THE RUSSIAN ACADEMY OF AGRICULTURAL SCIENCES

Far Eastern Research Institute of Agriculture, Karl Marx str. 105A kv. 167, Khabarovsk, 680009, Russian Federation.

Prospective varieties of spring wheat of Primorskey selection.

Ivan M. Shindin and Olga V. Lokteva.

In the Russian far east, spring wheat selection historically has been at three breeding sites Primorskey, Blagoveshenskey, and Khabarovskey. At the Primorskey breeding grounds, the selection of spring wheat is made by the Primorskey Research Institute of Agriculture.

From natural and climactic viewpoints, the Primorskey site is more harsh than the others. In addition to a lack of moisture and warmth in the spring and early sumer because of the proximity to the Pacific Ocean, monsoons occur here more frequently than at the other sites. The monsoon climate causes high moisture, plant diseases, lodging, and preharvest sprouting. Breeding cultivars for this area is quite a difficult task. The primary method of production of new cultivars is by hybridizing spring wheats with the best winter wheats adapted to the local climate. Descriptions of six, prospective new cultivars are in Tables 1 and 2.

Institute of Agricultural Employment of Reclaimed Land, Tver, 171330, Russian Federation.

Genealogical analysis of spring bread wheat cultivars included on the Russian Official List ­ 1997.


S.P. Martynov and T.V. Dobrotvorskaya.

Genealogical analysis of genetic diversity of modern Russian bread winter wheat cultivars.

We have determined the overall pattern of relationships within Russian winter bread wheats based on a pedigree analysis.

Eleven new cultivars were released in 1998: Don-95 (Zernogradka-3 / Donetskaya-46), Kroshka (Spartanka / Lutescens-4238-h-151 // Lutescens-4238-h-151), Kruiz (Chernozemka-153 / Pavlovka), Kupava (Massiv / Lutescens-90-ac-21-10), Orenburgskaya-105 (Ershovskaya-3 / Yuzhanka), Orenburgskaya-14 (Mironovskaya-808 / Albidum-114 (Lutescens-2-16) // Krupnokolosaya), Pobeda-50 (Lutescens-4473-h-144-10 / 5835-h-427 // Lutescens-4473-h-144-10), Smuglyanka (Saratovskaya-10 // Mironovskaya-808 / PV-18 /3/ Mironovskaya-808 / (HUN)I-392272), Snezhinka (Lutescens-5536 / Priboi // Chaika), Zernogradka-9 (Line 639-81 / Khersonskaya-552 (Line-1763-85) // (Line-1997-85) Line-292-80 / Line-463-76), and Zhirovka (Kavkaz / T. miguschovae // Bezostaya-1 /3/ Lutescens-4473-h-122 /4/ Lutescens-4473-h-122).

The Russian Official List 1998 includes a total of 100 winter wheat cultivars. With the exception of four cultivars with unknown or questionable pedigrees (Albidum-12, Belgorodskaya-12, Komsomolskaya-75, and Kulundinka), 96 cultivars were used for the analysis. Using the Genetic Resources Information System (GRIS 3.2), similarity of cultivars was examined using coefficients of parentage (COP) that were calculated for all pair combinations (4,560) of cultivars from pedigree information. The COP values were clustered by a hierarchical agglomerative algorithm of mean connection. A dendrograph was drawn based on the cluster analysis.

The first branch of the dendrograph produced three clusters: A, containing 47 cultivars with a within cluster mean COP of 0.20; B, containing 44 cultivars with a COP = 0.21; and a small cluster C, containing three cultivars with a COP of 0.31. Only two cultivars, Kabardinka and Polovchanka, were not in any cluster. All cultivars belonging to cluster A are derivatives of Bezostaya-1, whose genetic contribution is on average 41.6 %. More than half of the cultivars also are descendants of Mironovskaya-808, but its mean contribution is small, 7.2 %. Therefore, cluster A was named the 'Bezostaya-1 cluster'. Except for Kazanskaya-84, all cultivars belonging to cluster B are descendants of Mironovskaya-808, whose genetic contribution averages 39.8 %. Three-quarters of them also are derivatives of Bezostaya-1, but its mean contribution is less, 25.7 %. Consequently, cluster B was named the 'Mironovskaya-808 cluster'. The small C cluster was named the 'WAH cluster', because the cultivars in this cluster, Zvezda, Sibirskaya niva, and Stavropolskaya kormovaya are derivates of wheat-Agropyron hybrids. All modern cultivars are descendants of Bezostaya-1 and/or Mironovskaya-808, except Sibirskaya niva and Stavropolskaya kormovaya.

