The Strategy for Marker-Assisted Breeding of Anthocyanin-Rich Spring Bread Wheat (Triticum aestivum L.) Cultivars in Western Siberia

In wheat, anthocyanin pigments can be accumulated in pericarp tissues (under control of the Pp genes) resulting in purple-colored grain. In the current study, a strategy, based on the use of molecular and morphological markers, was applied to create purple-grained bread wheat cultivars adapted to the West Siberian region. The breeding scheme started from crossing of recipients (elite cultivars and lines) with donor lines carrying dominant alleles of the complementary genes Pp3 and Pp-D1. The F2 hybrids passed three-step marker-assisted selection, and those having dominant Pp-D1Pp-D1Pp3Pp3 genotypes were backcrossed with the recurrent parents. The desired BC1F2-3 progenies were selected using morphological marker, while BC1F3 also passed through field evaluation. At this stage, 120 lines were selected and planted in individual 1 m2 “breeding nursery (BN) plots” for assessment of heading dates, duration of vegetation period, resistance to powdery mildew, stem and leaf rusts, protein and gluten content, as well as productivity. After these investigations, a total of 17 promising anthocyanin-rich purple-grained lines characterized by multiple resistance and having best yield/quality characteristics were finally candidates for selection of commercial cultivars adapted to the West Siberian climate and suitable for functional food production.


Introduction
In the late 1980s, a new concept in nutritional science, known as functional foods, emerged in Japan and spread over the world. The main idea was improving health benefits of foods to prevent a wide range of human diseases [1]. Research programs were initiated to determine nutritional value of food ingredients. Furthermore, breeding programs were developed to improve crop cultivars for producing functional foods. Special attention was paid to wheat as a main staple food in many countries. One promising direction for improving wheat is the use of breeding blue-and purple-grained cultivars with an increased content of anthocyanins, which are biologically active substances known for both plant protection against severe environments and positive effect on human health [2][3][4]. They possess antioxidant, anti-inflammatory, hypoglycemic, antimutagenic, and neuroprotective activities; they can prevent cardiovascular disease, metabolic syndrome, different types of cancer, as well as they are beneficial for eye vision [5,6]. For example, in vitro studies have demonstrated that wheat or rye bread fortified with anthocyanin-rich berries caused a delay in the glycemic response after encoding for regulatory factor with bHLH domain, was suggested as a gene candidate for Pp3 [31]. The functional difference of dominant and recessive alleles of this gene is due to the copy number variation of tandem repeat sequence in its promoter [32]. The complementary gene Pp-1, required for activation of anthocyanin biosynthesis having pleiotropic effect on pigmentation of other parts of wheat plant, encodes the regulatory factor from the R2R3-MYB family [33,34].
Using molecular-genetic mechanisms underlying the phenotype of the purple-grained wheat, we developed a strategy for accelerated breeding of anthocyanin-rich bread wheat cultivars. Here, we describe the strategy and demonstrate its successful application for development of purple-grained breeding lines of spring bread wheat, adapted to conditions of the West Siberian region, resistant to a wide range of unfavorable biotic stresses and abiotic environmental factors, and finally, suitable for modern responsible farming.

Plant Material and Breeding Strategy
To get anthocyanin-rich cultivars adapted to the West Siberian region we used marker-assisted transfer of dominant alleles of the regulatory genes Pp3 and Pp-D1 from donor lines to elite cultivars or breeding lines having potential for production in this region. MAS was applied twice for selection of genotypes Pp-D1Pp-D1Pp3Pp3 as follows: (1) in F 2 (by using molecular and morphological marker) for immediate crossing with a recipient line and (2) in BC 1 F 2 /F 3 (by morphological markers only) ( Figure 1).
The functional difference of dominant and recessive alleles of this gene is due to the copy numbe variation of tandem repeat sequence in its promoter [32]. The complementary gene Pp-1, required fo activation of anthocyanin biosynthesis having pleiotropic effect on pigmentation of other parts o wheat plant, encodes the regulatory factor from the R2R3-MYB family [33,34].
Using molecular-genetic mechanisms underlying the phenotype of the purple-grained wheat we developed a strategy for accelerated breeding of anthocyanin-rich bread wheat cultivars. Here we describe the strategy and demonstrate its successful application for development of purple grained breeding lines of spring bread wheat, adapted to conditions of the West Siberian region resistant to a wide range of unfavorable biotic stresses and abiotic environmental factors, and finally suitable for modern responsible farming.

