Belarus, and the Belarusian State Agricultural Academy was demonstrated.
Based on comparison of R2R3 Myb genes in Solanaceae (S. lycopersicum: Ant1, Ant2, S. melongena: Myb1, C. annuum: Myb113-like1 and Myb113-like2) and Brassicaceae crops, a search for orthologous sequences was The sequences encoding Myb114 TF in Brassica oleracea and Brassica rapa was found to be the closest in a nucleotide structure to the previously investigated genes in Solanaceae. Тhе polymorphism in the promoter region of the Capsicum annuum Myb113-like1 gene that regulates anthocyanin biosynthesis: an additional 148 bp repeat and 2(1) bp insertion in the forms with impaired anthocyanin synthesis in fruits was studied. A relationship between the presence of an insert in the promoter (Myb113-like1pr+148) and polymorphisms in the exon regions of Myb113-like1delTand Myb113-like2C/Аgenes associated with impaired anthocyanin synthesis was established. A number of polymorphisms of the Myb114 gene in vegetable crops of the cabbage family (Brassica oleracea, Brassica rapa), which closely correlate with high/low accumulation of anthocyanins in leaves, were identified. In B. oleracea, SNPs that lead to the replacement of two amino acids located in the region of DNAbinding domains were found, which leads to a change in the efficiency of binding of this transcription factor and the promoters of structural biosynthesis genes. A protein sequence encoded by the Myb114 gene in accessions of the leafy turnip (B. rapa) with a high accumulation of anthocyanins in the leaves differed from the accessions without anthocyanin accumulation in the leaves by five amino acids, while the regions of DNA-binding domains were the same in the forms with various anthocyanin accumulation.
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