Genetic variability for waxy genes in Argentinean bread wheat germplasm

Leonardo Sebastián Vanzetti
Grupo de Biotecnología y Recursos Genéticos
INTA EEA Marcos Juárez
Ruta 12 S/N, (2580) Marcos Juárez
Córdoba, Argentina
Tel: 54 3472 425001
Fax: 54 3472 425001
E-mail: lvanzetti@mjuarez.inta.gov.ar

Laura Alicia Pflüger
Instituto de Recursos Biológicos CIRN-INTA
INTA Castelar
Las Cabañas y Los Reseros, 1712 Castelar
Buenos Aires, Argentina
Tel: 54 11 4621 1819/0840
Fax: 54 11 4621 1819/0840
E-mail: lpfluger@cnia.inta.gov.ar

Marta Rodríguez-Quijano
Unidad de Genética
Departamento de Biotecnología
Escuela Técnica Superior de Ingenieros Agrónomos
Universidad Politécnica 28040
Madrid España
Tel: 34 91 336 5716
Fax: 34 91 543 4879
E-mail: mquijano@bit.etsia.upm.es

José Maria Carrillo
Unidad de Genética
Departamento de Biotecnología
Escuela Técnica Superior de Ingenieros Agrónomos
Universidad Politécnica 28040
Madrid España
Tel: 34 91 336 5716
Fax: 34 91 543 4879
E-mail: josem.carrillo@upm.es

Marcelo Helguera*
Grupo de Biotecnología y Recursos Genéticos
INTA EEA Marcos Juárez
Ruta 12 S/N, (2580) Marcos Juarez
Córdoba, Argentina
Tel: 54 3472 425001
Fax: 54 3472 425001
E-mail: mhelguera@mjuarez.inta.gov.ar

*Corresponding author

Financial support: Grant from the Government of Argentina, FONCyT – INTA PICTO 12948.

Keywords: characterization, molecular markers, Triticum aestivum L., starch, waxy genes, wheat.

Amylose and amylopectin are the two polysaccharides that constitute starch in bread wheat and the enzyme GBSSI (Granule-bound starch synthase I), also known as waxy protein, is responsible for amylose synthesis in storage tissues. Decrease of the amylose content in starch has been associated with the lack of waxy protein(s). In this work, different sets of PCR markers were used to characterize the genetic variability of waxy loci from 103 Argentinean bread wheat cultivars. For the Wx-A1 locus, Wx-A1a and a novel molecular allele designed Wx-A1g were detected. Wx-B1 locus showed three alleles (Wx-B1a, Wx-B1b, Wx-B1e), and Wx-D1 locus showed only the Wx-D1a allele. Novel single-locus allele specific markers for Wx-A1b, Wx-B1b and Wx-D1b null alleles were also described. To our best knowledge this is the first study focused to characterize the genetic variability for waxy genes in bread wheat cultivars from South America.

Starch is a major component of wheat grain, accounting up to 65-70% of the dry matter in mature grain. Wheat starch is a polymer composed of two types of glucose carbohydrates: amylose, a lineal α-1,4 glucan, and amylopectin, a lineal α-1,4 glucan containing α-1,6 branch points. The granule-bound starch synthases (GBSSI or waxy proteins), are the enzymes responsible for amylose synthesis in storage tissues (Yamamori et al. 1994). Because bread wheat (Triticum aestivum L.) is an allohexaploid species (2n= 6x= 42, genomic formula AABBDD) it has three different waxy proteins (Wx-A1, Wx-B1, Wx-D1), encoded by three different genes: Wx-A1, Wx-B1, Wx-D1; located on chromosome arms 7AS, 4AL and 7DS, respectively (Nakamura et al. 1993a). Bread wheats with one or two non-functional (null) waxy genes produce starch with significant lower levels of amylose (partial waxy starch) (Nakamura et al. 1993b; Vrinten et al. 1999; Wickramasinghe and Miura, 2003). Partial waxy starch is a desirable trait in the development of wheat cultivars suitable for certain types of noodles (Epstein, 2002; Liu et al. 2003). Around the world several wheat collections have been characterized searching for null waxy proteins by 1D or 2D SDS-PAGE (Rodríguez-Quijano et al. 1998; Demeke et al. 2000; Urbano et al. 2002) and PCR markers (Briney et al. 1998; Boggini et al. 2001; Nakamura et al. 2002; Urbano et al. 2002). In wheat, PCR markers can co-amplify two or three waxy genes simultaneously making difficult its use in marker-assisted selection (MAS); this is the case of PCR markers developed by Briney et al. (1998), McLauchlan et al. (2001), Nakamura et al. (2002), Urbano et al. (2002). Therefore, the development of single-locus allele specific markers for waxy null alleles is a desirable goal for MAS. In this study we report the  development of single-locus allele specific markers for waxy nulls alleles suitable for MAS programs, and the characterization of the genetic variability of the Wx-A1, Wx-B1 and Wx-D1 loci in Argentinean bread wheat cultivars using molecular markers as a tool.