Plant Material and Breeding Strategy
To get anthocyanin-rich cultivars adapted to the West Siberian region we used marker-assisted transfer of dominant alleles of the regulatory genes Pp3 and Pp-D1 from donor lines to elite cultivar or breeding lines having potential for production in this region. MAS was applied twice for selection of genotypes Pp-D1Pp-D1Pp3Pp3 as follows: (1) in F2 (by using molecular and morphological marker for immediate crossing with a recipient line and (2) in BC1F2/F3 (by morphological markers only ( Figure 1).  line i:S29PpD1Pp3 PF with cv Purple Feed (PF) introgressions in chromosomes 2A and 7D, carrying, respectively, the Pp3 and Pp-D1 genes, responsible for purple pigmentation of grain was used as a donor. The three-step selection of the homozygous F 2 plants for the target genes based on the coleoptile pigmentation assessment (i), donor-like homozygous SSR-loci linked to the Pp3 (ii), and Pp-D1 (iii) genes was carried out. The selected plants underwent backcrossing with the recurrent parents. The BC 1 F 2 generation with dark-red coleoptiles were self-pollinated to get BC 1 F 3 seeds that were collected separately from individual plants and the resulted families were evaluated in the field conditions. The individual plant from the families without segregation of the pericarp color with resistance to mildew and rusts diseases were selected, and then their grain quality and agronomic traits were characterized in BC 1 F 4 .
A description of the parental recipient materials is given in Table 1. Near-isogenic lines, i:S29Pp-1DPp3 PF (abbreviation-i:S29 PF ) and i:S29Pp-1DPp3 P (i:S29 P ), inherited dark-red coleoptile color and purple pericarp color from their donor spring breeding lines, Purple Feed (k-49426, Canada) and Purple (k-46990, Australia), respectively, and were used as donors of the anthocyanin grain pigmentation. The lines were both created in the genetics background of red-grained spring bread wheat cultivar Saratovskaya 29 (S29) having uncolored pericarp. Derivatives carrying both complementary Pp-D1 and Pp3 were created by [35]; the introgressions of purple-grained donors' chromatin were mapped to the short arm of chromosome 7D and the long arm of chromosome 2A, respectively ( Figure A1) [36].

Molecular Markers
Gatersleben wheat microsatellite (GWM) markers [39,40] flanking target genes Pp3 (on chromosome 2A) and Pp-D1 (on chromosome 7D) ( Figure A1 and Table 2), shown previously to be efficient in MAS-based development of wheat lines with desired Pp alleles [41], were used for screenings. DNA was extracted from young leaves of the parental and individual progeny plants, according to a procedure described by Plaschke et al. [42]. The conditions of PCR are described in Röder et al. [39]. Amplicons were separated through either 2% or 5% ACTGene agarose gels (ACTGene, Inc., Piscataway, NJ, USA).

Morphological Markers
Before planting F 2 and BC 1 F 2 plants, the coleoptile color was assessed by sowing seeds in Petri dishes and growing them for one week on filter paper at 20 • C under a 12 h photoperiod in a "Rubarth Apparate" growth chamber (RUMED GmbH, Laatzen, Germany). Dark-red coleoptile color was used as a morphological marker for Pp-D1 (according to Gordeeva et al., 2015 [41]). To select homozygous families in BC 1 F 2 /F 3 , purple pericarp color was used as the morphological marker. Phenotyping of BC 1 F 3 material and further selection were carried out from May 2018 in the breeding nursery (BN) and the experimental fields at the Omsk State Agrarian University (55 • Figure A2).

