The cuticular wax inhibitor locus Iw2 in wild diploid wheat Aegilops tauschii: phenotypic survey, genetic analysis, and implications for the evolution of common wheat.

BACKGROUND: Cuticular wax production on plant surfaces confers a glaucous appearance and plays important roles in plant stress tolerance. Most common wheat cultivars, which are hexaploid, and most tetraploid wheat cultivars are glaucous; in contrast, a wild wheat progenitor, Aegilops tauschii, can be glaucous or non-glaucous. A dominant non-glaucous allele, Iw2, resides on the short arm of chromosome 2D, which was inherited from Ae. tauschii through polyploidization. Iw2 is one of the major causal genes related to variation in glaucousness among hexaploid wheat. Detailed genetic and phylogeographic knowledge of the Iw2 locus in Ae. tauschii may provide important information and lead to a better understanding of the evolution of common wheat.
RESULTS: Glaucous Ae. tauschii accessions were collected from a broad area ranging from Armenia to the southwestern coastal part of the Caspian Sea. Linkage analyses with five mapping populations showed that the glaucous versus non-glaucous difference was mainly controlled by the Iw2 locus in Ae. tauschii. Comparative genomic analysis of barley and Ae. tauschii was then used to develop molecular markers tightly linked with Ae. tauschii Iw2. Chromosomal synteny around the orthologous Iw2 regions indicated that some chromosomal rearrangement had occurred during the genetic divergence leading to Ae. tauschii, barley, and Brachypodium. Genetic associations between specific Iw2-linked markers and respective glaucous phenotypes in Ae. tauschii indicated that at least two non-glaucous accessions might carry other glaucousness-determining loci outside of the Iw2 locus.
CONCLUSION: Allelic differences at the Iw2 locus were the main contributors to the phenotypic difference between the glaucous and non-glaucous accessions of Ae. tauschii. Our results supported the previous assumption that the D-genome donor of common wheat could have been any Ae. tauschii variant that carried the recessive iw2 allele.

Background

Cuticular wax production on aerial surfaces of plants has important roles in various physiological functions and developmental events; the wax prevents non-stomatal water loss, inhibits organ fusion during development, protects from UV radiation damage, and imposes a physical barrier against pathogenic infection [1-4]. The trait, the coating of leaf and stem surfaces with a waxy whitish substance, is called glaucousness. In common wheat (Triticum aestivum L., 2n = 6x = 42, genome constitution BBAADD), dominant alleles W1 and W2, control the wax production and have been assigned to chromosomes 2B and 2D, respectively [5,6]. Additionally, dominant homoeoalleles for non-glaucousness, Iw1 and Iw2, have also been mapped to the short arms of chromosomes 2B and 2D, respectively [6-9]. Wheat plants with either the w1, w2, Iw1 or Iw2 allele show the non-glaucous phenotype, indicating that W1 and W2 are functionally redundant for the glaucous phenotype and that a single Iw dominant allele is sufficient to inhibit the glaucous phenotype even in the presence of a W1 or W2 allele [3,6]. Wax composition in wheat plants with one Iw dominant allele is biochemically different from that in glaucous plants of any genotype; ß-diketones are completely absent from extracts of cuticular wax from Iw plants, while aldehydes and primary alcohols are very abundant in these extracts [3,10]. A fine map around the Iw1 region on 2BS was constructed using an F2 population of tetraploid wheat (Triticum turgidum L., 2n = 4x =28, BBAA), and three markers tightly linked to Iw1 were developed [10,11]. A high-resolution map of Iw2 on 2DS has been developed in hexaploid wheat, and two markers tightly linked to Iw2 were also developed [11]. Comparative mapping of Iw1 and Iw2 shows that the two loci are homoeologous to each other and orthologous to the same chromosomal region of Brachypodium distachyon (L.) P. Beauv. [11]. Recently, a third wax-inhibitor locus Iw3 was identified on chromosome 1BS from wild emmer wheat [12], and a fine map of the Iw3 locus is available [13]. Iw2 is located on 2DS in Aegilops tauschii Coss. (2n = 2x = 14, DD), which is diploid and the progenitor of the D-genome of common wheat [14], but to our knowledge, a high-resolution genetic map of the Iw2 region in Ae. tauschii has not been constructed.

Common wheat is an allohexaploid species derived from interspecific hybridization between tetraploid wheat with a BBAA genome and Ae. tauschii. Most cultivated varieties of tetraploid wheat are glaucous, even though non-glaucous types are frequently found among wild tetraploid accessions [6,15]; this variation indicates that the glaucous phenotype might have been a target of artificial selection during the domestication of tetraploid wheat. Glaucous accessions of Ae. tauschii are found in the area ranging from Transcaucasia to the southern coastal region of the Caspian Sea [5,16]. Almost all varieties of common wheat carry W1 and W2 and lack Iw1 and Iw2; therefore, the D-genome donor of common wheat is assumed to have had the recessive iw2 allele [5]. Glaucous Ae. tauschii accessions have the W2 and iw2 alleles. Non-glaucous accessions of Ae. tauschii that have the W2 and Iw2 alleles have been recovered from a wide distribution range in central Eurasia [5]. Moreover, discovery of a non-glaucous Ae. tauschii accession with the w2 recessive allele has not yet been reported.

Therefore, analysis of the Iw2 locus may provide important information that improves our understanding of the evolution of common wheat. Population structure analyses of Ae. tauschii indicate that the whole species Ae. tauschii can be divided into three major genealogical lineages, tauschii lineage 1 (TauL1), TauL2, and TauL3, and that genetically genomes of TauL2 accessions are most closely related to the D genome of common wheat [17-19]. Recently, a whole-genome shotgun strategy was used to generate a draft genome sequence of Ae. tauschii that has been published; this draft anchors 1.72 Gb of the 4.36 Gb genome to chromosomes [20]. A physical map of the Ae. tauschii genome that covers 4 Gb is also available [21]. The objectives of this study were (1) to examine the natural variation in glaucousness among a species-wide set of Ae. tauschii accessions, (2) to use F2 populations of Ae. tauschii accessions and synthetic hexaploid wheat lines to fine-map Iw2 locus on 2DS, (3) to develop molecular markers that are closely linked to Iw2 based on chromosomal synteny between barley and wheat chromosomes, and (4) to provide novel insights into the evolutionary relationship between the Ae. tauschii genome and the D genome of common wheat on the basis of the detailed genetic and phylogeographic knowledge of the Iw2 chromosomal region.