Plant materials

A set of 103 bread wheat cultivars (T. aestivum L.) from Argentina (Table 1) was screened using a set of molecular markers to assess the frequency of different Wx-A1, Wx-B1 and Wx-D1 alleles. Seed stocks were obtained from INTA Marcos Juárez and/or INTA Castelar Wheat Germplasm Collections (Argentina). Australian wheat cultivars Bodallin, Cadoux, Eradu, Gamenya, Halberd and Reeves were used as controls carrying Wx-B1b allele (Briney et al. 1998).The Spanish wheat landrace Mariñar (accession BG-018258) was used as control carrying Wx-B1e allele (Rodríguez-Quijano et al. 1998). The Chinese wheat cultivar Komugi Norin was used as control carrying Wx-A1b allele and the Australian breeding line DHWx12 was used as triple-null control (Wx-A1b / Wx-B1b / Wx-D1b) (Shariflou and Sharp, 1999). Nullitetrasomic lines of cv. Chinese Spring, N7AT7B, N4AT4B, N7BT7D and N7DT7B were used to validate the genome specificity of the designed primers.

DNA extraction, primer design, PCR reactions and sequencing

Genomic DNA from leaves of single plants was isolated as described before (Weining and Langridge, 1991). Different primer combinations based on previously published sequences were used to amplify preferentially different alleles from Wx-A1, Wx-B1 and Wx-D1 genes (Murai et al. 1999; Vrinten et al. 1999). Details about primer sequences, amplified loci and cycling conditions are in Table 2. PRIMER3 program (Rozen and Skaletsky, 2000) was used for primer design. Primers #4F/#4R were developed by McLauchlan et al. (2001). The PCR reactions were performed in a MJ Research thermocycler model PTC 100 in a 25 μl reaction mixture. Each reaction consisted of 1X Taq polymerase buffer (Promega Corp. Madison WI), 1.0 U Taq DNA polymerase (Promega), 200 μM of each dNTP (Promega), 0.2 μM of each primer, and 100-150 ng of wheat genomic DNA as template, Magnesium Chloride concentrations are detailed in Table 2. Following amplification with primers Wx-A1L/Wx-A1R, 10 μl of PCR products were directly digested with restriction enzymes Hind III (New England Biolabs Inc. Beverly MA), by adding 5 units of enzyme to the PCR products and incubating for 90 min at 37ºC. Direct PCR fragments and digested products were separated by electrophoresis on 2% agarose gels in 1X SB Buffer (Brody and Kern, 2004), stained with Ethidium bromide [0.5 g/L] and visualized by UV exposure. DNA sequencing was performed directly from PCR fragments purified using Wizard SV Gel and PCR clean-Up System Kit (Promega) using the amplification primers in both directions. Detected mutations were confirmed by sequencing of PCR fragments from at least two independent PCR reactions.

Waxy protein extraction and electrophoresis

In a subset of 53 bread wheat cultivars (underlined in Table 1) starch granule-bound proteins (including waxy protein) were extracted from embryoless grains, and later separated by SDS-PAGE as described by Rodríguez-Quijano et al. (1998). Protein patterns were visualized by silver stain using the Silver Express kit (Invitrogen Corp. Carlsbad CA).

