Characterization and Phylogenetic Analysis of Α-Gliadin Gene Sequences Reveals Signiﬁcant Genomic Divergence in Triticeae Species

c Indian Academy of Sciences

RESEARCH ARTICLE

Characterization and phylogenetic analysis of α-gliadin gene sequences reveals signiﬁcant genomic divergence in Triticeae species

GUANG-RONG LI1, TAO LANG1, EN-NIAN YANG2, CHENG LIU1 and ZU-JUN YANG1∗

1School of Life Science and Technology, University of Electronic Science and Technology of China, Chengdu, Sichuan 610054, People’s Republic of China 2Crop Research Institute, Sichuan Academy of Agricultural Sciences, Chengdu, Sichuan 610066, People’s Republic of China

Abstract Although the unique properties of wheat α-gliadin gene family are well characterized, little is known about the evolution and genomic divergence of α-gliadin gene family within the Triticeae. We isolated a total of 203 α-gliadin gene sequences from 11 representative diploid and polyploid Triticeae species, and found 108 sequences putatively functional. Our results indicate that α-gliadin genes may have possibly originated from wild Secale species, where the sequences contain the shortest repetitive domains and display minimum variation. A miniature inverted-repeat transposable element insertion is reported for the first time in α-gliadin gene sequence of Thinopyrum intermedium in this study, indicating that the transposable element might have contributed to the diversification of α-gliadin genes family among Triticeae genomes. The phylogenetic analyses revealed that the α-gliadin gene sequences of Dasypyrum, Australopyrum, Lophopyrum, Eremopyrum and Pseudoroengeria species have amplified several times. A search for four typical toxic epitopes for celiac disease within the Triticeae α-gliadin gene sequences showed that the α-gliadins of wild Secale, Australopyrum and Agropyron genomes lack all four epitopes, while other Triticeae species have accumulated these epitopes, suggesting that the evolution of these toxic epitopes sequences occurred during the course of speciation, domestication or polyploidization of Triticeae.

[Li G.-R., Lang T., Yang E.-N., Liu C. and Yang Z.-J. 2014 Characterization and phylogenetic analysis of α-gliadin gene sequences reveals signiﬁcant genomic divergence in Triticeae species. J. Genet. 93, 725–731]

Introduction the α-gliadins are the most abundant, comprising 15–30% of the wheat seed proteins. The cereal plants including wheat, maize and rice are impor- Since the gliadins generally contribute to the viscosity and tant food crops for the daily intake of proteins, vitamins, extensibility of dough, a great deal of research attention has minerals and fibres worldwide. The total protein contents of focussed on genetic studies of wheat gliadins over the last cereal seeds vary from about 10–15% of the grain dry weight, decades (Gianibelli et al. 2001). It is well understood that the with about half of the total being storage proteins (Shewry α-gliadin proteins are encoded by members of a multigene and Tatham 1990). In addition to their nutritional value, family. Gene loci (Gli-2)forα/β-type gliadin subfamilies cereal seed proteins also influence the utilization of the grain are clustered on the short arm of wheat chromosomes 6A, in food processing (Shewry and Halford 2002). The major- 6B and 6D (Shewry and Tatham 1990; Anderson and Greene ity of seed storage proteins are prolamins. All prolamins of 1997). However, the number of gliadin proteins determined the Triticeae (wheat, barley and rye) were assigned to three by two-dimensional polyacrylamide gel electrophoresis (2-D broad groups: sulphur-rich (S-rich), sulphur-poor (S-poor) PAGE), was considerably less than the estimated gene copy and high molecular weight (HMW) prolamins. The S-rich numbers by genomic analysis (Anderson et al. 1997;Islam prolamins included subgroups of γ -gliadins, α/β-gliadins et al. 2003). It is shown that such differences were presum- and B-type and C-type low molecular weight (LMW) sub- ably caused by genes’ differential amplification along the units of glutenin (Shewry and Tatham 1990). Among them, course of genome evolution (Gu et al. 2004a, b). Recently, various gliadin genes have been cloned and sequenced which provided opportunities to understand the molecular ∗ For correspondence. E-mail: [email protected]. structures and genomic organization of wheat gliadins by Keywords. α-gliadin; celiac disease epitopes; genomic evolution; molecular divergence; Triticeae.

