Hereditas 145: 204Á211 (2008)

Molecular characterization of LMW prolamines from Crithopsis delileana and the comparative analysis with those from ZHI-FU GUO1,2, PAN DONG1, XIANG-YU LONG1, YU-MING WEI1, LI-JUN ZHANG2 and YOU-LIANG ZHENG1 1Triticeae Research Institute, Sichuan Agricultural University, Yaan, Sichuan, PR China 2Key Laboratory of Agricultural Biotechnology of Liaoning Province, Shenyang Agricultural University, Shenyang, PR China

Guo, Z.-F., Dong, P., Long, X.-Y., Wei, Y.-M., Zhang, L.-J. and Zheng, Y.-L. 2008. Molecular characterization of LMW prolamines from Crithopsis delileana and the comparative analysis with those from Triticeae. * Hereditas 145: 204Á211. Lund, Sweden. eISSN 1601-5223. Received January 9, 2008. Accepted July 18, 2008

Using the homologous primers designed from known sequences of low-molecular-weight glutenin subunits (LMW-GS), four LMW prolamine gene sequences, designated as LMW-GSK1 (EU283813), LMW-GSK2 (EU283814), LMW-GSK3 (EU283815) and LMW-GSK4 (EU283816), were isolated from wheat-related diploid species Crithopsis delileana, among which LMW-GSK3 and LMW-GSK4 were partial gene sequences. These LMW prolamine gene sequences from C. delileana had the similar typical primary structures to the known LMW glutenin subunit genes in wheat and its related species. The 5? flanking region of the LMW-GSK1 contained two cis-elements EM (5?-TGTAAAGT-3?) and GLM (5?-GGCGAGTCAT-3?). The deduced amino-acid sequence of the LMW-GSK1 consisted of a conserved signal peptide with 20 residues, a N-terminal region with 15 residues, a repetitive domain with 80 residues and a C-terminal region with 182 residues. Several in-frame stop codons were found in the coding sequences of LMW-GSK2 and LMW-GSK4, indicating that the LMW-GSK2 and LMW- GSK4 could be the putative pseudogenes. Phylogenetic analysis indicated that LMW-GSK1, LMW-GSK2 and LMW-GSK4 were closely related to the LMW-GS genes from A, D and R genomes, while LMW-GSK3 could be clustered together with X03103 from H genome. It was the first time that the coding genes of LMW prolamines on K genome of C. delileana were characterized.

You-Liang Zheng, Triticeae Research Institute, Sichuan Agricultural University, Yaan, CN-625014 Sichuan, PR China. E-mail: [email protected].

Wheat glutenins are divided into two unequal groups the tribe of Triticeae were thought to have a common with the predominant low-molecular-weight glutenin evolutionary origin (SHEWRY and TATHAM 1990). The subunits (LMW-GS) and the high-molecular-weight extensive genetic variations of LMW-GS in the glutenin subunits (HMW-GS) on the basis of their relatives of wheat had been proposed as a source of mobility in SDS-PAGE (PAYNE 1987). The HMW genes for wheat to improve the properties of end-use glutenins play a crucial role in determining the quality quality (D’OVIDIO and MASCI 2004). The LMW-GS of wheat due to its contribution to the dough visco- genes from wild species are also very useful in the elastic properties, while the LMW-GS are associated evolutionary analysis of Triticeae species (SHEWRY and with dough resistance and extensibility. Some allelic TATHAM 1990; YAN et al. 2006). forms of LMW-GS show even greater effects on these Crithopsis delileana (Schult) Roshev (2n2x14, properties than HMW-GS (PAYNE et al. 1987; GUPTA KK), which is the only member of the Crithopsis et al. 1989; ANDREWS et al. 1994; CORNISH et al. 2001; Jaub. Et Spach. in Triticeae, is a wild diploid species. D’OVIDIO and MASCI 2004). However, the role of The distribution of C. delileana extends from northern LMW-GS is poorly characterized due to the large of Africa to the southwest of Asia (SAKAMOTO 1973; numbers of LMW-GSs with similar mobility in SDS- LO¨ VE 1984; FREDERIKSEN 1993). The phylogenetic PAGE analysis (CIAFFI et al. 1999; LEE et al. 1999; relationships between this species and the other species ZHAO et al. 2004). in Triticeae had been investigated with morphology, LMW-GS are not only present in cultivated wheat, cytology and molecular markers (HSIAO et al. 1995; but also in its wild related species. LMW-GS or CATALAN et al. 1997; PETERSEN and SEBERG 1997; proteins clearly similar to LMW-GS had been re- LINDE-LAURSEN et al. 1999), whereas the comprehen- ported in the genera (OBUKHOVA et al. 1997), sive relationships have not been established. In the Dasypyrum (BLANCO et al. 1991), Elytrigia (GUPTA previous investigations we have characterized two and SHEPHARD 1990), (barley) (ATIENZA HMW prolamine genes from this species, and de- et al. 2002; HOU et al. 2006) and Secale (rye) (SHANG scribed the phylogenetic relationships with its related et al. 2005). The prolamine storage protein genes in species in Triticeae (GUO et al. 2005). In this study, the

