Advances in Environmental Biology Zahra Maryami, Arash Fazeli, Ali-Ashraf Mehrabi

Advances in Environmental Biology, 8(7) May 2014, Pages: 2012-2016
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Advances in Environmental Biology
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Identification of Variation for Wx-D Genome in Wheat and Its Ancestor
Zahra Maryami, Arash Fazeli, Ali-Ashraf Mehrabi
Agronomy and Plant Breeding Department, Faculty of Agriculture, Ilam University, Ilam, Iran.
ARTICLE INFO
Article history:
Received 28 February 2014
Received in revised form 19
April 2014
Accepted 23 April 2014
Available online 5 June 2014
Key words:
Wheat, Waxy gene, Wx-D Genome
and Aegilops
ABSTRACT
Waxy protein encoded by three genes (Wx-A, Wx-B and Wx-D) in wheat that have
important role in starch quality. In order to indentify of variation for Wx-D gene in
wheat and its ancestor used a primer to amplify small portion of Wx-D gene from 23
samples from different spices. Our result indicated that there is variation based on size
amplification in this region of Wx-D among samples. Ae.Umbelllulata and
Ae.ovatashow some insertion and size band more than 204 bp.Ae.cylandrica and
Ae.crassa have different genome and ancestor but show similar size. To investigate of
phylogenetic for Wx-D gene used new methods such as sequencing is necessary to
identify allelic variation among samples.
© 2014 AENSI Publisher All rights reserved.
To Cite This Article: Zahra Maryami, Arash Fazeli, Ali-Ashraf Mehrabi., Identification of Variation for Wx-D Genome in Wheat and Its
Ancestor. Adv. Environ. Biol., 8(7), 2012-2016, 2014
INTRODUCTION
The main component in wheat flour is starch, which is formed of two type’s glucose polymers: amylose
and amylopectin [1,2], amylose is the linear amylose (23-35%) and amylopectin is the highly branched
amylopectin (68-75%) to the total starch [3]. The ratio of polymers is important as it affects properties of the
starch such as gelatinization, pasting and gelation which are determinants of the quality of the end produce of
the flour or starch from bread wheat [4,5] and amylose is an important flour component [6]. Waxy protein
(GBSSI) is one of the most important determinants of starch synthesis of cereals [7] which are located on the
group-7 chromosomes of each genomes [8,9]. In bread wheat (TriticumaestivumL. ssp. aestivum; 2n = 6x = 42,
AABBDD), three waxy proteins, one for each genome, have been identified. Each waxy protein is controlled by
one waxy gene (Wx-A1, Wx-B1and Wx-D1), located on chromosome 7AS, 4AL (translocated from 7BS) and
7DS, respectively [10]. And often difficult to identify genetic variation for waxy proteins, because the
molecular weights of these proteins are very similar [11]. However more recently the search for new alleles has
been extended to ancient wheat’s [11-17], but Polymorphism studies in relative wheat species, as the Aegilops
genes, have been very limited [14, 18].
The Aegilops genus is the wild ancient wheat and involves 23 species di-tetra and hexaploid [19]. The
origin of this genus probably is the South Caucasus, Diploid species with limited dispersion, while tetra and
hexaploid species have more Climate compatibility [20] which plays an important role in the evolution of
cultivated wheat. Also have important role for Formation bread and durum wheat’s. The Aegilops species are
important source of new waxy variants [21]. In several studies suggest that AegilopstauschiiCoss (2n=2x=14,
DD) Contain the D genome and is the donor of D genome in bread wheat [20, 22] Also been reported this
genome of wheat originated in southeast or southwest of the Caspian Sea [23, 24] contain resistance genes to
low temperatures [25, 26] the dryness [27] and salinity [28]. Resistance to diseases and insects, means the
greater diversity of wheat’s to rage proteins and is unease useful resource for wheat breeding [29] Waxy gene
sequences have beennecessary to study the origin and phylogeny of other poaceae species, including wheat [11,
18, 19, 21, 30] then the analysis of the variation Wx- D gene in the Aegilopsgenus cloud be important to study
evolutionary relationships within this genes and between it and bread wheat.
The aim of the current study was the molecular characterization of Wx-Dgene present in eight Aegilops
species and its comparison with Wx-D in bread wheat which potentially could be an important source for
improving starch quality and also provides valuable information on the phylogeny of Wx-D in different species.
MATERIALS AND METHODS
Plant material:
Twenty-three samples from eight species Aegilopscollected from the West and North West of Iran (in table
1). Seeds from each samples planted in pots with 10 diameters and leaf sample from 4-6 leaves have been
collected and immediately storage in -20 C for more study.
