Development and investigation of common wheat lines of winter cultivar Bezostaya 1 with combinations of dominant alleles of VRN-1 loci

Гены чувствительности к яровизации (VRN) являются основ­ ными генетическими системами, определяющими продол­ жительность вегетационного периода в целом, а также длительность основных этапов органогенеза. К настоя­ щему времени для локусов VRN-1 описан ряд аллелей и разработаны аллель­специфичные праймеры, позволяю­ щие проводить быструю идентификацию аллельного состава у сортов и линий мягкой пшеницы. Установлено неодинаковое влияние различных аллелей локусов VRN-1 на продолжительность вегетационного периода, однако исследований, касающихся влияния комбинации различ­ ных аллелей на время колошения, недостаточно. Получе­ ние полных наборов всех возможных генотипов по разным аллелям доминантных генов VRN на одном генетическом фоне необходимо для более глубокого изучения генетиче­ ских эффектов генов VRN на продолжительность вегета­ ционного периода и отдельных фаз развития, а также на продуктивность. Поскольку большинство современных сортов России несет доминантные аллели двух генов VRN (Vrn-A1a и Vrn-B1a или Vrn-B1c), нами была поставлена за­ дача – получить линии, сочетающие доминантные аллели Vrn-A1a с Vrn-B1a и Vrn-B1c на генетическом фоне озимого сорта Безостая 1 (Без1 Vrn-A1a/Vrn-B1a и Без1 Vrn-A1a/ Vrn-B1c). С использованием известных аллель­специфич­ ных праймеров для локусов Vrn-A1 и Vrn-B1 в поколении F2 были выделены гомозиготные растения. У полученных линий с комбинацией двух доминантных аллелей локусов VRN-1 достоверно уменьшалась продолжительность перио­ да «кущение – первый узел», который представляет собой ключевой этап, определяющий продолжительность веге­ тационного периода и периода от всходов до колошения по сравнению с изогенными линиями по сорту Безостая 1 с доминантными аллелями Vrn-B1a и Vrn-B1c. По сравнению с изогенной линией с доминантным аллелем Vrn-A1a у полу­ ченных линий также произошло уменьшение продолжи­ тельности этих фаз развития, однако различия были недо­ стоверны. Достоверных различий по продолжительности VRN genes, determining wheat sensitivity to vernalization, are the main genetic system that defines the duration of the entire growing period and the durations of the main organogenesis phases. To date, several alleles have been described for VRN-1 loci, and allele­specific primers have been developed that allow rapid identification of allelic spectra in common wheat vari­ eties and lines. The unequal influence of different alleles of VRN-1 loci on the duration of the growing period has also been shown; however, there is little information on the effect of the combination of different alleles on heading time. In develop­ ing genotypes having different alleles of dominant VRN genes on the base of the same genetic background, it is necessary to study the genetic effects of VRN genes on the duration of the growing season and the individual developmental phases, as well as on productivity. Most varieties presently grown in Russia carry the dominant alleles of two VRN-1 genes: Vrn-A1a and Vrn-B1a or Vrn-B1c; thus, the task was to create lines com bining the dominant alleles of Vrn-A1a with Vrn-B1a and Vrn-B1c against the genetic background of the winter variety Bezostaya 1 (Bez1 Vrn-A1a/Vrn-B1a and Bez1 Vrn-A1a/Vrn-B1c). Homozygous plants were isolated in the F2 generation by using known allele­specific primers for the Vrn-A1 and Vrn-B1 loci. The durations of the tillering–first node period, which is the key stage determining growing duration, and the period from shoots to heading were significantly reduced in lines with a combination of two dominant alleles of VRN-1 loci compared to isogenic lines of Bezostaya 1 with the dominant alleles Vrn-B1a and Vrn-B1c. The duration of these developmental phases also decreased in the obtained lines as compared to the isogenic line containing the dominant Vrn-A1a allele, but the differences were not significant. No substantial differences were found in the duration of other growing phases in lines with two dominant alleles of the VRN-1 loci as compared to isogenic lines of Bezostaya 1.

