Molecular evolution of dimeric α-amylase inhibitor genes in wild emmer wheat and its ecological association

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    ssBioMed CentBMC Evolutionary Biology

    Open AcceResearch articleMolecular evolution of dimeric -amylase inhibitor genes in wild emmer wheat and its ecological associationJi-Rui Wang1, Yu-Ming Wei1, Xiang-Yu Long1, Ze-Hong Yan1, Eviatar Nevo2, Bernard R Baum3 and You-Liang Zheng*1,4

    Address: 1Triticeae Research Institute, Sichuan Agricultural University, Yaan, Sichuan 625014, China, 2Institute of Evolution, University of Haifa, Mt. Carmel, Haifa 31905, Israel, 3Agriculture and Agri-Food Canada, Eastern Cereal and Oilseed Research Centre, Ottawa, Ontario K1A 0C6, Canada and 4Key Laboratory of Crop Genetic Resources and Improvement in Southwest China, Ministry of Education, Sichuan Agricultural University, Yaan, Sichuan 625014, China

    Email: Ji-Rui Wang - wangjirui@gmail.com; Yu-Ming Wei - ymwei@sicau.edu.cn; Xiang-Yu Long - yuxianglong006@163.com; Ze-Hong Yan - grmb@sicau.edu.cn; Eviatar Nevo - nevo@research.haifa.ac.il; Bernard R Baum - baumbr@agr.gc.ca; You-Liang Zheng* - ylzheng@sicau.edu.cn

    * Corresponding author Equal contributors

    AbstractBackground: -Amylase inhibitors are attractive candidates for the control of seed weevils, as these insects are highlydependent on starch as an energy source. In this study, we aimed to reveal the structure and diversity of dimeric -amylase inhibitor genes in wild emmer wheat from Israel and to elucidate the relationship between the emmer wheatgenes and ecological factors using single nucleotide polymorphism (SNP) markers. Another objective of this study wasto find out whether there were any correlations between SNPs in functional protein-coding genes and the environment.

    Results: The influence of ecological factors on the genetic structure of dimeric -amylase inhibitor genes was evaluatedby specific SNP markers. A total of 244 dimeric -amylase inhibitor genes were obtained from 13 accessions in 10populations. Seventy-five polymorphic positions and 74 haplotypes were defined by sequence analysis. Sixteen out of the75 SNP markers were designed to detect SNP variations in wild emmer wheat accessions from different populations inIsrael. The proportion of polymorphic loci P (5%), the expected heterozygosity He, and Shannon's information index inthe 16 populations were 0.887, 0.404, and 0.589, respectively. The populations of wild emmer wheat showed greatdiversity in gene loci both between and within populations. Based on the SNP marker data, the genetic distance of pair-wise comparisons of the 16 populations displayed a sharp genetic differentiation over long geographic distances. Thevalues of P, He, and Shannon's information index were negatively correlated with three climatic moisture factors, whereasthe same values were positively correlated by Spearman rank correlation coefficients' analysis with some of the otherecological factors.

    Conclusion: The populations of wild emmer wheat showed a wide range of diversity in dimeric -amylase inhibitors,both between and within populations. We suggested that SNP markers are useful for the estimation of genetic diversityof functional genes in wild emmer wheat. These results show significant correlations between SNPs in the -amylaseinhibitor genes and ecological factors affecting diversity. Ecological factors, singly or in combination, explained a significantproportion of the variations in the SNPs, and the SNPs could be classified into several categories as ecogeographicalpredictors. It was suggested that the SNPs in the -amylase inhibitor genes have been subjected to natural selection, andecological factors had an important evolutionary influence on gene differentiation at specific loci.

    Published: 24 March 2008

    BMC Evolutionary Biology 2008, 8:91 doi:10.1186/1471-2148-8-91

    Received: 3 September 2007Accepted: 24 March 2008

    This article is available from: http://www.biomedcentral.com/1471-2148/8/91

    2008 Wang et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.Page 1 of 14(page number not for citation purposes)

  • BMC Evolutionary Biology 2008, 8:91 http://www.biomedcentral.com/1471-2148/8/91

    BackgroundWild emmer wheat, Triticum dicoccoides, the progenitor ofbread and pasta wheats, presumably originated in andadaptively diversified from, northeastern Israel into theNear East Fertile Crescent [1]. In this center of diversity,wild emmer wheat harbors rich genetic diversity andresources [1]. Previous studies in T. dicoccoides and othercereals have shown significant nonrandom adaptivemolecular genetic differentiation at single and multilocusstructures in either protein-coding regions or randomlyamplified polymorphic DNAs among micro-ecologicalenvironments [2,3]. It was also determined that wildemmer wheat is genetically variable and that the geneticdifferentiation of populations included regional and localpatterns with sharp genetic differentiation over short dis-tances [4]. Genetic polymorphisms of - and -amylase inwild emmer wheat have been characterized, and it wasfound that diversity of climatic and edaphic natural selec-tion, rather than stochasticity or migration, was the majorevolutionary force driving amylase differentiation [5].

