Physiological and genetic features of the rate of development of modern varieties of soft wheat (Triticum aestivum L.)

Fayt V., Hubich О. , Nahuliak O., Balashova І., Fedorova V., Zelenina H. 1, 3 – 6 Selection and Genetic Institute — National Center of Seed Science and Variety Studies, 3 Ovidiopolska Doroha, Odesa, 65036, Ukraine, 2 Odesa National Agrarian University, 13 Panteleimonivska Str., Odesa, 65012, Ukraine e-mail: faygen@ukr.net, lgubich97@gmail.com, ibalashova@ukr.net, fedgen@ukr.net, gzelenina@ukr.net ORCID: 0000-0001-9994-341X, 0000-0002-1002-9697, 20000-0001-7855-1134, 0000-0001-5251-9611, 00000002-76924117

In numerous recent scientific and promotional publications on alternative wheat in Ukraine, unfortunately, there is no complete research on the physiology of development (ontogenesis) and the genetics of alternative genotypes [13,14,15]. This often does not determine the difference between true alternative and short-term winter varieties or spring cold-resistant genotypes.
The purpose of the study was to assess the levels of vernalization needs, photoperiodic sensitivity, frost resistance, and identification of bread wheat by alleles of Vrn-1 and Ppd-1 genes.
Materials and methods of researches. Varieties Afina, Lastochka, Pallada, Yarka, L897Я23 (Krasnodar Scientific Research Institute of Agriculture named after P.P. Luk'ianenko, Russia), Khutorianka, Zymoiarka (Institute of Plant Physiology and Genetics National Academy of Sciences of Ukraine, Kyiv), Solomiia (Kherson State Agrarian Univercity, Kherson), Shestopolivka (farm "BOR") were used as starting breeding material which are described by their authors as alternative [16][17][18][19]. Also were used variety Demir 2000 (which, according to the description of the Center for Genetic Resources of Ukraine, is alternative) and F2 populations from the diallelic crossings of this varieties and crossing with almost isogenic monogeneously dominant by the Vrn-1 genes of the Mironovskaya 808 or Skorospelka 3b populations. In various experiments, the winter variety Borvii of the PBGI-NCSCI breeding and alternative almost isogenic breeding line of the Mironovskaya 808 variety according to the Vrn-B1a gene (hereinafter Mironovskaya 808 Vrn-B1a) were used as a controls.
Seeds of varieties and F2 populations from diallelic crossing were germinated at room temperature. Five-day seedlings were vernalized in the special chamber at + 2 °C and for a duration of day 12 hours: for varieties -40, 30, 20, 10 days; for F2 populations -40 days. After the end of the vernalization, the seedlings were planted under artificial conditions in light phytotron chambers, ten plants in 5-liter vessels and one part of each variety was grown under a 16hour prolonged day and the rest and F2 populations -under a 12-hour shortened day. At the same time, not vernalizated five-day seedlings were planted under prolonged and shortened days. The reaction to the vernalization of a particular sample was determined by comparing the average date of its earing in two adjacent variants of the previous vernalization. The difference between the average duration of the period before earning of the variety after the vernalization under growing on the shortened and elongated day characterized its level of photoperiodic sensitivity.
Genetic analysis of half-diallelic F2 hybrids by photoperiodic sensitivity was performed by this method [20]. The distribution of F2 populations into the phenotypic classes of plants, which sooner and later earned under shortened day, was carried out according to the date of earning of the first plant of the Mironovskaia line 808 Vrn-B1a. Multiplex PCR with gene-specific primers was used to determine alleles of Ppd-D1a and Ppd-D1b [21].
Hybridological analysis by type of development and distribution of F2 populations into the phenotypic classes of spring and winter plants was performed by the this method [22], with the only difference being that the plants were grown in 5-liter pots (ten plants per pot) at the growing area.