Methods

Plant materials and phenotype evaluation

In all, 210 Ae. tauschii accessions were used in this study [22]. Their passport data, including geographical coordinates, have been provided in previous reports [23,24]. Previously, 206 of the Ae. tasuchii accessions were grouped into the three lineages, TauL1, TauL2, and TauL3, based on DArT marker genotyping analysis [19]. Of the 210 accessions, 12 were previously identified as subspecies strangulata based on the sensu-strico criteria [25,26]. Seeds from two Ae. tauschii hybrid F2 populations (n = 116 from each population) were sown in November 2011; one F2 population resulted from a cross between KU-2154 (non-glaucous) and KU-2126 (glaucous), the other from a KU-2003 (non-glaucous) by KU-2124 (glaucous) cross. In the 2012–2013 season, 169 additional F2 individuals of the KU-2154/KU-2126 population were grown to increase the size of the mapping population.

Previously, 82 synthetic hexaploid wheat lines were produced from crosses between a tetraploid wheat (T. turgidum subspecies durum (Desf.) Husn.) cultivar Langdon (Ldn) and 69 Ae. tauschii accessions [26,27]. These synthetic hexaploid wheat lines were used for crossing and phenotypic studies conducted in a glasshouse at Kobe University. Ldn shows the glaucous phenotype and is homozygous for the iw1 allele [10]. Each synthetic hexaploid thus contained the A and B genomes from Ldn and one of many diverse D genomes originating from the Ae. tauschii pollen parents. In the present study, four F3 plants derived from one F2 plant of each synthetic hexaploid were grown individually during the 2007–2008 season in pots that were arranged randomly in the glasshouse; these 276 F3 plants were used for crossing and phenotypic observation. The following three pairs of synthetic hexaploids were used to generate three F2 mapping populations: Ldn/PI476874 (non-glaucous) and Ldn/KU-2069 (glaucous), Ldn/IG126387 (non-glaucous) and Ldn/KU-2159 (glaucous), and Ldn/KU-2124 (glaucous) and Ldn/IG47259 (non-glaucous). The first population (Ldn/PI476874//Ldn/KU-2069) comprised 106 F2 individuals grown in the glasshouse during the 2009–2010 season. Seeds from the other two populations were sown in November 2011, with the numbers of individuals in each being 100 (Ldn/KU-2159//Ldn/IG126387) and 82 (Ldn/KU-2124//Ldn/IG47259).

For analysis of the D genome of common wheat, 17 landraces collected in Iran were supplied from the National BioResource Project (NBRP) KOMUGI (http://www.shigen.nig.ac.jp/wheat/komugi). These Iranian landraces—KU-3097, KU-3098, KU-3121, KU-3126, KU-3136, KU-3162, KU-3184, KU-3189, KU-3202, KU-3232, KU-3236, KU-3274, KU-3289, KU-10393, KU-10439, KU-10480, and KU-10510—each showed the glaucous phenotype.

Glaucousness was evaluated based on the presence or absence of wax production on the surface of peduncles and spikes in both Ae. tauschii and synthetics. Wax production was clearly visible and whitish.

Genotyping and construction of linkage maps

To amplify PCR fragments containing molecular markers, some of which were simple sequence repeats (SSRs), total DNA was extracted from leaves of the parental strains and F2 individuals. For SSR genotyping, 40 cycles of PCR were performed using 2x Quick Taq HS DyeMix (TOYOBO, Osaka, Japan) and the following conditions: 10 s at 94°C, 30 s at the appropriate annealing temperature (72, 73, or 75°C), and 30 s at 68°C. The last step was a 1-min incubation at 68°C. Information on SSR markers and the respective annealing temperatures was obtained from the NBRP KOMUGI web site (http://www.shigen.nig.ac.jp/wheat/komugi/strains/aboutNbrpMarker.jsp) and the GrainGenes web site (http://wheat.pw.usda.gov/GG2/maps.shtml). PCR products were resolved in 2% agarose or 13% nondenaturing polyacrylamide gels and visualized under UV light after staining with ethidium bromide. The MAPMAKER/EXP version 3.0b package was used for genetic mapping [28]. The threshold for log-likelihood scores was set at 3.0, and genetic distances were calculated with the Kosambi function [29].

Each polymorphism at the Ppd-D1 locus on 2DS was detected with allele-specific primers and methodology described by Beales et al. [30]. A common forward primer, Ppd-D1_F (5′-ACGCCTCCCACTACACTG-3′), and two reverse primers, Ppd-D1_R1 (5′-GTTGGTTCAAACAGAGAGC-3′) and Ppd-D1_R2 (5′-CACTGGTGGTAGCTGAGATT-3′), were used for this PCR analysis. PCR products amplified with Ppd-D1_F and Ppd-D1_R2 detected a 2,089-bp deletion in the 5′ upstream region of Ppd-D1 that is indicative of the photoperiod-insensitive Ppd-D1a allele [30]. EST-derived sequence-tagged site (STS) markers on 2DS, TE6, and WE6 were also used for genotyping; two STS markers for each locus, and these markers were previously developed along with the Iw2-linked markers [7]. The amplified PCR products were separated via electrophoresis through a 2% agarose or 13% nondenaturing polyacrylamide gel and then stained with ethidium bromide.