Journal of Genetics, Vol. 93, No. 3, December 2014 725 Guang-Rong Li et al. comparing them with other cereal prolamins (Kawaura Based on the published α-gliadin gene sequences (NCBI et al. 2005). Among them, the α-gliadins gene sequences accession numbers: AJ130948 and TAU51308), a pair of from wheat and its ancestral species containing A, B and primers P1 (5-CCACAAATCCAACATG(A/T)AGACCTT D genomes have been thoroughly investigated (van Herpen (C/T)(A/C)T-3)andP2(5-TTCCCATG(C/T)TTGAACTA et al. 2006;Xieet al. 2010; van den Broeck et al. 2010; (G/C)TATAGGT(C/A)G-3) were designed for amplifying Qi et al. 2011). There are a few reports that describe the the complete sequences of α-gliadin genes. P1 was located α-gliadins of wild Triticeae species including Dasypyrum at 200-bp upstream of the translation start codon, whereas (Li et al. 2009), Lophopyrum and Elymus (Wu et al. 2010). primer P2 was located at about 100-bp downstream from the However, these data are not enough to represent a general stop codon. picture for the origin of the α-gliadins during the evolution Polymerase chain reaction (PCR) was carried out using and speciation of different Triticeae genomes. iCycle thermal cycler (Bio-Rad, Hercules, USA). Each PCR Unfortunately, the α-gliadins seem to be the major initia- reaction mixture (100 μL) contained 300 ng template, tors of celiac disease (CD), which affect as many as one in 0.2 mM of each dNTPs, 1 μM of each of the two primers, 10 every 300 people (Shewry et al. 1992). Gluten specific T-cell μL10× PCR buffer and 5 U high fidelity Ex Taq DNA poly- receptors in the small intestine play an important role in pro- merase (Takara, Shiga, Japan). The PCR was programmed ducing the inflammatory response in CD. It is also known for 2 min at 95◦C followed by 35 cycles of 94◦Cfor1min, that patients are generally sensitive to more than one gluten 60◦Cfor1minand72◦C for 2 min 30 s. After amplification, peptide. Multiple T-cell epitope motifs have been identified the final extension step of 10 min at 72◦C was carried out. in α-gliadins and γ -gliadins as well as in glutenins (van de The PCR product was analysed on 1% agarose gels. Wal et al. 1998; Koning 2003; Vader et al. 2003), in which specific native gluten peptides can bind to HLA-DQ2/8, and Cloning, sequencing and comparative analyses of α-gliadin the interaction of DQ2/8 represents the most significant asso- genes ciation with CD. A large variation exists in the amount The target PCR products were purified from the agarose gel α of CD4 T-cell stimulatory peptides present in -gliadins using QIAquick Gel Extraction kit (Qiagen, Valencia, USA). γ and -gliadins and glutenins among diploid, tetraploid and The fragment was ligated into a pGEM-T easy plasmid hexaploid wheat accessions (Molberg et al. 2005). Studies vector (Promega, Madison, USA), and used to trans- have suggested that the four known T-cell stimulatory pep- form competent cell of Escherichia coli DH5α strain. tides are nonrandomly distributed across the wheat A, B and Fifteen positive clones were sequenced on an automatic D genomes (Van Herpen et al. 2006). This raises an impor- DNA sequencer (TaKaRa Biotech, Dalian, China). The tant question about the evolution and distribution of the toxic nucleotide sequences were assembled and the sequence epitopes among the Triticeae species during their specia- alignment among different α-gliadin alleles from our stud- tion and polyploidization, and how one can possibly select ies and Aegilops tauschii α-gliadin gene sequences from and breed wheat varieties without toxic epitopes suitable for NCBI GenBank (HM188546 to HM188559) was car- consumption by CD patients. ried out using Bioedit 7.0 (Ibis Therapeutics, Carlsbad, USA). In this study, isolation and systematic analysis of the α- gliadin genes was carried out in several Triticeae species rep- Phylogenetic and nucleotide diversity analysis resenting different genomes, and our results provided new α insights into the Triticeae genome evolution with respect to The complete amino acid sequences of -gliadins were used polyploidy, pattern and dynamics of gene superfamily and for multiple alignment and phylogenetic analysis. The align- their influence on gene epitopes product. ment data were converted to mega format using the MEGA (molecular evolutionary genetics analysis) program (Kumar et al. 2004). Neighbour-joining (NJ) tree was constructed Materials and methods and the divergent times of different genes or genomes were estimated using MEGA4. Based on the full-ORF (open Plant materials reading frame) α-gliadin genes, the NJ tree was constructed Australopyrum retrofractum, Dasypyrum villosum, Thinopy- and the divergence was estimated using the molecular clock rum intermedium, Lophopyrum elongatum,andPseudo- of 6.5×10−9 substitutions per synonymous site per year roengeria spicata were kindly provided by Dr Harold (Gaut et al. 1996). Bockelman, National Plant Germplasm System, USDA-ARS, Aberdeen, Idaho, USA. Secale cereale, S. sylvestre and S. Analysis of CD-toxic epitopes valvovii were maintained at Key Laboratory for Plant Gene- The reported T-cell stimulatory epitopes glia-α (QGSFQP- tics and Breeding, Sichuan Agricultural University, China. SQQ), glia-α2 (PQPQLYPQ), glia-α9 (PFPQPQLPY) and glia-α20 (FRPQQPYPQ) have their own position in the DNA preparation and PCR amplification wheat α-gliadin protein (Van Herpen et al. 2006). We Genomic DNA was isolated from 1 to 2-g leaves of single searched for the perfect matches of the four epitopes in plant which was about 4–6 weeks old (Yang et al. 2006). the obtained full-ORF α-gliadin genes, and investigated the

