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Sel’skokhozyaistvennaya Biologiya [Agricultural Biology], 2010, № 5, p. 36-41.
UDK 633.11:577.114:631.52:577.2.08
ABOUT OPTIMIZATION OF MOLECULAR LABELING OF WHEAT Waxy-GENES FOR MAS-SELECTION
M. V. Klimushina, P.Yu. Kroupin, M.G. Divashuk, G.I. Karlov
Science Research Center of Molecular Biotechnology, Russian State Agrarian University - MTAA named after K.A.Timiryazev,
Moscow 127550, Russia, e-mail: [email protected]
Received April 29, 2010 S u m m a r y
The results of the investigations show that the two molecular markers for Wx-Al and Wx-Bl loci can be recommended for marker-assisted selection of wheat. They have been found to be suitable both for searching for the null-alleles among the variety of Russian cultivars and for MAS-selection for reduction of amylase content in grain. Using the selected markers the authors have developed the wheat lines containing two null-alleles of Wx-genes, which can be applied as donors to transfer these alleles to other wheat cultivars.
Key words: marker-assisted selection, molecular markers, Waxy-genes, bread wheat.
Soft wheat (Triricum aestivum L.) is one of most common food crops in the world. Wheat caryopses contains scratch - a component determining baking and technological properties of flour, whose structure includes both linear (amylose) and branchy (amy-lopectin) polysaccharides (1). Amylose and amylopectin are usually located in the plastids in the form of discrete water-insoluble granules. Wheat starch contains 15-25% amylose and 75-86% amylopectin (2); wheat forms enriched with one of these polysaccharides were obtained by breeding work. The ratio between contents of amylose and amylopectin determines differences in temperature of gelatinization, viscosity of starch glue, its texture and ability to form gel, as well as resistance to mechanical impact and influence of acidic medium (3).
The key enzyme in a synthesis of endosperm amylose is a granule-bonded starch synthetase I (GBSS I) encoded by so-called Waxy genes (4). The studied Wx (Waxy) mutants of maize, wheat, rice and barley didn’t carry GBSS I; amylose content in them was very low or it was absent, while the starch consisted of amylopectin of a wild type (5).
Three homeologous wheat genes GBSS I are located in chromosomes 7AS (Wx-A1), 4AL (Wx-B1) and 7DS (Wx-D1) (6). Should GBSS I not be formed, this suggests a presence of null alleles of Waxy-genes in wheat. Wheat with null alleles for one or two Wx-genes is called as part- Waxy wheat. The greatest impact on amylose content and quality of grain products has been established for Wx-B1 -genes, then Wx-D1 and Wx-A1 (7). In wild nature, the full- Waxy wheat hasn’t been found, as it requires a simultaneous presence of the three mutant genes (7).
The presence of one or two null Wx-alleles results in wheat grain as a number of properties important for production of noodles and biscuits (8). Thus, preparation of starch noodles requires a lot of wheat flour with high-volume swelling, high viscosity peak and rapid gelatinization, which is typical for varieties with one or two null alleles of Wx-genes and low amylose content. Reducing the content of amylose has a positive effect on bread-baking qualities, as it contributes to formation of lush and crunchy wheat flakes in dry corn breakfasts (starch of a wild type causes brittleness and crumbling of the product) and increases shelf life of bakery products (it is amylose that gets bread stale) (9).
The grain of Waxy-wheat with one or two null alleles is the most efficient raw for ethanol production owing to low temperature of starch gelatinization. After boiling of the corn at 85 °C, starch structure becomes significantly disrupted, which makes starch chains easily accessible to enzymes, reduces energy consumption and makes possible using Waxy-starch in production of biofuel. Waxy-wheat has higher enzymatic estimation compared to conventional wheat and maize as it takes less time to complete fermentation (9).
Thus, the selection of Waxy- and part- Waxy wheat varieties for use in food industry is one of promising directions. At the same time, this process is hindered by presence in the genome of three homeologous Waxy-genes with identical function (5, 6), as well as by problem of identification of part-Waxy forms and mutant gene loci.
At present time, there are different methods of identification of Waxy-mutants: staining of starch granules with a solution of I2-KI, RVA-analyzers (rapid visco analyzer), for example, RVA-StarchMaster2 (“Newport Scientific”, USA), as well as the differential scanning calorimetry, eg. DSC 204 F1 Phoenix (“Netzsch”, Germany), spectroscopy of near-infrared spectral range, one-dimensional SDS-electrophoresis of Waxy-proteins in polyacrylamide gel (SDS-PAGE) and the system of two-dimensional electrophoresis (2D-PAGE) (10). However, these methods can detect only full-mutant forms, or they are very difficult, time consuming and expensive.
Marker-assisted selection (MAS) is the most promising technique for creating soft wheat forms with targeted quality of starch (11). In this regard, polymerase chain reaction (PCR) can be effectively used for identification of carriers of GBSS I null alleles among many wheat samples. Mutations in three Wx-genes were studied at molecular level, which put the basis for development of DNA-markers identifying null alleles of Wx-gene (4, 8), but testing of these markers for MAS purposes hasn’t been performed yet.
