Identification of DNA Markers of Anthocyanin Biosynthesis Disorders Based on the Polymorphism of Anthocyanin 1 Tomato Ortholog Genes in Pepper and Eggplant

The aim of this research was the identification of DNA markers to Antocyanin 1 (Ant1) tomato orthologs in 30 Capsicum annuum, 30 Solanum melongena and 1 Solanum aethiopicum accessions with different anthocyanin accumulation. A search for orthologs to the Ant1 allele in the GenBank database revealed the following closest nucleotide sequences of Myb113-like TF: in C. annuum, mRNA XM_016689227, mRNA NM_001324618; in S. melongena, mRNA KT259043. In Capsicum spp. samples, 4 SNP and a single base deletion in exon 3 of the Myb113-like 1 allele and 2 SNP in exon 4 of the Myb113-like2 allele were detected in forms with the impaired anthocyanin synthesis, based on their phenotype. In S. melongena samples, a 6 bp deletion at the end of exon 1 of the Myb-1 gene was detected in accessions with a green fruit coloration. A 26 bp deletion between intron 1 and exon 2, 11 SNP in exon regions and a 52 bp insertion at the beginning of intron 2 were detected in forms with a white fruit coloration. A 9-nucleotide insertion and 11 SNP in exon 3 of the Myb-1 gene were detected in the S. aethiopicum accession with no anthocyanin accumulation in fruits. Optimal SCAR and CAPS markers were developed to identify a number of genetic polymorphisms of Myb-like TF genes that might cause the impaired anthocyanin synthesis in pepper and eggplant. CAPS Myb 113AccI and dCAPS Myb 113-AciI markers allow to identify C.annuum accessions without an anthocyanin coloration of fruits. SCAR MybMel and CAPS Mybmel-PstI markers allow to identify S. melongena accessions with white and green fruit colorations. Samples with different allelic polymorphism combinations were selected as models for the further study of the genetic regulation of anthocyanin accumulation in various parts of plants and for improved plant breeding. Open Access Received: 29 November 2019 Accepted: 31 May 2020 Published: 05 June 2020 Copyright © 2020 by the author(s). Licensee Hapres, London, United Kingdom. This is an open access article distributed under the terms and conditions of Creative Commons Attribution 4.0 International License. Crop Breeding, Genetics and Genomics 2 of 18


INTRODUCTION
Anthocyanins belong to a group of flavonoids. These biologically active compounds are derived from benzo-γ-pyrone and are based on a phenylpropene skeleton consisting of C6-C3-C6-carbonic units [1].
Being one of the forms of secondary plant metabolites, anthocyanins do not only provide a particular coloration for various parts of plants, but also determine resistance both to abiotic and biotic stress factors. The ever-growing attention to this area of research stems from the antioxidant and antimicrobial properties of certain enzymes consumed by humans with plant foods [1,2]. The Solanaceae is a proper model allowing to study the genetic regulation mechanisms of anthocyanin accumulation due to the wide inter-and intra-species polymorphism of their accumulation in various parts of plants.
To date, the structure and functions of these compounds have been well-studied, and the regulation mechanisms determining their accumulation and depending on environmental conditions and genetic structure have been shown. A review article by Y. Liu et al. [2] has compiled investigation results on the biosynthesis and degradation mechanisms of these compounds based on more than 130 scientific articles' analysis, a significant part of which gives consideration to the genetic regulation of such mechanisms.
The allelic polymorphism study of these regulatory genes is important due to the fact that the structural genes of an anthocyanin biosynthetic pathway function under the control of a regulatory complex called MYB-bHLH-WD40 (MBW) [2], and R2R3Myb-activators are key elements of this complex [2].
Genetic databases provide data on the gene sequences of a number of Myb factors that determine the accumulation of various anthocyanin forms [3,4].
Literary sources provide information about the specifics of regulatory genes' expression under various environmental conditions [5,6] and depending on the genetic allelic combination [7]. Genetic polymorphism of the Anthocyanin1 gene has been studied in tomato and a marker to identify the Aft phenotype has been developed [7]. However, the genetic polymorphism of these genes in pepper and eggplant remains poorly explored. Thus, a relationship between the Capsicum Anthocyanin 2 activity and its promoter region's polymorphism has been established (LTR retrotransposon insertion defined as Ca-nLTR-A) [8].
In view of the above, our investigation aimed to identify DNA markers identification of DNA markers that allow to differentiate samples by anthocyanin accumulation.

MATERIALS AND METHODS
The objects of research were collection pepper accessions provided by Based on the DNA typing results, optimal DNA markers were selected to identify the polymorphisms of accessions with no anthocyanin accumulation.
A search for homologous Myb TF nucleotide sequences was performed using the BLAST program (Basic Local Alignment Search Tool) on the server of the US National Center for Biotechnology Information [10].

RESULTS
A search for orthologs to the tomato allele Ant1 (EF433416) in the GenBank Database (Supplementary Table S1

Value
Due to the fact that the nomenclature of Myb-orthologous genes in different Solanaceous species is not consistent [2] and is also based on Vavilov's law of homologous series in the hereditary variation [11], the selection of sequences for the study was carried out according to the

DATA AVAILABILITY
All data generated from the study are available in the manuscript or supplementary files.

AUTHOR CONTRIBUTIONS
OB and AK developed the experiment; KY performed DNA sequencing; OB, TN, NN, and KY carried out field and DNA-typing experiments; OB, TN, and NN, and KY designed tables and figures; OB and AK analyzed the data obtained and wrote the paper.

CONFLICTS OF INTEREST
The authors declare that they have no conflict of interest.