Sunflower Genetic Collection at the Vavilov Institute of Plant Industry

Lines with uniform morphological characters

The sunflower collection at VIR totals 2,780 accessions that include 2,230 accessions of cultivated (Helianthus annuus L.) and 550 of wild sunflower accessions belonging to 24 species (5 annual and 19 perennial).

The cultivated sunflower collection is represented by local varieties, landraces, and cultivars of national and foreign breeding programs, as well as populations collected during explorations. It also includes the first cms-based hybrids which are preserved as hybrid populations and lines. The collection is composed of lines developed as a result of studying interspecific hybrids, varietal diversity, and repeated self-pollination, or interspecific hybridization. The genetic collection includes 189 lines with different morphological characters; 120 fertility restorer lines; 20 cms lines and their fertile analogs; 46 lines with genes for resistance to races 330, 710, and 730 of downy mildew (Plasmopara halstedii (Farl.) Berl. and de Toni); and 90 of the 362 lines have seed storage protein markers for polymorphic variants.

Lines that show no segregation of morphological characters in several subsequent generations

We have obtained a series of natural mutants with different expressions of one and the same character. For example, some lines differ in terms of intensity of anthocyanin coloration of vegetative and generative organs exhibited by only one line, VIR 364. The most numerous is the group of lines with upper branching. Branches form in the upper third of the stem and may be short like in VIR 397, or long as in VIR 721. Lines also exist that branch throughout the stem which may be compact (VIR 636) or spreading (VIR 702). Although genetic control of branching has been repeatedly investigated by several researchers (Putt, 1964; Kovačik and Škaloud, 1986; Miller and Fick, 1997; Gavrilova and Anisimova, 2003; Gavrilova et al., 2005), there does not seem to be a consensus of the gene control for the different branching types. This character is important in restoration lines for restoring pollen fertility in cms forms, as branching lines flower longer and produce more pollen, thus facilitating a longer period of cms pollination for commercial hybrid seed production. Some lines have very short petioles (VIR 708), or a very long reflected petiole (VIR 746), or no petiole at all (KG 49). Both plant habit and cultivation techniques depend on the petiole shape, especially at early stages of cultivar or hybrid development. Data on both recessive and dominant inheritance of Mendelian genes for the erect leaf character have been discussed by Gavrilova and Anisimova (2003).

The practical use of morphological characters with known genetic control such as the dark-green (Gr) and pale-green (gr) leaf color, white seed color, indentation and strong venation (vs) of the leaf blade, leaf blade knobbiness and asymmetry (As), erect petiole (Er), anthocyanin color (A), lemon (l) and orange (la) ray flower color are used as markers in heterotic breeding and for controlling line purity when they are maintained and multiplied for seed increase or during seed production (Gavrilova and Anisimova, 2003). The heterotic effects in commercial hybrid sunflower express itself in both seed yield and plant height. In order to produce hybrids with the optimal plant height (150–180 cm), dwarf lines can be used. A genetic analysis of the material obtained by crossing dwarf lines between themselves and a tall cv. Peredovik has identified three types of dwarfness. The first type is VIR 272 that acts in such a way that plant height is reduced due to the significant shortening of internodes, as well as increased internode number, hence extending the vegetation period (Table 1).

Dwarfness in VIR 272 is determined by the dw1 dw 2 genes with intermediate inheritance and recessive epistatic interaction (Yesaev, 1998; Gavrilova et al., 1999). A decreased internode length is due to the reduced cell size (Yakovleva, 2006). The average number of leaves in VIR 272 is 41 for about a 60-cm tall plant. The same number of leaves has been recorded for the tall cv. Gigant with plant height over 3.5 m. The second type of dwarfness is determined by the additive interaction of alleles of no less than three short stem genes (sht1 sht2 sht3) in VIR 319 and VIR 328. The third type of dwarfness is illustrated in VIR 253, VIR 500, VIR 501, and VIR 648. Control of this character is based on the polygenic action of no less than three semi-dwarf genes with intermediate inheritance (sd1 sd2 sd3). Triple and even larger stem shortening in the standard cv. Peredovik occurs at the expense of significant internode shortening. In this case, the number of leaves may be reduced to 15–17 (compare with 35–37 in cv. Peredovik) (Yesaev, 1998; Gavrilova et al., 1999). The number of leaves and hence the number of internodes determines the duration of the “germination-to-flowering” phenophase, since formation of a larger number of leaves requires more time. Therefore, the lines with a large number of leaves have a longer vegetation period. All dwarf lines have smaller achene and root system than those in cv. Peredovik. However, the head size is not related to dwarfness, since the lines studied included VIR 319, VIR 328, and VIR 648 with a fairly large head (18–20 cm in diameter). The smallest head recorded was 8–10 cm for VIR 501.