The large clusters A and B are subdivided into several subclusters at the higher relationship level equal with the midpoint COP between half-sibs and full-sibs (COP = 0.35). The A cluster (Bezostaya-1 cluster) has six subclusters. Subcluster A1 contains derivatives of Odessa and/or Balkan wheats (Albatros odesskii, Gorlitsa, Dakha, Krasnodarskaya-90, Leda, Nika Kubani, Obrii, Odesskaya-51, Olimpia-2, Rufa, Snezhinka, Soratnitsa, Stepnaya-7, Sfera, Ukrainka odesskaya, Khersonskaya-86, Yubileinaya-75, and Yuna). Subcluster A2 includes descendants of Krasnodarskaya-46 (Demetra, Kupava, and Umanka). In subcluster A3, derivatives of Donskaya polukarlikovaya (Donskaya yubileinaya, Donshchina, Eika, Zernogradka-6, Zernogradka-8, Yugtina, Donskaya bezostaya, and Murat) are predominant. The small, A4 subsluster was composed of descendants of Mironovskaya-10 (Komsomolskaya-56 and Saratovskaya-90). Subcluster A5 consists of Krasnodarskaya-39 and its derivates, Zhirovka, Kroshka, Kruiz, Ofeliya, Pobeda-50, Skifyanka, Spartanka, and Ekho. Subcluster A6 includes descendants of Donetskaya-5, Don-95, Donetskaya-46, and Tarasovskaya-87.

The B cluster (Mironovskaya-808 cluster) consists of five subclusters. Subcluster B1 includes Mironovskaya-808 and its close relatives Akhtyrchanka, Bezenchukskaya-380, Berezina, Zarya, Inna, Imeni Rapoporta, Mironovskaya-808 uluchshennaya, Omskaya ozimaya, Moskovskaya-70, Moskovskaya-642, Moskovskaya nizkostebelnaya, Nemchinovskaya-52, Niva, Pamyati Fedina, Prikumskaya-36, Smuglyanka, Chernozemka-212, and Yantarnaya-50. The mean contribution of Mironovskaya-808 is 58 %, twice its contribution in other subclusters. The subcluster B2 contains derivatives of Ilichevka (Volgogradskaya-84, Mironovskaya-25, Mironovskaya-61, and Mironovskaya poluintensivnaya). Subcluster B3 includes of Mironovskaya yubileinaya and its descendants Bagrationovskaya, Lutescens-72, Meshinskaya-2, Mironovskaya-27, Severodonskaya-5, Severodonskaya-12, Tarasovskaya-29, and Kharkovskaya-92. The B4 subcluster consists of descendants of both Yugoslavian cultivars (Sava and Biserka) and Bezostaya-1 (Don-85, Don-93, Kolos, and Kolos Dona). Subcluster B5 is composed of derivatives of the hard winter wheat Albidim-114, Bazalt, Volzhskaya-16, Ershovskaya-10, Zimorodok, Kazanskaya-84, Kinelskaya-4, Lutescens-9, and Orenburgskaya-14.

Except for the two feed wheats, Kabardinka and Stavropolskaya kormovaya, cultivars included in the Official List were split into three nearly equal quality groups: strong (34 cultivars), valuable or good fillers (30), and weak (30). The estimate of diversity in the different marked classes showed that the average COP among strong, valuable, and weak wheats differed significantly at the 0.0001 probability level (c2 = 75.6, df=6). Compared to the valuable and weak classes, the strong wheat had more full- and half-sibs, but a smaller share of unrelated pairs (Table 1).

