Сельскохозяйственная биология, 2012, № 5, с. 88-99.

doi: 10.15389/agrobiology.2012.5.88eng

УДК 633.854.78:631.522/.524:575.222.73

THEORETICAL AND APPLIED ASPECTS OF INTERSPECIFIC HYBRIDIZATION IN SUNFLOWER

I.N. Anisimova, V.A. Gavrilova

The development of N.I. Vavilov concepts on distant hybridization as a way for broaden-ing cultivated plants diversity is demonstrated on the example of sunflower. The review of studies on the crossability of cultivated sunflower with wild Helianthus species, the inheritance of characters in hybrid generations, the peculiarities of the genome structural and functional reorganizations in in-terspecific hybrid progenies is given. The practical results of the crosses between H. annuus and perennial Helianthus species are analyzed; the diverse aspects for application of this approach in sunflower breeding and genetics are discussed. With the use of distant hybridization a new model for investigation of nuclear-cytoplasmic interaction genetic mechanisms is suggested.

Keywords: sunflower, species, interspecific hybridization, CMS-Rf genetic model.

Distant hybridization (interspecific and intergeneric) is the method of obtaining new genomes and genotypes that express combination of valuable traits of parental species. Theoretical fundamentals of distant hybridization were significantly improved by N.I. Vavilov in respect to its role in plant selection. Emphasizing the advances of plant industry derived through distant hybridization, he wrote in 1932: “Among the common tasks of genetics ... we primarily highlight distant hybridization, the development of issues related to applicability of distant crosses, hybridization of different species and genera. The most fascinating tasks of a breeder and agronomist ... are largely associated with distant hybridization; a combination of the most interesting features in one variety… is determined in many cases namely by the applicability of distant hybridization” (1). The development of methods of distant hybridization has become one of major scientific activities of the Department of Genetics established by N.I. Vavilov in 1925 in the All-Russia Research Institute of Plant Industry (VIR) (2). The first head of it was G.D. Karpechenko, whose classic works on the theory of distant hybridization today are the golden fund of world science (3). The study of genetic and cytogenetic mechanisms of compatibility / incompatibility of species is still one of major tasks of this department of VIR.
Actual breeding achievements confirm the ideas of N.I. Vavilov about the special role of distant hybridization in increasing genetic diversity of cultivated plants (4, 5). In his view, distant hybridization is primarily the way to improve cultural crops by the transfer of useful traits for immunity to diseases, pests and environmental stress. The common regularities in distribution of immunity determinants discovered by N.I. Vavilov underlie later studies aimed at detection of resistant types among the diversity of cultivated plants and their wild relatives. He also found that “the most contrasting differences in immunity show the plants cytogenetically distinct as different species” and “plant species immune to one disease are often resistant to many other diseases (broad-spectrum resistance, or groups immunity)” (6) .
The regularities in distribution of immunity determinants discovered by N.I. Vavilov were applied in breeding sunflower, the most important oilseed crop. Vavilov’s works exemplified a resistance of perennial sunroot Helianthus tuberosus and annual sunflower H. argophyllus against rust fungus Puccinia helianthi (7). Today, there are described many diseases of cultivated sunflower caused by parasitic fungi, the most damaging of which are Phomopsis helianthi, Sclerotinia sclerotiorum, Botrytis cinerea,  Plasmopara halstedii, and rust. Along with it, the most harmful pathogens are Alternaria helianthi, Phoma macdonaldii, Verticillium dahliae, and a parasitic plant broomrape (Orobanche cumana).
In 1938 N.A. Schibrya defined practical tasks of interspecific hybridization of Helianthus (8): the prior task was increasing the immunity of cultivated sunflower (annual species H. annuus) by introducing it from high-resistant H. tuberosus, and the second task was obtaining the hybrid H. annuus ½ H. tuberosus as perennial sunflower producing green mass, tubers and seeds (9). As noted by other researchers, a limited gene pool of cultivated sunflower can be essentially improved using interspecific hybridization (10-14). Today, wild Helianthus species are considered as a valuable source of cytoplasmic male sterility (CMS), restoration of pollen fertility, resistance to pests and adverse environmental conditions, the determinants for unique compositions of fatty acids in oil and other valuable properties (10 - 18).
