Protein markers for increasing efficiency of Triticeae Dum. genetic resources utilisation in breeding
A.Konarev , V.Konarev, N.Gubareva, T.Peneva, I.Gavrilyuk and N.Alpatyeva
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To serve as an effective basis for improvement of cultivated plants, genetic diversity stored in gene banks, should be carefully and comprehensively evaluated and characterised (investigated). Collections should be rationally organised. Each accession should be identified and registered. Preservation of the genetic composition of accessions is also included into the category of basic problems. The objective of a gene bank is to maintain an accession unchanged as regards its genetic composition. It means preserving not only the sample as such, but together with its valuable properties, in particular adaptive ones, etc. Understanding the genetic structure of biodiversity (relationships inside a gene pool or between structural elements of genetic diversity) is an important goal of genebank activities. All above-mentioned directions should be developed to facilitate the use of germplasm for improvement of cultivars. Protein markers (PM) are successfully used in VIR since 1969 for increasing utilisation efficiency of Triticeae Dum. genetic resources in plant breeding. Genetic resources of Triticeae are studied in the following aspects:

  1. structure of biodiversity (intraspecific relations and interspecific relationships, genome analysis);
  2. identification and registration of genetic diversity and preparation of catalogues and data bases based on protein formulae;
  3. identification of duplicates, development of core collections;
  4. genetic integrity control;
  5. authorship rights control (for gene banks).

Serological markers have been successfully used in genome analysis of Triticum L., Aegilops L., Elytrigia Desf., Elymus L., Agropyron Gaertn. Genetic differentiation of wheat, oat and rye biodiversity based on prolamin polymorphism was carried out. Genetic diversity of the most economically important Triticeae species was registered on prolamin patterns, catalogues and a data base of protein (prolamin) formulae were composed.

INTRODUCTION

Global plant genetic resources are considered all over the world as the basic source for improvement of agricultural crops in the coming decades. The creation of sources and donors of important properties, i.e. organization of prebreeding work, is in most cases based on world genetic resources or collections of cultivated plants and their wild relatives.
Disclosure of the potential of genetic resources for the basic biological and selection properties provides a genetic base for realisation of selection programs in various directions. As a whole, the pre-breeding work includes all stages of work with plant germplasm from collecting, maintenance and study up to the legal aspects of authorship over the donors and sources of valuable characters.
Traditional approaches are founded on morphological characters which have some limitations. Proteins as primary products of gene expression reveal small changes (mutations and so on) inaccessible to visual analysis. Protein markers (PM) are, as a rule, inherited codominantly and analysis of a genotype is possible immediately by the protein phenotype. PM are successfully used in VIR for solving theoretical and applied problems of introduction, study, storage, reproduction, identification and registration of Triticeae genetic resources. Special attention is given to the development of effective tools for breeding, variety testing and seed production.

Seed proteins in identification and registration of the Triticeae gene pool.

The most significant element in the work of a botanist, geneticist or breeder is identification of species, varieties, biotypes and other biological systems. Identification simplifies documentation of a gene pool of cultivated plants and their wild relatives with the aim of its registration, preservation and effective usage in breeding. It is necessary to reveal and isolate desirable genotypes from complex natural varietal and hybrid populations. Identification of varieties and lines is especially topical for intensification of breeding and seed production, which demand high accuracy and efficiency of seed control (1).
In order to develop a flexible and reliable nomenclature and system of pattern recording, practically all intraspecific (or intrageneric) variability of a given protein marker should be investigated. Due to this total investigation of specific and generic biodiversity, almost all possible locations of protein components can be identified. Cultivars, wild growing populations, landraces from world collections have to be analysed. It was implemented in the Vavilov Institute, and this principle was laid in the base of nomenclatures and systems of recording electrophoretic components for many crops. This approach was first developed for wheat and then spread on all Triticeae and other crops (2).
Wheat gliadin pattern was taken as a basis. The pattern was divided into four zones. Within a zone the main possible positions of components were numbered to the start. By means of such etalon pattern, any variety or biotype of Triticeae may be recorded in the form of prolamin formula. By now cultivated and wild-growing gene pools of wheat, rye, barley and other representatives of Triticeae have been written down in the form of protein formulae; catalogues and data bases of such formulae were released.