The cultivars Bezostaya-1, Bezostaya-4, Mironovskaya-808, and Mironovskaya-264 contribute high milling and baking quality. The mean contribution of Bezostaya-1 is greatest among the strong wheats (39 %) and least among the weak (29 %). The mean contribution of Mironovskaya-808 is greatest in the group of weak wheats (26 %) and is 20 % among strong wheats. Similar results of the contribution of these parents were indicated by the cluster analysis. The Bezostaya-1 and Mironovskaya-808 clusters contained 23 (49 %) and 11 (25 %) strong wheats, respectively. Bezostaya-1 probably has a greater high-combining ability for quality than Mironovskaya-808.

The average COP among wheat cultivars developed at different institutes and within growing regions can estimate the spatial change of genetic diversity. The results of a genealogical analysis (Table 2) show that the diversity between cultivars created at different Russian and Ukrainian institutes differs significantly at the P = 0.0001 level (c2 = 98.0, df = 15). Cultivars developed at the Rostov, Krasnodar, and Mironovka institutes are less similar than those bred in Moscow and southern Ukraine. Three-quarters of the Moscow cultivars and most of the Odessa cultivars have a close relationship at the half-sib level, whereas the average COP among wheats from Krasnodar or Rostov was much less and equal to the midpoint between the half- and quarter-sibs. The large area of the Volga and Ural regions, including the Tatarstan, Bashkiria, Samara, and Saratov oblasts, contributes to the high diversity among wheat cultivars from these regions.

In addition to a quantitative estimate of the diversity in cultivars with different origins, the qualitative differences between breeding programs was determined, and the most frequent ancestors are listed in Table 3. The contribution of important ancestors is region-specific. The contributions of Bezostaya-1 and Odesskaya-16 decrease, whereas those of Mironovskaya-808 increase at higher latitudes. For example, the contributions of Bezostaya-1 and Mironovskaya-808 are equal to 0.40 and 0.09, respectively, in the Krasnodar oblast (45° N.), whereas their values in the Moscow oblast (56 N.) are 0.09 and 0.46.

The average COP within groups of cultivars recommended for different growing regions was calculated (Table 4). The regions of Russia differ significantly (at P = 0.0001, c2 = 291.8, df=24) for genetic diversity. The North-Western, Central, and Volga-Vyatka regions are quite low, because the mean similarity level is near the average between full- and half-sibs. This situation is dangerous, because it creates a narrow genetic base that may increase a crop's latent vulnerability to disease. The average COPs among cultivars recommended for the Central Chernozem, Northcaucasian, and Middle- and Sub-Volga regions are half as much. The genetic diversity of wheats in those areas is higher. Most of cultivars are unrelated or have distant relationships at the quarter-sib level.

The genetic diversity of Russian winter wheats increased to a great extent in the 1980s and especially the 1990s. This increase can be explained by the use of foreign material from Europe (Sava, Zlatna Dolina, Biserka, Sremica, Balkan, Partizanka, Novosadska Rana 2, Zagorka, Rubin, Produttore, Maris Templar, and TP-114-65A); the U.S. (Red River 68 and Atlas 66); and CIMMYT (Lerma Rojo 64, PV 18, and Siete Cerros 66).

Three conclusions are made on the basis of the genealogical analysis of winter wheat cultivars included in Russian Official List 1998.

1. The cultivar set has a cluster structure. At the quarter-sib level, the majority of cultivars belong to two large clusters, Bezostaya-1 and Mironovskaya-808. These two clusters can be subdivided into five or six subclusters at the half-sib level.
2. The diversity among strong winter wheats is significantly less than the diversity among fillers and weak cultivars. Thus, the quest to find new sources of good quality remains a high priority.
3. Diversity is low in cultivars proposed for use in the North-Western, Central, and Volga-Vyatka regions. This situation may cause economic losses because of the consequences of uniform susceptibility to disease.