DOI: 10.1111/j.2008.0018-0661.02051.x Hereditas 145 (2008) Molecular characterization of LMW prolamines from Crithopsis 205

LMW prolamine gene sequences from C. delileana UV light. The targeted DNA fragments were purified were characterized and compared with those of and ligated into the pGEMT-Easy vector (Promega), previously known genes. and then the positive clones were screened and sequenced.

MATERIAL AND METHODS Sequence analyses material Sequence analysis was conducted by using programs deposited in the NCBI network (http://www. Two accessions of Crithopsis delileana, which were ncbi.nlm.nih/gov). Primer Premier 5 software (Premier originally collected from Denmark, were used for the Biosoft International 1999) was used for all the primer characterization of LMW prolamine genes. Two designs. The nucleotide and deduced amino acid hexaploid wheats Chinese spring and CY12 were sequences were aligned using the CLUSTAL W 1.81 used as the reference of glutenins. (THOMPSON et al. 1994). Using the neighbor-joining SDS-PAGE analysis (NJ) method, the phylogenetic tree was constructed under the MEGA software (KUMAR et al. 2001). The The extraction of single seed glutenins and SDS- complete deletion option was adopted with respect to PAGE analysis were carried out according to YAN gaps in the aligned sequences, and the evolutionary et al. (2002). distances were measured by calculating p-distances for Designation of primers specific for LMW prolamines each pair of aligned sequences. genes Based on sequence alignments of the known LMW- RESULTS GS genes in GenBank and EMBL (accession numbers Compositions of HMW and LMW prolamines are AY263369, AY299485, AY585350, AY585354, U86026, U86027, X07747, X13306 and AJ519835), The glutenins of Crithopsis delileana were visualized as the primers were designed by using Primer Premier protein bands by SDS-PAGE (Fig. 1). Only one band 5 software (Premier Biosoft International 1999) (Table with the similar electrophoresis mobility to the HMW 1). These primers could amplify the 5?-flanking region glutenin subunit 1By8 of hexaploid wheat was de- and the entire coding regions of LMW-GS genes. tected in the HMW glutenins region of common wheat. In our previous investigations, this band had DNA extraction and PCR amplification been characterized as the only HMW prolamine in C. Genomic DNA was extracted from young leaves using delileana (GUO et al. 2005). A number of bands were a previously published procedure (WANG et al. 2004). detected in the corresponding LMW glutenins region PCR reactions were performed in a total reaction of common wheat, and they were likely LMW volume of 25 ml containing 50 ng of genomic DNA, prolamines or gliadins present in C. delileana. 1Taq DNA polymerase buffer, 1.5 mM MgCl ,0.5 2 Isolation of LMW prolamines mM each primer, 200 mM each dNTP and 1 U Taq DNA polymerase with high fidelity (Tiangen). The Owning to that the two C. delileana accessions had the cycling parameters were 948C for 4 min to pre- identical protein pattern in SDS-PAGE analysis, one denature, followed by 40 cycles of 948C for 30 s, accession was used for the further investigation. In 588C for 40 s and 728C for 1 min, and a final extension genomic PCR reactions, the amplification products at 728C for 7 min. The PCR products were electro- could be obtained by using 12 out of 16 pairs of phoresed on 1.2% agarose gels and visualized under primers (data not shown). The PCR products with the