Corresponding Author: Arash Fazeli, Agronomy and Plant Breeding Department, Faculty of Agriculture, Ilam University,
Ilam, Iran.
E-mail: [email protected]
Zahra Maryami et al, 2014
Advances in Environmental Biology, 8(7) May 2014, Pages: 2012-2016
DNA extraction and PCR amplification:
DNA genomics was extracted from the leaf samples according Doylle et al. [44] method with some
modification.The primers designed by Shariflou et al [29] were used to amplify the smallregion of Wx-D gene:
Wx-D F (5-ATAGGCACAACCCCTAAC-3) and Wx-D R (5- CGCTCCCTGAAGAGAGAAAGAA-3). PCR
reactions were performed in a total volume of 25μL containing 50 ng genomic DNA (3 μl), 1.5μM MgCl2, 2.5
μL10x PCR Buffer, 1.5 μL 1m M d NTP , 10 pmol of each primer(1μL) ,14.2 μLdd water and 0.3μL of
TaqDNA Polymerase . The PCR cycle consisted of an initial 4-min denaturation at 94C0, followed by 35 cycles
of 94C0 for 45 sec, 54C for 30 sec, 72C0 for 2 min, and 1 cycle of 72C0. A 5 μL aliquot of the PCR mixture
were resolved in 1.5% agarose gels, and the bands were visualized by ethidium bromide staining.
RESULTS AND DISCUSSION
Results:
The PCR amplification of the Wx-D 1 gene was carried out in genomic DNA with the specific primers
designed by Shariflou et al. [32]. This permitted the simultaneous amplification of the two waxy genes (A and
D). Results from the amplification of this region for Wx-D1 genefrom 8 Aegilops species was amplified in one
fragment [Fig 1]. The variation was seen among spices was as less as within the species that evaluated and in
addition the Aegilopsamplicons were same from the common wheat ones used as control. The size of the WxD1a ranged from 204bp found in Ae.Tauschii, Ae. Triuncialis, Ae.cylandrica , Ae. Umbelllulata ,Ae.ovataand
Ae.caudat that show similar size with Wx-D1a in common wheat. Also, results indicated that all Ae.Tauschii
show similar size with wheat. But, in Ae.Umbelllulata and Ae.ovata have some insertion and size band more
than 204 bp.Ae.cylandrica and Ae.crassa that have same genome and ancestor show similar size.
Discussion:
We know quality in wheat is correlated with waxy proteins, which Waxy proteins increased starch quality
in wheat. In the last 20 years, the waxy proteins have been the major subject of some studies, mainly Focused
on the search for presence of null alleles [33, 34] orthe mutations [35-39].in the previous studies , null mutation
at Wx-D Locus (Wx-D1b) by Ainsworth et al. [9] in AegilopsTauschii and new waxy allele(Wx-D1g) By
Guzman et al. [16] in spelt wheat identified, that could lead decreasing of amylose content also have been
identify molecular characterization of a new waxy allele [11, 14-16, 21, 40] which could be useful in modern
wheat breeding programs. Aegilops genus are neglected crop that could be used in the quality breeding of
modern bread wheat, because are related to wheat and are important sources of variation in waxy genes.Yan., et
al [18] and Li.,et al [25] Used waxy genes to study the relationships among the species of the Aegilops and
Triticum genera. Recently, Ortega, et al [21] used sequencing method to identify phylogenetic relationships
among Aegilops spices for waxy genes and concluded that there are high variations among them. Variation
observed among spices based on size for Wax D1 for Ae.caudatameans that this sample has CC genome that
may be some portion of this genome through evolution deleted for this region, before study by Long-Dou., et al
(2009) validated our results. Also, U, C and M genomes are the ancestor of the D genome to Ae.Tauschii
thatshow variation among Ae.Tauschii samples, so, previous study by Dvorak et al. [41] and Zeng [5] and
Wang et al. [43], Also Ortega et al. [21] reported U, C and M genomes are related to Ae.Tauschii.
Conclusions:
The present study suggests that that exploiting the variation of waxy –D1 in new genetic resources is
necessary to understanding the genetic diversity and phylogenetic analysis. Also allelic diversity of the Wx-D
within the Aegilopsgenus could be greater than described for the common wheat.Although the expression and
effect of these allelic diversity needs to be tested in the genetic pool of modern wheat and in enzyme activity
that thus to the starchquality. In this study used different samples that show little variation based on size
amplification in Wx-D except for Ae. Caudate that show small deletion for this region, so for more information
suggested used sequencing methods for this region and also complete Wx-D gene in order to identify
phylogenetic relationship and diversity in Wx-D gene. Also, using sequencing methods identify deleted and
insertion region in Ae.cuadata and Ae.Umbelllulata and Ae.ovata that maybe help breeder in quality breeding
programs.