726 Journal of Genetics, Vol. 93, No. 3, December 2014 Characterization of α-gliadin genes in Triticeae presence and absence of the epitopes among the α-gliadin other Secale species including S. cereale and S. valvovii genes. did not give positive PCR results. The nucleotide comparison showed that the 10 S. sylvestre sequences belong to the Results α-gliadin gene sequences, in which no pseudogenes were found (table 1). This is the first report on the α-gliadin gene Cloning of α-gliadin genes sequences in Secale species. The amino acid sequence align- The PCR primers, P1/P2, were used to amplify the com- ment indicated that these Secale α-gliadin gene sequences plete sequences of α-gliadin genes from the representa- contained eight conserved cysteine residues, and a short glu- tive genus of Australopyrum, Dasypyrum, Lophopyrum, tamine repeat in Q1 and Q2 regions (figure 1). We cloned Thinopyrum, Eremopyrum, Agropyron, Pseudoroengeria and the α-gliadin gene sequences from both diploid D. villosum Secale. Besides the nine diploid species, two polyploid and D. breviaristatum species, and found that the glutamine species, namely, tetraploid Dasypyrum breviaristatum and number in Q2 region varied highly, in which D. villosum pos- hexaploid Thinopyrum intermedium were also included in sessed 12–24 (averaged 18), diploid D. breviaristatum con- the analysis (table 1). The PCR gave a product of 750–1300 tained only 2–11 (averaged 6). The α-gliadin gene sequences bp. Total 300 unique clones from all species were sequenced. of Th. bessarabicum exhibited high percentage of pseudo- After removing the low-quality and duplicated sequences, genes (15/17). Among the α-gliadin gene sequences from the 203 sequences were deposited into GenBank (table 1 in representing species Ps. spicata (St genome), Lo. elongatum electronic supplementary material at http://www.ias.ac.in/ (E genome), Au. retrofractum (W genome), Ag. desetorum jgenet/). The nucleotide comparison of the entire sequences (P genome) and Er. bonaepartis (F genome), there is high showed a high degree of homology with wheat α-gliadin variation of glutamine residues in Q2 region, Er. bonaepartis sequences. In each sequence, TATA box was commonly had the longest glutamine residues (averaged 24), while found at 92–96 bp upstream of the ATG start codon, and the Au. retrofractum contained six glutamine residues polyadenylation element was present at 72–80 bp down- (table 1). stream of the stop codon. Sequence prediction indicated that the 108 sequences include the complete ORF of genes α (table 1). The 95 sequences although displaying structural The -gliadin genes from polyploid species similarity to the full-ORF genes were pseudogenes since Out of the 28 tetraploid Dasypyrum breviaristatum α-gliadin they comprised of typical in-frame premature stop codons genes, 12 were pseudogenes (table 1). The tetraploid D. bre- resulted from the transition of T by C at the first base of viaristatum α-gliadin sequences contained 8–10 glutamine glutamine codon (CAA, CAG). The deduced amino acid residues, while in Q2 region, possessed 10–28 (averaged sequence of the α-gliadin gene sequences represented a pre- 16.3). Compared to the diploid D. breviaristatum based sumptive mature protein with 186–297 residues. All full on the α-gliadin sequences variation, it is likely that the ORFs gave rise to the general structure of α-gliadin pro- tetraploid D. breviaristatum displayed large genomic diver- tein given by van Herpen et al. (2006), which consists of a gences, which supported our previous hypothesis of evo- short N-terminal signal peptide (S) followed by a repetitive lution of Dasypyrum species (Li et al. 2009;Liuet al. domain (R) and a longer nonrepetitive domains (NR1 and 2010). NR2), separated by two polyglutamine repeats (Q1 and Q2). Totally, 20 Th. intermedium α-gliadin genes sequences were identified, among which 12 were pseudogenes (table 1). In particular, we found a 1314 bp α-gliadin The α-gliadin genes from diploid species gene sequence (HM452949) from Th. intermedium.The Among the Secale species, the primers P1/P2 can amplify sequences alignment revealed that it encoded 284 amino the target products from the wild S. sylvestre only, while acids (figure 1 in electronic supplementary material). In

