Biological and economic characteristics of the allotetraploid with genomic formula DDAuAu from the cereal family

The synthesis  of new  allopolyploid  cereal  genotypes is an important task aimed  at involving new  genetic resources in breeding programs. Diploid species of the genera Triticum and Aegilops – bread  wheat  relatives – are an important source of agronomically  valuable traits. A tetraploid synthetic  with genomic formula DDAuAu was obtained by N.A. Navruzbekov through crossing Aegilops tauschii Coss. and Triticum urartu Thum. ex Gandil. The purpose of this work was to study  the  chromosomal composition and  biological  and  commercially  important traits of the  tetraploid. Cytogenetic analysis using fluorescent in situ hybridization  showed the  presence of all chromosomes of the D genome in the chromosomal complement of the synthetic. By means  of stepwise  vernalization, the winter habit was established for the tetraploid synthetic with the optimum vernalization requirement of 45 days. Under greenhouse conditions, two groups of genotypes were found whose flowering dates differed by 6.5 days, which may indicate an allelism at the Vrn-3 locus. The coloring of various organs of the tetraploid plant, such as coleoptile,  stem, anthers,  and glumes  of the spike, was revealed. The coloration  of the aleurone layer of the grain may indicate that the donor species T. urartu is a carrier of the Ba gene that controls its blue color. A new morphotype of leaf pubescence was found. In terms of productivity, the tetraploid is comparable to bread wheat. Grains are characterized by a supersoft structure and high wet gluten  content, from 39–45 to 65 %, in the field and greenhouse conditions, respectively. Thus, the tetraploid can be used to create  new wheat  genotypes as a source  of untapped genetic diversity, as well as a new genetic model  for studying  the patterns of evolution  of polyploid plants.


Introduction
The highland areas of the Caucasus are natural habitats of diploid species of the genera Triticum and Aegilops, which gave rise to cultural allopolyploid species of wheat. In the second half of the 20th century, many attempts were made to synthesize the new types of allopolyploids in order to engage in breeding and, subsequently, in economic acti vity, the new genetic resources from diploid species of the above mentioned genera. The tetraploid synthetic with a genomic formula DDA u A u was obtained by N.А. Nav ruzbekov (1982) by crossing Aegilops tauschii Coss and T. urartu Thum. ex Gandil. The character of meiosis was studied in amphidiploid and the karyotype was shown to do be stabile (Aminov, Navruzbekov, 1985). A similar hybrid with the participation of other initial samples of these species was named T. erebuni Gandul. (Gandilyan, 1984). These tetraploids, as well as the tetraploid species T. palmovae G. Ivanov (A b A b DD) carrying the genome A from the species T. boeoticum Boiss., are very limitedly involved in the researches of both applied and fundamental nature. Only the information about the use of two species as donors of fungal disease resistance genes in common wheat is available (Babayants et al., 2012;Davoyan et al., 2018). The biological characteristics of these tetraploid has not yet been conducted in more details. At the Dagestan experi mental station of VIR allotetraploid of N.A. Navruzbekov with the genomic formula A u A u DD (AT Navruzbekov) is being maintained and successfully propagated. In this work, the chromosomal composition and biological and economically important traits were studied.

Material and methods
The tetraploid was obtained by a direct hybridization of diploid species Ae. tauschii and T. urartu Thum. ex Gandil. followed by colchicine treatment of the roots of F 1 hybrids (Navruzbekov, 1982).
Tetraploid plants were studied under natural conditions in the city of Derbent (Dagestan) at the experimental station of the VIR branch (St. Petersburg) and under the hydroponic greenhouse conditions of the Institute of Cytology and Genetics of the Siberian Branch of the Russian Academy of Sciences (Novosibirsk). The field study was conducted in a dry subtropical climate of Dagestan, in the southern plane zone. The average annual temperature of the area is +1…-1.4 °C with an annual amount of precipitation of 400-500 mm, 80-90 % of which falls in the autumnwinter period. Summer is hot and dry. Productivity indicators were studied in 2018 on a random sample of 20 plants. Plants were analyzed individually; spike morphology and productivity were assessed on the main spikes. In the hydro ponic greenhouse the plants were grown on artificial soil, expanded clay, with daily double top dressing with Knop solution and under 14hour lighting; day/night tempera tures are 18/20 °C before and 20/22 °C after tillering. To determine the growth habit, the plants were grown without vernalization and under 30, 45, and 60 days of vernalizing at +4 °C. The seedlings were sown in bathtubs filled with expanded clay, 7-10 plants in a row with a length of 1 m.