The genus Helianthus includes 51 species, 14 of which are annual diploids (2n = 34) including cultivated sunflower H. annuus (section Helianthus), and 37 –perennials with different ploidy levels (sections Atrorubentes and Ciliares) (19). Annual wild species (2n = 34) can be crossed with cultivated sunflower, which results in fertile offspring at any direction of crosses. The hybrids of cultivated sunflower and annual wild species are the valuable source material for obtaining CMS donors (20-22) and breeding lines including the carriers of resistance genes (15, 23). Perennial species are especially valuable for breeding, as they can be donors of determinants not present in annual species, in particular, immunity to diseases and pests. Under normal conditions, perennial and annual species of Helianthus are cross-incompatible or they can be crossed with great difficulty and the hybrid offspring is sterile (10, 24, 25). In definition of G.D. Karpechenko (3), such crosses are incongruent. The analysis of crossability along with the data of cytogenetic and molecular genetic studies have revealed peculiarities of interspecific interactions and genomic structure of the genus. It was found that cross-incompatibility of annual and perennial Helianthus species is the result of differences in their genomic structure (24, 27). According to a recent hypothesis of French researchers, all Helianthus species have a common C-genome; H-genome is common for annual species, P-genome – for perennial species, and A-genome is specific to the section Atrorubentes (28).
Cross-incompatibility is an obstacle for efficient use of perennial wild species in breeding sunflower. Various techniques were used to overcome it: selection of partners, exposure to thermal stress, preliminary vegetative convergence, multiple pollination (27). In the last decade, such hybrids were successfully obtained through biotechnology (embryo culture, protoplast fusion) (29-34). It was also reported about some approaches to restore fertility in hybrids between cultivated sunflower and perennial Helianthus species, such as backcrossing: pollination of sterile F1 plants by pollen mix of cultivated sunflower  resulting in fertile BC1. This approach was used in hybridization of H. annuus and perennial diploid H. mollis, and it was found to be very time-consuming and low-effective (35). However, it has allowed a successful introgression into annual sunflower of genes affecting plant architecture (short internodes peculiar to H. mollis).  Gene maps of resulting samples show the location of introgressed fragments in two linkage groups (36).
Diploidization was another strategy of breeding sunflower based on classical works of G.D. Karpechenko (37). First artificial amphidiploid sunflower was obtained by R.C. Jackson and B.G. Murrey (38), and later this method was used many times (39). In particular, amphidiploids resistant to broomrape were created through crosses of cultivated annual sunflower and perennial species (40). However, this technique hasn’t become commonly adopted, because the hybrid offspring often expressed a major defect – reversion to diploid state (bypassing the stage of aneuploidy) and resulting loss of valuable genetic determinants transferred from wild species.
G.V. Pustovoit has done a lot for breeding sunflower for broad-spectrum immunity by the method of distant hybridization. She performed her works in the All-Russia Research and Development Institute of Oil-Producing Crops (VNIIMK, Krasnodar) and she first managed to obtain reciprocal interspecific hybrids between cultivated sunflower and H. tuberosus, H. tomentosus, H. subcanescens, H. scaberimus, H. mollis (10, 11). These annual highly-fertile samples manifested an intermediate phenotype, the number of chromosomes similar to the cultivated sunflower (2n = 34) regardless of another parent’s ploidy, and resistance to pathogens acquired from wild species. The 22nd generation of these samples was used to create in VNIIMK the new varieties of sunflower Lider, Berezansky etc. with high oil content and immunity to diseases. Today, hybridization of cultivated sunflower with perennial wild species is conducted in different countries, which has provided a valuable source material for breeding including the lines with introgressed resistance to sclerotinia (41) and alternaria (42), drought resistance (43) and other important features.
VIR scientists have created a number of interspecific hybrids, whose maternal form was a single CMS line of cultivated sunflower and paternal – various perennial species with different ploidy levels (H. mollis, H. maximiliani, H. laetiflorus, H. trachelifolius, H . angustifolius, H. occidentalis, H. strumosus, H. rigidus, H. giganteus, H. grosseserratus, H. divaricatus, H. hirsutus, H. decapetalus and H. tomentosus) (44-46). The progeny of these crosses was quite diverse: fertile seed-producing perennials with a habit distinct from both parents; annual plants phenotypically close to wild annual sunflower; hybrids whose progeny F1 and F2 were split to cultivated and wild-type plants in features of pollen fertility, branching, the presence of pubescence and anthocyanin coloration; the hybrids not splitting in F2-F8 and morphologically similar to cultivated sunflower except the unique form of branching.