Marker-Assisted Selection
The crosses and number of plants obtained and assessed during MAS breeding (according to the scheme described in the Section 2 and illustrated in Figure 1) are given in Table 3.
Homozygous plants having the donor-like microsatellite alleles linked to the Pp3 gene (as an example, microsatellite genotyping results are given at Figure A3) were selected and further genotyped by the markers linked to the Pp-D1 gene. Finally, the homozygous plants having Pp-D1Pp-D1Pp3Pp3 genotype were selected and backcrossed with the recurrent parental cultivars (Table 3).
A low number of F 2 seeds was obtained in crosses of cv Aina with both donors and Tobol'skaya with i:S29 PF . Among their offspring, a large percentage of sterile bush-shaped plants was observed. This can be related to hybrid dysgenesis in these crosses.
The BC 1 F 1 hybrids were planted and self-pollinated to obtain BC 1 F 2 grains. Around 120 seeds of each combination were germinated and assessed for coleoptile color. Eighty seedlings with the dark-red coleoptile color were taken for each combination and further planted to obtained BC 1 F 3 seeds. The BC 1 F 3 seeds were collected separately from individual plants. The only seeds with purple color from individual plants were planted in particular 1 m 2 "BN plots", in the experimental field at the Omsk State Agrarian University. The individual BC 1 F 3 plants that were resistant to powdery mildew and rust fungi diseases from each plot, which did not have segregation by purple grain color trait, were collected to get BC 1 F 4 . In total, 120 BC 1 F 4 lines were selected for field testing.

Field Screening and Characterization of the Lines
The BC 1 F 4 lines were planted in individual 1 m 2 "BN plots" in the experimental field at the Omsk State Agrarian University, in May, and harvested in August 2019, and were assessed in respect to the heading dates, duration of vegetation period, resistance to powdery mildew, stem and leaf rusts, protein and gluten content, and productivity (Table A1). The data on the best lines evaluated in the 1 m 2 "BN plots" are presented in Table 4. The lines were different in grain protein content, ranging from 12.22% for the line 307 (background of cv Tobol'skaya), having low productivity 318 g/m 2 (less than all checks), to 19.18% for the line 264 (background of cv Element 22) characterized by higher productivity 546 g/m 2 than its recurrent parent cv Element 22 (Table A1). The highest productivity was shown by line 272 (background of STRU 0732) with a yield of 852 g/m 2 and 16.73% protein content. Unfortunately, this line was highly susceptible to stem rust infection.
The biotic resistance of bread wheat plants promotes photosynthesis and grain filling, which significantly improves grain quality and yield. For all the lines, the degree of resistance to powdery mildew varied among the genotypes. Most of the lines showed susceptibility to stem rust and resistance to leaf rust. Only nine lines were moderately resistant to stem rust as follows: late maturing lines 238, 239, 249, 255, 260, and 266 (background of cv Element 22), and medium maturing lines 275, 279, and 281 (background of BW49880). The lines derived from STRU 0732 showed the worst results and were discarded. The lines derived from cv Tobol'skaya were resistant to leaf rust, but susceptible to stem rust. Tobol'skaya is a medium late high-yielding cultivar of the steppe ecotype, characterized by high drought tolerance, good grain quality, and resistance to head smut. The lines derived from it, however, did not demonstrate high productivity and protein content.