    The estimates of molecular diversity derived from PCR-based techniques such as amplified restriction fragmentlength polymorphism (AFLP), microsatellites (shortsequence repeats or SSR), single nucleotide polymor-phism (SNP), and sequence comparisons are several-foldhigher than enzymatic diversity [6]. A substantial privateand public effort has been undertaken to characterizeSNPs tightly associated for genetic diversity. SNPs areidentified in ESTs (expressed sequence tags), thus the pol-ymorphisms could be directly used to map functional andexpressed genes, rather than DNA sequences derived fromconventional RAPD and AFLP techniques, which are typi-cally not functional genes [7-9]. The majority of SNPs incoding regions (cSNPs) are single-base substitutions,which may or may not result in amino acid changes. SomecSNPs may alter a functionally important amino acid res-idue, and these are of interest for their potential links withphenotypes [10].

    -Amylase is a family of enzymes that hydrolyze -D-(1,4)-glucan linkages and play an important role in thecarbohydrate metabolism of many autotrophic and heter-otrophic organisms [11]. Heterotrophic organisms use -amylase primarily to digest starch in their food sources[12]. Several kinds of -amylase and proteinase inhibitorsin seeds and vegetative organs act to regulate the numbersof phytophagous insects [13-15]. -Amylase inhibitorsare attractive candidates for the control of seed weevils asthese insects are highly dependent on starch as an energysource [16]. In cereal seeds, -amylase inhibitor proteinswith 120130 amino acids, which include trypsin inhibi-tors, as well as -amylase inhibitors, can be grouped into

    dimeric -amylase inhibitor has been well characterized.For weevil control, -amylase inhibitors could be manip-ulated through plant genetic engineering. However, manyinsects have several -amylases that differ in specificity,and successful utilization of a food source is dependenton the expression of a -amylase for which there is no spe-cific inhibitor [12]. The dimeric -amylase inhibitor geneswere located on chromosome 3BS and 3DS; there was noknown evidence of a homoeologous locus or loci on chro-mosome 3AS of the polyploid wheats [18,19]. Therefore,the tetraploid wheats, which are lacking the D genome,have only the inhibitor genes on chromosome 3BS [19].

    Evolutionary pressures of various kinds have often beenhypothesized to cause active and rapid evolutionarychanges. In a co-evolving system of plant-insect interac-tions, plants synthesize a variety of toxic proteinaceousand nonproteinaceous molecules for their protectionagainst insects [20,21]. Proteinase inhibitors are thereforea potential model system in which to study basic evolu-tionary processes, such as functional diversification [22].

    It is well established that multiple forms of proteins areactive on exogenous or endogenous -amylases in thewheat kernel, and proteinaceous dimeric -amylaseinhibitors could function against -amylase from variousorigins [23]. It is known that the bulk of wheat albuminsconsist of a few amylase iso-inhibitor families that arevery likely phylogenetically related and coded by a smallnumber of parental genes [24]. The -amylase inhibitorshave long been proposed as possible important weaponsagainst pests whose diets make them highly dependent on-amylase activity. In vitro and in vivo trials using -amy-lase inhibitors, including those made under field condi-tions, have now fully confirmed their potential forincreasing yields by controlling insect populations [16].

    Two conflicting views confront ecologists and evolution-ary biologists on the degree of symmetry in interactionsbetween plants and phytophagous insects [25]. The sym-metrical view holds that insects and plants have strongeffects on one another's evolutionary and ecologicaldynamics. The asymmetrical view acknowledges thatplants have major effects on insects but claims that insectsseldom impose significant effects on plants [25]. Plantdefense mechanisms have been the subject of intenseinvestigation [26]. The genome shaping events and proc-esses occurring at dimeric -amylase inhibitor gene locifrom the B and S genomes of wheat and Aegilops sectionsitopsis, respectively, have been characterized. A Phyloge-netic Median-Joining network of the haplotypes and aneighbor-joining tree analysis have indicated that theinhibitor gene sequences from common wheat and T. dic-Page 2 of 14(page number not for citation purposes)

    one large family on the basis of the homology betweentheir amino acid sequences [17]. In this family, the

    occoides are closely related to those from Ae. speltoides [27].However, little is known about their evolution under the

  • BMC Evolutionary Biology 2008, 8:91 http://www.biomedcentral.com/1471-2148/8/91

    influence of ecology. The molecular diversity of -amylaseinhibitor genes, as well as their divergence among 16 pop-ulations of wild emmer wheat from Israel, was investi-gated to gain insight into the correlation between plantdefense proteinaceous inhibitors and ecological factors.