F2 and following generations manifested the appearance of similar morphological types regardless of their perennial parent species. The collection of introgressed sunflower lines includes the samples derived by preliminary selection among the progeny of interspecific hybrids F1 and among the progeny of test crosses with sterile maternal form. These lines express phenotypic homogeneity and a number of valuable biological traits, presumably transferred from the perennial parent – a unique morphotype, pathogen immunity, and the capability of pollen fertility restoration in CMS-carriers. Already in F1 interspecific hybrids of sunflower showed stabilization of chromosomal set at diploid level, regardless of ploidy of paternal species (16, 47, 48). Weight of 1000 seeds of the introgression lines varied from 31 to 80 g. Husk content in the hybrids ranged from 23 to 43%, and in six ones it was less than 30%. Though, kernel size and husk content in these lines were inferior to those of standard variety Peredovik, while oil content (the main estimate of oilseed quality) varied from 48,0 to 56,3%, i.e. by an average 1,9% higher than in the standard. Except for the two samples, oil content in kernel of the hybrids exceeded 50%, and in five of them it was 55%. One valuable line originated from the hybrid 114 ½ H. giganteus showed complete immunity to phomopsis during three years (2009-2011) in conditions of the Kuban Experimental Station of VIR (Krasnodarsky Krai).
Sunflower forms obtained through distant hybridization are early-ripening, with high combining ability for seed yield in crosses with CMS line. Many of them show recessive form of branching and are suitable as a pollen fertility restorers in CMS lines during obtaining commercial hybrids. Some hybrid combinations produce the offspring showing non-branching, completely restored pollen fertility and seed yield exceeding that of the standard variety Peredovik. Through backcrosses with a CMS line it was derived a one-headed hybrid very tall (180-190 cm) and quite uniform in height, with large heads of about 25 cm diameter. This success was largely associated with hybridization using CMS lines the donors of genes for autofertility instead of self-incompatible cultivars. In  autofertile samples capable to overcome the incompatibility, this process occurs much easier even in distant crosses (16, 46).
Chronologically, there are two stages of research of sunflower hybrids derived through the crosses of cultivated sunflower with perennial Helianthus species. The first stage (mid-1980ies) was investigation of causes of genomic incompatibility in crosses between cultivated sunflower with perennial species and searching for ways to overcome it. In this period were performed studies of meiosis in hybrids, eg. works of Bulgarian School headed by the Professor J. Georgieva-Todorova (25). The second stage of research coincided with the introduction of modern molecular genetic technologies. Molecular labeling made it possible to determine the nature of these hybrids, many of which were previously considered as pseudohybrids. However, the mechanisms of introgressions and characteristics of genome transformation in interspecific hybrids of sunflower are still poorly studied. Today, the analysis of DNA polymorphism allows characterization of the hybrids and detection of determinants for valuable traits introgressed from wild species. In particular, the presence in the genome of hybrids H. annuus ½ H. mollis of fragments introgressed from paternal perennial species was confirmed using RAPD-, RFLP-and AFLP-markers (30, 31, 36). Other methods used in such studies were RAPD (49), SSR (50, 51), AFLP (41), and Southern blot hybridization (52).
Peculiarities of structural and functional modifications in genomes of interspecific hybrid sunflower were studied using the polymorphism of electrophoretic spectra of seed storage protein 11S globulin (helianthinin) (53). Perennial and annual Helianthus species differ in componential composition of electrophoretic spectrum of helianthinin encoded by at least three loci - HelA, HelB and HelC. In F1 of hybrids between H. annuus and annual wild species and forms (regardless of cross direction) it was observed the co-dominant expression of helianthinin polypeptide variants of both parents, and in F2 the trait was subject to Mendelian pattern of splitting.
A different type of inheritance of this trait was found in interspecific hybrids between cultivated sunflower and perennial species. Electrophoretic spectra of helianthinin extracted from the seeds of F1 interspecific hybrids were identical to the spectrum of maternal line or they included new variants not peculiar to parental forms. Such diversity was observed in following generations as well. Six different types of helianthinin spectrum (phenotypes) were identified in the group of 83 introgression lines. The spectrum similar to that of the maternal annual parent was found in 35% lines. In 60% progenies of the interspecific hybrids, the spectrum reflected changes in components encoded by the locus HelC. All types of electrophoretic spectrum of seed storage proteins revealed in the studied hybrids had been previously described in cultivated sunflower.