Fig. 1: Amplification products of the Wx-D gene in Aegilops species.
Zahra Maryami et al, 2014
Advances in Environmental Biology, 8(7) May 2014, Pages: 2012-2016
Table 1: Sample of the Aegilops species used in the study.
Number
Spiceses
1
Aegilops Tauschi sspstrangulata
2
Aegilops Tauschi isspTauschii
3
AegiloTauschiissp
Tauschii
4
Aegilops Tauschii sspTauschi
Genome
DD
DD
DD
Ploidy
2X
2X
2X
Origin
orth iran- Babolsar
North iran- Rasht
North iran- Rasht
DD
2X
North iran- GilanAmlosh
North iran- Rasht
North iran- Rasht
West iran- Malayer
West iran- Malayer
West iran- Tuyserkan
West iran- Ilam
West iran- Ilam
Ilam -Badreh
West iran- Ilam
West iran- Ilam
Iran
Iran
Badreh-Ilam
Ilam-Malekshahi
west iran- IlamDarehshar
west iran- IlamDarehshar
west iran- IlamDarehshar
West iran- Ilam
West iran- Ilam
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
Aegilops Tauschii sspTauschi
Aegilops Tauschii sspTuschi
Aegilops triuncialis
Aegilops triuncialis
Aegilops triuncialis
Aegilops neglecta
Aegilops neglecta
Aegilops crassa
Aegilop scrassa
Aegilops crassa
Aegilops cylandrica
Aegilops cylandrica
Aegilops umbellulata
Aegilops umbellulata
Aegilops umbellulata
DD
DD
UUCC
UUCC
UUCC
UUMM
UUMM
DcDcMM
DcDcMM
DcDcMM
CCDD
CCDD
UU
UU
UU
2X
2X
4x
4x
4x
4x
4X
4X
4X
4X
4X
4X
2X
2X
2X
20
Aegilops ovata
UUMM
4X
21
Aegilops ovate
UUMM
4X
22
23
Aegilops caudate
Aegilops caudate
CC
CC
2X
2X
REFERENCES
[1]
[2]
[3]
[4]
[5]
[6]
[7]
[8]
[9]
[10]
[11]
[12]
[13]
Bouatrous Yamina, Bousba Ratiba, Djekoun Abed alhamid, Ykhlef Nadia, 2012. Evaluation of salinity
tolerance of wheat (Triticum durum Desf) at the level cellular and plantlet, Advances in Environmental
Biology, 6(4): 1381-1391.
Baldwin, PM., 2001. Starch granule-associated proteins and polypeptides: a review. Starch/Staerke, (53):
475-503.
Faghani Elham, Ramzan Ali Khavari-Nejad, Ghasem Hosseini Salekdeh and Farzaneh Najafi, 2012.
Evaluation of Cuticular Wax Deposition, Stomata and Carbohydrate of Wheat Leaves for Screening
Drought Tolerance, Advances in Environmental Biology, 6(13): 4035-4040.
Behnam Habibi Khaniani, Mohammad Reza Bihamta, Mohammad Esmail Hassani, Godarz Najafian and
Farrokh Darvish, 2012. Identification of a null allele at the Wx-B1 locus in some of Iranian bread wheat
genotypes, Advances in Environmental Biology, 6(10): 2586-2589.
Zeng, M., C.F. Morris, I.L. Batey, C.W. Wrigley, 1997. Sources of variation for starch gelatinization,
pasting, and gelation properties in wheat. Cereal Chem, (74): 63-71.
Maningat, C.C., P.A. Seib, S.D. Bassi, K.S. Woo, G.D. Lasater, 2009. Wheat starch: production
properties, modification and uses. In: BeMiller J, Whistler R (eds) Starch technology, 3rd edn. Elsevier,
New York, pp: 441-510.
Nakamura, T., M. Yamamori, H. Hirano, S. Hidaka, 1993. Production of waxy (amylose-free wheat’s.
Mol Gen Genet, 248: 253-259.
Chao, S., P.J. Sharp, A.J. Worland, E.J. Warham, R.M.D. Koebner, M.D. Gale, 1989. RFLP-based
genetic maps of wheat homoeologous group 7 chromosomes. TheorAppl Genet, 78: 495-504.