Table 1. The primer sequences and their location in LMW glutenins.

Name Sequences (5?03?) Location in genes

Lmw-gsF1 TTGTAGAAACTGCCATCCTTTACAT Â-610 bp upstream from start codon Lmw-gsF2 TGCTCTATAGACGTCAGTTTATCTT Â-550 bp upstream from start codon Lmw-gsF3 CGAGCATATCCTAACAGCCCA Â-450 bp upstream from start codon Lmw-gsF4 CCAAAAGTACGCTTGTAGCTAGT Â-200 bp upstream from start codon Lmw-gsR1 AGGAAGGTCTTCATGGTGG/AATTG start site Lmw-gsR2 TTGCATGGGTTTAGCTGCTG near the C-terminal domain start site Lmw-gsR3 TTTCTTATCAGTAGA/GCACCAACTCC end site Lmw-gsR4 GTCACCGCTGCATCGACATAT exactly 3? end sequences 206 Z.-F.Guo et al. Hereditas 145 (2008) bcd were 1490, 1111, 591 and 640 bp, respectively, and they a had been deposited in GenBank under the accession 1 numbers EU283813 to EU283816. 2 5 7 7 8 8 HMW-GS Comparison of LMW prolamines 12 10 Sequence analysis showed that four gene sequences (i.e. LMW-GSK1, LMW-GSK2, LMW-GSK3 and LMW-GSK4) had the similar typical primary struc- tures to the known LMW glutenin subunits in wheat, LMW-GS and they contained the partial 5? flanking region (Fig. 3, 4). However, LMW-GSK3 and LMW-GSK4 were partial gene sequences. The in-frame stop codons were found in coding region of LMW-GSK2 and LMW- GSK4 (Fig. 4). The mutation resulting from ‘‘TATA’’ Fig. 1. SDS-PAGE analysis of the glutenins in C. delileana. to ‘‘CATA’’ was found in the second TATA box of 5? Lanes a and b were hexaploid wheat Chinese spring and flanking region of LMW-GSK4. Therefore, LMW- Chuanyu12, respectively. Lanes c and d were glutenins of GSK2 and LMW-GSK4 could be the pseudogenes. C. delileana. The nontailed arrowheads indicated the HMW prolamines. The 5? flanking region of LMW-GSK1 had the similar but not identical structures to the other three appropriate size from 600 bp to 1500 bp were purified sequences in present study and the known LMW-GS and cloned into the pGEMT-Easy vector, and the sequences from Triticeae (Fig. 3). Besides four general recombinant clones with the expected PCR fragments cis-elements with two TATA box, one CAAT box and were screened and sequenced. By sequence analysis one AGGA box, the 5? flanking region of and comparisons, four representative gene sequences, LMW-GSK1 contained two cis-elements EM (5?- designated as LMW-GSK1, LMW-GSK2, LMW- TGTAAAGT-3?) and GLM (5?-GGCGAGTCAT-3?). GSK3 and LMW-GSK4, were obtained. LMW- These sequence motifs were required for endosperm- GSK1, LMW-GSK2, LMW-GSK3 and LMW-GSK4 specific gene expression.

AGGA box TATA box

Lmw-gsR3 Fig. 2. Nucleotide and deduced amino-acid sequences of the LMW-GSK1. Primers sequences are underlined. Hereditas 145 (2008) Molecular characterization of LMW prolamines from Crithopsis 207

Fig. 3. Alignment of the partial 5’ flanking sequences of LMW prolamines from C. delileana and other known gene sequecnes. ‘‘Á’’, consensus sequences; ‘‘.’’, deletions.