The observations were carried out for to the following traits: the day number from seedlings to flowering, the presence of pigmentation and pubescence on the plant organs, spike shape and awnedness, productivity indicators, technologi cal properties of grain and flour.
Fluorescent in situ hybridization (FISH) was performed according to a previously published procedure . For the identification of chromosomes, pSc119.2 and (Bedbrook et al., 1980) and pAs1 (Rayburn, Gill, 1986) probes labeled with the nicktranslation reaction were used. The pubescence of the leaf surface was studied in micro photographs of the folds of boot leaves according to the procedure described previously (Genaev et al., 2012). Grain was studied for the nine technological indicators using the methods recommended for variety testing of agricultural crops in Russia (Methods of State Tests of Crops, 1988), with modifications for small portions of grain. Raw gluten content was determined manually from the whole meal us ing a micromethod based on the state standard of the Rus sian Federation (State Standard R 544782011…, 2012). The diameter of the flour particles (μm) was determined on a PSH4 device. Physical properties of dough: tenacity (mm), extensibility (mm), the balance of dough and flour strength (u. a.) were determined on a Chopin alveograph with 50g mixer. Gluten content was determined in grain grown in greenhouse and under field conditions, the other parameters -in grain of field origin grown in Dagestan.

Results
Chromosomal composition. The analysis of the metaphase chromosomes of a tetraploid sample by the FISH method was carried out with the pSc119.2 and pAs1 probes, which are most often used to identify chromosomes in wheat and a number of other cereals including the donors of the B and D genomes of polyploid wheat -Ae. speltoides Tausch. and Ae. tauschii Coss. (Badaeva et al., 1996;Schneider et al., 2003). The pSc119.2 probe hybridizes mainly with the chromosomes of the B genome of polyploid wheat, and the pAs1 sites are localized mainly on the chromosomes of the D genome. In the result of the study the sample was found to be a tetraploid synthetic wheat (2n = 28, DDAA), car rying 14 chromosomes of the D genome in the karyotype (Fig. 1). The sites of hybridization with pAs1 were also found at the ends of the long arms of one of the Agenome chromosome pair, which does not contradict the results of (Badaeva et al., 2015). The presence of the separate pAs1 blocks on different chromosomes of diploid species wheat were showed. The pSc119.2 probe is localized on the short arms of chromosomes 2D and 5D, which corresponds to the previously obtained data for Ae. tauschii (Badaeva et al., 1996). The Fig. 1 also contains a pair of submetacen tric chromosomes of genome A with pSc119.2 signals in subtelomeric regions of short arms. According to previous studies, pSc119.2 sites on the A chromosomes are few. In tetraploid and hexaploid wheats this tandem repeats can be localized at 5AS and 4AL chromosomes (Schneider et al., 2003;Kubaláková et al., 2005). In diploid wheat, pSc119.2 almost does not occur. However, E.D. Badaeva et al. (2015) reported on the localization of this probe on a long arm, presumably on 2A chromosome, in one of the samples of T. boeoticum. Consequently, it is quite possible that the sample of T. urartu that participated in the obtaining of Navruzbekov's AT carries pSc119.2 sites on the short arms of one of the pairs of submetacentric chromosomes, of genome A possibly 5A.
Determination of growth habit. Growth habit and pace of development determine the adaptation of the genotype to environmental conditions through the timely formation of generative organs and successful seed reproduction. Under the conditions of Dagestan, tetraploid was sown in the fall. Due to the mildness of the climate it was not pos sible to determine the genetic status of vernalization genes. An experiment in the greenhouse showed that without vernalization and with 30day vernalization, the plants do not pass to the generative phase. The most effective terms of vernalization were 45 and 60 days. This suggests that the sample carries the recessive alleles of the Vrn-A1 and Vrn-D1 genes. The winter growth habit was also noted in synthetic allotetraploid T. palmovae (DDA b A b ) (Ivanov, 1984). The vernalized plants studied in the autumn and spring seasons under greenhouse conditions separated into two groups according to the flowering time. In the first group, the number of days was 40.5, while in the second group it was 46.8 days (the differences are significant at p = 0.0014). This may indicate that the sample carries dif ferent alleles of the Vrn-3 locus in the chromosomes of the 7 th homoeological group responsible for the "fine tuning" of the time of transition to flowering during the growing season, or other minor loci responsible for the timing of flowering per se.