Wx-A1 locus

Three sets of PCR primers considering proximal and central portions of Wx-A1 gene were used. Primers Wx-A1F/Wx-A1R amplify a 671-bp fragment from the proximal region of the Wx-A1 locus in plants carrying the Wx-A1a allele and a 652-bp fragment in plants with the Wx-A1b allele described by Vrinten et al. (1999). The amplification products from Wx-A1a (671-bp) and Wx-A1b (652-bp) alleles can not be easily separated in agarose gels, therefore, we found a polymorphic Hind III restriction site in the sequence from the Wx-A1a allele that divides the 671-bp fragment into two fragments of 495-bp and 176-bp (Figure 1, lanes 4 to 11), this restriction site is not present into the Wx-A1b sequence (Figure 1, lanes 1 and 2). The described markers are an effective tool to select Wx-A1b allele in marker-assisted selection breeding. When primers Wx-A1F/Wx-A1R were used to evaluate the genetic variability in the selected 103 Argentinean bread wheats, all the samples showed the Wx-A1a allele.

Primers #4F/#4R were initially designed to detect the null Wx-B1b allele (McLauchlan et al. 2001). These primers co-amplify exons 5 and 6 of the three homoeoalleles of Wx-A1, Wx-B1 and Wx-D1 genes and were used to detect genetic variability in this portion of the Wx-A1 locus. In Figure 2a, the larger fragment (299-bp) belongs to the D genome, the intermediate (257-bp) to the A genome and the smaller one (227-bp) to the B genome. When this marker was used to evaluate the genetic variability in Argentinean germplasm, 78 cultivars (76%) showed the 257-bp fragment associated with the Wx-A1 locus (Wx-A1a allele) (Figure 2b lines 4, 5, 6 and 7), and 25 (24%) showed absence of the 257-bp fragment (Figure 2b lines 8, 9, 10 and 11), which is an unexpected high proportion of putative Wx-A1 null alleles. We also found that DHWx12 (Wx-A1b control) amplified the 257-bp fragment of the Wx-A1a allele (Figure 2b lane 2).

To confirm that result, local cultivars were tested with a third set of primers Wx-A1-specific that included #4F/#4R primers region (Wx-A2L/Wx-A2R), and surprisingly, all of them amplified a 491-bp fragment. The specificity of Wx-A2L/Wx-A2R primers for Wx-A1 locus was confirmed by the lack of amplification of the 491-bp fragment in N7AT4B Chinese Spring nullitetrasomic line. Sequence comparison of PCR products amplified with Wx-A2L/Wx-A2R primers from local cultivars previously scored as “nulls” for Wx-A1 locus using #4F/#4R primers, showed two silent single nucleotide polymorphisms (SNPs), one of them located in the annealing site of the primer #4R (Figure 5). The primer #4R has an additional mismatch at position 17 (starting from the 3' end), as #4F/#4R primers amplify simultaneously Wx-A1, Wx-B1 and Wx-D1 genes, these two mismatches (one in the Wx-A1g sequence, one in the #4R primer) would favour Wx-B1 and Wx-D1 amplification against Wx-A1g. This is not a real “null” allele, so we propose to designate it as Wx-A1g (GeneBank accession DQ431232).

Wx-B1 locus

When using #4F/#4R primers, 81 tested cultivars (79%) showed the 227-bp PCR fragment for the Wx-B1a allele (Figure 2b lanes 4, 5, 8 and 9) and 22 (22%) showed absence to the 227-bp PCR fragment (Wx-B1b allele, Figure 2b lanes 6, 7, 10 and 11). Wheat cultivars Gamenya, Cadoux, Reeves and Bodallin (Wx-B1b controls), and unexpectedly, the landrace Mariñar (control Wx-B1e, Rodríguez-Quijano et al. 1998) (Figure 2b lane 3) showed absence to the 227-bp PCR fragment.

To confirm that result a second combination of Wx-B1-specific primers (Wx-B1L/Wx-B1R) was included in the analysis. This marker amplifies a 461-bp fragment in bread wheats with Wx-B1a allele and no PCR fragments are observed in wheats carrying Wx-B1b allele (nulls). With this marker, 81 of 103 cultivars (78%) showed the 461-bp PCR fragment and were scored as Wx-B1a as before (Figure 3 lanes 1, 5 and 6), but only 17 cultivars (16%) showed absence of the 461-bp fragment and were scored as Wx-B1b (Figure 3 lanes 7 and 8). The 5 remaining cultivars (5%), including the control Wx-B1e landrace Mariñar, amplified a slightly larger fragment 495-bp long (Figure 3 lanes 2, 3 and 4).