  1. Usage of protein markers for testing THE genetic constituTION of AN ex situ collection
    1. The collection of the Vavilov Institute comprises a lot of accessions of old varieties (winter bread wheat) and forms with a high level of population polymorphism as compared with modern varieties. The diversity of old varieties and forms is an important source of genetic variability and consequently of valuable traits for wheat improvement. The task of genebanks is to identify, register and - store the whole genetic variability of these unique forms. It is known that in the course of long-term storage and reproduction, original populations lose part of their genotypes. One of our goals was to estimate gliadin polymorphism of old wheat varieties and to determine the level of population dynamics during reproduction and storage. Changes in genotype composition during seed multiplication in 1989-1991 were shown for some bread wheat landraces. Simultaneously it was discovered that separate genotypes of these old varieties had lost their germination ability with different rate after 2 to 8 years of storage (3). Practically, heterogenic populations (landraces, old varieties and original forms) require identification of separate genotypes and organization of their individual storage.
    2. Stored in collection of the Vavilov Institute are the original cultivars of winter bread wheat (Mironovskaya 10, Soladin, Burgas 2, Orlandi, Neuzucht 14/14, WRN 48/49, Aurora, Caucasus, Bezostaya 2, Lovrin 10, Hamlet, Linos etc.) carrying the genetic material of chromosome 1R from rye and their reproductions. Gliadin and glutenin PAG electrophoresis was used to test the genetic stability of such cultivars. Gliadin components w2 31 4 g5 encoded by the polygenic Sec 1 locus were used for detection of 1RS chromosome part. Glutenin components controlled by the Sec 3 locus are used as the markers of 1RL. Above-mentioned markers of 1RS and 1RL were discovered simultaneously in Mironovskaya 10, Soladin, Burgas 2, Orlandi, Neuzucht 14/14, WRH 48/49. That means substitution of 1R by 1B. The others are characterised by translocation T1BL-1RS. Cultivars Hamlet and Linos were stable after reproduction, as shown by protein markers. Cultivars Mironovskaya 10, Feldkrone, Perseus, Aurora, Caucasus, Bezostaya 2, Lovrin 10, Burgas 2, Saladin were also stable and corresponded to the originals by 80-90%. However, some cultivars underwent considerable changes (witnessed by protein markers) after reproduction. These changes may be as follows: elimination of 1RS, structural modification of wheat and rye chromosomes, mechanical admixtures and so on. Our results proved the need for strict control of the genuineness and integrity of such accessions stored and reproduced in gene banks and the efficiency of using protein markers for these purposes.

  2. Analysis of Triticeae germplasm based on prolamin polymorphism
    1. Genetic diversity of Triticum, Aegilops, Erytrigia, Secale, Hordeum collections stored in VIR was characterised by prolamin polymorphism. The main goals were: identification and registration of the diversity in the form of prolamin formulae, identification of duplicate accessions, formation of core-collections, and differentiation of biodiversity. Genetic differentiation of Triticum spelta L. germplasm based on gliadin polymorphism is one of the latest examples of this work.
    2. Spelt wheat is a hexaploid wheat with genome composition AABBDD. The germplasm collection of this crop maintained in VIR consists of 170 accessions originated from all principal regions of its cultivation in modern and former times. Polymorphism of seed storage proteins (gliadins) was used to characterise the spelt wheat genetic diversity. Of 170 accessions analysed, 71 were characterised by one specific type of gliadin electrohporetic pattern and identified as monomorphic. Other 99 accessions comprised from two up to eight biotypes with different gliadin patterns, and they were identified as polymorphic. Totally 42 gliadin pattern types were revealed for 86 accessions from different European countries, and 29 pattern types – for 50 accessions from Azerbaijan and Central Asia (Tadjikistan, Turkmenistan, Uzbekistan). The spelt germplasm collection maintained at VIR was registered in the form of a database of the protein formulae. The methods of cluster and principal component analyses made it possible to divide the spelt germplasm collection into several genetic groups. Groups of accessions from Germany and from some other European countries, and also groups from Spain and Tadjikistan were classified most precisely. Iranian accessions did not form a distinctive group based on the analysis of principal components. In cluster analysis, four Iranian accessions formed a subgroup and were been combined with the accessions from Tadjikistan and Morocco. The groups of accessions identified by means of gliadin markers basically corresponded to the ancient centres of spelt wheat cultivation, which were named the global centres of spelt diversity. Eco-geographical classification of this crop was proposed by P.M.Zhukovskii (4). V.F.Dorofeev et al. (5) distinguished European (subsp. spelta) and Asian (subsp. kuckuckianum) subspecies of T.spelta. The first subspecies comprises two eco-geographical groups – proles bavaricum (accessions from Germany and Switzerland) and proles ibericum (accessions from Spain). These researchers did not divide the Asian subspecies into separate groups. Revealed by gliadin analysis German and Spanish genetic groups corresponded to the above-mentioned eco-geographical groups of subsp. spelta. The differentiation of European and Asian spelt revealed by gliadins may serve as a basis for more detailed eco-geographical and taxonomic classification of this crop. We identified some accessions which can be regarded as duplicates or "genetically very close accessions". If their resemblance is confirmed by other methods, these accessions can be transferred into the rank of accessions with a rarer reproduction cycle.

