77. Figure 1. PCR products of the varieties Bánkúti 1201, Chinese Spring and Cheyenne obtained with primers specific for 1Dx5 HMW-GS gene. Figure 2.
Euphytica 119: 75–79, 2001. © 2001 Kluwer Academic Publishers. Printed in the Netherlands.
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Identification, cloning and characterisation of a HMW-glutenin gene from ´ 1201 an old Hungarian wheat variety, B´ankuti A. Juh´asz1 , L. Tam´as2 , I. Karsai1 , G. Vida1 , L. L´ang1 & Z. Bedö1 1 Agricultural
Research Institute of the Hungarian Academy of Sciences, Martonv´as´ar, Hungary; 2 Department of Plant Physiology, Lor´and Eötvös University, Budapest, Hungary
Key words: cysteine residue, HMW-glutenin, storage proteins, wheat
Abstract Despite its good functional properties, the variety Bánkúti 1201 has been found to possess 2 + 12 or 3 + 12 allele composition on chromosome 1D. In order to determine the reason for its quality traits a gene-specific PCR technique was applied in preliminary experiments to examine the HMW glutenin allele composition of the variety. In the course of the analysis a fragment characteristic of Bánkúti 1201 was identified and the nucleotide sequence was determined. This showed the presence of a 1Ax2 ∗ gene variant which, despite near homology, differed from the original 1Ax2 ∗ gene at one important point. At 1181 bp of the 1Ax2 ∗ sequence nucleotide exchange was observed which is the middle nucleotide of the TCT–TGT base triplet, involving the exchange of serine for cysteine. The gene was designated 1Ax2 ∗B . The presence of an extra sulphydryl group, like that of the extra cysteine in the 1Dx5 gene, facilitates the formation of further disulphide bonds, might lead to an improvement in gluten quality characters.
Introduction The use of populations of old landraces and varieties in plant breeding has numerous advantages when the aim is to increase genetic variability. Selection from varieties with traditionally good quality and the use of genetically heterogeneous populations as gene sources play an important role in developing and improving new genotypes. The landraces of Eastern Europe have no small part in breeding for quality. Red Fife, which was one of the parents of the Canadian spring wheat variety Marquis, and the Bánát landraces, which are found, for example, in the pedigree of Bezostaya 1, all originate from the eastern edge of the Carpathians. Bánkúti 1201, developed in the first half of the 20 t h century from a cross between Marquis and Bánkúti 5, enjoyed great popularity in Hungary, and variants of this variety were also used as basic breeding stock in the neighbouring countries (Bedö et al., 1995; Vida et al., 1998). In the late sixties the old Hungarian varieties were gradually removed from cultivation and their popu-
lations are only preserved in gene banks. Like other old Hungarian varieties, Bánkúti 1201 is a genetically heterogeneous population consisting of lines with various storage protein compositions. When the population was divided into lines on the basis of HMW glutenin subunit composition several allelic variants were discovered on all three chromosomes. One of the most remarkable things about this variety is that despite having mainly subunits 2+12 (Bedö, 1994; Bedö et al., 1995; Hänsel et al., 1994; Vida et al., 1998) or 3 + 12 (Kárpáti et al., 1994) on chromosome 1D, it has excellent technological properties. The old Hungarian wheat varieties generally have high protein contents (above 15%) with a gluten content of over 40%, but fairly large gluten extensibility values. Their SDS sedimentation values are also high and they can be classed in the A1–A2 category on the basis of farinograph water uptake (Bedö et al., 1995; Vida et al., 1998). The experiments were aimed at discovering whether there was any explanation at the molecular level for the good technological properties of Bánkúti
76 1201 despite its 2 + 12 subunit composition on chromosome 1D.
PRISMTM Dye Terminator Sequencing Kit (PE Applied Biosystem). The EMBL and Genbank data bases and the GCG program package were used to evaluate the sequencing data.
Materials and methods PCR analysis
Results and discussion
a. Fifty-three lines of a Bánkúti 1201 population were tested with a primer pair (Prim51: 5 GCCTAGCAACCTTCACAATC-3 and Prim52: 5 GAAACCTGCTGCGGACAAG-3 ) specific for the N terminal region of the HMW 1Dx5 gene (D’Ovidio et al., 1994), Chinese Spring (– 7 + 8 2 + 12) and Cheyenne (2∗ 7 + 9 5 + 10) were used as controls. One of the primers (Prim52) contains a base triplet (TGT) coding for the amino acid cysteine, which is capable of forming disulphide bridges. The product specific for the HMW 1Dx5 gene is 450 bp in length and the specificity of the reaction is given by the Cys base triplet found at the beginning of the repetitive section of the 1Dx5 gene. According to D’Ovidio the primer pair is suitable for the distinction of 2 + 12 genotypes, since in the presence of a sufficiently large quantity of template DNA (over 100 ng) the 450 bp product is not amplified and a DNA fragment measuring 1160 bp is formed. This product is characteristic of genotypes possessing the 1Dx2 + 1Dy12 allele composition (D’Ovidio & Anderson, 1994). b. PCR analysis to determine the sequence around the binding site of Prim52 In order to clarify the sequence of the gene section around the binding site of Prim52 a primer pair was compiled based on the experimental and known sequence data with the aid of the Prime (GCG – Genetics Computer Group, Madison, Wis., USA) program. These primers have the sequences 5 -GCAACAATCAACACAAGAG-3, which binds to the known Ax2 ∗ sequence at 978 bp, and 5 TTGCTCCCCTTGTCTTTG-3 , which binds to the Ax2∗ gene sequence at 1299 bp.