VRN genes, determining wheat sensitivity to vernalization, are the main genetic system that defines the duration of the entire growing period and the durations of the main organogenesis phases.To date, several alleles have been described for VRN-1 loci, and allelespecific primers have been developed that allow rapid identification of allelic spectra in common wheat vari eties and lines.The unequal influence of different alleles of VRN-1 loci on the duration of the growing period has also been shown; however, there is little information on the effect of the combination of different alleles on heading time.In develop ing genotypes having different alleles of dominant VRN genes on the base of the same genetic background, it is necessary to study the genetic effects of VRN genes on the duration of the growing season and the individual developmental phases, as well as on productivity.Most varieties presently grown in Russia carry the dominant alleles of two VRN-1 genes: Vrn-A1a and Vrn-B1a or Vrn-B1c; thus, the task was to create lines com bining the dominant alleles of Vrn-A1a with Vrn-B1a and Vrn-B1c against the genetic background of the winter variety Bezostaya 1 (Bez1 Vrn-A1a/Vrn-B1a and Bez1 Vrn-A1a/Vrn-B1c).
Homozygous plants were isolated in the F 2 generation by using known allelespecific primers for the Vrn-A1 and Vrn-B1 loci.The durations of the tillering-first node period, which is the key stage determining growing duration, and the period from shoots to heading were significantly reduced in lines with a combination of two dominant alleles of VRN-1 loci compared to isogenic lines of Bezostaya 1 with the dominant alleles Vrn-B1a and Vrn-B1c.The duration of these developmental phases also decreased in the obtained lines as compared to the isogenic line containing the dominant Vrn-A1a allele, but the differences were not significant.No substantial differences were found in the duration of other growing phases in lines with two dominant alleles of the VRN-1 loci as compared to isogenic lines of Bezostaya 1. T he duration of the growing period in common wheat is an important adaptive trait, which determines plant productivity and resistance to biotic and abiotic stress factors: drought, low temperatures, diseases, and pests (Stelmakh, 1990;Worland, 1996;Snape et al., 2001;Cockram et al., 2007;Kamran et al., 2014).The main genetic systems that initiate the transition of wheat plants from the vegetative to the generative stage of development are the genes for vernalization (VRN ) and photoperiod sensitivity (PPD) (Snape et al., 2001;Kamran et al., 2014).