Table 1. The α-gliadin sequences from 11 species.

Species Genome notation Ploidy Full ORF Pseudogene Length of ORF Length of Q1 Length of Q2

S. sylvestre R2n =14 10 0 558–570 3 (3) 6 (6) A. retrofractum W2n =14 12 6 720–910 8 (3–14) 6 (2–11) A. desertorum P2n =14 9 7 870–915 10 (8–14) 18 (12–24) E. bonaepartis F2n =14 8 8 834–969 10 (7–11) 24 (18–29) D. breviaristatum Vb 2n =14 8 6 810–954 8 (3–14) 6 (2–11) D. villosum V2n =14 10 6 846–897 10 (8–14) 18 (12–24) L. elongatum E2n =14 16 4 855–884 18 (6–24) 8 (7–9) P. spi cat a St 2n =14 10 18 812–1073 6 (3–8) 13 (6–28) T. bessarabicum J2n =14 2 15 855–872 8 (7–9) 5 (3–7) T. intermedium JJsSt 2n =42 7 13 831–861 5 (3–8) 5 (3–10) D. breviaristatum VbVb 2n =28 16 12 867–900 8 (7–10) 16 (8–23)

Journal of Genetics, Vol. 93, No. 3, December 2014 727 Guang-Rong Li et al.

Figure 1. Alignment of the deduced amino acid sequences for the representing α-gliadin full ORFs. The accession number and the genome formula are shown on the left. ‘–’ represent deletions of amino acid residues. Q1 and Q2 represent the two polyglutamine repeats. The boxed letters are conserved cysteine residues. the untranslated region (3 UTR) downstream of stop DQ217663, DQ267478 and AY695367) as outgroups. As codon of HM452949, we found a miniature inverted-repeat shown in figure 2, it is clear that the γ -gliadins were transposable element (MITE) Stowaway transposon inser- closely related to α-gliadin genes. Among the α-gliadin tion. The secondary structure of this element showed that the gene sequences, the Secale α-gliadin gene sequences were four inverted repeat regions formed a more-or-less hairpin- first separated, and D. breviaristatum and Au. retrofractum like structure (figure 2 in electronic supplementary mate- α-gliadin genes were categorized as the second subgroup. rial). It is implied that the transposon elements were involved Based on the estimated evolutionary rates, it is possible in the amplification of α-gliadin genes family during the that the diploid D. breviaristatum α-gliadin sequences have polyploidization of Thinopyrum species. diverged around 21 million years ago (Mya) from Secale α-gliadin. The sequences from D. villosum, Lo. elongatum, Th. bessarabicum, Ps. spicata and Ae. tauschii α-gliadin Phylogenetic analysis of α-gliadin sequences were clustered as the third subgroup. The Th. bessarabicum A total of 81 representative α-gliadin gene sequences from α-gliadin sequences showed close relationship (less than diploid species of Triticeae tribe were used to construct 4 Mya) to those sequences of wheat D-genome ancestor a phylogenetic tree by MEGA3 using genes of γ -gliadin Aegilops tauschii. It is most likely that they have undergone (FJ441103 and FJ595936) and LMW-GS (AY542896, convergent evolution after their speciation. It is interesting to