SARATOV VAVILOV STATE AGRICULTURAL ACADEMY

Department of Biotechnology, Selection and Genetics, 1 Teatralnaya Sg., Saratov 410600, Russian Federation.

Effects of Rht-genes on high-temperature strength and drought resistance in spring bread wheat.

Yu.V. Lobachev.

The taller cultivar Saratovskaya 29 and its NILs and the short-stemmed backcross lines (Rht1, Rht5, Rht8, Rht14, RhtML, and RhtR) were studied between 1996-98 under dryland conditions (Lobachev et al. 1998). A drought was present in the Volga Region in 1998. The last recorded drought in this region was 100 years ago. No measurable precipitation occurred during the vegetative season, and average day temperatures frequently exceeded the average from previous years. Estimates of drought resistance and high-temperature tolerance of the cultivar Saratovskaya 29 and its short-stemmed NIL were made in the field. Maximum differences were observed in dryland conditions between field experiments in 1997 and 1998. The coefficients of high-temperature tolerance and drought resistance (CHTDR) were determined as percentages between values for 1998 and 1997. The effects of the Rht genes on CHTDR were calculated as a difference between the CHTDR for the recipient cultivar and the CHTDR for the short-stemmed NIL with the Rht gene. A difference exceeding 5 % between the CHTDR for the recipient cultivar and that for a line was significant.

Wheat yield under drought and high temperatures decreased an average of 4.5 times. In these conditions, some characteristics were affected to a lesser degree, i.e., harvest index, quantity of spikes/unit area, quantity of spikelets/spike, and 1,000-kernel weight. The average effects of CHTDR were over 70 %. Stalk height and number of grains/spike were influenced by the drought to the greatest degree, with reductions of 60 % and 40 %, respectively. Drought and heat influenced productivity of grain, and spike and grain weights, with CHTDR averaging between 20-30 %.

The cultivar Saratovskaya 29 was characterized by the following CHTDRs (in %): plant height, 58.4; grain yield, 26.2; harvest index, 89.4; spikes/unit area, 72.1; spikelets/spike, 71.1; grains/spike, 39.8; spike weight, 25.0; weight of grains/spike, 23.1; and 1,000-kernel weight, 78.8. The Rht genes did not influence CHTDRs for plant height and number of spikelets/spike. The genes Rht1 and RhtML decreased the CHTDRs for grain yield by 12.0 and 5.9 %, respectively. The remaining genes did not significantly influence these parameters. Genes Rht1, Rht5, Rht8, and RhtML decreased the CHTDRs for the harvest index by 22.4, 23.3, 15.0, and 15.2 %, respectively. All Rht genes positively influenced the high-temperature tolerance and drought resistance of wheat for the number of ears/unit area, and the genes Rht5, Rht14, and RhtML increased the CHTDRs by 5.5, 17.2, and 7.8 %, respectively. The genes Rht1 and Rht5 decreased the CHTDR for the number of grains/spike by 11.3 and 5.0 %, respectively, but Rht8 gave an increase of 8.2 %. All genes had a positive influence on the CTSDRs of spike weight. Increases were 6.3 % for Rht8, 8.3 % for Rht14, and 5.8 % for RhtR. The majority of Rht genes did not influence the CHTDRs of grains/spike weight, except for Rht8 (a 7.7 % increase) and RhtR (a 6.9 % increase). The CHTDRs for 1,000 kernel weight decreased only with Rht8 (10.0 %), RhtML (8.7 %), and RhtR (7.1 %).

Under intensive drought and heat, differences in grain yield between the recipient cultivar and the short stemmed NIL with genes Rht1, Rht5, and RhtML were observed. These genes negatively influence the high temperature strength and drought resistance to different degrees in spring bread wheat, although the negative effects of these genes on the CHTDR of grain yield did not exceed 3-12 %.

As a whole, Rht genes had an unequal influence on high-temperature strength and drought resistance in spring bread wheat. The degree of negative influence on the CHTDR for grain yield increases as following: Rht1, RhtML, Rht5, Rht8, and Rht14. The gene RhtR positively influenced the CHTDR of grain yield.