Ainsworth, C., J. Clark, J. Balsdon, 1993. Expression, organization and structure of the genes encoding
the waxy protein (granule-bound starch synthase) in wheat. Plant MolBiol, 22: 67-82.
Yamamori, M., T. Nakamura, T.R. Endo, T. Nagamine, 1994. Waxy
protein deficiency and
chromosomal location of coding genes in common wheat. TheorAppl Genet, 89: 179-184.
Guzma´n, C., J.B. Alvarez, 2012. Molecular characterization of a novel waxy allele (Wx-Au1a) from
TriticumurartuThum. exGandil. Genet Resour Crop Evol, 59: 971-979.
Yamamori, M., T. Nakamura, T. Nagamine, 1995. Polymorphism of two wax proteins in the emmer group
of tetraploidwheat, Triticumdicoccoides, T. dicoccum, and T. durumPlant Breed, 114: 215-218.
Urbano, M., B. Margiotta, G. Colaprico, D. Lafiandra, 2002. Waxy proteins in diploid, tetraploid and
hexaploidwheats. Plant Breed, 121: 465-469.
Zahra Maryami et al, 2014
Advances in Environmental Biology, 8(7) May 2014, Pages: 2012-2016
[14] Caballero, L., L.M. Martı´n., J.B. Alvarez, 2008a. Genetic diversity in Spanish populations of
Triticumspelta L. (escanda): example of an endangered genetic resource. Genet Resour Crop Evol, 55:
675-682.
[15] Guzma´n, C., L. Caballero, J.B. Alvarez, 2009. Variation in Spanish cultivated einkorn wheat
(Triticummonococcum L. sspmonococcum) as determined by morphological traits and waxy proteins.
Genet Resour Crop Evol, 56: 601-604.
[16] Guzma´n, C., L. Caballero, J.B. Alvarez, 2010. Genetic variation for waxy proteins and amylose content
in Spanish spelt wheat (Triticumspelta L.). Genet Resour Crop Evol, 57: 721-725.
[17] Guzma´n, C., L. Caballero, L.M. Martı´n, J.B. Alvarez, 2012a. Waxy genes from spelt wheat: new alleles
for modern wheat breeding and new phylogenetic inferences about the origin of this species. Ann Bot,
110: 1161-1171.
[18] Yan, L., M. Bhave, 2000. Sequences of the waxy loci of wheat: utility in analysis of waxy proteins and
developing molecular markers. Biochem Genet, 38: 391-411.
[19] Kilian, B., K. Mammen, E. Millet, R. Sharma, A. Graner, F. Salamini, K. Hammer, H. O¨zkan, 2011.
Aegilops. In: Kole C (ed) Wild crop relatives: genomic and breeding Resources. Cereals. Springer, New
York, pp: 1-76.
[20] Kerber, E.R., G.G. Rowland, 1974. Origin of the free threshing character in hexaploid wheat. Can J Genet
Cytol, 16: 145-154.
[21] Ortega, R., J.B. Alvarez. C. Guzman, 2013. Characterization of the Wx- gene in diploid Aegilops species
and its potential use in wheat breeding. Genet Resour Crop Evol, DOI 10.1007/s10722-013-0040-y .
[22] McFadden, E., E.R.S. Sears, 1946. The origin of Triticumspelta and its free-threshing hexaploid relatives.
J Hered, 37: 81.
[23] Nakai, Y., 1979. Isosyme variation Aegilops and Triticum IV origin of the common wheats revealed from
the study on esterase isozymes in synthysizedwheats . Jpn J Genet, 54: 175-189.
[24] Nishikawa, K., Y. furuta and T. Wada, 1980. Genetic studies on alpha-amylase isozaymes in wheat III
Intraspecific variation in Aegilopssquarrosa and the birthplace of hexaploid wheat. Jpn J Genet, 55: 325336.
[25] Li, W., Z. Gao, W Xiao, Y.M. Wei, Y.X. Liu. G.Y. Chen, Z.E. Pu, H.P. Chen, Y.L. Zheng, 2012.
Molecular diversity of restriction enzyme sites, Indels and upstream open reading frames (uORFs) of 50
untransalted regions (UTRs) of Waxy genes in Triticum L. and Aegilops L. species. Genet Resour Crop
Evol, 59: 1625-1647.
[26] Lelley, T., M. Stachel, H. Garusgerabel and J. Vollmann, 2000. Analysis of relationships between
Aegilopstauschi the Dgenome of wheat utilizing microsatellites. Genome, 43: 661-668.