Frost resistance was assessed by freezing seedlings at -12 0 C and using the "beams" method at -16 0 C [23] in the freezing chamber. Winter hardiness was assessed in the field by accounting for plants in autumn and in spring season. Dates of spiking of individual plants were marked in the phytotron and at the vegetation area during the growing season for determine the period before earning. In the field, the date of spiking was marked visually in the presence of 75% of the spiked plants in the area.
Statistical processing of the obtained results was carried out according to conventional methods of variance and correlation analysis, criterion  2 [24].
Research results. Comparison of the period duration before the spiking of alternative varieties plants after vernalization of different duration (term) under prolonged (PD) and shortened (SD) day of the climatic chambers of the phytotron indicated a significant influence of the genotype on the specified trait (Table 1). If as a criterion for evaluating the reaction to vernalization use the fact of earning or not earning a specific genotype after pre-vernalization of a certain duration, then the studied varieties can be divided into two groups. Varieties of the first group Afina, Zymoiarka, Lastivka, Pallada, Solomiia, Khutorianka, Yara, L897Я23 and control alternative line Mironovskaya 808 Vrn-B1a spiked by 41.6 days (Afina) -89.9 days (Pallada) in conditions of PD regardless of the previous vernalization of different duration (40-10 days) and even in its absence. In the conditions of SD, the earning of the varieties Afina, Zymoiarka, Lastivka, Pallada, Solomiia, Khutoryanka, Yara, L897Y23 were noted at 43.1 days (Afina) -97.5 days (Lastivka) both in the variant without vernalization, and after 10-40 days of the previous vernalization. However, in the absence, as well as in the vernalization of 10 and 20 days, the spiking of plants of the almost isogenic line Mironovskaya 808 Vrn-B1a (alternative genotype, control) was not observed in the conditions of SD. The gradual reduction of the vernalization duration to 30, 20, 10 days and to its complete absence contributed to the increase in the duration of the spiking period by 2.1 days (Solomiia) -37.5 (Pallada) days in the variance without vernalization compared to the 40-day vernalization in the conditions PD and 8.3 days (Solomiia) -41.3 (Yara) days in the conditions of SD. However, these genotypes differ in response to vernalization. Thus, in the artificial conditions of the phytotron chambers, the variety Solomiia almost did not respond to the vernalization by the acceleration of development (shortening the duration of the period to the earning). Zymoyarka and Khutoryanka varieties are smaller, and Lastivka -to a greater accelerated the development after 10 days of vernalization. The Afina and Yara varieties and the L897Я23 line responded significantly even to the 20-day vernalization. Of all the genotypes, the Pallada variety and the Mironovskaya 808 Vrn-B1a control line, which responded for 30 days of vernalization with the acceleration of the ear, are slightly different.
The varieties of the second group -Shestopalivka and Demir 2000 -responded to the vernalization similar to the control winter variety Borvii. The need for vernalization of the Borvii was 40 in the conditions of PD, and 30 days -of SD . Earning of varieties Shestopalivka and Demir 2000 were noted both in the PD and in the SD only after 30 days of vernalization. At the same time, in both cases, the 40-day vernalization significantly accelerated the spiking of these varieties by 16.9 -21.3 and 18.4 -19.7 days, respectively. Hybridological analysis of F2 populations from test-cross alternative varieties with monogeneously dominant Vrn-A1a or Vrn-B1a or Vrn-D1a genes by type of development (spring and winter) revealed five groups of varieties with different genetic control of type development (Table 2). In particular, in the genotypes Khutorianka and Zymoiarka, two genes Vrn-A1a and Vrn-B1a of type development were identified. The alternative type of development of other varieties that have been spiked in the previous experiment without vernalization is controlled by a single gene. Solomiia has only the dominant allele Vrn-A1a, Pallada, Yara, as well as the control line Mironivska808 Vrn-B1a, -only the allele Vrn-B1a, Lastivka, Afina and line L897Я23 -only the allele Vrn-D1a. The Shestopalivka and Demir 2000, as well as the winter Borvii, which spiked only after 30-40 days vernalization, are carriers of only recessive alleles of all three genes of the orthologous series Vrn-1 (genotype Vrn-A1b Vrn-B1b Vrn-D1b).