Reference.

Lobachev YuV, Zavarzin AI, and Vertikova EA. 1998. Effects of six Rht genes in spring bread wheats of the Volga Region. AnnWheat Newslet 44:190-191.

VAVILOV INSTITUTE OF GENERAL GENETICS

Gubkin str. 3, 117809 Moscow, Russian Federation.

The distribution of hybrid necrosis genes in T. turgidum subsp. dicoccum genotypes.

V.A. Pukhalskiy and E.N. Bilinskaya.

Triticum turgidum subsp. dicoccum is one of the most ancient, cultivated, wheat species. The data on the center of origin of this species are conflicting. Vavilov (1935) described two centers of origin, a Mediterranean and an Abyssinian. Later, Sinskaya (1969) showed the Near East to be the major primary center of origin and from here, the species spread worldwide. Several distribution pathways have been suggested (Sinskay 1969): 1. from the Near East to the Balkan peninsula, then along the Danube River to central Europe, and from there via France to Spain; 2. from the Balkan peninsula to the Volga and Kama regions of Russia; and 3 from the Near East to Iran, Egypt, and Ethiopia, and then into India. All of these suggestions were based on the botanic studies of T. turdigum subspecies. Genetic data are scarce. We postulated that the frequency of hybrid necrosis genes and the strength of their alleles are suitable genetic markers for describing wheats from different parts of the world.

Materials and methods. We studied 147 samples of T. turgidum subsp. dicoccum obtained from the Vavilov Institute of Plant Industry (St. Petersburg). The common, spring wheat cultivars Marquillo (genotype Ne1Ne1ne2ne2), and Balaganka (ne1ne1Ne2Ne2), and the common, winter wheat cultivars Mironovskaya 808 (ne1ne1Ne2Ne2), and Felix (Ne1Ne1ne2ne2) were used in crosses as testers. F1 and F2 hybrids were grown in the field. The manifestation of hybrid necrosis was recorded at different growth stages.

Results and discussion. Our data (see Tables 1 and 2) confirmed that the absence of the Ne2 gene in T. turgidum subsp. dicoccum, and this is typical of all tetraploid wheats. The genotype ne1ne1ne2ne2 prevailed (83.0 %). The worldwide distribution of hybrid necrosis genes is shown in Table 1.

In the Near East, the center of origin of T. turgidum subsp. dicoccum, populations without hybrid necrosis genes (noncarriers) predominated ( 92.8 % ), whereas Ne1 carriers constituted only 7.2 %, consistent with the distribution of the species from the Balkan peninsula to Europe. However, populations from central Europe (the Netherlands, Germany, Sweden, and Poland) differed somewhat from the populations of France, Italy, and Spain. In the central European group, Ne1ne2 genotypes occur at a frequency of 13.3 %, whereas the frequency was 23.1 % in the other group. The frequency of Ne1ne2 genotypes in the Volga region also was 13.0 % (the same as that in central Europe). The geographical relationship between wheats of Africa (Ethiopia and Morocco) and India was confirmed, because the frequency of Ne1ne2 genotypes from these areas was 57.1 %. These findings coincide with those of Tsunewaki and Nakai (1973) who showed that the frequency of the Ne1ne2 genotype in Ethiopian wheats was 59 %. The analysis of Ne1 alleles demonstrated that weak (w) and moderately strong (m) alleles (see Table 2) prevailed in the cultivars examined. Strong alleles were found only in wheats from Ethiopia, France, and the Volga region.

Table 2. Necrotic genotypes in T. turgidum subsp. dicoccum varieties. Accessions indicated with an 's' have stong Ne1 alleles, those with an 'm' are moderately strong, and those with a 'w' are weak alleles. The country of origin is indicated in parentheses.

