Allelic diversity of puroindoline genes at the Ha locus in the core-collection of hexaploid wheat accessions conserved by NBRP-Wheat

Hiroyuki Tanaka^1*, Yuma Ohnishi¹, Shotaro Takenaka², Miyuki Nitta², Taihachi Kawahara², Shuhei Nasuda²

¹ Laboratory of Plant Genetics, Faculty of Agriculture, Tottori University, Tottori 680-8553, Japan

² Laboratory of Plant Genetics, Graduate School of Agriculture, Kyoto University, Kyoto 606-8502, Japan

^*Corresponding author: Hiroyuki Tanaka (E-mail: htanaka@muses.tottori-u.ac.jp)

Abstract

Allelic diversity of puroindoline genes Pina-D1 and Pinb-D1 was investigated by nucleotide sequencing of the PCR-amplified genes in the core-collection of 163 hexaploid wheat accessions conserved by NBRP-Wheat. Of the total, 99 accessions possessed both of wild-type gene sequences, Pina-D1a/Pinb-D1a. One and 5 of the previously reported mutations were found in the Pina-D1 and Pinb-D1 genes, respectively.

The most prevalent mutation was Pinb-D1b, being present in one third of accessions with mutant alleles. The Pinb-D1p allele was found predominantly in Afghanistan. All puroindoline alleles found in Triticum aestivum were shared by the other Tiriticum species.

Key words: hexaploid wheat, grain hardness, allelic diversity, puroindoline

Introduction

Grain hardness is an important end-use quality characteristics of wheat. This trait is mainly controlled by a single locus, called Hardness (Ha) (Symes 1965; Baker 1977), which is located on the short arm of chromosome 5D (Mattern et al. 1973; Law et al. 1978). The genes encoding friabilin were tightly linked to the Ha locus (Jolly et al. 1993; Sourdille et al. 1996; Campbell et al., 1999).  Friabilin are composed mainly of two proteins, puroindoline a and b, which are encoded by the Pina-D1 and Pinb-D1 genes (Morris, 2002).  All soft wheat varieties examined so far carry the wild-type puroindoline alleles that have been designated Pina-D1a and Pinb-D1a (Giroux and Morris, 1997, 1998; Morris, 2002; Bhave and Morris, 2008a, b). Hard wheat varieties have specific mutations in either of the Pina-D1 or Pinb-D1 genes, or they lack the genes (Morris, 2002; Morris and Bhave, 2007; Wang et al., 2008). In this study, we investigated puroindoline genotypes of the core-collection of hexaploid wheat accessions developed by the National BioResource Project-Wheat.

Materials and methods

We examined 163 accessions of the core-collection of hexaploid wheat (Triticum aestivum, T. compactum, T. macha, T. spelta, T. sphaerococcum, T. vavilovii) develpped and conserved by National BioResource Project-Wheat, Japan (Takenaka et al., in preparation, Table 1). Total genomic DNA was extracted from young leaves by the CTAB method (Kim et al., 1997) and used as template for PCR. The PCR primers used to amplify the puroindoline genes were those reported by Gautier et al. (1994) as follows: 5’-ATGAAGGCCCTCTTCCTCA-3’ and 5’-TCACCAGTAATAGCCAATAGTG-3’ for the amplification of the full-length (447 bp) Pina-D1 gene, and 5’-ATGAAGACCTTATTCCTCCTA-3’ and 5’-TCACCAGTAATAGCCACTAGGGAA-3’ for the amplification of the full-length (447 bp) Pinb-D1 gene. PCR amplification was performed using TaKaRa Ex Taq DNA polymerase (2.5 U, TaKaRa) in 100 µl of reaction buffer (TaKaRa, 2 mM MgCl₂) containing 100 ng of genomic DNA, 200 µM of each dNTP and 40 pmol of each primer. The PCR conditions were 93ºC for 4 min followed by 35 cycles of 94ºC for 60 s, 53ºC for 90 s and 72ºC for 120 s. A final cycle with an extension of 10 min at 72ºC completed the reaction. The DNA amplification was performed using a C1000^TM Thermal Cycler (Bio-Rad). A 10 µL aliquot of the product was separated in 1.5% (w/v) agarose gel, stained with ethidium bromide and visualized using UV light. A 1 µL aliquot of the product was used for direct DNA sequencing on a DNA sequencer (Applied Biosystems 3130xl Genetic Analyzer) using the BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems). The sequencing primers were those described above for primary gene amplification (Gautier et al., 1994). The nucleotide sequence results were called by Sequence Scanner v1.0 (Applied Biosystems) and further analyzed using GENETYX v12 (GENETYX).