The deduced amino-acid sequence of the LMW- DISCUSSION GSK1 consisted of a conserved signal peptide with 20 Comparisons of the promoter sequences of various residues, a N-terminal region with 15 residues, a cereal prolamine genes have identified a conserved repetitive domain with 80 residues and a C-terminal region located Â300 bp upstream of the transcrip- region with 182 residues (Fig. 2, 4). The deduced N- tional start, and this region has been named the terminal sequence of LMW-GSK1 was METICNP, endosperm box (KREIS et al. 1985). In Triticeae which were different with any other deduced N- species, the conserved region contains two different terminal sequences of LMW-GS genes from other conserved motifs, called the endosperm motif (EM) species in Triticeae (Table 2, Fig. 4). and the GCN4-like motif (GLM) (MU¨ LLER and KNUDSEN 1993). The deletion analysis of a wheat Evolutionary relationships LMW glutenin (LMWG-7D7) promoter demonstrated that a fragment located from positions Â326 to Â160 The 5? flanking regions and the deduced amino acid and containing the bifactorial endosperm box was sequences of LMW prolamines from C. delileana were essential for expression in tobacco endosperm (COLOT aligned with the corresponding regions of the known et al. 1987; HAMMOND-KOSACK et al. 1993). The 5? LMW-GS from wheat and its related species, and used flanking region of LMW-GSK1 from C.delileana to determine the evolutionary relationships. The contained two cis-element motifs EM (5?- phylogenetic tree, based on the alignment of 5? TGTAAAGT-3?) and GLM (5?-GGCGAGTCAT-3?). flanking regions, showed that the LMW-GSK1, It could be necessary for specific expression of LMW- LMW-GSK2 and LMW-GSK4 from K genome were GS1. closely related to the corresponding genes from A and The present of the in-frame stop codon could lead to D genomes, whereas the LMW-GSK3 could be the silence of the prolamine genes. It mostly was clustered together with X03103 from H genome by resulting from similar CAG (Q) to TAG (stop) and an interior paralleled branch (Fig. 5A). The numerous CAA (Q) to TAA (stop) mutations (FORDE et al. 1985; variation of the repetitive domain is not amenable RAFALSKI 1986; BUSTOS et al. 2000; WAN et al. 2002; to multiple alignments (FLAVELL et al. 1989; ALLABY GUO et al. 2005). In the deduced amino-acid sequences et al. 1999). Therefore, only the conserved signal of LMW-GSK2 and LMW-GSK4, two in-frame stop peptide, N-terminal region and C-terminal region of codons were found in the coding regions at position 523 all sequences were aligned for the phylogenetic ana- bp and 124 bp with mutations resulting from CAA (Q) lyses (Fig. 4). It indicated that LMW-GSK1 and to TAA (stop) and CAG (Q) to TAG (stop), respec- LMW-GSK2 were closely related to the genes from tively. They could lead to the silence of LMW-GSK2 R and D genomes (Fig. 5B). and LMW-GSK4.HALFORD et al. (1989) analyzed the 208 Z.-F.Guo et al. Hereditas 145 (2008)

Fig. 4. Comparison of the deduced amino acid sequence of the LMW prolamines of C. delileana with other known LMW-glutenin subunits and B-hordein from Triticeae. The R region represented the repetitive domain, which were removed. The nontailed arrowheads indicated the positions of the eight cysteine residues. CterI, CrII and CterIII represented the sub-regions of C-terminal domain. The asterisk indicated the in-frame premature stop codon in amino acid sequence of EU283814.

Table 2. The genes of LMW-GS and B-hordein from Triticeae used in this study.