Development of additional markers linked to Iw2

In our previous studies, we conducted deep-sequencing analyses of the leaf and spike transcriptomes of two Ae. tauschii accessions that represented two major lineages, and discovered more than 16,000 high-confidence single nucleotide polymorphisms (SNPs) in 5,808 contigs [31,32]. Contigs with the SNPs were searched with blastn against Ae. tauschii genome sequences [20] and barley genome sequences [33]; these genome sequences included high-confidence genes with an E-value threshold of 10−5 and hit length ≥ 50 bp, fingerprinted contigs, and whole genome shotgun assemblies.

To choose scaffolds for Ae. tauschii sequences throughout the Iw2 chromosomal region, all the genes contained in each scaffold were searched with blastn against the barley genomic sequence using parameters described above. Scaffolds containing at least one gene aligned on the distal region of chromosome 2HS (between 3.66 Mb and 5.51 Mb) were considered possible candidates for marker development. Scaffolds without genes were anchored based on respective results from the blastn searches against the barley genome. First, high-confidence SNPs [31,32] plotted in this 2HS chromosomal segment were used for marker development to refine the target region. Next, SciRoKo version 3.4 [34] was used with search mode setting “mismatched; fixed penalty” to identify additional SSR markers in sequence data of candidate scaffolds. Additional SNPs were also identified on candidate scaffolds by sequencing approximately 700 bp of amplified DNA of two Ae. tauschii accessions, KU-2154 and KU-2126. The nucleotide sequences were determined using an Applied Biosystems 3730xl DNA Analyzer (Applied Biosystems, Foster City, CA, USA), and SNPs were found via sequence alignments constructed and searched with GENETYX-MAC version 12.00 software (Whitehead Institute for Biomedical Research, Cambridge, MA, USA).

For genotyping, total DNA was extracted from leaves taken from each of the 210 Ae. tauschii accessions and the 17 Iranian wheat landraces. SSR amplification and detection of polymorphisms at these loci were conducted as described above. The identified SNPs were then further developed into cleaved amplified polymorphic sequence (CAPS) or high resolution melting (HRM) markers. The primer sequences for each SNP marker and any relevant restriction enzymes are summarized in Additional file 1. PCR and subsequent analyses were performed as described previously [31,32,35].

Blast analysis of the Ae. tauschii genes relative to the Brachypodium genome

Nucleotide sequences and annotation information of the selected Ae. tauschii scaffolds were analyzed with reference to the Ae. tauschii draft genome data, which was published by Jia et al. [20]. Reference sequences from Brachypodium [36] were searched against the National Center for Biotechnology Information (NCBI) NR protein database using the blastx algorithm with an E-value cut-off of 10−3.

Association analysis of the linked markers with glaucousness

The Q + K method was conducted using a mixed linear model (MLM) function in TASSEL ver 4.0 software [37] for an association analysis by incorporating phenotypic and genotypic data and information on population structure. In a previous report, the Bayesian clustering approach implemented in the software program STRUCTURE 2.3 [38] was used with the setting k = 2 to predict the population structure of the Ae. tauschii accessions [19]. The Q-matrix of population membership probabilities was served as covariates in MLM. Kinship (K) was calculated in TASSEL based on the genotyping information of the 169 DArT markers for the 206 Ae. tauschii accessions [19]. We performed the F-statistics and calculated the P-values for the F-test, and the threshold value was set as 1E-3 for the significant association. We omitted the target markers from the association analysis when their minor allele frequencies were less than 0.05.

Results

Wax production variation among Ae. tauschii accessions and among synthetic wheat lines

Of the 210 Ae. tauschii accessions examined, only 20 (9.5%) exhibited the glaucous phenotype and produced whitish wax on the surfaces of peduncles and spikes (Figure 1A-D, Additional file 2). Wax production for each accession was completely consistent between the Fukui and Kobe environments. Each glaucous accession belonged to Ae. tauschii subspecies tauschii; in other words, none belonged to Ae. tauschii subspecies strangulata; the geographic distribution of glaucous accessions was limited to the area that spans from Transcaucasia to the southern coastal region of the Caspian Sea (Figure 1H). In the eastern habitats (central Asia, Afghanistan, Pakistan, India, and China) of the species range, no glaucous accession was found. Of the 20 glaucous accessions, 19 belonged to the TauL2 lineage, and only one (IG127015 collected in Armenia) belonged to the TauL1 lineage (Additional file 2).

Figure 1

Variation in cuticular wax production among accessions. (A,B) Non-glaucous accessions of Ae. tauschii. PI508262 and KU-2075 are classified as subspecies tauschii and subspecies strangulata, respectively. (C,D) Glaucous accessions of Ae. tauschii. (E) A tetraploid wheat cultivar Langdon. (F) A synthetic hexaploid wheat line with the non-glaucous phenotype: the line was derived from an interspecific cross between Langdon and a non-glaucous Ae. tauschii accession, KU-2078. (G) A synthetic hexaploid wheat line with the glaucous phenotype; the line was derived from an interspecific cross between Langdon and a glaucous Ae. tauschii accession, KU-2156. (H) Geographical distribution of glaucous-type accessions in Ae. tauschii. The Ae. tauschii accessions were classified into three genealogical lineages, TauL1, TauL2, and TauL3 [19].

Of the 82 synthetic wheat lines that we examined, 15 exhibited whitish wax production on the peduncle and spike surface (Figure 1E-G), whereas no wax production was evident in any of the 67 other lines (Additional file 2). Of the 15 lines that showed the glaucous phenotype, 13 were produced by crossing Ldn with glaucous Ae. tauschii accessions, and each of the 67 non-glaucous lines was produced by crossing Ldn with a non-glaucous Ae. tauschii accession. Notably, two synthetic lines, Ldn/KU-2104 and Ldn/KU-2105, exhibited the glaucous phenotype even though their parental Ae. tauschii accessions were non-glaucous.