Hybrid sunflower was derived in Justus Liebig University (Giessen, Germany) by crossing the line HA89 with di-, tetra- and hexaploid species in field conditions with following culturing embryos in artificial medium (30).  These plants had similar changes in electrophoretic spectra of components encoded by the locus HelC, as well as the inheritance of morphological traits. The progeny of interspecific crosses was distinguished to 5 different morphological types known as mutants of cultivated sunflower (16). So, hybrid progenies of different variants of crosses between cultivated sunflower and perennial species were found to have obvious similarities in morphological traits and electrophoretic spectra of seed storage proteins, which can be regarded as the expression of parallelism and illustration of Vavilov’s law of homologous series in hereditary variation (54).
Using random oligonucleotide primers that differentiate genetic material of perennial and annual Helianthus species (28) has revealed individual fragments supposedly introgressed from the genome of perennial species, along with fragments not known in parental forms possibly due to genomic modifications in hybrids. Genomic instability of introgression lines was found by RAPD-analysis even after years of inbreeding (in generations F8-F12). According to hybridological analysis, one introgression line has unstable locus for basal branching trait (55). The successful introgression of Rf1 gene from perennial species in the genome of interspecific hybrids was confirmed using SCAR-marker (56).
Genomic modifications of interspecific hybrids can be assessed by PCR analysis of nucleotide sequences encoding certain functionally important proteins. The lengths of amplified fragments in cultivated sunflower and perennial species of Helianthus were clearly different. However, in all hybrids the lengths of amplification products for the most of loci were similar to those of the maternal line of cultivated sunflower. So, there was a question – whether the mentioned gene introgressed into the hybrid genotype from the wild species? The answer was found in primary nucleotide sequences of H. annuus and perennial species: they have significantly different composition of nucleotide sequences of encoding (gene for storage albumin SFA8) and non-encoding (SCAR-marker of Rf1locus) fragments of the genome. At the same time, nucleotide sequences of homologous fragments in both hybrid and annual species were either identical or included many single nucleotide substitutions (57, 58).
Molecular genetics of interspecific hybrids of sunflower was analyzed by means of biotechnology techniques (culture of embryos, protoplast fusion); the progeny of interspecific crosses regardless of their origin, were determined as partial, or “asymmetric” hybrids, and the greater share of their genomes was inherited from cultivated sunflower. Somatic hybrids resulting from electro-fusion of protoplasts of H. annuus and H. maximiliani have isoenzymes typical to cultivated sunflower. The introgressed fragment of the wild species’ genome was detected only by one of 10 random primers (59). Knowing the impossibility of non-sexual (apomictic) origin of these hybrids, asymmetric structure of their genomes can be explained by modifications and elimination of wild-type chromosomes in early stages of callus development. Monitoring of meiosis in F1 hybrids between annual sunflower and perennial Helianthus has shown the formation of uni-, tri- and tetravalents, but bivalents occurred most frequent, and it was observed correct segregation of chromosomes in anaphase (25). The presented data indicate that modification of genomes (or haploidization) during sexual hybridization of cultivated sunflower with perennial Helianthus species occurs in early pre-meiotic and possibly post-zygotic stages.
The comparative analysis of polymorphisms of helianthinin and amplified DNA fragments allows assuming that variability of the progenies of interspecific sunflower hybrids isn’t random, but possibly associated with “genomic shock” of interspecific hybridization and its consequences at molecular genetic level (60). Modifications in hybrid genomes may be also associated with  mobile genetic elements triggered by distant hybridization (55, 61).
One of the most important achievements of interspecific hybridization of sunflower is obtaining new donors of CMS; according to the literature, most of 72 known CMS donors were derived through distant hybridization (20-22, 46, 62). New donors of CMS can reduce the probability of epiphytoties associated with unification of cytoplasm. However, modern sunflower hybrids intended for large-scale use are known to be developed primarily on the base of the only CMS donor PET1 derived upon the hybrid H. petiolaris ½ H. annuus (63). Such a limited use of new genetic systems CMS-Rf in breeding work can be explained by the lack of confident Rf genes for each type of cytoplasm, because genetic mechanisms of pollen fertility restoration are poorly studied yet.
Cytoplasmic male sterility (CMS) was discovered by M. Rhodes in maize in 1931 (64), and simultaneously – by M.I. Khadzhinov in VIR Department of Genetics who though didn’t publish his results. Presently, CMS is described in more than 150 species (65, 66) while a wide variety of its phenotypical expression (67). CMS can serve as the best model of maternal inheritance in plants. The discovery of nuclear genes – restorers of pollen fertility (Rf) that can suppress CMS phenotype allows using the systems CMS-Rf as models of interactions between nuclear and mitochondrial genomes. Unfortunately, molecular and genetic aspects of such interactions are still relatively poorly understood due to the lack of appropriate model objects.