Coloration of plant organs. Flavonoid pigments were synthesized at various stages of ontogenesis by various plant organs of the tetraploid synthetic. Their role in life processes is associated with adaptive responses to many abiotic and biotic stresses. The anthocyanin pigmentation was found on the coleoptile, stem and anthers (Fig. 2, a, b). These dominant traits in common wheat are controlled by Ra, Pc-1 and Pan-1 loci in the chromosomes of the 7 th homoeological group (Khlestkina, 2012). Bright and stable color indicates a good expression of these genes in tetraploid. Among these genes, the genes of the 7D chromosome have the highest expression in bread wheat and are probably also determined by the D genome in the tetraploid. The ripened grain had a very rare blue color of the aleurone layer both in the field and in greenhouse conditions (see Fig. 2, c). Previously, the Ba gene that de termines this trait was localized on chromosome 4A of the species T. boeoticum (Singh et al., 2007). No such genes were found in the D genome (Khlestkina, 2012). Thus, it can be assumed that the species T. urartu, which donated the genome to the synthetic tetraploid AT Navruzbekov, is the carrier of the dominant allele of this gene.
The tetraploid had colored spike glumes; the trait was well manifested in the field conditions but rather weakly in the greenhouse (see Fig. 2, d ). This dominant trait in polyploid wheat is controlled by a series of homoeoallelic genes Rg-1 in the chromosomes of the 1 st homoeological group (Khlestkina et al., 2006 the occurence of this trait has not been specifically studied. However, it is known that among the varieties of bread wheat the dominant allele is more common in elevated areas and in areas with a cold climate (Yakubtsiner, Savit sky, 1947). The allele of Rg-D1 gene (chromosome 1D) which determines the graysmoky (subsp. cesium) color of spikelet glumes is characterized of bread wheat. Earlier it was shown that Ae. tauschii species carries another than in bread wheat varieties allele of this gene, which gives a dark brown color (Pshenichnikova et al., 2005). Morphobiological characteristics of the ear. Interest ingly, that the studied genotype is close to a bread wheat in terms of the spike shape and density (see Table 1). It is well known that the A genome of the hexaploid precursor of bread wheat T. spelta carries the Q gene, which deter mines the speltoid form of the spike (Matsuoka, 2011). It can be assumed that the A u A u genome of the tetraploid under study carries an allele of this gene that does not affect the shape of the ear, and its relatively poor threshability is more closely linked to the Tg gene in chromosome 2D (McIntosh et al., 2013). The spike of Navruzbekov's AT is awned which is generally characteristic of wild and poorly cultivated relatives of common wheat.
Leaf blade pubescence and the presence of wax in plants. The presence of pubescence on the leaf blades was a characteristic feature of the AT Navruzbekov. Its morphol ogy differed from the pubescence in bread wheat (Fig. 3). It was long, tough to the touch and rather rare. The adaptive significance of this trait is associated with a protection from damage by leafeating insects, as well as with the influence on the energy balance and gas exchange of the leaf by changing the thickness of the air boundary layer adjacent to its surface (Schuepp, 1993). The pubescence protects the photosynthetic apparatus of the leaf from excessive solar radiation and is especially common in plants whose growing areas are steppes, deserts, high moun tains and tundra (Johnson, 1975). To date, the genes of bread wheat have been identified in chromosomes 4B and 7B (McIntosh et al., 2013), as well as in chromosome 5A of the diploid and tetraploid species T. monococcum and T. timopheevii (Jing et al., 2007;Pshenichnikova et al., 2019). Such genes have not yet been found for the D genome. We assume that the studied genotype is the carrier of a new gene from this genome. Earlier, we showed that among the samples of Ae. tauschii some carry the longest leaf pubescence; this expression level of the trait was inherited by the allopoly ploid T. palmovae, which carries the D and A b genomes (Pshenichnikova et al., 2017). The plants of the sample studied were completely wax less. All einkorn wheats are the same (Tsunewaki, Ebona, 1999). No the inhibitor gene has been yet detected respon sible for wax formation on einkorn wheat organs, however, the inhibitor gene is known on chromosome 2D (McIntosh et al., 2013).