Accession no. N-terminal sequence Locus Size of putative protein (residues) Origin Reference

EU283813 METICNP Glu-K 297 C.delileana This study EU283814 METSCIP Glu-K pseudogene C.delileana This study EU283815 METICIP Glu-K pseudogene C.delileana This study EU283816 METSCIP Glu-K partical sequence C.delileana This study AB062868 MDTSCIP Glu-A3 303 T.aestivum IKEDA et al. 2002 U86026 METSCIP Glu-A3 298 T.aestivum CASSIDY et al. 1998 X07747 Á Glu-A3 356 T.aestivum PITTS et al. 1988 Y14104 METSHIP Glu-B3 350 T.durum D’OVIDIO et al. 1997 AJ519835 METRCIP Glu-D3 304 T.aestivum CHARDOT et al. 2002 AB062851 METSHIP Glu-D3 365 T.aestivum IKEDA et al. 2002 AY585350 METSCIS Glu-D3 304 Ae.tauschii JOHAL et al. 2004 AY585355 METSRV Glu-D3 344 Ae.tauschii JOHAL et al. 2004 U86028 METSCIS Glu-D3 303 T.aestivum CASSIDY et al. 1998 AY829368 METSRV Glu-R 353 S. sylvestre SHANG et al. 2005 AY724438 Á Glu-Ee 239 L. elongatum LUO et al. 2005 X03103 Á Glu-H 293 H. vulgare FORDE et al. 1985 Hereditas 145 (2008) Molecular characterization of LMW prolamines from Crithopsis 209

therefore subjected to determine their phylogenetic relationships (SHEWRY and TATHAM 1990; AKHUNOV et al. 2003). The alignment of homogeneous genes of storage protein had been widely used to reveal the evolutionary relationships of Triticeae species (GUO et al. 2005; LONG et al. 2006; YAN et al. 2006; WANG et al. 2007). In the present study, the analysis of evolutionary relationship indicated that the LMW prolamine genes from C. delileana were most closely related to those of corresponding genes on A, D and R genomes. The deduced amino acid sequences of the LMW prolamines of C. delileana were different from the LMW glutenin representatives by substitutions, inser- tions and/or deletions involving single amino acid residues or motifs. These rearrangements could be originated during the evolution of Triticeae (AKHUNOV et al. 2003). Comparison of the sequences indicated that the genes encoding prolamines have diverged during the evolutionary process of Triticeae species, although they originated from a common ancestor (KREIS et al. 1985). These results suggested that wild species of Triticeae could be a source of new storage protein genes, including prolamines for the improvement of common wheat.

Acknowledgements Á The authors thank Bernard Baum (Eastern Cereal and Oilseed Research Centre, Agriculture and Agri-Food Canada) for critical review of the manu- script. This work was supported by the National High Technology Research and the Development Program of China (863 program 2006AA10Z1F8 and 2006AA10Z179), the Key Technologies R&D Program of China (2006 BAD01A02-23 and 2006BAD12B02), and the FANEDD Fig. 5. The evolutionary relationships of LMW-GS genes project (200357 and 200458) from the Ministry of Education, from C. delileana and other species in Triticeae based on the China. Y.-M. Wei was supported by the Program for New alignments of the 5’ flanking sequences (A) and the amino Century Excellent Talents in the Univ. of China (NECT-05- acid sequences (B). 0814). function of the upstream regions of a silent and an REFERENCES expressed wheat seed protein genes in transgenic tobacco. It was indicated that another possible cause Allaby, R. G., Banerjee, M. and Brown, T. A. 1999. Evolution of the high molecular weight glutenin loci of for not being expressed is inactivation of promoter. In A, B, D and G genomes of wheat. Á Genome 42: 296Á307. the second TATA box of 5? flanking region of LMW- Akhunov, E. D., Goodyear, J. A., Geng, S. et al. 2003. The GSK4, the mutation resulting from ‘‘TATA’’ to organization and rate of evolution of the wheat genomes ‘‘CATA’’ could also lead to the silence of LMW-GSK4. are correlated with recombination rates along chromo- some arms. Á Genome Res. 13: 1Á11. In the past 10 years, numerous molecular phyloge- Andrews, J. L., Hay, R. L., Skerritt, J. H. et al. 1994. HPLC netic analyses in Triticeae species had been conducted and immunoassay-based glutenin subunit analysis: on data from chloroplast DNA markers, high-copy screening for dough properties in wheats grown under nuclear genes, and low- or single-copy nuclear genes different environmental conditions. Á J. Cereal Sci. 20: 203Á215. (HSIAO et al. 1995; KELLOGG and APPELS 1995; Atienza, S. G., Alvarez, J. B., Villegas, A. M. et al. 2002. MASON-GAMER and KELLOGG 1996). The high Variation for the low-molecular-weight glutenin subunits degree of sequence homology among all genes of in a collection of Hordeum chilense. Á Euphytica 128: 269Á storage protein from wheat and its related species 277. Bustos, A. D., Rubio, P. and Jouve, N. 2000. Molecular implied that they arose from the duplication of a single characterisation of the inactive allele of the gene Glu-A1 ancestral sequence. The sequences of these genes were and the development of a set of AS-PCR markers for 210 Z.-F.Guo et al. Hereditas 145 (2008)