The 495-bp PCR fragment was amplified from the local cultivar Buck Poncho and sequenced (GeneBank AY954026). Sequence comparison of that fragment and the Wx-B1a allele showed nine SNPs in intron 5; six SNPs in exons 5 and 6, including four silent mutations and two amino acid changes in exon 5 (Ser to Asn and Arg to Met) and a 34-bp insertion in intron 5 that explain the different size (Figure 6). In addition, SDS-PAGE analysis in cultivars carrying Buck Poncho allele showed a Wx-B1 protein that migrate slightly slower than Wx-B1a, having the same size of the Wx-B1e allele in Mariñar (Figure 7). Therefore, PCR fragment size and waxy protein band size were used as arguments to assign the 495-bp PCR fragment amplified with primers Wx-B1L/Wx-B1R from the local cultivar Buck Poncho to the Wx-B1e allele.

Wx-D1 locus

For the molecular characterization of the Wx-D1 locus two set of primers were used. The set of primers Wx-D1L/Wx-D1R was developed to detect the Wx-D1b allele described by Vrinten et al. (1999) in the cultivar Bai Huo. These primers amplified a 930-bp fragment from the distal region of the wild type Wx-D1a allele and also included a 588-bp deletion present in the Wx-D1b allele. The Wx-D1b control breeding line DHWx12 showed the mutation described by Vrinten et al. (1999) in Bai Huo (Figure 4 lane 1). When this molecular marker was used to evaluate the genetic variability in 103 Argentinean bread wheat cultivars, all tested samples showed the Wx-D1a allele. (Figure 4 lanes 2 to 11).

Primers #4F/#4R were also used to characterize the genetic variability of the Wx-D1 locus and no molecular variability was detected. SDS-PAGE protein analysis showed the GBSSI subunit corresponding to the Wx-D1 locus in all tested cultivars, confirming previous data observed with PCR markers (data not shown). The lack of variability observed in the Wx-D1 locus in comparison with Wx-A1 and Wx-B1 loci agreed with previous data (Graybosh et al. 1998).

Urbano et al. (2002) expressed that waxy protein polymorphism in wheat is not very high, especially when compared with other group of proteins, such as storage proteins of wheat kernels. A possible explanation could be that most of the initial characterization studies of waxy proteins in wheat were performed by SDS PAGE of GBSSI proteins from starch granules, and with this technology small differences between proteins might be underestimated. In this context, molecular markers can be a valuable tool to characterize the genetic variability of waxy genes at DNA level, as sequences from Wx-A1, Wx-B1, Wx-D1 genes are available (Murai et al. 1999). Moreover, the polyploid nature of bread wheat and the high homology between A, B and D genomes, are difficult issues to overcome in the development of single-locus allele specific markers because of the always latent possibility of coamplifying homologous alleles. This is the case of markers for waxy genes developed by Briney et al. (1998), McLauchlan et al. (2001), Nakamura et al. (2002) and Urbano et al. (2002).

In this work we used allele-specific mutations to develop single-locus allele specific markers for Wx-A1, Wx-B1, Wx-D1 genes. As expected, the most frequent alleles in waxy genes from tested wheat cultivars were the wild types (Table 1). Moreover, additional genetic variability in Wx-A1 and Wx-B1 loci was detected.

Wx-A1g is a new allele whose origin is probably CIMMYT, as the most ancient reference stocks in this work are Pampa INTA and Las Rosas INTA (year of release 1984) which are selections from CIMMYT germplasm and Klein Salado (year of release 1985) which is a selection from local and CIMMYT germplasm. As previously expressed, mutations associated with changes in aminoacid composition were not detected in partial Wx-A1g sequence, suggesting that protein function has not been affected in this allele. In line with these arguments, waxy protein patterns in cultivars scored as Wx-A1a and Wx-A1g were identical, different from Wx-A1c allele with a slightly altered isoelectric point and from Wx-A1b and Wx-A1f which showed real null protein alleles (Yamamori et al. 1994; Saito et al. 2004). Wx-A1d and e alleles were described in the bread wheat relatives T. dicoccoides (Wx-A1d) and T. durum (Wx-A1e), respectively (Yamamori et al. 1995) as protein bands with sizes different from Wx-A1a, which is not the case of Wx-A1g.