    ResultsIsolation of the ORF of dimeric -amylase inhibitorsUsing two cloning primers, genomic PCR amplificationswere conducted, and one desired DNA band was detectedin each accession of wild emmer wheat. Cloning the frag-ments yielded 244 positive clones from 13 accessions(randomly selected from 10 populations), which weresubsequently sequenced (data not shown). Only three outof 244 dimeric -amylase inhibitor genes had a commonthree bp deletion, and those three genes were obtainedfrom one accession derived from Mt. Hermon, whereasthe other cloned fragments had 426 bp long (data notshown). It was predicted that all of the 426-bp sequenceswould encode functional dimeric -amylase inhibitors.Alignment of the gene sequences from emmer wheat withsequences from the species of Aegilops section Sitopsis(including Ae. speltoides, Ae. bicornis, Ae. longissima, Ae.searsii, and Ae. sharonensis), Ae. tauschii, einkorn wheats,and common wheat clearly indicated that the emmerwheat sequences were derived from the B genome [27].

    SNP and haplotype analyses of dimeric -amylase inhibitor genesThe frequency of SNPs in the dimeric -amylase inhibitorgenes in emmer wheat was 1 out of 5.7 bases, which washigher than the SNPs observed for kunitz-type -amylaseinhibitor and -amylase/subtilisin inhibitor genes in bar-ley and dimeric -amylase inhibitor genes in commonwheat [28-30]. Among the 426 nucleotides, there were351 conserved positions and 75 variable positions amongthe 244 -amylase inhibitor genes sequenced from 13accessions.

    A total of 74 haplotypes were revealed by sequence analy-sis (Figure 1); 53 of these were each found in only a singlesequence. Haplotype 41 was observed at the highest fre-quency, i.e., in 38 gene sequences, followed by haplotype27 in 33 sequences (Figure 1).

    The relationship between SNPs and amino acid changesin the -amylase inhibitor proteins is summarized inTable 1. The 75 SNPs resulted in 38 amino acid substitu-tions. The position of each SNP in the sequence, whetherthe predicted change was synonymous (silent) or non-synonymous (replacement), was determined. Forty per-cent of SNPs were found to occur at the third codon posi-tion, and as expected, most of these were synonymous

    than 95% of the non-synonymous changes (Table 1). Intotal, 60% of the SNPs resulted in non-synonymouschanges.

    Primer design and SNP mining of wild emmer wheatUsing the information from the 75 SNPs identified in the-amylase inhibitor genes, 16 primers (combined withthe reverse cloning primer, R, as SNP markers) were suc-cessfully designed to detect the SNPs in 205 accessionsfrom 18 populations. The primers, with the SNP (bold let-ters) at the 3' end and an extra mismatched nucleotide(underline) on the third nucleotide from the end arelisted in Table 2. A total of 14 SNPs were detected with the16 SNP markers from position 19 to 288 of the -amylaseinhibitor gene, and the size of the amplified fragmentsranged from 158 to 426 bp. The data was then organizedin terms of genotypic frequencies ("0" or "1") to assess thepopulation structure.

    There were only 5 and 2 accessions from Yehudiyya andAchihood, respectively. Thus, the data for Yehudiyya andAchihood were not used in further analyses. Positive frag-ment frequency for each primer in the 16 populations islisted Additional file 1.

    Genetic diversity and distance of -amylase inhibitor genesSome genetic parameters of the 16 populations of wildemmer wheat are summarized in Table 3. The proportionof polymorphic loci P (5%), the expected heterozygosityHe, and Shannon's information index of the 16 popula-tions of wild emmer wheat were 0.887, 0.404, and 0.589,respectively. The values of He ranged from 0.182 to 0.437,and the population of Kokhav Hashahar had the highestvalue of He (0.437), followed by the population of Rosh-Pinna, whereas the population from Daliyya was charac-terized by the lowest He value of 0.182.

    The genetic distances (D) were calculated for comparisonsof all 16 populations based on the positive fragment ofSNP markers among all population pairs (see Additionalfile 2). The highest genetic distance (0.263) was obtainedbetween populations of Kokhav Hashahar and Daliyya,whereas the most related populations were Qazzrin andGamla with a genetic distance of 0.017. However, lower Dvalues (< 0.050) were observed between some popula-tions from different areas, and, for the most part, the esti-mates of D value were geographically independent. Largegenetic distances and sharp genetic differentiation overlong geographic distances could be found. For example,Kokhav Hashahar i...