Genotype Ne1Ne1ne2ne2
VIR-1725 m (Germany)**	VIR-15011 m (Poland) *	VIR-21582 s (France)
VIR-5154 m (Ethiopia)	VIR-15837 m (Marocco)	VIR-21585 (France)
VIR-6382 w (Volga region)	VIR-15840 m (Marocco)	VIR-22481 (Volga region)
VIR-6413 w (Georgia)	VIR-15847 w (Marocco)	VIR-38885 (Yugoslavia)
VIR-6436 (Georgia)	VIR-16814 (Azerbaijan)	VIR-41035 (Georgia)
VIR-7490s (Volga region)	VIR-18969 s (Ethiopia)	VIR-44154 (India)
VIR-13893 m (Ethiopia)	VIR-19256 (Ethiopia)	VIR-44167 (India)
VIR-13925 w (Daghestan)	VIR-20541 m (Spain)	VIR-46466 (India)
VIR-46482 m (India)

Genotype ne1ne1ne2ne2
VIR-81 (Germany)	VIR-13011 (Volga region)	VIR-21177 (Spain)
VIR-417 (Volga region)	VIR-13085 (Northern Caucasia)	VIR-21309 (Italy)
VIR-859 (Volga region)	VIR-13482 (Armenia)	VIR-21416 (Italy)
VIR-1726 (Germany)	VIR-13634 (Armenia)	VIR-38885 (Yugoslavia)
VIR-6436 (Georgia)	VIR-16814 (Azerbaijan)	VIR-21433 (Germany)
VIR-1730 (Germany)	VIR-13636 (Armenia)	VIR-21633 (Armenia)
VIR-6246 (Volga region)	VIR-13646 (Armenia)	VIR-21961 (Germany)
VIR-16387 (Georgia)	VIR-13648 (Armenia)	VIR-21993 (Armenia)
VIR-6391 (Azerbaijan)	VIR-13654 (Armenia)	VIR-23036 (Yugoslavia)
VIR-6412 (Georgia)	VIR-13664 (Armenia)	VIR-23637 (Armenia)
VIR-6461 (Georgia)	VIR-13665 (Armenia)	VIR-23645- (Armenia)
VIR-6534 (Germany)	VIR-13669 (Armenia)	VIR-25516 (Volga region)
VIR-7146 (Iran)	VIR-13927 (Northern Caucasia)	VIR-27885 (Georgia)
VIR-7355 (Volga region)	VIR-13930 (Northern Caucasia)	VIR-27887 (Georgia)
VIR-7356 (Volga region)	VIR-13962 (Armenia)	VIR-30091 (Azerbaijan)
VIR-7494 (Volga region)	VIR-14039 (Armenia)	VIR-30095 (Azerbaijan)
VIR-7497 (Volga region)	VIR-14043 (Armenia)	VIR-30728 (Volga region)
VIR-7498 (Volga region)	VIR-14118 (Armenia)	VIR-31602 (Azerbaijan)
VIR-7504 (Germany)	VIR-14169 (Armenia)	VIR-33116 (Armenia)
VIR-7505 (Germany)	VIR-14236 (Bulgaria)	VIR-33153 (Volga region)
VIR-7506 (Iran)	VIR-14322 (Iran)	VIR-35890 (the Netherlands)
VIR-7508 (Volga region)	VIR-14380 (Turkey)	VIR-36527 (Sweden)
VIR-7514 (Volga region)	VIR-14928 (India)	VIR-38816 (Yugoslavia)
VIR-7517 (Volga region)	VIR-14999 (Poland)	VIR-38889 (Yugoslavia)
VIR-9228 (Volga region)	VIR-15000 (Poland)	VIR-38904 (Yugoslavia)
VIR-9934 (Volga region)	VIR-15001 (Poland)	VIR-38917 (Yugoslavia)
VIR-10355 (Georgia)	VIR-16843 (Azerbaijan)	VIR-40030 (Yugoslavia)
VIR-10456 (Volga region)	VIR-16894 (Azerbaijan)	VIR-40170 (Azerbaijan)
VIR-10460 (Armenia)	VIR-17633 (Armenia)	VIR-40606 (Volga region)
VIR-11398 (Spain)	VIR-17980 (Armenia)	VIR-42065 (Volga region)
VIR-11400 (Armenia)	VIR-18616 (Armenia)	VIR-43771 (Ethiopia)
VIR-11704 (Azerbaijan)	VIR-18971 (Ethiopia)	VIR-43813 (Azerbaijan)
VIR-11740 (Armenia)	VIR-19475 (India)	VIR-43872 (Armenia)
VIR-11742 (Armenia)	VIR-20410 (Spain)	VIR-44924 (Iran)
VIR-11744 (Armenia)	VIR-20546 (Spain)	VIR-45514 (India)
VIR-11750 (Armenia)	VIR-20638 (Spain)	VIR-45541 (Iran)
VIR-11870 (Georgia)	VIR-20967 (Turkey)	VIR-45542 (Iran)
VIR-11876 (Georgia)	VIR-20968 (Turkey)	VIR-45543 (Iran)
VIR-12133 (Bulgaria)	VIR-20989 (Turkey)	VIR-45544 (Iran)
VIR-12134 (Bulgaria)	VIR-20993 (Turkey)	VIR-45545 (Iran)
VIR-12136 (Bulgaria)	VIR-21171 (Spain)
VIR-12992 (Georgia)	VIR-21172 (Spain)
* the gene was first identified by A. Zeven (1981) and later in our studies.