The origin of Wx-B1b allele in local germplasm is also unclear, as it was detected in cultivars developed using CIMMYT germplasm (Prointa Puntal, Klein Escorpión, Klein Chajá) and old local germplasm (Klein Rendidor, year of release 1954). The cultivars scored in this study as Wx-B1b showed lack of PCR products with two different sets of primers (#4F/#4R, Wx-B1L/Wx-B1R) and lack of the diagnostic Wx-B1a protein fragment by SDS-PAGE. This data suggest the presence of a large mutation similar to the null Wx-B1b allele described by Vrinten et al. (1999) in the cultivar Kanto 107. This mutation is the most frequent null mutation for waxy genes in wheat, having been detected in germplasm from Asia, Europe and North America (Saito et al. 2004) but not from South America, being this study the first report. Unfortunately the extend of the Wx-B1b deletion has not been established yet; therefore,  it is uncertain if the size of the putative deletion observed in local cultivars carrying Wx-B1b allele is the same that the Wx-B1b allele described by Vrinten et al. (1999) in the cultivar Kanto 107. Further fine mapping studies focused on WxB1b allele can answer this question.

In the case of the Wx-B1e allele, the most probable origin is Buck Poncho, a selection from local germplasm released in 1986, because all the other cultivars carrying Wx-B1e allele share Buck Poncho in their pedigree. The Wx-B1e allele is difficult to detect with markers #4L/#4R developed by McLauchlan et al. (2001) and by GBSS pattern detection in SDS-PAGE because of fragment overlapping. Cultivars with the Wx-B1e allele using #4L/#4R primers will generate fragments of 261-bp, which is very close to the 257-bp of Wx-A1a allele, and they will be probably scored as "nulls" for Wx-B1 locus in agarose gels. The Wx-B1e allele is also difficult to be detected by SDS-PAGE because its mobility is similar to the Wx-D1a allele (lower mobility than Wx-B1a) (Demeke et al. 2000; Marcoz-Ragot et al. 2000; Yamamori and Quynh, 2000). In this work we describe a novel single-locus allele specific marker that accurately detects the Wx-B1e allele in agarose gels.This marker is a valuable tool to develop isogenic lines for Wx-B1e allele to evaluate the effect of amino acid changes detected in exon 5 of the Wx-B1e allele in amylose/amylopectin ratio.

The importance of the identification of new forms of waxy protein is related to a reduction in the amylose content found in genotypes carrying these mutations (null mutations) (Nakamura et al. 1993b). In this work we have detected one allele (Wx-B1b) with a deleterious effect in protein function, and a second allele (Wx-B1e) carrying several mutations whose effect in protein function still have to be elucidated. The Wx-B1b allele was detected in 16% of wheat cultivars. These cultivars carrying partial waxy starch can be an attractive target in the development of local adapted cultivars, suitable for certain specialties like dry white chinese noodles (Liu et al. 2003). No null alleles for Wx-A1 or Wx-D1 loci were detected in our bread wheat collection, which agree with data observed in European germplasm (Marcoz-Ragot et al. 2000). The vast majority of germplasm carrying Wx-A1b or Wx-D1b alleles is from Turkey, Korea, Japan (Yamamori et al. 1994) and probably, China. The molecular markers single-loci allele specific for nulls Wx-A1b, and Wx-D1b alleles are being used to introgress foreign null Wx-A1b and Wx-D1b alleles in local germplasm by marker assisted selection programs in order to develop adapted wheats with partial and total waxy starchs with different levels of amylose content.

L. Vanzetti expresses his gratitude to FONCYT-INTA for a PICTO fellowship during this work which is part of his PhD Thesis. The authors want to express their gratitude to Paola Romina Aurelia for her excellent technical assistance, to Beatriz Formica for providing the wheat seeds used in this study and to Jorge Dubcovsky for his valuable suggestions in the writing of this paper.

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