[27] Gororo, N.N., H.A. Eagles, R.F. Eastwood, M.E. Nicolas R.G. flood, 2002. Use of Triticumtauschii to
improve yield of wheat in low yielding environments.Euphytica, 123: 241-254.
[28] Shah, S.H., J.F. Gorham, B.P. orster and R.J. Wyn Jones, 1987. Salt tolerance in the Triticeae: the
contribution of the D genome to cation selectivity in hexaploid wheat . J Exp Bot, 38: 254-269.
[29] Lubbers, E.L., K.S. Gill, T.S. Cox and B.S. Gill, 1991. Variation of molecular markers among
geographically diverse accessions of Triticumtauschii. Genome, 34: 354-361.
[30] Mason-Gamer, R.J., C.F. Weil, E.A. Kellogg, 1998. Granule- boundstarchsynthase: structure, function,
and phylogenetic utility. MolBiolEvol, 15: 1658–1673.
[31] Fortune, P.M., K.A. Schierenbeck, A.K. Ainouche, J. Jacquemin, J.F. Wendel, M.L. Ainouche, 2007.
Evolutionary dynamics of waxy and the origin of hexaploidSpartina species (Poaceae).
MolPhylogenetEvol, 43: 1040-1055.
[32] Shariflou, M.R., P.J. Sharp, 1999. A polymorphic microsatellite in the 3' end of ‘waxy’ genes of wheat,
Triticumaestivm. Plant Breed, 118(3): 275-277.
[33] Yamamori, M., T. Nakamura, T.R. Endo, T. Nagamine, 1994. Waxy protein location of coding genes in
common wheat. TheorAppl Genet deficiency and chromosomal, 89: 179-184.
[34] Rodrı´guez-Quijano, M., M.T. Nieto-Taladriz, J.M. Carrillo, 1998. Polymorphism of waxy proteins in
Iberian hexaploidwheats. PlantBreeding, 117: 341-344.
[35] Vrinten, P., T. Nakamura, M. Yamamori, 1999. Molecular characterization of waxy mutations in wheat.
Mol Gen Genet, 261: 463-471.
[36] Saito, M., M. Konda, P. Vrinten, K. Nakamura, T. Nakamura, 2004. Molecular comparison of waxy null
alleles in common wheat and identification of a unique null allele. TheorAppl Genet, 108: 1205-1211.
[37] Saito, M., T. Nakamura, 2005. Two point mutations identified in emmer wheat generate null Wx-A1
alleles. TheorAppl Genet, 110: 276-282.
[38] Monari, A.M., M.C. Simeone, M. Urbano, B. Margiotta, D. Lafiandra, 2005. Molecular characterization
of new waxy mutants identified in bread and durum wheat. Theoretical and Applied Genetics, 110: 14811489.
Zahra Maryami et al, 2014
Advances in Environmental Biology, 8(7) May 2014, Pages: 2012-2016
[39] Yanagisawa, T., C. Kiribuchi-Otobe, H. Yoshida, 2001. An alanine to threonine change in the Wx-D1
protein reduces GBSS I activity in waxy mutant wheat. Euphytica, 121: 209-214.
[40] Yamamori, M., C. Guzman, 2012. SNPs and an insertion sequence in five Wx-A1 alleles as factors for
variant Wx-A1 protein in wheat. Euphytica, 192: 325-338.
[41] Dvorˇa´ k.J., H.B. Zhang, 1992. Reconstruction of the phylogeny of the genus Triticum from variation in
repeated nucleotide sequences. TheorAppl Genet, 84: 419-429.
[42] Long-Dou, L.U., Cai-Ling. Hou, Long. Chen, Gui-Hong. Yin, Chuan-Liang Deng, Wu-Jun1. Gao, XuQin. Yang, Guang-Xuan. Tan, 2009. Molecular identification on Waxy genes in wheat using multiplePCR.Hereditas (Beijing), 31(8): 844-848.
[43] Wang, S., X. Li, K. Wang, X. Wang, S. Li, Y. Zhang, G. Guo, F.J. Zeller, S.l.K. Hsam, Y. Yan, P.
Gustafson, 2011. Phylogenetic analysis of C, M, N, and U genomes and their relationships with Triticum
and other related genomes as revealed by LMW-GS genes at Glu-3 loci. Genome, (54):273–284.
[44] Doyle, J.J. and J.L. Doyle, 1990. Isolation of plant DNA from fresh tissue. Focus, 12: 13-15.