Discussion
Breeding programs aimed at obtaining anthocyanin-rich wheat cultivars are under way in many countries. A major goal in breeding of purple wheat is to develop high-yielding cultivars adapted to regions of their cultivation, disease resistant, and having good baking and milling quality [45]. This time-consuming process took fourteen years to breed the first commercial purple hard bread wheat cv CDC Primepurple at the University of Saskatchewan (USask) in Canada by Dr. Pierre Hucl. [19]. In the current work, the accelerated breeding strategy based on the achievements by studying molecular-genetics mechanisms for regulation of anthocyanin biosynthesis in wheat grain [30] was proposed and successfully applied to breed promising anthocyanin, rich lines of spring bread wheat adapted to growing conditions of the West Siberian region. The region is characterized by extreme continental climate with frequent droughts (two to three years out of five years), often early summer droughts, severe cold and long winters (150-170 days), relatively short warm summers, low precipitation, sharp temperature fluctuations throughout the year, and even during the day [46]. The climatic conditions of Western Siberia are generally suitable for growing spring-type wheats. Despite the low agro-climatic potential, an abundance of sunlight and heat during the growing season makes the region applicable for production of high-quality wheat grain [47]. However, due to climate warming and, as a consequence, the emergence of aggressive races of fungal diseases, there are significant fluctuations in grain yield from year to year. Cultivars resistant to stem rust occupy about 10-15% of all wheat-cultivated areas in the region [48]. For effective genetic protection of wheat plants, the diversity of cultivated cultivars, in terms of resistance types and genes, plays an important role. The growing of resistant cultivars is the most effective and environmentally friendly approach, as this method eliminates the need to use fungicides and reduces the cost of production. Thus, the main directions of wheat breeding programs in this zone is the development of early maturing, highly productive, resistant to biotic and abiotic stresses spring wheat cultivars. As the purple-grained wheat isogenic donor lines i:S29 P and i:S29 PF having good levels of grain protein were susceptible to disease, and therefore had low yields [29], the breeding strategy was aimed at developing cultivars not only accumulating anthocyanins in grain but also resistant to fungal diseases and adaptive for growing conditions for this region.
Application of the molecular markers substantially facilitates the selection of the plants with the required genotype and finally speeds up the breeding process [49]. It is especially efficient in case of the grain pericarp color, which is inherited with maternal tissue and has a complex control by two complementary dominant genes, since only 6.25% of the F 2 progeny can be used for backcrossing. The breeding strategy for development of anthocyanin-rich cultivars was based on the transfer of the regulatory genes Pp3 and Pp-D1 controlling purple color of wheat grain and mapped on the chromosomes 2A and 7D, respectively, into background of elite cultivars and promising breeding lines adapted for growing conditions of Western Siberia. The critical point of the strategy was the selection of the homozygous plants for the Pp genes in the F 2 generation after crossing the donors with the recurrent cultivars/lines. However, before this critical step, it was possible to reduce the cost for molecular screening by selection of progeny with at least one dominant Pp-D1 allele based on a morphological marker, i.e., dark-red coleoptile color due to pleotropic effect of this R2R3-Myb encoding gene on the short arm of chromosome 7D. The use of this morphological marker reduced the progeny size to be analyzed with microsatellite markers by 25%. The remaining F 2 progeny was reduced by 75% based on analysis of microsatellites flanking the Pp3 gene. Finally, one-third of the plants selected with microsatellites flanking the Pp-D1 gene were suitable for backcrossing. These were Pp-D1Pp-D1Pp3Pp3 genotypes. Today instead of microsatellites flanking the Pp3 gene, one intragenic marker can be potentially used, since Jiang et al. [32] described promoter region polymorphism between Pp3 and pp3 alleles representing, respectively, expressed and silent TaMyc1 (syn. of Pp3) gene, known earlier from Shoeva et al. [31].
The resulting BC 1 F 3-4 lines were characterized by different intensities of the anthocyanin pigmentation of grains that depended on the genotype. The lightest grain color was in progeny derived from STRU0732, the darkest in BW49880-derived lines. The grain color of the latter line was even deeper than the donor line ( Figure A4). The difference can be explained by genes modifying the color. A probable explanation for BW49880-derived lines can also be an increased content of iron and zinc in the grain of this line affecting the formation of stable chelate bonds with anthocyanins [50,51].
We used a breeding scheme without backcross beyond BC 1 . This scheme which produced progeny with three quarters of the genetic material from recipient has been demonstrated earlier to be efficient to select proper recipient-like lines by consequent field screenings [52]. Especially, since Saratovskaya 29 background in the donor lines is a source of good quality and productivity. Therefore, the main purpose of the backcrossing was to overcome its sensitivity to diseases. The development of the rust resistant cultivars is the focus of the breeding program in Western Siberia and worldwide. The late maturing lines created in the background of cv Element 22 showed the high level of resistance to leaf and stem rusts, inherited from the cv Element 22 which is one of the few cultivars with effective group resistance against all local pathogen populations of stem and leaf rusts [48,53]. The cultivar has wheat-rye translocation 1BL.1RS with the Sr31 gene, co-segregated with Lr26, and Yr9. At present, African stem rust strain Ug99, originated in Uganda, overcame resistance underlined by the Sr31 gene [54]. In Element 22, this gene work is complimented by the Sr35 gene transferred from diploid wheat T. monococcum [55]. Together they provide a high level of resistance to Ug99. Many of the purple-grained lines developed using Element 22 genetic background had higher productivity and grain protein content than its check cv Element 22.
The intermediate maturing purple-grained lines derived from Aina showed resistance to leaf rust, which was connected to the Lr24 gene derived from Thinopyrum ponticum located on chromosome 3DL [56], which always presents with the Sr24 gene with the low level of effectiveness to stem rust. Recently, a new pathotype of Ug99 with virulence to Sr24 has been identified in Kenya [57]. However, the genes Lr24 and Sr24 retain their effectiveness in most regions of Russia and Kazakhstan [48,58]. The cultivar is highly drought resistant, able to withstand a prolonged absence of precipitations and high temperatures and is resistant to lodging. Solid stem of cv Aina protects against harmful insects. Therefore, the best purple-grained lines derived from Aina were also chosen for future tests.
Early maturing lines developed using BW49880 as recipient were resistant to local populations of leaf and stem rusts under condition of Western Siberia. The resistance could be associated with the slow rusting Sr2 gene complex, Sr2+Yr30+Lr27+Pbc (pseudo-black chaff), located on chromosome 3BS [59,60]. The Sr2-complex originating from the cv Hope could be identified by the pseudo-black chaff or brown necrosis phenotype on the glumes and below the nodes [61], that were observed on the glumes of the purple-grained lines developed in the background of BW49880 during their ripening ( Figure A5). This complex reduced epidemic development and provided durable and adult plant resistance to a broad spectrum of rusts including Ug99 in the CIMMYT-derived spring wheat germplasm [62]. The lines developed in the genetic background of BW49880 also demonstrated good productivity and high protein content.
The lines derived from cv Tobol'skaya were resistant to leaf rust, but susceptible to stem rust; cv Tobol'skaya is a medium late high-yielding cultivar of the steppe ecotype, characterized by high drought resistance, good grain quality, and resistance to head smut. The lines did not show good results in productivity and protein content as compared with the parental cultivar and were rejected from the further testing.
Thus, the purple-grained lines in the background of cvs Aina, Element 22, and the line BW49880 were selected for further evaluation and potential release as new spring wheat commercial cultivars for Western Siberia.