The genetic collection of more advanced generation germplasm includes sunflower lines from the 5th–27th generation with all possible mutations of all morphological characters. As a result of our genetic analysis, 33 genes have been identified (Table 2) with genetic control of 16 morphological characters discovered in 18 lines from the collection (Gavrilova and Anisimova, 2003; Gavrilova et al., 2005).

In order to determine the degree of resistance to new downy mildew races in sunflower lines in the VIR collection, genotypes resistant to this pathogen have been selected in the field based on observations performed over several years. They were tested in the immunity laboratory of the Pustovoit All-Russian Research Institute of Oil Crops (VNIIMK) for resistance to races 330, 710, and 730 which have spread in the Krasnodar Territory and Rostov Province in recent years (Antonova et al., 2011). Forty-three lines were found to be resistant to race 330, 13 lines showed simultaneous resistance to two races, and 12 lines demonstrated resistance to three downy mildew races (Table 3). In addition to resistance to three downy mildew races and Phomopsis, VIR 249 can also restore fertility of the cms РЕТ1 cytoplasm as a male parent when used for breeding commercial sunflower hybrids.

Lines with certain uniform biochemical characters showing no segregation for several generations

The sunflower seed protein fraction includes two main components, namely the salt-soluble protein 11S globulin (helianthinin) and the water-soluble 2S albumins which differ by their molecular mass, amino acid composition, and physicochemical properties. The majority of lines in the genetic collection have been analyzed using 11S globulin enzyme electrophoretic banding pattern (Anisimova et al., 1991; Anashchenko et al., 1992; Anisimova et al., 2004). Polymorphism of seed 2S albumins has been thoroughly studied in seven lines using a complex of biochemical methods (Anisimova et al., 1995), polymorphism of SFA7 and SFA8 proteins, the main methionine-rich components of the albumin fraction investigated in 100 lines (Anisimova et al., 2003), and polymorphism of proteolytic enzyme inhibitors studied in 70 lines (Konarev et al., 2000).

The analysis of segregation in F₂ and F_a (F_a is F₁×Parents=F_b) populations from crosses of inbred lines differing in helianthinin composition has shown that polymorphism is determined by the allelic variation in at least three loci: HelA, HelB, and HelC (Table 2). The hybridological analysis identified polymorphic alleles in each of these loci (Table 4). It has been shown by dihybrid crosses that the HelA locus is inherited independently from HelB and HelC, while HelB and HelC demonstrated a linkage: the frequency of recombination in F₂ from two different cross combinations did not exceed 24%, while in the test cross [(VIR 130×VIR 104)×VIR 130], the value was 19%. It confirms the localization of both genes in the same linkage group. In many cases, the presence of these alleles was associated with the line origin (Table 2). For instance, the presence of HelC_c or HelC_b alleles indicated, as a rule, the presence of genetic material from wild forms. Such lines include VIR 104, HA61, RHA273, and RHA274. The HelВ_b allele, which is characteristic of helianthinin from the accession k-2266, was also found in the inbred lines created from it (Anisimova et al., 2004).

Ninety inbred lines out of 188 analyzed possessed the characteristic helianthinin polypeptides. Besides the above allelic helianthinin variants, other electrophoretic variants were observed. Line VIR 387 lacked the major polypeptide band 10, while lines VIR 130, VIR 649, and VIR 302 lacked variant 4 in the helianthinin banding patterns.

The electrophoretic variant of the SFA8 protein was found in five lines (VIR 130, VIR 365, VIR 666, VIR 676, and VIR 262) which differed from the variants present in all other lines by its PAG mobility (Tris–Tricine–SDS electrophoretic buffer, рН 8.8) and isoelectric point mobility (Anisimova et al., 2003). Codominant inheritance was observed in the F₁ from the cross VIR 130 × VIR 104, which was characterized by different variants of SFA8, while segregation in the F₂ showed that the normal and variant SFA8 were encoded by alleles at the same locus (Table 4).

The mutation that led to the appearance of the SFA8 protein variant has been identified. A comparative analysis of SFA encoding nucleotide sequences revealed that the protein is encoded by a small multigenic family. A single nucleotide substitution in the encoding region of the gene was found in the lines possessing the SFA8 variant. Such a substitution changes the molecule conformation, isoelectric point value, and, consequently, electrophoretic PAG mobility (Anisimova et al., 2010).