Mapping of the Iw2 locus in Ae. tauschii and synthetic wheat

Two F2 populations of Ae. tauschii and three F2 populations from the synthetic wheat lines were analyzed to map the loci that control inhibition of wax production. Each F1 plant used for the five cross combinations exhibited the non-glaucous phenotype. In each F2 population, the ratio of non-glaucous to glaucous individuals was 3:1; these findings were statistically significant and consistent with Mendelian segregation of alleles of a single gene (Table 1). These results indicated that a single genetic locus was associated with the phenotypic difference between non-glaucous and glaucous surfaces on peduncles and spikes, and that allele conferring the non-glaucous phenotype was dominant and the allele conferring the glaucous phenotype was recessive.

Table 1

Segregation analysis of the non-glaucous phenotype in the five F mapping populations

F 2 population	N	Non-glaucous type	Glaucous type	χ 2 value*	P value
KU-2003/KU-2124	116	89	27	0.184	0.668
KU-2154/KU-2126	116	78	38	3.724	0.054
Ldn/KU-2159//Ldn/IG126387	100	71	29	0.853	0.356
Ldn/KU-2124//Ldn/IG47259	82	65	17	0.797	0.372
Ldn/PI476874//Ldn/KU-2069	106	77	29	0.314	0.575

*Expected segregation ratio was 3:1.

A single locus that controlled inhibition of wax production in Ae. tauschii was mapped to the same region of the short arm of chromosome 2D in each F2 mapping population (Figure 2). In the KU-2003/KU-2124 population, the locus that controlled inhibition of wax production, together with the loci for 25 SSR markers and Ppd-D1, was assigned to chromosome 2D, and the map length was 230.0 cM with an average inter-loci interval of 8.85 cM. In the KU-2154/KU-2126 population, the locus that controlled inhibition of wax production, together with 14 SSR and 2 STS markers and Ppd-D1, was assigned to chromosome 2D, and the map length was 175.4 cM with average inter-loci spacing of 10.32 cM. In the three synthetic wheat populations, Ldn/KU-2159//Ldn/IG126387, Ldn/KU-2124//Ldn/IG47259, and Ldn/PI476874//Ldn/KU-2069, the locus that controlled inhibition of wax production was mapped to a similar position on the short arm of chromosome 2D (Figure 2). In these three synthetic wheat populations, the locus that controlled inhibition of wax production was mapped together with 11 to 13 SSR markers, 0 to 2 STS markers, and Ppd-D1; additionally, the map lengths ranged from 79.4 to 93.8 cM with an average inter-loci spacing of 4.96 to 8.53 cM.

Figure 2

Linkage maps of on chromosome 2D. Two and three mapping populations were generated for Ae. tauschii and synthetic hexaploid wheat, respectively. Genetic distances are represented in centimorgans to the left of each chromosome.

WE6 and TE6 are EST-derived STS markers that are linked to Iw2 in two mapping populations [7,9]. In three of our mapping populations, linkage of the non-glaucousness loci to WE6 and TE6 were confirmed. Thus, the position of one locus that controlled inhibition of wax production in Ae. tauschii corresponded to the well-known wax inhibitor gene, Iw2, on chromosome 2D [6,7]. Therefore, hereafter, all glaucousness-related loci mapped in this study were considered to be identical to Iw2.

Fine mapping of the Iw2 locus

The high-confidence SNPs derived from Ae. tauschii RNA-seq data have been plotted onto barley chromosomes [32], and physical map information for the barley genome is available [33]. Additionally, physical map information for Ae. tauschii and 16,876 scaffolds that constitute 1.49 Gb from the draft Ae. tauschii genome sequence are anchored to the Ae. tauschii linkage map [20,21]. The RNA-seq-derived SNP information [31,32] was used to map seven high-confidence SNPs, represented as Xctg loci in Figure 3, throughout the Iw2 chromosomal region in the KU-2154/KU-2126 F2 population. Of the seven Xctg loci, four were located within the 8.8 cM chromosomal region immediately surrounding Iw2. Nucleotide sequences of the four cDNAs corresponding to these Xctg loci were used as queries to select the carrier scaffolds from Ae. tauschii sequences. We selected the Ae. tauschii scaffolds that mapped near the Xctg-carrying Ae. tauschii scaffolds based on synteny between the wheat and barley genomes and the barley physical map [39]. In all, 18 Ae. tauschii scaffolds were assigned in silico to an area of the Ae. tauschii genome that corresponded to the Iw2 region in the physical map of barley chromosome 2H (Figure 3). Using a previously developed physical map of the Ae. tauschii 2DS chromosome [21], we mapped six Ae. tauschii scaffolds in silico to the corresponding region in the 2DS physical map. Nucleotide sequences of the selected scaffolds were used to design CAPS or SSR markers for each scaffold, and the markers that were polymorphic between KU-2154 and KU-2126 were then mapped in the F2 population (Figure 3).

Figure 3

Comparison of the linkage map, which contains the scaffolds, with the physical maps of barley and . The Ae. tauschii scaffolds were assigned to regions of the barley physical map of chromosome 2H [33]. An Ae. tauschii physical map with the mapped scaffolds [21] is represented. Scaffold positions (Mb) and numbers [20,21] are shown on the left and right of each chromosome, respectively.

Of the selected scaffolds, 23 were mapped to the Iw2 chromosomal region on 2DS, and the remaining three scaffolds were assigned to other chromosomes. In the KU-2154/KU-2126 population with 115 F2 individuals, the Iw2 locus was mapped within the 1.1 cM interval between the most closely linked markers (Figure 3). A dominant marker (S51038-8), derived from the Ae. tauschii scaffold 51038 sequence, was located 0.2 cM distal to Iw2, and the WE6 SSR marker was located 0.9 cM proximal to Iw2. Five co-dominant markers, derived from two Ae. tauschii scaffolds 10812 and 82981, co-localized with Iw2. The marker order in the KU-2154/KU-2126 linkage map was generally conserved with that in the barley 2H physical map. However, barley scaffold 9655 was more closely linked to the barley Iw2 ortholog than were two corresponding Ae. tauschii scaffolds, 13577 and 33766, to the tauschii Iw2 ortholog; this positioning indicated that a local inversion had occurred in the region proximal to Iw2 during the divergence between barley and tauschii.