HMW glutenins of wheat. Á Theor. Appl. Genet. 100: protein genes in transgenic tobacco. Á Plant Sci. 62: 207Á 1085Á1094. 216. Blanco, A., Resta, P., Simeone, R. et al. 1991. Chromosomal Hammond-Kosack, M., Holdsworth, M. and Bevan, M. location of seed storage protein genes in the genome of 1993. In vivo footprinting at the endosperm box of a low Dasypyrum villosum. Á Theor. Appl. Genet. 82: 358Á362. molecular weight glutenin gene in wheat endosperm. Cassidy, B. G., Dvorak, J. and Anderson, O. D. 1998. The Á EMBO J. 12: 545Á554. wheat low-molecular-weight glutenin genes: characteriza- Hou, Y. C., Liu, Q., Long, H. et al. 2006. Characterization of tion of six new genes and progress in understanding gene low-molecular-weight glutenin subunit genes from Hor- family structure. Á Theor. Appl. Genet. 96: 743Á750. deum brevisubulatum ssp. turkestanicum. Á Biol. Bull. 33: Catalan, P., Kellogg, E. A. and Olmstead, R. G. 1997. 35Á42. Phylogeny of subfamily based on Hsiao, C., Chatterton, N. J., Asay, K. H. et al. 1995. chloroplast ndhF gene sequences. Á Mol. Phylogenet. Phylogenetic relationships of the monogenomic species Evol. 8: 150Á166. of the wheat tribe, Triticeae (Poaceae), inferred from Ciaffi, M., Lee, Y. K., Tamas, L. et al. 1999. The low- nuclear rDNA (internal transcribed spacer) sequences. molecular-weight glutenin subunit proteins of primitive Á Genome 38: 211Á223. wheats. III. The genes from D-genome species. Á Theor. Ikeda, T. M., Nagamine, T., Fukuoka, H. et al. 2002. Appl. Genet. 98: 135Á148. Identification of new low-molecular- weight glutenin Chardot, T., Do, T., Perret, L. et al. 2002. Heterogeneity of subunit genes in wheat. Á Theor. Appl. Genet. 104: 680Á genes encoding the low molecular weight glutenin sub- 687. units of bread wheat. Á In: Renard, D., Delle, V. G. and Johal, J., Gianibelli, M. C., Rahman, S. et al. 2004. Popineau, Y. (eds), Plant biopolymer science: food and Characterization of low-molecular-weight glutenin genes non-food applications. R. Soc. Chem. 24Á30. in Aegilops tauschii. Á Theor. Appl. Genet. 109: 1028Á Cornish, G. B., Bekes, F., Allen, H. M. et al. 2001. Flour 1040. proteins linked to quality traits in an Australian doubled Kumar, S., Tamura, K., Jakobsen, I. B. et al. 2001. MEGA2: haploid wheat population. Á Aust. J. Agric. Res. 52: 1339Á Molecular evolutionary genetics analysis software. 1348. Á Arizona State Univ., Tempe. Colot, V., Robert, L. S., Kavanagh, T. A. et al. 1987. Kellogg, E. A. and Appels, R. 1995. Intraspecific and Localization of sequences in wheat endosperm protein interspecific variation in 5S RNA genes are decoupled genes which confer tissue-specific expression in tobacco. in diploid wheat relatives. Á Genetics 140: 325Á343. EMBO J. 6: 3559 3564. Á Á Kreis, M., Shewry, P. R., Forde, B. G. et al. 1985. Structure D’Ovidio, R. and Masci, S. 2004. The low-molecular-weight and evolution of seed storage proteins and their genes, glutenin subunits of wheat gluten. Á J. Cereal Sci. 39: 321Á with particular reference to those of