This work was aimed at solution of following tasks: choosing, approbation and testing of DNA-markers for null alleles of Waxy-genes best suitable for MAS-selection, identification of such alleles in samples of strong wheat varieties and creation of forms carrying multiple null alleles.
Technique. The objects of study were 40 varieties and lines of winter soft wheat (the collection was kindly provided by prof. Netsvetaev V.P., the Belgorod Research and Development Institute of Agriculture).
DNA was isolated according the method of Bernatzky and Tanksley (1986) with modifications (12). Primers were synthesized by the company "Synthol" (Moscow).
PCR amplification was performed under the following conditions:
Wx-A1, primers AFC and AR2 (12) 1 cycle — 95 °C, 5 min; 32 cycles—95 °C, 30 s;
65 °C, 30 s; 72 °C, 2 min; 1 cycle — 72 °C, 7 min; storage at 4 °C;
Wx-A1, primers Wx-Alb-F-MH and Wx-Alb-R-MH (13) Wx-A1, primers Sun1F and Sun1R (14)
Wx-B1, primers BDFL and BRDR (12)
Wx-B1, primers Wx-B1F and Wx-B1R (15)
Wx-D1, primers Wx-D1-2-F and Wx-D1-2-R (7)
Wx-D1, primers Wx-D1-1-F and Wx-D1-1-R (16) Wx-D1, primers BDFL and DRSL (12)
1 cycle — 95 °C, 3 min; 40 cycles — 94 °C, 45 s;
55 °C, 30 s; 72 °C, 1 min; 1 cycle — 72 °C, 7 min; storage at 4 °C;
1 cycle — 94 °C, 3 min; 30 cycles — 94 °C, 45 s;
58 °C, 30 s; 72 °C, 1 min; 1 cycle — 72 °C, 7 min; storage at 4 °C;
1 cycle — 95 °C, 5 min; 32 cycles — 95 °C, 30 с;
55 °C, 30 с; 72 °C, 2 min; 1 cycle — 72 °C, 7 min; storage at 4 °C;
1 cycle — 94 °C, 3 min; 30 cycles — 94 °C, 60 s;
58 °C, 60 с; 72 °C, 30 s; 1 cycle—72 °C, 7 min; storage at 4 °C;
1 cycle — 95 °C, 3 min; 40 cycles — 94 °C, 30 s;
55 °C, 45 s; 72 °C, 1 min; 1 cycle — 72 °C, 7 min; storage at 4 °C;
1 cycle — 95 °C, 3 min; 40 cycles — 94 °C, 30 s;
55 °C, 45 s; 72 °C, 1 min; 1 cycle — 72 °C, 7 min; storage at 4 °C
1 cycle — 95 °C, 5 min; 32 cycles — 94 °C, 30 s;
58 °C, 30 s; 72 °C, 1,5 min; 1 cycle — 72 °C, 7 min; storage at 4 °C.
PCR products were separated by electrophoresis in 2% agarose gel with 0,5 XTris-borate buffer (field strength 6 V/cm). 100 bp DNA Ladder (“Fermentas”, Lithuania) was used as a marker of sizes.
Screening of the collection of wheat lines and varieties was carried out using the optimized systems for molecular labeling of Wx-gene alleles. The varieties carrying mutant alleles of Waxy gene were isolated and crossed with each other. To obtain a splitting F2 population, F1 plants were self-pollinated. Hybrid nature of F1 and splitting processes in the F2 population were revealed by the optimized systems of molecular labeling.
Statistical processing of data was performed by the method of ^-correspondence (13).
Results. The advantages of PCR technique -ability to identify part- Waxy samples and mutant alleles of GBSS I at relatively low expenses - were appreciated during the identification of Waxy- and part- Waxy- wheat forms.
Upon the analysis of literature data, the authors determined the molecular markers suitable for identification of different alleles of GBSS I.
Gene Waxy-A1. A co-dominant CAPS-marker for null allele of Wx-A1b has been developed by M. Helguera et al. (14). After PCR with an appropriate primer, the size of amplicons in normal alleles and in null alleles differed insignificantly and could be identified only by polyacrylamide gel electrophoresis or capillary electrophoresis. That’s why amplification should be followed by restriction visualizing the differences in agarose gel.
M.R. Shariflou et al. (15) suggested the primers - sequences flanking the microsatellite repeat near 3'-end of cDNA sequence of Waxy-A1. A high polymorphism of microsatellite repeats allows using this marker for identification of allelic variants of the gene Waxy-A 1 (15).
Using the sequences of mutant and normal Waxy-alleles, T. Nakamura et al. (16) have constructed the co-dominant marker based on primers which provide the amplification of fragments from all homeologous alleles of Waxy-genes. Particularly, for alleles Wx-A1a a wild type, these are the fragments of 389 bp length and for null allele - 370 bp (16).