Next, F2 individuals of the KU-2154/KU-2126 population and 12 markers from five Ae. tauschii scaffolds were used to construct a fine map of Iw2 (Figure 4A). Based on this linkage map, Iw2 was located within the 0.7 cM between Xctg216249/S51038-8 and WE6 and co-localized with five markers derived from two scaffolds, 10812 and 82981. Each of the five scaffolds was 63 to 334 kb in length and included one to 16 putative protein-coding genes [20,21]; marker positions of each scaffold are indicated in Figure 4B. Of the 12 markers, eight were derived from intergenic regions, the other four from open reading frames.

Figure 4

Assignment of protein-encoding genes found on the scaffolds around to orthologs on chromosomes. (A) Linkage map of the region around Iw2 generated with 285 F2 individuals. Genetic distances (cM) are shown on the left, and markers on the right. (B) The figure shows the positions of putative genes and mapped markers in the Ae. tauschii scaffolds anchored to the Iw2 region. (C) The Iw2-orthologous regions on Brachypodium chromosomes based on the blastx search of anchored Ae. tauschii genes. Brachypodium genes are shown on the right, and their position (kb) on the left.

In all, 36 genes were evident on the five scaffolds, and gene annotation could be confirmed for 27 of the 36 genes (Table 2). Of these 27 Ae. tauschii genes, 10 putatively encoded cytochrome P450 monooxygenase proteins, and eight encoded disease-related proteins. Additionally, genes encoding laccase, agmatine coumaroyltransferase, receptor kinase, and cell number regulator 2-like were found on the two scaffolds that co-localized with Iw2.

Table 2

Colinearity between and in the syntenic genomic regions around

Ae. tauschii gene	Brachypodium gene	Annotation
AEGTA20795	Bradi1g15030.1	cytochrome p450 85a1
AEGTA20794	Bradi1g15030.1	cytochrome p450 85a1
AEGTA25164	Bradi1g15030.1	cytochrome p450 85a1
AEGTA22963	Bradi1g15030.1	cytochrome p450 85a1
AEGTA20793	Bradi1g15030.1	cytochrome p450 85a1
AEGTA20792		f-box domain containing protein
AEGTA04539		hypothetical protein F775_04539
AEGTA09742	Bradi1g15010.1	probable fructokinase-1-like
AEGTA20791	Bradi2g39120.1	hypothetical protein F775_20791
	Bradi2g39100.1
AEGTA32301	Bradi3g18920.1	hypothetical protein F775_32301
AEGTA20790		cytochrome p450
AEGTA20789		cytochrome p450 monooxygenase cyp71d70
AEGTA20788		cytochrome p450
AEGTA09741	Bradi2g27777.1	cytochrome p450 71c4
AEGTA09740	Bradi5g01360.1	sulfotransferase 16-like
	Bradi4g37480.1
	Bradi3g03460.1
AEGTA32300	Bradi2g10230.2	deleted in split hand split foot protein 1
	Bradi2g10230.1
AEGTA24906	Bradi5g01180.1	brown planthopper-induced resistance protein 1
AEGTA19771	Bradi3g02290.1	laccase-15-like
	Bradi3g02300.1
	Bradi3g02370.1
	Bradi4g11840.1
AEGTA19772	Bradi4g36820.1	agmatine coumaroyltransferase-2-like
	Bradi3g02310.1
	Bradi4g36850.1
AEGTA43098		protein da1-related 1-like
AEGTA33234	Bradi5g01167.1	disease resistance protein rpm1
AEGTA19773		l-type lectin-domain containing receptor kinase -like
AEGTA09277		cytochrome p450 84a1
AEGTA17544	Bradi5g01167.1	disease resistance protein rpm1
AEGTA08264	Bradi5g01160.1	protein da1-related 1-like
AEGTA03281		cell number regulator 2-like
AEGTA17543	Bradi1g30630.1	cell number regulator 2-like
	Bradi3g46930.1
	Bradi5g12460.1
AEGTA17542	Bradi1g33650.1	serine threonine-protein kinase receptor
	Bradi1g05890.1
	Bradi1g75950.1
	Bradi3g41060.1
AEGTA17439	Bradi5g01135.1	probable pectate lyase 15-like
AEGTA17438	Bradi5g01110.1	disease resistance rpp13-like protein 1-like
	Bradi5g01080.1
AEGTA17437	Bradi5g01070.1	disease resistance rpp13-like protein 1-like
	Bradi5g01080.1
	Bradi5g01110.1
AEGTA17436	Bradi5g01080.1	disease resistance rpp13-like protein 1-like
	Bradi5g01110.1
AEGTA17435	Bradi5g01110.1	disease resistance rpp13-like protein 1-like
	Bradi5g01070.1
	Bradi5g01080.1
AEGTA17434	Bradi5g01080.1	disease resistance rpp13-like protein 1-like
	Bradi5g01110.1
AEGTA23449		hypothetical protein F775_23449
AEGTA03244		hypothetical protein F775_03244