728 Journal of Genetics, Vol. 93, No. 3, December 2014 Characterization of α-gliadin genes in Triticeae

Figure 2. A phylogenetic tree generated from the nucleotide sequence alignment of the Triticeae α-gliadin genes. The wheat LMW-GS and γ -gliadin genes were used as outgroups. The bar indicates the estimated divergence time by sequences variation. show that the α-gliadin genes from Au. retrofractum, D. bre- the deletion of a glutamine (Q) at both glia-α2 and glia-α9 viaristatum, Lo. elongatum, Er. bonaepartis and Ps. spicata epitopes, the presence of arginine (R) instead of proline (P) clustered into two different groups (figure 2). We conclude at Glia-α20 region and the changes of several amino acids at that the α-gliadin genes showed divergent evolution to give the position of glia-α region. Both Au. retrofractum and Ag. rise to several orthologues among species of the Triticeae. desetorum genomes lack toxic epitopes, suggesting that the toxic epitopes evolved during the divergence of Agropyron species. Therefore, the results demonstrate that a set of epi- Analysis of CD-toxic epitopes topes are totally absent in the α-gliadin sequences from Aus- Four epitopes, glia-α (QGSFQPSQQ), glia-α2 (PQPQLY tralopyrum (figure 3 in electronic supplementary material). PQ), glia-α9 (PFPQPQLPY) and glia-α20 (FRPQQPYPQ), The sequences of diploid D. breviaristatum in the first group were used as queries in searches and perfect matches were have no epitopes, while all diploid D. villosum and most of obtained for full-ORF genes as well as for the pseudo- D. breviaristatum in the second group contained the epitope genes. The single nucleotide polymorphism (SNP) resulted Glia-α. Similarly, the first group of Ps. spicata (St) genome in an amino acid change in a particular epitope. Among the lack epitope Glia-α, the rest with full-ORF genes contained obtained 10 full-ORF Secale α-gliadin genes, none of the epitope Glia-α with different frequency. Therefore, the four epitopes related sequences were found, indicating the distribution and variation of the four sets of epitopes were in- complete lack of the toxic epitopes in the putative ancestry deed distinct from the genomes, which provided probabilities α-gliadin genes. All ORFs from Australopyrum displayed to select nontoxic germplasm suitable for CD patients.

Journal of Genetics, Vol. 93, No. 3, December 2014 729 Guang-Rong Li et al.