Results and Discussion

Pina-D1 and Pinb-D1 primer sets successfully amplified an expected single band (447 bp) in 149 and 149 out of 163 accessions, respectively. The rest 14 and 14 accessions were assumed to have null alleles of Pina-D1 and Pinb-D1, respectively. Ninety-nine accessions possessed wild-type alleles, Pina-D1a/Pinb-D1a. DNA sequencing of the Pina-D1 gene identified one of the well characterized mutations, Pina-D1l (Gazza et al., 2005). As for Pinb-D1, five previously reported mutations were identified (Pinb-D1b (Giroux and Morris, 1997), Pinb-D1c (Lillemo and Morris, 2000), Pinb-D1d (Lillemo and Morris, 2000), Pinb-D1p (Xia et al., 2005) and Pinb-D1ab (Tanaka et al., 2008)). All accessions were classified according to their puroindoline haplotypes and geographic origin (Table 1). The Pinb-D1b allele was the most prevalent mutation, being present in one third of accessions with mutations. Interestingly, this allele is the most frequent mutation among wheat varieties of all over the world (Cane et al., 2004; Xia et al., 2005; Ikeda et al., 2005; Chang et al., 2006; Chen et al., 2006; Lillemo et al., 2006; Pickering and Bhave, 2007; Tanaka et al., 2008). The Pinb-D1p allele was found predominantly in Afghanistan. Tanaka et al. (2008) reported that the distribution of the allele Pinb-D1p may be associated with the so-called ‘Silk Road’, the ancient trade and cultural transmission route between China and the Mediterranean Sea. This allele was also present in Pakistan and Afghanistan but was not found in Japan. In this study, the allele Pinb-D1p was found in one accession in China but was not found in Japan, which is in accordance with the previous study (Tanaka et al. 2008). The absence of the allele in Japan could be explained by assuming that the common wheat in small population size with a limited amount of genetic diversity was introduced to Japan. All of the puroindoline alleles found in this study, have been reported in T. aestivum (genome constitution AABBDD) and/or Aegilops tauschii (DD). We found these alleles in the other Triticum species. Therefore, if the polyphylogenetic origin of hexaploid wheat species is assumed, these alleles should have been established in Ae. tauschii before the allopolyploidization event in Triticum species. Alternatively, if we assume limited number of hexaploidization events, recurrent mutations in Pina-D1 and Pinb-D1 genes have occurred both in Ae. tauschii and hexaploid wheat.

References

Baker RJ (1977) Inheritance of kernel hardness in spring wheat. Crop Science 17: 960-962.

Bhave M, Morris CF (2008a) Molecular genetics of puroindolines and related genes: allelic diversity in wheat and other grasses. Plant Molecular Biology, 66: 205–219.

Bhave M and Morris CF (2008b) Molecular genetics of puroindolines and related genes: regulation of expression, membrane binding properties and applications. Plant Molecular Biology, 66: 221–231.

Campbell KG, Bergman CJ, Gualberto DG, Anderson JA, Giroux MJ, Hareland G, Fulcher RG, Sorrells ME, Finney PL (1999) Quantitative trait loci associated with kernel traits in soft £ hard wheat cross. Crop Science 39: 1184–1195.

Cane K, Spackman M, Eagles H (2004) Puroindoline genes and their effects on grain quality traits in southern Australian wheat cultivars. Aus J Agric Res 55:89-95.

Chang C, Zhang H, Xu J, Li W, Liu G, You M, Li B (2006) Identification of allelic variations of puroindoline genes controlling grain hardness in wheat using a modified denaturing PAGE. Euphytica 152:225-234.

Chen F, He ZH, Xia XC, Xia LQ, Zhang XY, Lillemo M, Morris CF (2006) Molecular and biochemical characterisation of puroindoline a and b alleles in Chinese landraces and historical cultivars. Theor Appl Genet 112: 400-409.