The duration of the period before spiking of alternative varieties in the conditions of prolonged (PD) and shortened (SD) days after temporal vernalization, days
Reducing the day duration to 12 hours led to an increase in the period duration of the spiking of all varieties, regardless of the duration of the pre-vernalization (Table 1). In Demir 2000 and Pallada varieties, the conditions of the shorted day even contributed to acceleration of the development rate by 0.5 -1.8 days with 40-, 30-day vernalization. According to the degree of spiking delay in the conditions of SD compared with PD varieties can be divided into two groups. Afina, L897Y23, Pallada, Solomiia, Yara, which did not respond or spiked under conditions of SD with a delay up to 3.2 days with 40 days vernalization and up to 15.2 days in the variant without vernalization can be characterized as low sensitive to the photoperiod [25].
Shepopalivka and Demir 2000 can also be included to this group of varieties, as well as control winter Borvii. The differences between PD and SD variants for these three genotypes after 40 days vernalization were 8.8, 1.8 and 15 days, respectively. The varieties of the second group (Lastivka, Khutorianka, Zymoiarka) showed significantly higher photoperiodic sensitivity. The difference in the duration of the period before spiking between the variants of PD and SD of these genotypes after 40 days vernalization was 9.0 -11.3 days. With the decrease in the duration of the pre-vernalization, the response to the 12-hour day increased in variety Lastivka up to 21.4 -29.5; Khutorianka -up to 27.4 -34.1 and Zymoiarka -up to 36.8 -41.6 days. This value is like to that in the highly sensitive line Mironivska 808 Vrn-B1a, whose developmental delay in the conditions of SD compared to PD at 40-30 days vernalization was 39.2 -45.7 days.

The need for vernalization (NV), photoperiodic sensitivity (PhPS) and genotypes of modern alternative varieties by genes of the orthologous series Vrn-1 and Ppd-1
Genetic analysis of photoperiodic sensitivity (early : late plants) of F2 populations derived from crossing alternative varieties according to the half-diallellic scheme confirmed the absence of differences in the genetic control of photoperiodic sensitivity, as in the group of low sensitive to photoperiod and also in group of high sensitive to photoperiod. However, splitting in F2 populations into earler or later spiking plants in most combinations of the crossing of 4 high sensitive to the photoperiod varieties (Lastivka, Khutorianka, Zymoiarka and Mironivska 808 Vrn-B1a control line) on the one hand, with 8 varieties sensitive to the photoperiod (Demir 2000, Solomiia, Pallada, Yara, Afina, Shestopalivka, L897Y23, and winter Borvii), on the other hand, significantly corresponded to that with differences of parents by one gene.
The diversity of alternative varieties by alleles of Vrn-1 and Ppd-1 genes did not significantly affect on differences in winter-hardiness genotypes under field conditions in 2015/2016 (Table 3) The freezing of seedlings at -12°С revealed significant differences between frost varieties. The duration of the day during quenching has a significant impact on the formation of frost resistance of the two hands. In general, the level of frost resistance of varieties during hardening under PD conditions (33% of living plants) is lower than that under hardening under DM conditions (47% of living plants). The freezing of seedlings at -12С revealed significant differences between alternative varieties by frost resistance. The duration of the day during quenching has a significant impact on the formation of frost resistance for alternative varieties. In general, the level of frost resistance of varieties during hardening under PD conditions (33% of live plants) is lower than that under hardening under SD conditions (47% of live plants). Estimates of frost resistance of alternative varieties in the two hardening variants do not significantly coincide (r = + 0.71). As a result, some varieties (L897Y23, Afina, Pallada, Shestopalivka) formed higher frost resistance by 3 -23% during the hardening of plants under PD conditions. Other varieties (Solomiia, Demir 2000, Lastivka, Mironivska 808 Vrn-B1a), on the contrary, showed a higher resistance to negative temperatures by 13 -30% when hardened under conditions of SD. Regardless of the day duration during hardening, higher frost resistance is inherent in winter varieties Borvii and Shestopolivka. Spring varieties with Vrn-B1a or Vrn-D1a genes were lower in winter by 10 -42%, and the carriers of the Vrn-A1a gene or Vrn-B1a gene were completely died. High level of frost resistance (71%) of the control line Mironovskaya 808 Vrn-B1a is close to the level of the control winter variety Borvii (85%) when hardening in the conditions of SD (actually in the field conditions of Ukraine autumn and winter day is less than 12 hours), so only the interaction of the dominant Vrn-B1a gene with the recessive alleles Ppd-A1b, Ppd-B1b and Ppd-D1b contributes to significant delay in the development under shortened natural day of autumn and winter and, as a consequence, the formation of better winter hardeness and frost resistance of alternative varieties.