Two types of inhibitors, trypsin (TI), and bifunctional inhibitors of trypsin/subtilisin (T/Sl) have been found in sunflower seeds (Konarev et al., 2000). Inhibitor bands were found to be polymorphic in different inbred lines. The F₂ from a cross between VIR 670 × VIR 648 differed by the presence/absence of three different variants (a–c) of trypsin/subtilisin inhibitors (based on isoelectric focusing) has been analyzed regarding segregation at their encoding loci, i.e. T/Sla, T/Slb, and T/Slc. According to the results of hybridological analysis, all three loci are localized in one linkage group. The distance between T/Sla and T/Slb loci was 32% (in recombination units), while 23% between T/Slb and T/Slc.

Lines homozygous for phenotypical manifestation of certain characters and molecularly marked lines

Seventeen VIR cms lines have been produced from hybrids between a single source, H. petiolaris L. (cms PET1) obtained by Leclercq, P. in 1968 (Leclercq, 1969), and an unknown pedigree, H. annuus L. (Table 5). For instance, VIR 114 was produced by crossing VIR 104 (cms РЕТ) with cv. Sputnik (bred at the Armavir Station). Parental forms of VIR 104 are the cms source from Leclercq and cv. Armavirsky 1813. VIR 116 was created from an American line cms НА 234, which in turn had been obtained from a hybrid between a cms source from a wild sunflower from Texas and cv. Smena (bred at VNIIMK). The Vympel cultivar was used as the paternal parent for VIR 116. Pedigrees of both lines feature a cms source obtained from an interspecific hybrid between the wild annual and cultivated sunflower and also a Russian variety. The НА 89 line was from the USA, while VK-2086 was from VNIIMK.

The VIR genetic collection includes two other types of cms, RIG0 obtained from a perennial wild species, H. pauciflorus Nutt. (rigidus) (Cass.) Desf., and PEF based on H. petiolaris Nutt. ssp. fallax Heiser. Sterile analogs of VIR109 and VIR151 have been created based on cms РЕТ1 and RIG0 (Gavrilova et al., 2005).

The VIR collection contains 120 fertility restorer lines obtained using three different methods. In the first, lines were obtained through backcrossing self-fertile lines with a source of Rf genes and subsequent control of the progeny using paired crossings. The second and most common way of producing such lines is by extraction from commercial hybrids (Rozhkova and Anashchenko, 1997). Since commercial hybrids are produced based on cms, all fertile progeny have Rf genes and sterile cytoplasms (Table 6). The third method is from interspecific hybrid lines. As a general rule, interspecific hybrids are produced using sterile maternal lines of cultivated sunflower (Table 7). Therefore, that fertility restorer lines from interspecific hybrids also have a sterile cytoplasm.

The collection at the fourth level is represented by lines with cms and pollen fertility restoration genes (Table 8). The atp9 and orfH522 molecular markers (Schnabel et al., 2008), which are specific for the mitochondrial genes associated with cms PET1, have been used to differentiate between the lines with fertile and sterile cytoplasm, as well as the difference between lines with cms PET1 (e.g. VIR 109, VIR 114, VIR 116, VIR 151, etc.) from cms of other types (Anisimova et al., 2011). It has been established that many pollen fertility restorer lines with Rf genes have sterile cytoplasm (VIR 364, VIR 365, and VIR 558, Table 8), since they have been produced through self-pollination of commercial F₁ hybrids (Table 8). Sterile cytoplasm makes it possible to control the Rf genes when reproducing the line without additional crosses. Pollen fertility restorer lines, based on self-fertile lines (VIR 740 and others) through backcrossing with the restorer lines and the further testing of Rf genes in paired crosses, have fertile cytoplasm (Anisimova et al., 2011).

Most lines restoring pollen fertility of cms have molecular markers (Horn et al., 2003) for the Rf₁ gene. However, two lines (VIR364 and VIR 365), which restore pollen fertility in F₁ hybrids quite well, have been found during field testing that do not have molecular markers for Rf₁. The absence of markers for these lines and the molecular-genetic analysis data on other Rf₁ donor lines allows one to speculate that other genes control pollen fertility restoration in genotypes of VIR 364 and VIR 365.

It has been observed that of the 38 analyzed Rf₁ gene donors, 4 are fertile based, 22 based on PET1, and 13 have sterile cytoplasm differing from PET1 (Table 8). All the mentioned lines restore pollen fertility in F₁ of hybrids with cms PET.