The Ae. tauschii scaffolds that included protein coding genes were used as queries to search the Brachypodium genomic information via a blastn search. Of the Ae. tauschii genes on the five scaffolds, 18 had obvious orthologs in the Brachypodium genome (Figure 4C). Putative orthologs of the Ae. tauschii genes from the four scaffolds were assigned to the 987 to 1068 kb region of Brachypodium chromosome 5. In addition, three Brachypodium paralogs (Bradi5g01220.1, Bradi5g01220.2, and Bradi5g01230.1) positioned in the 1133 to 1143 kb region were orthologous to an Ae. tauschii gene, AEGTA20985; additionally, Bradi5g01280.1 at 1186 kb was orthologous to AEGTA28084 in scaffold 6859. The locations of two Ae. tauschii genes, AEGTA20985 and AEGTA28084, were 3 and 3.9 cM, respectively, distal to Iw2 (Figure 3); therefore, the distal part of Iw2 showed chromosomal synteny to Brachypodium chromosome 5. Thus, the Iw2 chromosomal region on 2DS was generally syntenic to Brachypodium chromosome 5. However, putative orthologs of the Ae. tauschii genes from scaffold 43829 were assigned to Brachypodium chromosomes 1 and 2. Two paralogous Ae. tauschii genes, AEGTA19771 and AEGTA19772, on scaffold 10812 were orthologous to three paralogous Brachypodium genes (Bradi3g02290.1, Bradi3g02300.1, and Bradi3g02370.1) on Brachypodium chromosome 3. Therefore, the chromosomal synteny between Ae. tauschii and Brachypodium around the Iw2 orthologs was complex with regard to chromosome structure.

Iw2-linked marker genotypes in Ae. tauschii

To determine the genetic associations among the developed markers and glaucousness, 13 Iw2-linked PCR markers—including five CAPSs, five SSRs, one HRM, one insertion/deletion (indel), and one dominant (presence or absence) marker—were used to genotype the 210 Ae. tauschii accessions (Table 3). For eight of the 13 markers, the 210 accessions exhibited just two apparent alleles; additionally, the set of accessions exhibited just three distinct electrophoresis patterns—including the KU-2154-type, the KU-2126- type, and one other type—at one SSR marker for WE6. The other four SSR markers were highly polymorphic among the accessions; specifically, each marker gave rise to more than three distinct electrophoresis patterns.

Table 3

Association between -linked marker genotypes and glaucous versus non-glaucous phenotypes in 210 accessions of and the distribution of marker genotypes among Iranian wheat landraces

Marker name	Marker type	No. accessions	Glaucous phenotype ( N  = 20)			Non-glaucous phenotype ( N  = 190)			P -value for F -test in the association analysis a	Iranian wheat landraces ( N  = 17)
			KU-2154 -type	KU-2126 -type	Others	KU-2154 -type	KU-2126 -type	Others
C141566873	CAPS	210	0	20	0	91	99	0	0.403	KU-2126-type
S43829-13	SSR	210	4	5	12	65	22	102	8.20E-05	Other types
S43829-3	CAPS	206	17	2	0	184	3	0	-	KU-2154-type
S43829-12	SSR	207	0	6	14	136	37	14	1.55E-07	KU-2126-type (15)/Others (2)
Xctg216249	HRM	210	9	11	0	177	13	0	1.52E-04	KU-2154-type
S51038-8	Dominant	210	0	20	0	170	20	0	8.18E-10	KU-2154-type
S10812-12	CAPS	210	18	2	0	170	20	0	1.55E-05	KU-2126-type
S10812-14	Indel	197	0	20	0	145	32	0	8.66E-11	KU-2126-type
S10812-1	SSR	210	0	15	5*	135	0	55 (4)	3.26E-24	Other types
S10812-13	CAPS	206	0	20	0	180	16	0	9.92E-16	KU-2126-type
S82981-2	SSR	210	0	15	5*	136	0	54 (5)	1.95E-22	Other types
WE6	SSR	210	0	14	6*	59	56	75 (75)	0.169	Other types
Xctg202354	CAPS	210	0	20	0	113	77	0	0.041	KU-2126-type

The numbers of accessions for each genotype are represented in glaucous and non-glaucous phenotypes.

The numbers of non-glaucous-type accessions showing the genotype corresponding to the other one in the glaucous-type accessions are indicated in parenthesis.

*These accessions showed the same genotype different from KU-2154 and KU-2126.

aThe values were calculated based on a mixed linear model in the TASSEL ver. 4.0 software.

The association analysis showed that four SSR markers (S43829-13, S43829-12, S10812-1, and S82981-2), an HRM marker (Xctg216249), the dominant marker (S51038-8), an indel marker (S10812-14), and two CAPS markers (S10812-12, and S10812-13), co-localized with Iw2 in the Ae. tauschii linkage map, were significantly (P < 1E-3) associated with variation in glaucousness; in contrast, the other three genotyped markers were not significantly associated with variation in glaucousness (Table 3). The CAPS marker S43829-3 was removed from this association analysis because of the low-frequency (<0.05) allele. In particular, the KU-2126-type allele of the SSR locus S10812-1 was found only in 15 of the 20 glaucous accessions; moreover, none of the 190 non-glaucous accessions carried this KU-2126-type allele. The other five glaucous accessions carried a third allele of the S10812-1 locus. In 55 of the 190 non-glaucous accessions, only four carried the third allele of the S10812-1 locus, and the other 135 accessions carried different S10812-1 alleles. Of the four exceptional non-glaucous accessions that carried the third S10812-1 allele, two were KU-2104 and KU-2105, and these had each been used to generate a synthetic hexaploid wheat line Ldn/KU-2104 and Ldn/KU-2105, respectively; both synthetic lines showed the glaucous phenotype (Additional file 2). However, the phenotype of each synthetic hexaploid line (Ldn/KU-2074 and Ldn/KU-2079) derived from the remaining two of the exceptional accessions (KU-2074 and KU-2079) was non-glaucous. Therefore, phenotypic differentiation in glaucousness was almost completely explained by the allelic configuration at the S10812-1 locus in these natural populations of Ae. tauschii.

The 17 Iranian wheat landraces showed the KU-2154-type alleles at S43829-3, Xctg216249, and S51038-8, whereas they exhibited the KU-2126-type alleles at C141566873, S10812-12, S10812-14, S10812-13, and Xctg202354. In addition, these landraces exhibited various genotypes that differed from the allelic combinations found in Ae. tauschii accessions at WE6 and four SSR marker loci, S43829-13, S10812-1, S82981-2 (Table 3). At S43829-12, 15 landraces showed the KU-2126-type genotype, and two exhibited other genotypes.