Discussion of α-gliadin sequences occurred in these species. Mason- Gamer (2007) reported that unique MITE Stowaway family In the last 20 years, numerous molecular phylogenetic anal- were found in Australopyrum and Taeniatherum species. Sun yses of Triticeae species were based on data from chloroplast et al. (2009) also revealed that MITE insertion appeared DNA markers, high-copy nuclear genes and lower single- in low-copy nuclear RNA polymerase II (RPB2) genes of copy nuclear genes (Sun et al. 2009;Liuet al. 2010). Some Hordeum species. In this study, we also found a MITE inser- of the earlier datasets showed appreciable conflicts with one tion at the 3 UTR region of the Thinopyrum intermedium α- another (Kellogg et al. 1996). The phylogenetic relationships gliadin genes. It may represent a typical model for Triticeae among Triticeae genera are not consistent with a single bifur- genomic evolution, in which the transposable element cating tree, it should be possible to clarify reticulate patterns mediated mechanism may be related to the process of structural with the use of multiple gene trees, based on genomewide and functional evolution of genes (Gu et al. 2004b). markers (Mason-Gamer 2005, 2007). The availability of Studies have shown higher presence of T-cell stimula- large collections of wild and cultivated species and abundant tory epitopes (glia-α2/9) in the D genome species compared seeds storage proteins in their grains of Triticeae provided an to A and B genome species (Spaenij-Dekking et al. 2005). attractive subject to study genomic evolution using various The large differences that existed in the content of predicted known glutenin and gliadin genes. The general conserved T-cell epitopes (glia-α, glia-α2, glia-α9 and glia-α20) in amino acids MKTFLIL and FGIFGTN encoding sequences full-ORF genes and pseudogenes from all the three diploid can be used to produce PCR primers for the amplification of ancestors of polyploid wheat, namely Triticum monococcum the α-gliadin genes from Triticum species (van Herpen et al. (AA), T. speltoides (BB) and Ae. tauschii (DD) were deter- 2006). However, these primers are of limited use in the iso- mined (van Herpen et al. 2006). Our study revealed that the lation of α-gliadin genes in other wild Triticeae species due α-gliadin sequences from W genome of Australopyrum to its low specificity (Liu et al. 2010;Qiet al. 2011). In was apparently lacking these toxic epitopes, although their this study, we searched public databases of the published α- α-gliadin gene sequences have two groups (figure 2). It is gliadin sequences and found the conserved 5 and 3 untrans- possible that the Australopyrum species were diploid and lated gene regions. In this study, a pair of primers, P1/P2 was self-pollinating perennial taxon endemic to Australia and successfully designed to produce specific amplicon of the New Zealand (Connor et al. 1993). Most of the α-gliadin target α-gliadin genes from all the available species of genera genes from the St, E, F and V genomes were evolved to Pseudoroengeria, Australopyrum, Lophopyrum, Secale, have the glia-α epitope (figure 3 in electronic supplemen- Dasypyrum, Eremopyrum and Thinopyrum. We charac- tary material). It is worthwhile to postulate the evolutionary terized a number of α-gliadin gene sequences in different origin of the toxic epitopes after the divergence of α-gliadin Triticeae species which will shed light on the understanding gene sequences. The recent studies indicated that the toxic of evolution of the prolamin gene superfamily. epitopes in four Aegilops genomes were significantly less Xu and Messing (2009) showed that the youngest and than those of Triticum A, B, D and their progenitor genomes largest α-prolamins gene family arose about 22–26 Mya after (Li et al. 2012). It will be interesting to investigate in the split of the Panicoideae (including maize, sorghum and particular the accumulation of the T-cell stimulatory epitopes millet) from the Pooideae (including wheat, barley and oats) in cultivated Triticeae species with respect to evolution and and Oryzoideae (rice). Shewry and Tatham (1990) revealed adaptation (Overpeck et al. 2005). Meanwhile, the existence that α-gliadins are present only in wheat and its closely of large differences in distribution of toxic α-gliadin genes related species. We found that the α-gliadin gene sequences provides opportunities to select and breed wheat varieties are present in S. sylvestre species, but not in cultivated with less toxic epitopes suitable for consumption by CD Secale species. The Secale α-gliadin sequences contained patients. It is thus essential to study the distribution of the eight conserved cysteine residues and rather short glutamine gliadin gene structure among closely related species of wheat repeats in NR regions compared to the typical α-gliadin in the tribe Triticeae. gene sequences (figure 1). It is possible that the wild Secale α-gliadin gene sequences were an ancestral α-gliadin type Acknowledgements α which evolved in parallel from -prolamin ancestor of cereal We are grateful to Dr Niaz Ali, Hazara University, Mansehra, Pak- plants. istan, and two anonymous reviewers for their helpful comments to The extensive hybridization among the genera, lineage the manuscript, and thankful to the National Natural Science Foun- variation and polyploid introgression may contribute to the dation of China (nos. 31171542, 31101143), Fundamental Research Funds for the Central Universities of China (ZYGX2010J099, complexity of phylogenetic relationships among Triticeae ZYGX2011J101) for their financial support. genera and species (Mason-Gamer 2005). In this study, phylogenetic analysis indicated that the α-gliadin sequences from D. breviaristatum, Lo. elongatum and Ps. spicata References Australopyrum clustered in two different subgroups with Anderson O. D. and Greene F. C. 1997 The α-gliadin gene family. different divergence times (figure 2). The results suggested II. DNA and protein sequence variation, subfamily structure and that regardless the pollination type, the rapid divergence origins of pseudogenes. Theor. Appl. Genet. 95, 59–65.

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Received 18 March 2014, in ﬁnal revised form 22 June 2014; accepted 26 June 2014 Unedited version published online: 7 July 2014 Final version published online: 1 December 2014

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