wheat, barley and 339. rye. Oxford Surv. Plant Cell Mol. Biol. 2: 253 317. D’Ovidio, R., Simeone, M., Masci, S. et al. 1997. Molecular Á Á Lee, Y. K., Ciaffi, M., Appels, R. et al. 1999. The low- characterization of a LMW-GS gene located on chromo- molecular-weight glutenin subunit proteins of primitive some 1B and the development of primers specific for the wheats. The genes from A-genome species. Theor. Appl. Glu-B3 complex locus in durum wheat. Á Theor. Appl. Á Genet. 95: 1119Á1126. Genet. 98: 126Á134. Frederiksen, S. 1993. Taxonomuc studies in some annual Linde-Laursen, I., Frederiksen, S. and Seberg, O. 1999. The genera of the Triticeae (poaceae). Á Nord. J. Bot. 13: 481Á Giemsa C-banded karyotype of Crithopsis delileana 493. (Poaceae; Triticeae). Á Hereditas 130: 51Á55. Forde, J., Malpica, J. M., Halford, N. G. et al. 1985. The Lo¨ve, A. 1984. Conspectus of the Triticeae. Á Feddes Rep. 95: nucleotide sequence of an HMW glutenin subunit gene 425Á521. located on chromosome 1A of wheat. Á Nucleic Acids Long, H., Wei, Y. M., Yan, Z. H. et al. 2006. Classification Res. 13: 6817Á6832. of wheat low-molecular-weight glutenin subunit genes Flavell, R. B., Goldsbrough, A. P., Robert, L. S. et al. 1989. and its chromosome assignment by developing LMW-GS Genetic variation in wheat HMW glutenin subunits and group-specific primers. Á Theor. Appl. Genet. 111: 1251Á the molecular basis of breadmaking quality. Á BioTech- 1259. nology 7: 281Á285. Luo, Z., Chen, F., Feng, D. et al. 2005. LMW-GS genes in Gupta, R. B. and Shephard, K. W. 1990. Two-step one- elongatum and their potential value in wheat dimensional SDS-PAGE analysis of LMW subunits of breeding. Á Theor. Appl. Genet. 111: 272Á280. glutelin. 2. Genetic control of the subunits in species Mason-Gamer, R. J. and Kellogg, E. A. 1996. Chloroplast related to wheat. Á Theor. Appl. Genet. 80: 183Á187. DNA analysis of the monogenomic Triticeae: phyloge- Gupta, R. B., Singh, N. K. and Shepherd, K. W. 1989. The netic implications and genome-specific markers. Á In: cumulative effect of allelic variation in LMW and HMW Jauhar, P. P. (ed.), Methods of genome analysis in . glutenin subunits on dough properties in the progeny of CRC Press, p. 301Á325. two bread wheats. Á Theor. Appl. Genet. 77: 57Á64. Mu¨ller, M. and Knudsen, S. 1993. The nitrogen response of Guo, Z. F., Yan, Z. H., Wang, J. R. et al. 2005. Character- a barley C-hordein promoter is controlled by positive and ization of HMW prolamines and their coding sequences negative regulation of the GCN4 and endosperm box. from Crithopsis delileana. Á Hereditas 142: 56Á64. Á Plant J. 4: 343Á355. Halford, N. G., Forde, J., Shewry, P. R. et al. 1989. Obukhova, L. V., Generalova, G. V., Agafonov, A. V. et al. Functional analysis of the upstream regions of a silent 1997. A comparative molecular genetic study of glutelins and an expressed member of a family of wheat seed in wheat and Elymus. Á Genetica 33: 1001Á1004. Hereditas 145 (2008) Molecular characterization of LMW prolamines from Crithopsis 211