This work was partly supported by the Russian State Program " Frontiers in Genetics".

References.

Vavilov NI. 1935. Botanic-geographical principles of plant breeding. In: The Theoretical Bases of Plant Breeding. State Agricultural Publishing House. Moscow-Leningrad 1:17-74.

Sinskaya EN. 1969. Geographic History of Cultivated Flora. Kolos, Leningrad. 480 p.

Tsunewaki K and Nakai Y. 1973. Considerations on the origin and speciation of four groups of wheat from the distribution of necrosis and chlorosis genes. In: Proc 4th Inter Wheat Genet Symp (Sears ER ed). Columbia, Mo. pp. 123-129.

Zeven AC. 1981. Eighth supplementary list of wheat varieties classified according to their genotype for hybrid necrosis. Euphytica 30:521-539.

VAVILOV INSTITUTE OF GENERAL GENETICS, RUSSIAN ACADEMY OF SCIENCES *

Gubkin str. 3, 117809 Moscow, Russian Federation.

 

ENGELHARDT INSTITUTE OF MOLECULAR BIOLOGY, RUSSIAN ACADEMY OF SCIENCES **

Vavilov str. 32, 117984 Moscow, Russian Federation.

T.I. Odintsova *, T.I. Egorov **, and A.K. Musolyamov **.

Isolation and characterization of the high-molecular-weight glutenin subunits 17 and 18 from wheat isogenic line L-88-31.

High-molecular-weight glutenin subunits play an important role in wheat technological properties, the molecular basis for most of which remain unknown (Shewry 1995). The HMW-glutenins are encoded by genes of the Glu1 locus on chromososme group 1. Each locus consists of two genes coding for x- and y-type subunits, which differ in electrophoretic mobility. Multiple allelism was shown for HMW-glutenins.

Based on cDNA sequences, the structure of HMW-glutenin subunits was subdivided into three domains: the central domain with multiple repeats and N- and C-terminal domains. The distribution of cysteine residues in HMW glutenin subunits is of particular interest because of their specific role in the formation of the viscoelastic properties of the gluten. In all subunits, one cysteine residue is located in the C-terminal domain, whereas in the N-terminal region, three and five cysteine residues are found in the x- and y- subunits, respectively. In some HMW-glutenin subunits, additional cysteine residues are present.

The objective of this work was to isolate and characterize the HMW-glutenin subunits 17 and 18 from the isogenic line L-88-31 kindly provided by Dr. P. Shewry (Bristol University, UK). This line was used as a model, because it contains only the two subunits 17 and 18, which are the allelic variants of the HMW-glutenin subunits 7 and 9, respectively (Reddy et al, 1993) and are x- and y-type subunits. The gene for HMW-glutenin subunit 17 was isolated and sequenced (Reddy et al, 1993), and the amino acid sequence was deduced from the nucleotide sequence. Until now, HMW-glutenin subunit 18 had not been isolated or characterized. These subunits correlate with other high-quality characteristics.