Discussion

Natural variation for wax production in Ae. tauschii

Glaucousness is presumably among the components of the domestication syndrome in tetraploid wheat [5,6]. Therefore, glaucousness was apparently a target of artificial selection during tetraploid domestication and common wheat speciation; nevertheless, whether glaucousness is an adaptive trait in wild wheat species remains unclear. Cuticular wax on plant surfaces plays an important role in reducing water loss under drought stress conditions for Arabidopsis and rice [1,4], and observations in these other species indicate that relationships between glaucousness and drought stress tolerance are tight. Presence of either the Iw1 or Iw2 allele greatly reduces ß-diketones in the wax components of plants, resulting in a non-glaucous phenotype [3,10]. Comparative study of glaucousness-related genes in near-isogenic lines (NILs) of a common wheat cultivar (S-615) (BC10F3 generation; [6]) demonstrates that Iw alleles had a negative impact on drought tolerance [3]. However, another study of Iw1 in a NIL (BC2F3 generation) of common wheat did not detect an association between Iw1 genotype and water-use efficiency [10].

In this study, we used a set of 210 accessions that represented the entire geographical range of Ae. tauschii to examine natural variation in wax production among Ae. tauschii, and found 20 glaucous accessions that were collected in the area that spans from Transcaucasus to the southern-eastern coastal region of the Caspian Sea (Figure 1, Additional file 2). In a previous study of 176 Ae. tauschii accessions collected from 105 different habitats throughout Afghanistan, Pakistan, and Iran, 17 glaucous accessions were found in this same area that spans from Transcaucasus to the southern-eastern coastal region [16]. Therefore, our findings were fully consistent with previous observations.

Most glaucous accessions belonged to the TauL2 lineage (Additional file 2). TauL2 accessions derived from geographically wide-spread sites throughout the Transcaucasus/Middle East region; these sites represented the western habitats of Ae. tauschii [19]. TauL1 accessions were collected from sites widely distributed throughout the species range, and most TauL1 accessions showed a non-glaucous phenotype. Notably, one TauL1 accession (IG127015), collected in Armenia, showed a glaucous phenotype, and the collection site was located in the middle of an area where glaucous TauL2 accessions were collected (Figure 1). Genotyping data suggested that IG127015 had an Iw2 chromosomal region that was very similar to the Iw2 chromosomal region of the glaucous TauL2 accessions. One possible explanation for this observation is that IG127015 acquired the Iw2 chromosomal region from some glaucous individual of the TauL2 lineage. Such introgression could occur in the natural habitat where IG127015 was originally sampled and in experimental fields where the accession was propagated for several generations. Another explanation is that IG127015 became a wax producer through a de novo recessive mutation at the Iw2 locus; this scenario, however, is unlikely because the molecular marker genotypes in the Iw2 chromosomal region of IG127015 were largely identical to those in the Iw2 chromosomal region of glaucous TauL2 accessions (Table 3).

Whether the glaucous phenotype of the exceptional TauL1 accession was due to introgression of a glaucous allele from a glaucous TauL2 plant may be difficult to discern. Genome-wide marker analyses using SNP array and diversity arrays technology (DArT) systems indicated that TauL2 was clearly distinct and genetically differentiated from TauL1 [18,19]. This high level of differentiation indicates that the two genealogical lineages have been reproductively isolated, and that, under natural conditions, inter-lineage hybridization seems to have occurred only rarely [17,18]. Nevertheless, the presence of a glaucous-type TauL1 accession indicated that the hybridization between TauL1 and TauL2 might have occurred, but the number of hybridizations seems to be quite small. Further detailed study is required to clarify the past occurrence of the TauL1-TauL2 inter-lineage hybridization in Ae. tauschii.

Causal loci for variation in glaucousness among Ae. tauschii

Previous studies show that, in Ae. tauschii, the causal gene for the glaucous/non-glaucous phenotypic difference is Iw2, and that the genotypes of glaucous and non-glaucous accessions were W2W2iw2iw2 and W2W2Iw2Iw2, respectively [5,14]. The molecular markers tightly linked to Iw2 were very closely associated with glaucous versus non-glaucous phenotypic difference among the 210 accessions of Ae. tauschii (Table 3). Thus, the allelic difference at the Iw2 locus was the main contributor to the phenotypic difference between the glaucous and non-glaucous accessions of Ae. tauschii (Figure 2, Table 3). In common wheat, the markers derived from Bradi5g01180 and Bradi5g01160 are tightly linked to Iw2 as well as Iw1 [10,11]. Because the loci that control the glaucous versus non-glaucous phenotypic difference in Ae. tauschii mapped to the chromosome 2DS region where the common wheat Iw2 gene resides (Figure 4), the same Iw2 gene is likely involved in wax production in both Ae. tauschii and common wheat. Actually, although most SSR markers around the Iw2 region were highly polymorphic among the Ae. tauschii accessions and Iranian wheat landraces, three markers co-localized with Iw2 in Ae. tauschii S10812-12, S10812-14, and S10812-13; notably, each showed the KU-2126-type alleles in each of the 17 Iranian wheat landraces (Table 3). These results indicated that the Iranian wheat landraces, which exhibited the glaucous phenotype, had the iw2iw2 genotype.

Marker order and gene order around Ae. tauschii Iw2 was well conserved with those on barley chromosome 2HS and Brachypodium chromosome 5 (Figures 3 and 4). Similar chromosomal synteny between the Iw1 region on 2BS and Brachypodium chromosome 5 was recently reported based on mapping with common wheat populations [10,11]. In Ae. tauschii, scaffold information derived from the draft genome data were available for detailed analysis of chromosomal synteny at the Iw2 region. Chromosomal order of the selected scaffolds at Iw2 revealed the occurrence of a local inversion during divergence between barley and Ae. tauschii (Figure 3). Moreover, information of predicted genes in the scaffolds showed that putative translocations occurred during divergence between Brachypodium and Ae. tauschii (Figure 4). These results also indicated that several gaps existed between the Ae. tauschii scaffolds. Thus, colinearity among barley, Brachypodium and Ae. tauschii was observed in the Iw2 syntenic region, as was reported recently [11], but further screening of Ae. tauschii BAC clones may be required for construction of the complete physical map at Iw2.