  3. Genome Analysis of Wheat and Related Cereals
    1. Advantages of the immunological technique were explored in the study of phylogenetic relations between different genomes belonging to Triticum, Aegilops, Elytrigia, Elymus and Agropyron. Non-prolamin seed proteins of the Alcohol extract were successfully used as serological markers in the genome analysis of wheat and related cereals (2,6).
    2. Modern varieties of cultivated wheat belong mainly to two species: T.durum and T.aestivum. Polypliod wheats are traditionally divided into two evolutionary groups: turgidum group with genome formula AABB and timopheevii group (AAGG). Up to the present, there has been no common opinion on the origin of these genomes, especially of genome A. Originally, T.monococcum L. was considered as the donor of the first genome of polyploid wheat. Later it was assumed that wild einkorn T.boeoticum Boiss. is the source of genome A, where as Aegilops speltoides Tauch. or another species of the Sitopsis section is the source of genome B. The problem of wheat genomes has been discussed by many scientists, but it still remains unsolved. In the genome analysis of wheat and closely related cereals we used as serological markers a fraction of wheat albumins accompanying prolamins in an alcohol extract. This albumin fraction of seed proteins was a peculiar concentrate of genome-specific proteins (GSP). Methods of electrophoresis, immunodiffusion, affinity immune chromatography, enzyme-dependent immunosorbent test, thin-layer chromatography and others have been used for fractionation, purification and study of the component composition and nature of Triticum L., Aegilops L. and Elytrigia Desf. A correlation was established between the number and quantity of specific antigens of GSP and genetic interrelationships of cereal species or genomes (6). It was shown that the most active GSP antigens of cereal seeds are lipoproteins of cell membranes. Analysis of polyploid and diploid Triticum and Aegilops GSP showed that wild einkorn T.urartu Thum. was the phylogenetic donor of genome A in the turgidum-aestivum group of wheat species, while T.boeoticum was the donor for the first genome of the timopheevii group. A.V.Konarev et al. (7) were the first to publish information on the relationship of T.aestivum and T.durum genome A to wild einkorn T.urartu. Later this was confirmed by immunological (5), morphological (5) and molecular (8) methods. The proteins of wheat species from the turgidum-aestivum group revealed antigens typical for the genome of Ae.longissima, while the proteins of wheat with genome G revealed antigens typical for Ae.speltoides (1,2). It seemed likely, therefore, that Ae.speltoides (genome Bsp ) could be the source of genome G, whereas Ae.longissima could be source of genome B (1,2,6).
    3. Like Trriticum and Aegilops, the genera Elytrigia, Elymus and Agropyron include species of different ploidy levels: 2n=14,28,42,56,80. Serological markers (GSP) have helped to make a successful analysis of the interrelation between genomes belonging to genera Triticum, Aegilops, Elytrigia, Elymus and Agropyron (1,6). We used monospecific immune sera against following genome specific protein antigens: Antigen Ab (T.boeoticum genome), antigen Au (T.urartu), antigen B1 (Ae.longissima), antigen D (Ae.taushii), antigen Albumin 0,19 (Triticum, Aegilops species except T.boeoticum and T.monococcum) and other antigens (1). It was shown that some Elytrygia species, including E.elongata (2n=56,70), E.intermedia (2n=42), E.trichophora (2n=42) and E.iuncea (2n=42) possess antigens common for all three genomes of T.aestivum. Antigen 0,19 was also typical for them. Other Elytrigia and Agropyron species have common antigens only as far as one of these genomes is concerned. A series of Elytrigia and Agropyron species do not have wheat genome antigens or have the most common antigens. A representative of these species appear to be incompatible of poorly compatible in crosses with wheat species. Small degree of homology between the genomes of T.aestivum and Ag.yezoense was demonstrated. More than 80 species belonging to Elytrigia, Elymus and Agropyron were proved to be immunochemically distinctive from wheat genomes. It was shown that genetic compatibility between these species and wheat ones lies in their correspondence with the presence of protein antigens marking wheat genomes (1,6 ).
