Dihaploid plant production of red beet (Beta vulgaris subsp. vulgaris), homozygosity evaluation using isoenzymatic and NGS methods

Abstract Conditions of in vitro gynogenesis process in red beet was examined. A significant influence of the genotype on the gynogenesis process was demonstrated. Of the eight genotypes, 58.3% planted ovules regenerated embryo-like structures in breeding line 411, 2.1% in RA-10, RA-11, RA-12 breeding lines and 0.9% embryo-like structures in ‘Opolski’. For the gynogenesis induction, B5 medium containing 0.1 mgl⁻¹ 2,4-dichlorophenoxyacetic acid was the most effective from all tested media. On this medium, the highest number of gynogenetic embryo-like structures was obtained. Most of the plants were regenerated on MS medium supplemented with 30 g l⁻¹ sucrose, 0.2 mg l⁻¹ 6-benzylaminopurine and 1 mg l⁻¹ indole-3-acetic acid. Thirty nine percent of the regenerated plants acclimatized. Cytometric evaluation of the gynogenetic plants of four tested genotypes revealed that in three genotypes, 100% of tested plants were haploid. Plants showed diploid ploidy level in one genotype. Isoenzymatic analysis of gynogenetic plants demonstrated that 95% and 70% of examined populations were homozygotic for the phosphohexose isomerase isoenzyme and the aspartato aminotransferase isoenzyme, respectively. During the next generation sequencing, 93% of reads were successfully mapped, from which 83–85% were mapped in pairs. For 15% of pairs it was clear that the obtained sequence was fully homozygous, the rest of the readings were not unambiguous, but similar to the sequence of a homozygous base pair system.