Pitts, E. G., Rafalski, J. A. and Hedgcoth, C. 1988. ple sequence alignment through sequence weighting, Nucleotide sequence and encoded amino acid sequence position-specific gap penalties and weight matrix choice. of a genomic gene region for a low molecular weight Á Nucleic Acids Res. 22: 4673Á4680. glutenin. Á Nucleic Acids Res. 16: 11376. Wang, J. R., Yan, Z. H., Wei, Y. M. et al. 2004. A novel Petersen, G. and Seberg, O. 1997. Phylogenetic analysis of HMW glutenin subunit gene Ee1.5 from Elytrigia the Triticeae (Poaceae) based on rpoA sequence data. elongata (Host) Nevski. Á J. Cereal Sci. 40: 289Á294. Á Mol. Phylogenet. Evol. 7: 217Á230. Wang, J. R., Yan, Z. H., Jiang, Q. T. et al. 2007. Sequence Payne, P. I. 1987. Genetics of wheat storage proteins and the variations and molecular phylogenetic analyses of the effect of allelic variation on bread making quality. HMW-GS genes from different genomes in Triticeae. Á Annu. Rev. Plant Physiol. 38: 141Á153. Á Biochem. Syst. Ecol. 35: 421Á433. Payne, P. I., Seekings, J. A., Worland, A. J. et al. 1987. Allelic Wan, Y., Wang, D., Shewry, P. R. et al. 2002. Isolation and variation of glutenin subunits and gliadins and its effect characterization of five novel high molecular weight on breadmaking quality in wheat: analysis of F5 progeny subunit of glutenin genes from Triticum timopheevi and from Chinese SpringChinese Spring (Hope 1A). Á J. Aegilops cylindrical. Á Theor. Appl. Genet. 104: 828Á839. Cereal Sci. 6: 103Á118. Yan, Z., Wan, Y., Liu, K. et al. 2002. Identification of a Rafalski, J. A. 1986. Structure of wheat gliadin genes. Á Gene novel HMW gultenin subunit and comparison of its 43: 221Á229. amino acid sequence with those of homologous subunits. Sakamoto, S. 1973. Patterns phylogenetic differentiation in Á Chin. Sci. Bull. 47: 220Á225. the tribe Triticeae. Rep. Kihara Inst. Á Biol. Res. 24: 11Á Yan, Z. H., Wei, Y. M., Wang, J. R. et al. 2006. Character- 31. ization of two HMW glutenin subunit genes from Shang, H. Y., Wei, Y. M., Long, H. et al. 2005. Identification Taenitherum Nevski. Á Genetica 127: 267Á276. of LMW glutenin like genes from Secale sylvestre Host. Zhao, H. X., Wang, R. J., Guo, A. G. et al. 2004. Á Russian J. Genet. 41: 1372Á1380. Development of primers specific for LMW-GS genes Shewry, P. R. and Tatham, A. S. 1990. The prolamin storage located on chromosome 1D and moleculer characteriza- proteins of cereal seeds: structure and evolution. tion of a gene from Glu-D3 complex locus in bread wheat. Á Biochem. J. 267: 1Á12. Á Hereditas 141: 193Á198. Thompson, J. D., Higgins, D. G. and Gibson, T. J. 1994. Clustal-W- improving the sensitivity of progressive multi-