Conclusions
New high-yielding and resistant to fungal disease wheat cultivars with high anthocyanin content in grain is a resource for high added-value products such as healthy functional and dietary food. We demonstrated the efficiency of the breeding strategy which lasting only three years from the first cross between purple-grained donor and regional elite cultivar as recipient until the BC 1 F 4 lines ready for the state cultivar competitive testing. The anthocyanin-rich lines are characterized by increased productivity combined with resistance to the local populations of leaf and stem rust pathogens, which make them environmentally friendly and suitable for modern responsible farming.
Author Contributions: Crossing and evaluating with morphological markers, E.G. and T.K.; molecular marker screening, E.G.; field screenings and analysis of grain quality, V.S.; manuscript draft preparation, E.G. and V.S.; participation data interpretation and revising the manuscript critically, O.S.; coordinating the study, contributing to its conception and design, and revising the manuscript critically, A.M. and E.K. All authors have read and agreed to the published version of the manuscript.

Conflicts of Interest:
The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

Appendix A
Thus, the purple-grained lines in the background of cvs Aina, Element 22, and the line BW49880 were selected for further evaluation and potential release as new spring wheat commercial cultivars for Western Siberia.

Conclusions
New high-yielding and resistant to fungal disease wheat cultivars with high anthocyanin content in grain is a resource for high added-value products such as healthy functional and dietary food. We demonstrated the efficiency of the breeding strategy which lasting only three years from the first cross between purple-grained donor and regional elite cultivar as recipient until the BC1F4 lines ready for the state cultivar competitive testing. The anthocyanin-rich lines are characterized by increased productivity combined with resistance to the local populations of leaf and stem rust pathogens, which make them environmentally friendly and suitable for modern responsible farming.
Author Contributions: Crossing and evaluating with morphological markers, E.G. and T.K.; molecular marker screening, E.G.; field screenings and analysis of grain quality, V.S.; manuscript draft preparation, E.G. and V.S.; participation data interpretation and revising the manuscript critically, O.S.; coordinating the study, contributing to its conception and design, and revising the manuscript critically, A.M. and E.K.