In the genotyping analysis with Iw2-linked markers, non-glaucous accessions with the Iw2Iw2 genotype constituted the majority of all 210 accessions (Table 3). However, four non-glaucous accessions (KU-2074, KU-2079, KU-2104, and KU-2015) shared a genotype at S10812-1 (the most tightly linked marker) with five glaucous accessions (IG127015, KU-2106, KU-2158, KU-2159, KU-2160), indicating that these four non-glaucous accessions may have the iw2iw2 genotype in spite of the S10812-1 genotypes. In fact, synthetic hexaploids from hybrids between Ldn, which has the glaucous genotype (W1W1iw1iw1) [10], and two of the four non-glaucous accessions, KU-2104 and KU-2105, exhibited the glaucous phenotype (Additional file 2). In contrast, the phenotypes of all synthetic hexaploids derived from the KU-2074 and KU-2079, were non-glaucous. Accordingly, KU-2074 and KU-2079 seemed to have the Iw2Iw2 genotype even though they shared an S10812-1 genotype with the five glaucous accessions. Taken together, all this evidence indicated that Iw2 was the major gene that controls inhibition of wax production in Ae. tauschii.

As yet, no loss-of-function allele has been reported for W2, a major wax-producing gene in Ae. tauschii. In common wheat, however, some cultivars such as Chinese Spring and Salmon carry the recessive w2 allele [5]. Similarly, non-glaucous-type accessions with the w1 recessive allele have been discovered among wild emmer wheat [5]. Whether the recessive loss-of-function mutation occurred at the diploid level (i.e., in Ae. tauschii) or at the hexaploid level (i.e., in T. aestivum) is not known. Further studies are required to clarify the details of the genetic mechanism that underlies the wax production in Ae. tauschii.

Implication of the Iw2 variation in hexaploid wheat speciation

Based on a comparative genic analysis among common wheat and its ancestral species, Tsunewaki [5] suggested that common wheat, which is hexaploid, is the product of a hybrid cross that took place between a glaucous cultivated emmer wheat with the genotype W1W1iw1iw1 and a glaucous wild Ae. tauschii with the genotype the W2W2iw2iw2 genotype in the mountainous region near the southwestern coastal part of the Caspian Sea. Here, we found that, of 210 Ae. tauschii accessions, only 20 had the glaucous phenotype (Additional file 2) and that a dominant allele at the Iw2 locus were responsible for expression of the non-glaucous phenotype (Table 1). Furthermore, we found that, on the basis of the molecular-marker genotypes in the Iw2 chromosomal region and the phenotypes of the synthetic common wheat lines, virtually all non-glaucous accessions had the Iw2Iw2 genotype (Table 3, Additional file 2). A non-glaucous accession that had the iw2iw2 genotype was not found among the 210 accessions. This finding was notable because the double recessive w2w2iw2iw2 genotype, if present, would have also caused the non-glaucousness phenotype. The reason for the absence of any Ae. tauschii accession with the w2w2 iw2iw2 genotype from this collection was not clear, but this fact may indicate that functional W2 alleles confer some adaptive advantage under natural conditions. Taken together, the evidence from this study was consistent with the view that glaucous Ae. tauschii individuals that had the W2W2iw2iw2 genotype were involved in the origin of hexaploid common wheat.

Previous evidence based on isozyme variations and DNA marker polymorphisms is consistent with the hypothesis that the birthplace of hexaploid wheat is within a broad area ranging from Armenia to southwestern Caspian Iran [18,40-42]. The geographic range of the parent populations of glaucous Ae. tauschii accessions was very consistent with the region postulated in this hypothesis (Figure 1). However, the Ae. tauschii subspecies-strangulata has been postulated to be the D-genome donor of common wheat [43]. Of the 210 Ae. tauschii accessions that we examined, only 12 accessions have markedly moniliform spikes, and each of these were originally collected in the southeastern coastal Caspian region [25,26]. Taxonomically, these accessions could be classified as Ae. tauschii Coss. subspecies strangulata (Eig) Tzvel. Our data demonstrated that all these strangulata accessions, which were not glaucous, had the Iw2Iw2 genotype (Figure 1). On the assumption that the ancestral Ae. tauschii had the W2W2iw2iw2 genotype, this finding may suggest that the southeastern coastal Caspian populations of Ae. tauschii subspecies strangulata do not represent the direct descendants of the ancestral populations that gave rise to hexaploid common wheat.

Conclusions

Analysis of the Iw2 locus may contribute to improve our understanding of the evolution of hexaploid wheat. Of the 210 Ae. tauschii accessions, only 20 glaucous accessions were found in the area that spans from Transcaucasia to the southern coastal region of the Caspian Sea. Of the 82 synthetic wheat lines that we examined, 15 were glaucous, and each of the 67 non-glaucous lines was produced by crossing Ldn with a non-glaucous Ae. tauschii accession. Of the 15 glaucous lines, 13 were produced by crossing Ldn with glaucous Ae. tauschii accessions. The remained two accessions seemed to have the Iw2Iw2 genotype according to the genotyping analysis with the Iw2-linked markers. Therefore, allelic differences at the Iw2 locus on the short arm of chromosome 2D were the main contributors to the phenotypic difference between the glaucous and non-glaucous accessions of Ae. tauschii. Some molecular markers, such as S10812-1, closely linked to Iw2 were significantly associated with variation in glaucousness in Ae. tauschii. These results suggest that the D-genome donor of common wheat could have been any Ae. tauschii variant that carried the recessive iw2 allele.

Availability of supporting data

The data sets supporting the results of this article are included within the article and its supplementary files.