For lines selected from the Bánkúti 1201 population a fragment measuring approx. 1300 bp was formed as well as the 450 bp and 1160 bp products expected. On the basis of the fragment composition the population can be divided into 3 groups: Group A: 1160 bp + 1300 bp (e.g. B1201/B11), Group B: 450 bp + 1300 bp (e.g. B1201/B29), Group C: 450 bp + 1160 bp (e.g. B1201/B10). More than 80% of the lines examined contained this extra 1300 bp DNA fragment, which was not found in any of the control samples (Fig. 1). Sequencing was carried out on three clones from three independent PCR reactions of each of three lines of Bánkúti 1201 (B1201/B11, B1201/B20, B1201/B29), all of which were shown in preliminary tests to contain the 1300 bp fragment. Comparison with the sequences of the data bases (Fasta – GCG) showed the greatest similarity with the HMW glutenin genes. On the basis of nucleotide and derived amino acid sequences the fragment exhibited over 90% homology with 1Ax type genes. Compared with the sequences of the 1Ax type genes available in the data base, the 1300 bp fragment showed 100% similarity with the relevant section of the 1Ax2∗ gene. This result is in agreement with the results of earlier SDS-PAGE analyses, which indicated that Bánkúti 1201 contained the 1Ax2 ∗ protein subunit (Bed˝o et al., 1994; Vida et al., 1998). Due to the great extent of sequence homology between the various HMW glutenin genes, all the HMW glutenin alleles (1Ax2 ∗, 1Dx2, 1Bx7, 1By9, 1Dy12) of Bánkúti 1201 and the 1Ax1 and 1Dx5 alleles were tested to determine whether the primer pair was able to bind and whether it amplified the 1300 bp fragment. It was found that the primer Prim52 would only be able to bind at a distance of approx. 1300 bp from the binding site of Prim51 if the reaction conditions allowed 6 mismatches (Figure 2). The most important difference between genes 1Ax1 and 1Ax2 ∗, judging by the sequences available in the data bank, are that the repetitive section of gene 1Ax1 contains two insertions (one 18 nucleotides and the other 27 nucleotides in length) compared to 1Ax2∗. Since one of these insertions is located in the
Cloning and sequencing The purified PCR product was ligated into pGEMT vector (Promega) and then transformed into an Escherichia coli JM109 competent cell. Universal Forward and Reverse primers were used to determine which of the positive clones contained the 1300 bp insert. The sequencing was carried out with the aid of universal Forward and Reverse primers using the ABI
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Figure 1. PCR products of the varieties B´ank´uti 1201, Chinese Spring and Cheyenne obtained with primers specific for 1Dx5 HMW-GS gene.
Figure 2. Priming possibilities of the primers prim51 and prim52 determining a 1300 bp size fragment in different HMW-GS genes
section we examined, but was not found in the course of sequencing, this is further proof that the gene containing the fragment is a variant of 1Ax2 ∗ (Figure 4). The Prim51-Prim52 primer pair was also used to test a number of varieties indicated by SDS-PAGE analysis to contain the protein subunit 2 ∗ . The 1300 bp product was not formed either in the control variety Cheyenne (2 ∗ 7 + 9 5 + 10) or in any of the other genotypes containing the 1Ax2 ∗ HMW glutenin subunit (e.g. Bezostaya 1, Mv Magma, Mv Madrigál). Both PCR analysis and computer analysis of the sequences confirmed the hypothesis that the gene containing the 1300 bp fragment was a mutation of 1Ax2∗ , while on the basis of sequence identity with gene 1Ax2 ∗ the mutation occurred at the binding site
of Prim52. To check this, a primer pair was constructed, based on the results of sequencing, with the aid of which the nucleotide sequence of the DNA section near the 3 end of the 1300 bp fragment, i.e. near the binding site of Prim52, could be determined. The amplified fragment, measuring approx. 300 bp was cloned and then sequenced. When the sequence was compared with that of the HMW glutenin genes, 100% identity was observed with 1Ax2 ∗ except at a single point. The only difference was that at the 1181 bp point of the coding region of gene 1Ax2 ∗ there was a C–G point mutation, as the result of which the Prim52 primer was able to bind and the 1300 bp product was amplified (Figure 3). The point mutation also influenced the derived amino acid sequence. The triplet
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Figure 3. Comparison of the Prim52 primer, the B1201/B11 clone and the relevant section of the Ax2∗ gene
Figure 4. Comparison of the 1Ax type gene sequences (1Ax2∗ and 1Ax1) to 1Ax2∗B gene sequence from the variety B´ank´uti 1201
79 coded by TCT was modified to TGT, which is one of the nucleotide triplets coding for cysteine (Figure 4). In order to examine the effect of the gene containing an extra cysteine, correlations were sought between the fragment composition of the Bánkúti 1201 lines included in the PCR analysis and technological parameters. In both the 7 + 9 2 + 12 and the 7 + 8 2 + 12 backgrounds significantly higher sedimentation and gluten indexes were observed in lines containing both the 2∗ protein subunit and the 1300 bp product; this type made up approx. 75% of the tested lines. This result is confirmed by earlier data indicating that the allele composition of the individual lines was much more strongly correlated with parameters indicative of gluten quality than with those associated with gluten quantity. An analysis of lines with different HMW glutenin compositions revealed a significant difference between lines with the allele compositions 2 ∗ 7 + 9 2 + 12 and 1 7 + 9 2 + 12 and between those with 2 ∗ 7 + 8 2 + 12 and 1 7 + 8 2 + 12. All parameters associated with gluten quality exhibited higher values if the line possessed the 1Ax2 ∗ allele, so the values of the sedimentation and gluten indexes were higher and the gluten spread was smaller (Vida et al., 1998). It can be concluded from these results that the presence in Bánkúti 1201 of a mutant 1Ax2 ∗ gene, designated as 1Ax2 ∗B, has a positive influence on functional characters similar to that of the 1Dx5 gene.
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