    4. Own genome-specific antigens have been identified in proteins of the following diploid Elytrigia species: E.elongata (antigen E), E.stipifolia, E.ferganensis (antigen S), E.juncea (antigen J). Antigens marking genome E have been found in proteins of 28-56 and 70 chromosome E.elongata races and some of Elytrigia and Elymus species (1,6). Antigens marking genome S have been found in most of Elytrigia species, almost always in the absence of antigens of genome E. Antigens of genome S are present in the proteins of all Agropyron and many Elymus species. It should be noted that S-antigens distinctly differentiate Elymus species into two groups. Elymus species with genome S stand closer to Elytrigia species possessing genome S than to the species of their own genus (Elymus) which do not have this genome. Elymus species with S-antigens are well compatible with diploid Elytrigia species carrying genome S. All this agrees with the results of cytogenetic analysis and supports the opinion of those researches (9, 10) who suggest that only the species with genome S should be attributed to the Elymus genus. It has been suggested that only the forms that can be crossed with wheat should remain in the Elytrigia genus. Similar classification based on cytogenetic genome analysis was proposed (10), whereby Elytrigia species which could be crossed with wheat were separated from Agropyron genus. Antigens of genome J have been identified as traces in 28- and 42- chromosome races of E.juncea as well as in polyploid forms of E.elongata (2n=56,70), E.intermedia and E.trichophora (2n=42). Genome J unites the Elytrigia species closely related by their antigenic composition to wheat species, providing for easy crossability (6).
    5. Thus, comparative analysis of Elytigia, Elymus and Agropyron showed that their diploid species carried genomes E, S and J which had specific antigens-markers. Judging by protein antigens, in most cases these genomes showed similarity with wheat genomes either by einkorns (Au,Ab) or by Aegilops (Bl, D). Polyploid species may involve, together with above mentioned genomes E, S and J, the genetic material from "wheat" genomes. Thus, a comparative analysis of Triticum, Elytrigia, Elymus and Agropyron species by grain proteins-antigens gave a possibility to correct the genome composition and also to elucidate genomic interrelation between the species. The problem of relationship between Elytrigia and wheat species is of principal interest because Elytrigia is often involved in hybridisation to produce Triticum x Elytrigia hybrids. However, a set of Elytrigia species used in distant hybridisation is very limited. Most of Elytrigia and Elymus were never involved in crossing with wheat because of the lack of information on genetic proximity of the genomes. Our results (1) partially compensate for this deficiency.
    6. Above-mentioned data on the genomic origin have been obtained by serological methods which have advantages and also several limitations. It is impossible to make an objective evaluation of quantitative content and component composition of GSP with these methods. We have proved that these parametrs are the ones of fundamental importance in determining the degree of relationships between cereal species and genomes by antigen markers. In the recent years we have managed to develop a method of applying ELISA and immunoblotting techniques to GSP of Triticeae (12). The data derived from ELISA corresponded well with the results of double immunodiffusion. All this enhances the possibility of a more efficient genome analysis by GSP antigens and of solving the problems faced in the search for markers of valuable qualities and characters of cereals.

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