Mapping the glaucousness suppressor Iw1 from wild emmer wheat“PI 481521”

Zongchng Xu,Cuiling Yun,Jirui Wng,Dolin Fu,*,Jijie Wu,*

aState Key Laboratory of Crop Biology,Shandong Key Laboratory of Crop Biology,Shandong Agricultural University,Tai'an 271018,China

b Triticeae Research Institute,Sichuan Agricultural University,Chengdu 611130,China

1.Introduction

Epicuticular wax (EW) is an important surface structure on plants.In general,EW may affect water relations,protect plants from radiation,provide a physical barrier for toxic substances,enhance canopy reflectance,and increase grain yield [1–3].Epicuticular wax also plays important roles in plant defense against bacterial and fungal pathogens,and impacts plant–insect interactions[2,4].

Plant EW contains a variety of long chain-length hydrocarbons,such as alcohols,aldehydes and alkanes,each of which also contains various homologues[5].When compounds accumulate in the wax layer,especially those compounds enriched in one single homologue,they form ordered microcrystalline structures,which cause light-scattering effects and glaucousness [6].Therefore,plant EW can be grouped into non-glaucous and glaucous epicuticular waxes[7].Glaucous EW is associated with high concentrations of β-diketones,C29and C31hydrocarbons,primary alcohols,triterpene ketones,and esters within the EW hydrocarbon matrix [7–9].In xeric or semiarid plants,the glaucous EW improve water status under drought stress conditions[10].

In the Triticeae,many species have evolved with both glaucous and non-glaucous phenotypes,such as Aegilops tauschii [11],Hordeum vulgare [12,13],tetraploid wheat [14]and polyploid wheat[15,16].In wheat,major compounds such as β-diketones and hydroxy-β-diketones cause glaucousness[17,18].Glaucous phenotypes in wheat (Triticum aestivum L.)are mainly controlled by two wax production loci(W1 and W2)and closely associated inhibitor genes Iw1 and Iw2,that are epistatic to W1 and W2 [19].Wax composition was recently studied in six near-isogenic lines(NILs)varying in different W and Iw combinations in the genetic background of common wheat “S-615” [18].NILs W1W2iw1iw2,W1w2iw1iw2,and w1W2iw1iw2 are glaucous,whereas w1w2iw1iw2,W1W2Iw1iw2,and W1W2iw1Iw2 are non-glaucous.In general,the glaucous NILs are similar in wax load and wax composition,and β-diketones account for ca.60% of the total wax.However,the levels of β-diketones are dramatically reduced in the non-glaucous NILs,accounting for 8% of total wax in w1w2iw1iw2 and becoming undetectable in W1W2Iw1iw2 and W1W2iw1Iw2[18].

The wax production genes as well as the inhibitors were mapped on wheat chromosome arms 2BS and 2DS [19].The Iw1 locus is ca.2 cM distal to the W1 locus,whereas the Iw2 locus is ca.131 cM distal to W2.However,Iw1 and Iw2 are likely orthologs in wheat homologous group 2 [19,20].In wheat,two Iw loci,potential equivalents of Iw1 and Iw2,were mapped on the distal ends of the short arms of chromosome 2B and 2D [21,22].More recently,Adamski et al.[23] mapped the Iw1 gene within a sub-cM interval containing a single colinear gene in Brachypodium and rice (Oryza sativa L.).It was shown that Iw1 inhibits formation of β-and hydroxy-β-diketones in wheat EW on peduncles and flag leaf tissues.

In tetraploid wheat,several sets of chromosome substitution lines have been developed in durum cultivar “Langdon”(LDN,T.turgidum ssp.durum) [24–26].In the Langdon background,the substitution line LDNDIC521-2Bcarries a pair of 2B chromosomes from wild emmer wheat PI 481521 (T.turgidum ssp.dicoccoides,DIC) [26].Langdon has a glaucous phenotype,and LDNDIC521-2Bis non-glaucous.In this study,we report the mapping of the Iw1 locus using an F2population developed from Langdon and LDNDIC521-2B.

2.Materials and methods

2.1.Plant materials

This study was conducted on tetraploid wheat Langdon and LDNDIC521-2B(T.turgidum L.,2n = 4x = 28,AABB).Langdon was released by the North Dakota Agricultural Experiment Station in 1956 [27].LDNDIC521-2Bis a substitution line,in which the chromosomes 2B pair of Langdon is replaced by the homologous pair from the wild emmer accession “PI 481521”[26].Reciprocal crosses were made between Langdon and LDNDIC521-2B,and F1,F2,and F3plants were used to analyze the visual EW phenotypes.The parental lines for the study were obtained from USDA-ARS,Fargo,ND,USA.

2.2.Evaluation of glaucous and non-glaucous phenotypes

The visual EW phenotypes on flag-leaf sheaths,peduncles,and glumes were evaluated at the plant booting and heading stages.Glaucous EW is visible waxiness that contributes to the bluish color of organs; the transparent non-glaucous EW allows a natural reflection of green light from the investigated tissue surfaces.