Gautier MF, Aleman ME, Guirao A, Marion D, Joudrier P (1994) Triticum aestivum puroindolines, two basic cystine-rich seed proteins: cDNA sequence analysis and developmental gene expression. Plant Molecular Biology 25: 43–57.

Gazza L, Nocente F, Ng PKW, Pogna NE (2005) Genetic and biochemical analysis of common wheat cultivars lacking puroindoline a. Theor Appl Genet 110: 470-478.

Giroux MJ, Morris CF (1997) A glycine to serine change in puroindoline b is associated with wheat grain hardness and low levels of starch-surface friabilin. Theoretical and Applied Genetics 95: 857–864.

Giroux MJ, Morris CF (1998) Wheat grain hardness results from highly conserved mutations in the friabilin components puroindoline a and b. Proceedings of the National Academy of Sciences of the United States of America 95: 6262–6266.

Ikeda TM, Ohnishi N, Nagamine T, Oda S, Hisatomi T, Yano H (2005) Identification of new puroindoline genotypes and their relationship to flour texture among wheat cultivars. J Cereal Sci 41:1-6.

Jolly CJ, Rahman S, Korrt AA, Higgins TVJ (1993) Characterization of the wheat Mr 15000 ‘grain softness protein’ and analysis of the relationship between its accumulation in the whole seed and grain softness. Theoretical and Applied Genetics 86: 589–597.

Kim CS, Lee CH, Shin JS, Chung YS, Hyung NI (1997) A simple and rapid method for isolation of high quality genomic DNA from fruit trees and conifers using PVP. Nucleic Acids Research 25 (5): 1085-1086.

Law CN, Young CF, Brown JWS, Snape JW, Worland JW (1978) The study of grain protein control in wheat using whole chromosome substitution lines. Seed Protein Improvement by Nuclear Techniques. Vienna, Austria: International Atomic Energy Agency, pp. 483-502.

Lillemo M, Morris CF (2000) A leucine to proline mutation in puroindoline b is frequently present in hard wheats from Northern Europe. Theor Appl Genet 100: 1100-1107.

Lillemo M, Chen F, Xi X, William M, Peña RJ, Trethowan R, He Z (2006) Puroindoline grain hardness alleles in CIMMYT bread wheat germplasm. J Cereal Sci 44: 86-92.

Mattern J, Morris R, Schmidt JW, Johnson VA (1973) Location of genes for kernel properties in the wheat cultivar ‘Cheyenne’ using chromosome substitution lines. In: Sears ER and Sears LMS (eds) Proceedings of the 4th International Wheat Genetics Symposium. Columbia, MO: University of Missouri, pp. 703-707.

Morris CF (2002) Puroindolines: the molecular genetic basis of wheat grain hardness. Plant Molecular Biology 48: 633–647.

Morris CF and Bhave M (2007) Reconciliation of D-genome puroindoline allele designations with current DNA sequence data. Journal of Cereal Science, doi: 10.1016/jjcs2007.09.012.

Pickering P, Bhave M (2007) Comprehensive analysis of Australian hard wheat cultivars shows limited puroindoline allele diversity. Plant Sci 172: 371-379.

Sourdille P, Perretant MR, Charmet G, Leroy P, Gautier MF, Joudrier P, Nelson JC, Sorrells ME, Bernard M (1996) Linkage between RFLP markers and genes affecting kernel hardness in wheat. Theoretical and Applied Genetics 93: 580–586.

Symes KJ (1965) The inheritance of grain hardness in wheat as measured by particle size index. Australian Journal of Agricultural Research 16: 113-123.

Tanaka H, Morris CF, Haruna M, Tsujimoto H (2008) Prevalence of puroindoline alleles in wheat varieties from eastern Asia including the discovery of a new SNP in puroindoline b. Plant Genetic Resources: Characterization and Utilization 6: 142–152.

Wang L, Li G, Xia X, He Z, Mu P (2008) Molecular characterization of Pina and Pinb allelic variations in Xinjiang la ndraces and commercial wheat cultivars. Euphytica 164: 745-752.

Xia L, Chen F, He Z, Chen X, Morris CF (2005) Occurrence of puroindoline alleles in Chinese winter wheats. Cereal Chem 82: 38-43.