Plant gene pool and breeding
The main loci that determine sensitivity to the photoperiod (PPD-1) in wheat were mapped on the short arms of the second homoeologous group chromosomes: 2D, 2B, and 2A (Worland et al., 1998;Snape et al., 2001).
Common wheat has three homoeologous VRN-1 loci: VRN-A1, VRN-B1, and VRN-D1.They are located on the long arms of chromosomes 5A, 5B and 5D, respectively (Snape et al., 2001;Yan et al., 2003).The spring habit of development is determined by the presence of at least one dominant allele; the presence of all the three recessive vrn alleles defines the winter habit of wheat varieties.Cultivars bearing the dominant VRN-A1 allele are completely insensitive to vernalization, whereas cultivars with the dominant alleles VRN-B1 and VRN-D1 are slightly sensitive to vernalization (Pugsley, 1971).
For VRN-1 loci (VRN-A1, VRN-B1, VRN-D1), a series of different alleles was described, mutations resulting in changes in the structure of these alleles were cloned and characterized, and allele-specific primers were designed for rapid genotyping of common wheat varieties and lines (Yan et al., 2004;Fu et al., 2005;Milec et al., 2012;Shcherban et al., 2012).It was found that the dominant alleles of these loci had insertions or deletions in the promoter and the first intron.
It is known that wheat cultivars bearing the dominant Vrn-A1a allele are the earliest ripening.The Vrn-A1b allele, on the contrary, increases the duration of the heading period (Koval, Goncharov, 1998;Kamran et al., 2014).Genotypes with the dominant Vrn-B1c allele ripe earlier than genotypes with Vrn-B1a allele (Efremova et al., 2011).
It has been shown that various combinations of alleles of the VRN and PPD loci differently affect the lengths of the growing period and phases of plant development, as well as productivity.The earliest ripening genotypes are those with three dominant genes (VRN-A1, VRN-B1, VRN-D1); however, they tend to have lower yields, and, for this reason, they are seldom used in breeding.The habit of most Russian varieties (including Siberia), Europe, North America and Australia is determined by two dominant genes VRN-A1 and VRN-B1.These varieties ripe earlier, and they are more productive than varieties with one VRN gene, because such allelic composition of the VRN-1 genes allows the plants to sustain late spring and early autumn frosts (Stelmakh, 1998;Goncharov, 2004;Efremova et al., 2016).Genotypes with the dominant VRN-D1 gene are advantageous in regions with extreme conditions such as drought and high temperature during grain filling (Stel makh, 1993).
Isogenic lines created on the basis of winter varieties with known alleles of PPD genes play an important role in the detailed analysis of the VRN genes, which control the duration of the growing period (Stelmakh, Avsenin, 1983;Voronin, Stelmakh, 1985;Koval et al., 2001;Efremova et al., 2011).Study of such lines permits one to single out the effect of alleles of each of the VRN genes or different alleles of the same gene, as well as of their combinations, on the duration of the heading period (Merezhko, 1994;Koval, Goncharov, 1998;Stelmakh et al., 2000;Efremova et al., 2011), the duration of the main stages of organogenesis (Voronin, Stelmakh, 1985;Emtseva et al., 2013), and productivity and fitness (Voronin, 1988).In addition, isogenic lines are suitable models for studying the primary structure and expression of VRN alleles (Loukoianov et al., 2005;Shcherban et al., 2013).