Winter hardiness and frost resistance of plants in the tillering phase at -16 ° C and seedlings at -12 °C during quenching of alternative varieties under prolonged (PD) and shortened (SD) days, % of living plants
Alternative varieties differ significantly in the duration of period before spiking, both in autumn sowing and in sowing in December, February or March (Table 4). At the same time, this feature was significantly influenced by the sowing period in the conditions of 2012/2013 and 2014/2015. Afina, Yara, Pallada, L897Я23, Khutorianka, Solomiia, Lastivka, Mironivska 808 Vrn-B1a spiked in both years of study regardless of the sowing period. However, under the shortening of the duration of the period before the spiking of the varieties under sowing in December, February, March and, especially, in April, compared with the autumn sowing, the calendar dates of spiking in winter and spring sowing come much later. Even with sowing in February (19)(20)(21)(22)(23)(24)(25), the spiking of these genotypes was observed in 2013 with early warm spring by [13][14][15][16][17], and in 2015 -with late cold spring -by 8.3 -13.0 days later in compared to autumn sowing.
The main reason for this is the fact that all these genotypes are selected for and under the conditions of autumn sowing, perhaps even slightly later than optimal, but not in spring. Eating late will have a negative impact on the formation of elements of the crop structure and grain filling in the face of increasing drought in most years in the steppe. Spiking late will have a negative impact on the formation of elements of the crop structure and grain formation under increasing drought in most years in the steppe.
Demir 2000 and Shestopalivka varieties, as well as control winter Borvii, were sown on February 25, 2015, and the plants were spiked. In 2013, the aforementioned three varieties did not spiked at sowing on March 19 and later sowing dates, and Demir 2000 did not spiked at sowing on March 5. Probably, in these variants of the experiment, the duration of natural vernalization was insufficient to move to the generative path of development of these three genotypes. Unlike other spring varieties, the Zymoiarka responded to the spring 2013 sowing time, similar to winter Borvii. Under the sowing date March 19 and April 2, the plants of the variety Zymoiarka did not even form a tube, although in the phytotron its were spiked without prior vernalization both in the conditions of prolonged and shortened days. 4 In the south of Ukraine, alternative varieties have a rather high level of overwintering 87.6%. Hardening in the conditions of a shortened day helps to increase the level of frost resistance of seedlings of alternative varieties. The presence in the genotype of the varieties of two genes at once, Vrn-A1a and Vrn-B1a (Khutorianks, Zymoiarka) or only Vrn-A1a (Solomiia), and in some variants, only Vrn-D1a contributes to a significant reduction of frost resistance of seedlings and winter hardiness of alternative varieties.
Alternative varieties differ significantly in the duration of the period before spiking, regardless of the sowing period (October, December, February, March and April). At the same time, winter and spring sowing periods contribute to the shifting of spiking to much later calendar times compared to autumn sowing, which may negatively affect on yield formation of the alternative varieties.