Testing and optimization of PCR conditions have shown that the marker developed by M.R. Shariflou et al. (15) was unsuitable for screening and identification of null alleles in the studied samples. This system can only be used for molecular labeling of the null allele in later generations derived from parental lines, one of with is known to carry the identified null allele. Two other systems were found to be suitable for screening of the targeted null allele, but the technique proposed by M. Helguera et al. (14) required carrying out either capillary electrophoresis or subsequent restriction increasing the cost of performing tests. As a result, the molecular marker proposed by T. Nakamura et al. (16) was selected for use as the most effective and reliable.
Gene Waxy-B1. A. McLauchlan et al. (17) have developed a molecular labeling system for the locus Waxy-B1: in a wild type, it provided amplification of three DNA fragments, in forms with null allele - only two fragments (the lightest one absent). T. Nakamura et al. (16) proposed the primers based upon sequences of mutant and normal alleles of Waxy-gene. Fragments from all homeologous alleles of Waxy-genes could amplify with these primers, while forming by the wild-type Wx-B1a of amplicons 425 bp length and an absence of amplification in the case of null allele of Wx-B1b.
In the authors’ experiments performed under optimal conditions of PCR, applying the first of the listed systems was accompanied with atypical amplification, which could complicate interpretation of results at screening of the studied collection of wheat samples. Considering this fact, the molecular marker proposed by T. Nakamura et al. (16) was used for screening of Wx-B1 gene as the most reliable.
Gene Waxy-D1. M.R. Shariflou et al. (7) applied the primers providing amplification of the fragment 279 bp length at presence of the null allele of Waxy-D1, and 910 bp - for allele of a wild type. P. Vrinten et al. (18) separated the amplification products in 3% agarose gel with a consequent staining of null alleles of Waxy-D1 with ethidium bromide, which resulted in obtaining of a fragment smaller than in the wild type. Based upon sequences of mutant and normal Waxy-alleles, T. Nakamura et al. (16) have developed the co-dominant marker for alleles of Waxy-D1 (for null allele, the amplified fragment 1731 bp length was detected, for a wild type -2307 bp).
For the second and third systems, no amplification was observed by the authors. In general, this fact agrees with data of creators of these markers, who reported about their effectiveness only on plant material and alleles used in their breeding programs and researches. As a result, the molecular marker developed by MR Shariflou et al. (15) was considered to be the only suitable for screening of Wx-D1 gene in the studied collection.
Identification of carriers of null alleles among the collection samples and obtaining of hybrids. The screening of 40 wheat varieties using the selected primers has revealed only two samples (the varieties Starshina and Korotyshka) possessing the null alleles
for, respectively, Wx-A1 and Wx-B1 loci. In the studied collection, no forms with null allele of Wx-D1 gene were identified (all the varieties had the locus of a wild type). It is noteworthy, that the null allele of Wx-D1 gene is extremely rare in soft wheat.
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Electrophoregram showing the products of amplification of molecular markers Wx-B1 (A) and Wx-A1 (B) in splitting population F2: 1, 3, 4, 6, 7, 8 — the plants heterozygous or homozygous for the wild type allele of Wx-B1; 2, 5 — the plants homozygous for null allele of Wx-B1; 9 — the plants heterozygous for Wx-A1; 10 — the plants carrying the wild type allele of Wx-A1; 11 — the plants carrying homozygous null-allele of WX-A1. М — molecular-weight marker (100 bp DNA Ladder by “Fermentas”, Lithuania).
F1 hybrids were obtained from the cross Strarshina lA Korotyshka, and their hybrid nature for the loci Wx-A1 and Wx-B1 was confirmed using molecular markers. 203 plants of F2 resulting a self-pollination of F1 were also tested on presence of null allele loci of Wx-genes (Fig. A). Strictly speaking, the molecular marker Wx-B1 is not co-dominant, because it doesn’t provide the identification of heterozygous plants. However, the amplification from other Wx-genes was observed, which reliably proved the correctness of DNA isolation and PCR processes in the case of null allele as well. The estimated splitting of the studied F2 population was 3 (1 homozygote of a wild-type and 2 heterozygotes): 1 (homozygote for null allele), while the established ratio - 159:47 individuals, or 3,38:1. The actual ^2 was equal to 0,524, while the tabular value 3,841.
Since the main purpose of testing the splitting F2 population was identification of forms carrying double null alleles of Waxy-genes, the marker for Wx-A1 was applied only upon the plants with established homozygous null alleles of Wx-B1. In optimized conditions of electrophoresis (4% agarose gel, 3 V/cm), the amplified fragments were divided into three fractions (the authors of the method isolated two fractions) (16), which revealed both homozygous and heterozygous forms in the splitting population (Fig. B). Using these molecular markers, 10 plants with double null alleles of Wx-genes have been selected from 206 plants the analyzed F2 population.
Thus, two molecular markers for wheat Wx-A1 and Wx-B1 loci have been identified and proved to be equally suitable for screening of carriers of null al-
leles among samples of wheat varieties, as well as for use in marker-assisted selection (MAS) aimed at obtaining the forms with low amylose content. Using these markers, the authors have identified wheat forms carrying two null alleles of Wx-gene loci in the genome, which gives the prospects for their use as donors of these alleles in crosses with other wheat varieties.
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