A series of near-isogenic lines of winter Bezostaya 1 cv.(Bez1) possessing one dominant Ppd-D1a gene (determining weak sensitivity to day length (Worland et al., 1998)), with two different alleles of the VRN-B1 gene (Efremova et al., 2011), as well as on the VRN-A1a and VRN-D4 loci were raised at the Institute of Cytology and Genetics, Novosibirsk.In these lines, different alleles of the dominant VRN-B1 gene (Vrn-B1a and Vrn-B1c) were in the same genetic background, and this fact allowed more accurate identification of differences in the duration of the period from shoots to heading.With these lines, it was shown that the Vrn-A1a allele determined earlier heading than the Vrn-B1c and Vrn-B1a alleles, and the Vrn-B1c allele, in turn, reduced the duration of the period from shoots to heading as compared to the Vrn-B1a allele (Emtseva et al., 2013).However, since most modern commercial varieties in Russia carry the dominant alleles of two genes, Vrn-A1a and either Vrn-B1a or Vrn-B1c, it should be found out how the combination of different alleles of the two genes can affect the time before heading.Therefore, it is advisable to obtain lines with two alleles of these genes against the genetic background of Bez1 cv.This would allow a more detailed study of the contribution of VRN alleles to early maturity and productivity.It should be noted that all previously created isogenic lines and lines with two or three dominant VRN genes were obtained without taking into account the presence of alleles of the VRN genes and their role in controlling heading time.Therefore, the raise of complete sets of all possible genotypes for different alleles of the VRN-1 loci against the same genetic background is necessary for a more comprehensive study of the genetic effects of the VRN loci.The objectives of this work were (1) to obtain lines combining two different VRN-1 loci (Vrn-A1a/Vrn-B1a and Vrn-A1a/ Vrn-B1c) in one genotype on the base of previously obtained isogenic common wheat lines with dominant Vrn-A1a, Vrn-B1a and Vrn-B1c for the winter variety Bez1 (Efremova et al., 2011) and (2) to determine the effect of the combination of alleles the duration of individual development phases under the conditions of the forest-steppe zone of the Novosibirsk region.
DNA isolation and PCR.Genomic DNA was isolated from leaves of adult plants after digestion with proteinase K according to a previously described method (Edwards et al., 1991).All amplification reactions were carried out in a 25 μl volume containing 50-100 ng genomic DNA, reaction buffer (67 mM Tris HCl pH 8.8, 1.5 mM MgCl 2 , 18 mM (NH 4 ) 2 SO 4 , 0.01 % Tween 20), 0.2 mM each dNTP, 0.25 μM primer pair, and 1 unit of Taq polymerase.
Amplification of DNA was carried out according to our domestic protocols.The nucleotide sequences of the primers and PCR conditions are shown in Tables 2 and 3. Amplification products were resolved by electrophoresis in 1.5 % agarose gel in 1×TAE buffer with the presence of ethidium bromide.The gels were photographed under UV illumination with the Doc-Print II documentation system for gels (Vilber Lourmat).
Study of the duration of developmental phases.The durations of individual phases of development in the lines of common wheat with dominant alleles of the VRN-1 loci were studied in an experimental field of the Institute of Cytology and Genetics SB RAS at natural daylength (At 55°2ʹ N, 82°56ʹ E, the daylength in May-August is 17 h.) in 2017.The following phases of development were recorded: shooting, the emergence of the third leaf, tillering, the emergence of the first node, stem elongation, and heading.Tillering was recorded when the second shoot began to depart from the main shoot.The first node phase was recorded when the first node was palpable on the main shoot at a height of 1 cm above the soil surface.The stem elongation phase was recorded when the first node rose to a height of about 5 cm and the second node began to separate from it.Heading was recorded when the spike was completely out of the flag leaf.The dates of developmental stages were recorded for each plant individually, and the mean values were calculated.Twenty-five plants of each line were studied.
The statistical evaluation of the data was carried out with Microsoft Excel 2013.The statistical significance of the differences between mean values was assessed by Student's t-test (Rokitskii, 1974).

