Study of the progeny of sorghum mutants obtained using the CRISPR/Cas9 genetic construct directed at inducing mutations in the α-kafirin k1C5 gene

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Abstract
Site-directed mutagenesis using genetic constructs carrying the CRISPR/Cas system is an effective technology that is actively used to solve a variety of problems in plant genetics and breeding. One of these problems is to improve the nutritional value of grain sorghum, a high-yielding heat- and drought-tolerant cereal crop that is becoming increasingly important in the conditions of climate aridization. The main reason for the relatively low nutritional value of sorghum grain is the resistance of its storage proteins, kafirins, to proteolytic digestion. We have previously obtained mutants with improved kafirin in vitro digestibility using the CRISPR/Cas technology in grain sorghum variety Avance. The nucleotide sequence of one of the genes (k1C5) of the gene family encoding the signal polypeptide of 22 kDa α-kafirin was used as a target. The aim of this study was to investigate the manifestation of the main agronomically-important traits in the progeny of these mutants and inheritance of high in vitro protein digestibility, and also sequencing nucleotide sequences encoding the 22 kDa α-kafirin signal polypeptide in a number of plants from the T 0 generation and their T 1 progeny. It was revealed that four of the six studied T 0 plants, as well as their progeny, had the same mutation: a T→C substitution in the 23rd position of the nucleotide sequence of the k1C5 gene encoding the signal polypeptide, which led to a substitution of the coding triplet CTC→CCC (Leu→Pro). This mutation is located off-target, 3’ from the PAM sequence. It is suggested that this mutation may have arisen as a result of Cas9 nuclease errors caused by the presence of multiple PAM sequences located close to each other. It was found that the progeny of two of the three studied mutants (T 2 and T 3 families), grown in the experimental field conditions, differed from the original variety by a reduced plant height (by 12.4–15.5 %). The peduncle length, 1,000-grain mass, and grain mass per panicle did not differ from the original variety, with the exception of the progeny of the 2C-1.2.5b mutant, which had a reduced grain yield per panicle. Unlike the original variety, plants from the T 2 and T 3 generations had kernels with a modified type of endosperm (completely floury, or floury with inclusions of vitreous endosperm, or with a thin vitreous layer). The level of grain protein digestibility in the progeny of mutants 2C-2.1.1 #13 and 2C-1.2.5a #14 varied from 77 to 84 %, significantly exceeding the original variety (63.4 ± 2.3 %, p < 0.05). The level of protein digestibility from kernels with modified endosperm was higher than that of kernels with normal vitreous endosperm (84–93 %, p < 0.05). The reasons for the variation in endosperm texture in the progeny of the mutants and its relationship with the high digestibility of kafirins are discussed.

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