Economically important traits. For plant height wheat the amphidiploid may be attributed to the intermediate forms, its average straw length is 128.5 cm (see Table). The straw is tough, but lodging resistance is low (3 points of the scale). The length of the ear varies from 8.5 to 13.5 cm. The spike and flower glumes are tough, with poor threshing. Ear density is average. The average weight of grains per spike is 1.2 g, the maximum number of grains per spike is 39. Visual evaluation of grain plumpness was 4,1 points out of 10 possible (see Table). This suggests a lack of uniformity of grain surface (see Fig. 2, c). However, the grain is quite large: thousandgrain weight of the main spike was 38.1 g under field conditions (see Table), and during the technological analysis of grain yielded in 2017 and 2018 it consisted 33.7 g on average.
Technological properties of dough. Technological properties of grain are the most important breeding charac teristics that determine the production purpose of the harvested crop. For the first time in this work, the techno logical properties of synthetic tetraploid were studied. One of the main technological indicators in the classification of grain according to the production classes in Russia is gluten content in grain. This parameter was studied both in greenhouse and field grain. It turned out to be very high (Fig. 4). In the first case, the content was 65 %. In field conditions this figure was: in 2017 -39 %, in 2018 -45 %. Thus, A u A u DD tetraploid can be a source of improvement of this trait in breeding. The genotype may be characte rized as softgrained as the diameter of flour particles was 8.7 microns with a total grain vitreousness of 50 %. Such flour can be described as supersoft. Possibly, the tetraploid is the carrier of the dominant alleles of the Ha loci in both 5A and 5D chromosomes which synthesize the complete proteins PinA and PinB, providing the soft endosperm structure. This is characteristic of diploid ancestral species of wheat T. monococcum and Ae. tauschii (Gautier et al., 2000). It should be noted that the tetraploid species with the genomic composition BBAA completely lost the dominant alleles of the genes Pina-A1 and Pina-D1 and the DDA u A u tetraploid under study is unique in this respect. With its help it is possible to transfer into the bread wheat the dominant allele of Pina-A1 gene from 5A chromosome, lost in the process of polyploidization. The physical properties of flour (on alveograph) were characterized by relatively high elasticity but low extensibility. The ratio of these rheological parameters was 6.1, which brings the tetraploid closer to durum wheat with the genomic composition of BBAA.

Conclusion
Estimating the breeding value of AT Navruzbekov it can be concluded that due to the noted shortcomings (lodging, poor threshing, low productivity, etc.) its direct use in production is not advisable. At this stage, it is necessary to determine the possible ways of transferring the identified valuable traits to a genotype that is more adapted to industrial culti vation or to obtain a new improved form on the basis of the amphidiploid. At present, there is only one paper concern ing crossability of this tetraploid with certain samples of wheat, Aegilops and rye (Navruzbekov, 1984). It indicates that the best seed setting and germination is observed under hybridization with a cultivated emmer (over 80 %). With bread wheat this parameters are slightly lower. Seed setting under crossing with rye and various samples of Aegilops up to 10 %. These studies indicate the possibility of car rying out the breeding works with this tetraploid. Newly developed allopolyploids are of considerable interest for basic research. They simulate the processes of evolution occurring through polyploidy that is resulted in a simul taneous restructuring of the interactions between many genetic networks that existed in separate genomes. These processes largely determine both the adaptive capabilities and the future of the new genotype. The combination of the genomes A and B in cereals was very successful for the formation of a cytogenetically stable tetraploid, which adapted to environmental conditions and later gave rise to a large spectrum of species with the genomic formula BBAA. Later, the addition of the D genome was also evolutionarily favorable and gave the hexaploid T. aestivum, which spread widely across the globe.
A comprehensive study of artificial allopolyploids ob tained in previous years, including by means of new mo lecular genetic technologies, will make it possible to master new genetic resources for practical purposes and create new genetic models for studying the patterns of plant evolution through polyploidy. The results obtained in this work indi cate the prospects for a deeper genetic and breeding study of the Navruzbekovs' allotetraploid.