Results and discussion
Raise of common wheat lines of winter variety Bez1 with a combination of dominant alleles of the VRN-1 loci.Two near-isogenic lines, i: Bez1Vrn-B1a and i: Bez1Vrn-B1c, were crossed to the isogenic line i: Bez1Vrn-A1a.The resulting F 1 hybrids were self-pollinated.Homozygous plants with two dominant alleles of the VRN genes, Vrn-A1a/Vrn-B1a and Vrn-A1a/Vrn-B1c, were selected with known allele-specific primers for the VRN-A1 and VRN-B1 genes presented in Table 2 in the F 2 generation.Amplification of the dominant allele Vrn-A1a with the allele-specific primers VRN1AF and VRN1R revealed two fragments approximately 650 bp and 750 bp in size, and in the PCR of the recessive vrn-A1 allele one fragment of about 500 bp was obtained.Correspondingly, three fragments were amplified in heterozygous plants.
Twenty-eight plants were analyzed in the Vrn-A1a/Vrn-B1a combination and nineteen, in Vrn-A1a/Vrn-B1c.In the Vrn-A1a/Vrn-B1a combination, fourteen plants with two dominant alleles were isolated, five of them being homozygous, two with the dominant Vrn-A1a allele and recessive vrn-B1, ten plants with the dominant Vrn-B1a allele and recessive vrn-A1, and two winter plants with recessive alleles vrn-A1 and vrn-B1.In the Vrn-A1a/Vrn-B1c combination, nine plants with two dominant alleles were isolated.Four of them were homozy-gous, one plant had the dominant Vrn-A1a and the recessive vrn-B1 allele, eight plants had the dominant Vrn-B1a allele and recessive vrn-A1, and one plant showed the winter habit.
Thus, by using molecular markers already in the F 2 generation, we managed to isolate homozygous plants with two dominant alleles of the VRN-A1 and VRN-B1 genes.This approach is the most effective in isolating genotypes for a target gene, as it shortens the time for selecting the desired genotype significantly and permits one to determine the presence of VRN loci at early stages of plant development.At present, marker-assisted selection complements traditional methods, and it is widely used to introgress target genes and create near-isogenic lines, especially in the breeding of lines with genes that control the resistance to various types of stress (Leonova, 2013).
Determination the effect of the combination of dominant alleles in the VRN-1 loci on the duration of individual developmental phases in the forest-steppe zone of the Novosibirsk region.We studied the duration of individual developmental phases of the common wheat lines with two dominant alleles of the VRN-1 loci obtained in the present work, Bez1 Vrn-A1a/Vrn-B1a and Bez1 Vrn-A1a/Vrn-B1c, in an experimental field of the Institute of Cytology and Genetics with natural daylength in the spring of 2017.Isogenic lines of Bez1 with dominant alleles Vrn-A1a, Vrn-B1a, and Vrn-B1c were used as controls.The results are presented in Table 4 and Fig. 3.
Isogenic line i: Bez 1Vrn-A1a headed on day 42.Plants of isogenic lines with dominant alleles in the VRN-B1 locus headed later than the isogenic line with Vrn-A1a.The difference between the isogenic lines with the Vrn-B1a and Vrn-B1c alleles in this experiment was four days.The isogenic line with the Vrn-B1a allele headed on day 49, and the line with Vrn-B1c, on day 45.This result is consistent with the previous work (Emtseva et al., 2013).In the lines obtained in this work with the combination of two alleles of the VRN-1 loci (Bez1 Vrn-A1a/Vrn-B1a and Bez1 Vrn-A1a/Vrn-B1c), the period from shooting to heading was forty days, which was two days less than in the isogenic line with the Vrn-A1a allele (differences are not significant), and significantly shorter than in the isogenic lines i: Bez1 Vrn-B1a (nine days shorter) ( p ≤ 0.01) and i: Bez1 Vrn-B1c (five days shorter) ( p ≤ 0.05).
It was shown that varieties with two alleles, Vrn-A1a and Vrn-B1c, headed earlier than varieties with one dominant allele, Vrn-A1a or Vrn-B1c (Efremova et al., 2016).The durations of the main phases of development, which ultimately determine the duration of the period from shooting to heading, are shown in Fig. 3.The duration of the shoots -tillering period was approximately the same in all lines examined, 11-12 days.The length of the first node -heading period, which was 16-18 days, did not vary significantly either.Significant differences were observed only in the tillering -first node period.For lines with two dominant alleles of VRN-1 loci (Bez1 Vrn-A1a/Vrn-B1a and Bez1 Vrn-A1a/Vrn-B1c), the tillering-first node phases were 11 and 10 days, respectively.In comparison to the isogenic line with the Vrn-A1a allele, for which this phase lasted 13 days, the differences were insignificant.For isogenic lines i: Bez1Vrn-B1a and i: Bez1Vrn-B1c, the durations were 20 and 18 days, respectively.The combination of the dominant Vrn-A1a allele with Vrn-B1a and Vrn-B1c in the genotype resulted in a decrease of the tillering -first node phase compared to the isogenic lines i: Bez1Vrn-B1a and i: Bez1Vrn-B1c: nine and eight days respectively ( p ≤ 0.001).Several studies of the duration of developmental phases of substituted and isogenic common wheat lines with different alleles of the VRN-1 loci show that the key stage determining the duration of the vegetation period is just the length of the tillering-first node period (Voronin, Stelmakh, 1985;Pánková, Košner, 2004;Emtseva et al., 2013).

Table 1 .
Isogenic lines of Bez1 cultivar used to obtain lines with two dominant alleles of the VRN-1 loci

Table 3 .
PCR schedules with allelespecific primers for the VRN-A1 and VRN-B1 genes

Table 2 .
Primers for identifying alleles of the VRN-A1 and VRN-B1 genes in common wheat lines

Plant gene pool and breeding Vavilov Journal of Genetics and Breeding
• 2018 • 22 • 8

Table 4 .
Duration of developmental phases in common wheat lines with dominant alleles of theVRN-1 loci (field, 2017)