Materials and methods. The HMW-glutenin subunits 17 and 18 were isolated from the T. aestivum isogenic line L-88-31. An enriched HMW-gluenin preparation was obtained according to Marchylo et al (1989) by extraction of wheat flour with 50 % propanol and subsequent precipitation of the HMW-glutenins in 60 % propanol. The HMW-glutenin preparation was separated by RP-HPLC on an Aquapore RP-300 column (C-8, 300 nm, 4.6 x 220) with an acetonitrile gradient from 23 to 40 % B for 1 h at a flow rate of 0.5 ml/min. N-terminal sequencing was on a protein/peptide sequencer (Knauer, Berlin) supplied with a model 120A PTH-analyzer (Applied Biosystems, Foster City, CA). Selective isolation of cysteine-containing peptides was according to Egorov et al. (1994).

Results and discussion. Figure 1 shows the RP-HPLC separation of HMW-glutenin subunits 17 and 18.

As seen in Fig. 1, the HMW-glutenin subunits 17 and 18 are poorly resolved under these conditions. Changing the column type and elution time did not improve the resolution. The fractions obtained, designated 1, 2, and 3, were reduced, alkylated with 4-vinylpyridine, and subjected to rechromatography on the same column. The purified fractions were analyzed electrophoretically and by N-terminal sequencing. In fraction 1, HMW-glutenin subunit 18 prevailed, 17 predominated in fraction 2, and fraction 3 contained only the HMW-glutenin subunit 17. The N-terminal amino acid sequences of the fractions are as follows:
Fraction 1: EGEASR
Fraction 2: EGEASG
Fraction 3: blocked

Fig. 1. RP-HPLC of the enriched HMW-glutenin preparation from wheat isogenic line L-88-31.

In order to improve the separation of HMW-glutenin subunits 17 and 18, we alkylated the total HMW glutenin preparation with 4-vinylpyridine prior to RP-HPLC. the subunits were still poorly resolved, but this allowed us to isolate (not shown) and identify an a-amylase inhibitor, and its N-terminal sequence is as follows: SGPWMCYPGQAFQVPALPGC. This phenomenon is of particular interest, because a-amylase was found associated with LMW-glutenin subunits. In all probability, a specific mechanism exists for regulating activity of a amylase in wheat endosperm, and its components are somehow connected with LMW- and HMW-glutenin subunits.

To characterize the primary structure of HMW-glutenin subunits 17 and 18 in more detail, we isolated their cysteine-containing peptides by immobilization on thiopropyl sepharose 6B with subsequent RP-HPLC separation of the tryptic peptides (not shown). Because it was impossible to isolate both subunits in a pure form at the preparative scale, we used a mixture of both subunits for this purpose. The major peptide fractions were sequenced (see Table 1). For two of the peptide fractions (4 and 7), two sequences were obtained, and they could be assigned clearly to HMW-glutenin subunit 17 or 18. The results obtained indicate that the amino acid sequences around cysteine residues in HMW-glutenin subunits 17 and 18 are conserved and similar to 1Bx7 and 1By9.

References.

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Reddy P and Appels R. 1993. Analysis of a genomic DNA segment carrying the wheat high-molecular-weight (HMW) glutenin Bx17 subunit and its use as an RFLP marker. Theor Appl Genet 85:616-624.

Marchylo B, Kruger J, and Hatcher D. 1989. Quantitative reversed-phase high-performance liquid chromatographic analysis of wheat storage proteins as a potential quality prediction tool. J Cereal Sci 9:113-130.

Egorov TA, Musolyamov AK, Andersen JS, and Roepstorff P. 1996. The complete amino acid sequence and disulphide bond arrangement of oat alcohol-soluble avenin-3. Eur J Biochem 224:631-638.