Most of the CMS donors were obtained through interspecific hybridization (65, 68, 69). Merging foreign cytoplasmic and nuclear genomes can lead to mutations or chimaeric mitochondrial genes that almost always include fragments or copies of the known genes (e.g., genes encoding subunits of ATP synthase) and (or) unidentified sequences (70). Both phenomena of CMS as genomic barrier between the nucleus and cytoplasm, and genes-restorers of male fertility against CMS, are assumed as the most important evolutionary factors in development of species in flowering plants (71-73).
The analysis of molecular diversity in 29 samples of sunflower – CMS donors has revealed several types of CMS associated with the structure of mitochondrial DNA (mtDNA) (74). For many of these types, CMS is associated with expression of the new open reading frame of mitochondrial gene orfH522 co-transcribed with gene atp1. These data allowed to develop diagnostic PCR markers for identification of cytoplasmon type (75). Today, it has been determined a chromosomal location of five genes – restorers of male fertility in sunflower on the background of different types of cytoplasmon (76).
In higher plants, co-operation of the nuclear and organelles’ genomes is regulated by a large group of genes encoding proteins involved in anterograde / retrograde regulation that contain tandemly repeated sequences of 35 amino acid residues (pentatricopeptide repeats, PPR). PPR-proteins play an important role in processing and translation of organellic RNA (77, 78). Most of genetic determinants – restorers of pollen fertility are homologous in different plant species and belong to the family PPR-genes (79-82) as the subfamily of PPR-RFL genes (PPR-Restorer-of-Fertility-Like) (83). The distinctive feature of PPR-RFL-genes of higher plants is cluster location in the genome and the unique pattern of divergence of PPR-motifs (84). The diversity of nucleotide sequences in PPR-RFL-genes may be a cause of different alleles whose products are capable of specific interaction with products of mitochondrial genes associated with CMS phenotype. Recently, it has been identified in sunflower the presence of nucleotide polymorphism of a fragment associated with fertility restoration and containing PPR-motifs (86).
Unique introgression lines of sunflower were established in VIR on the basis of interspecific hybrids derived by crossing sterile line of H. annuus with perennial Helianthus species of different ploidy levels. These lines can be the effective model for studying the fundamentals of nuclear-cytoplasmic conflict caused by genomic incompatibility and overcoming it in distant hybridization. This group of lines is supposed to have the mitochondrial genome of a source maternal line, or the modified one.
In the lines’ progenies genotypes with restored male fertility were selected as from F1, therefore they carry different functional alleles of Rf genes that were introgressed from a perennial parent or resulted the modification of nuclear genome in a source maternal line triggered by distant hybridization. Besides, hybridization could initiate rearrangements in the mitochondrial genome of a source maternal lines. This assumption was confirmed by PCR-analysis with primers specific to orfH522 – the mitochondrial gene associated with CMS PET1. In some lines and in a number of hybrids in F1 it was observed the absence of the marker fragment or its length was changed. It’s expectable that such introgression lines may carry new alleles of nuclear Rf genes that compensate rearrangements in the mitochondrial genome caused by distant hybridization. Usually, identification of Rf  genes and studying the peculiarities of their expression are performed using test crosses with CMS lines and analysis of F1 progeny. Introgression lines of sunflower are the model objects whose genotype includes both aberrant mitochondrial genes – inducers of male sterility, and nuclear factors Rf – suppressors of the CMS-inducing genes. Using this approach for identification and assessment of Rf-genes obviates the necessity of any crosses. In addition, these introgressed lines carry the genes in a homozygous state, which is preferable for this sort of research because it prevents the potential impact of interallelic interactions on manifestation of the trait in hybrid plants.
Thus, the abovementioned achievements of distant hybridization of sunflower are consistent with ideas of N.I. Vavilov about the importance of this method for increasing genetic diversity of cultivated plants. The results of interspecific hybridization have significantly promoted the development of individual genetics of sunflower, and improved the knowledge about genomic composition of Helianthus, intra- and interspecific interactions within the genus. Today, application of distant hybridization should be focused primarily on the development of disease-resistant and high-yielding varieties and hybrids. The major targets of breeding sunflower include the transfer of genes for resistance to pests and stress factors from wild Helianthus into the genomes of cultivated species, and the creation of new genetic systems CMS-Rf. Along with a conventional use of CMS in breeding, this phenomenon can serve in a different aspect – as a model in studies of fundamental mechanisms of nucleus-cytoplasmic interactions in flowering plants, which involves both classical genetics and molecular genetics.

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