2.3.Polymerase chain reaction

Genomic DNA of leaf tissues was extracted from plants at the jointing stage using the Sarkosyl method [28].PCR amplifications were performed in 20 μL mixes containing 1×PCR buffer(1.5 mmol L-1MgCl2,0.2 mmol L-1each of dCTP,dGTP,dTTP,and dATP; Promega,Madison,USA),0.4 μmol L-1of both forward and reverse primers,100 ng DNA template,0.4 U of Taq DNA polymerase (Promega),and ddH2O.Amplifications were conducted in an ABI 9700 Thermal Cycler (Life Technologies,Grand Island,NY,USA).Amplification cycles included an initial denaturation (94 °C for 5 min); 40 cycles of denaturation(94 °C for 30 s),annealing(58 °C for 30 s)and extension(72 °C for 30 s); and a final extension (72 °C for 10 min).PCR products were separated on 6% PAGE gels and examined under UV light.

2.4.Development of gene-based markers on wheat chromosome 2B

Gene-based markers provide informative data for genetic mapping and comparative genomics.To develop this type of marker,we utilized DNA polymorphisms in the gene region,which normally corresponds to expressed sequence tags(EST)and transcriptome-derived single nucleotide polymorphisms(SNP).To differentiate the 2B chromosomes between durum and wild emmer,we genotyped Langdon and LDNDIC521-2Busing the wheat 90K iSelect SNP array,in which all SNP probes were generated from data mining of genomic sequences and wheat transcriptomes[29].PCR markers were developed from selected SNPs showing polymorphism between Langdon and LDNDIC521-2B.In addition,BE444541 and BE498396 were chosen to develop 2B-specific markers; these are two wheat ESTs belonging to the distal 6S deletion bin in the group 2 consensus map[30].Three closely linked EST markers recently developed for the Iw1 gene[23]were also integrated into current mapping effort.

2.5.Construction of a genetic linkage map

Data for the Langdon/LDNDIC521-2BF2population was used to construct a chromosome 2B linkage map.In addition to 10 gene-associated markers,40 simple sequence repeats (SSR)were chosen from those previously mapped to chromosome 2B [31–33].The resulting 19 polymorphic SSR markers were used to map the chromosome 2B inhibitor of wax production.The genetic map was created using JoinMap 4.0(Kyazma B.V.,Wageningen,Netherlands),with map distances being estimated by the Kosambi mapping function[34].

3.Results

3.1.Wild emmer “PI 481521” carries a dominant inhibitor of wax production

Langdon and LDNDIC521-2Bwere both non-glaucous at the seedling stage.When plants reached the early booting stage or the Feekes Stages 9–10 [35],Langdon gradually became glaucous,but LDNDIC521-2Bremained non-glaucous until maturity(Fig.1).We investigated the phenotypes on F1plants generated from reciprocal crosses between Langdon and LDNDIC521-2B;the phenotypes were consistent among spikes,peduncles,and flag-leaf sheaths at the heading stage.All three organs were non-glaucous suggesting the presence of a dominant Iw allele in LDNDIC521-2B.Because Langdon and LDNDIC521-2Bdiffered only in respect of chromosomes 2B,an initial hypothesis was that LDNDIC521-2Band its 2B chromosome donor PI 481521 carried at least one Iw gene on chromosome 2B,completely negating the glaucous appearance.

We further checked the phenotypes of F2plants and F3lines from the Langdon/LDNDIC521-2Bcross.In the F2generation,64 plants were non-glaucous whereas 21 were glaucous,fitting a single locus 3:1 Mendelian segregation ratio(χ2= 0.0039,df = 1,P ＞0.95).Among the 85 F3lines(15–20 plants per line),23 were homozygous non-glaucous,39 segregated and 20 were heavily glaucous like Langdon confirming segregation at a single genetic locus (χ21:2:1= 0.41,df = 2,P ＞0.80).Thus PI 481521 possesses a single dominant Iw allele on chromosome 2B,presumably Iw1 as reported in earlier studies in tetraploid wheat[14,20,23].

3.2.Chromosome 2B-specific markers differentiate Langdon and PI 481521

To map the dominant Iw allele from PI 481521,we initially screened 40 SSR markers associated chromosome 2B [31–33];19 were polymorphic between Langdon and PI 481521(Table 1).

To better differentiate the 2B chromosomes of durum and wild emmer,Langdon and the 2B substitution line LDNDIC521-2Bwere analyzed using the wheat 90K iSelect array[29],revealing 345 polymorphic SNPs (Table 2).To check the applicability of the SNPs we also genotyped wild emmer accession “DIC479” [36].Of the 345 polymorphic SNPs,263 were also polymorphic between Langdon and DIC479.The wheat survey sequence was recently made publically accessible by the International Wheat Genome Sequencing Consortium(IWGSC)[37],and in addition wheat consensus maps containing 40,267 SNPs were constructed using the wheat 90K iSelect genotyping array [29].Based on the wheat survey sequence and wheat 90K consensus maps,158 SNPs were anchored to chromosome 2BS,and 174 were anchored to 2BL.Of the 345 polymorphic SNPs,257 were placed on the 2B consensus map,including 122 on each arm and 13 in the centromere region.Three SNPs,including IWA7120(CJ685558),IWA2116 (CJ858592),and IWA1359 (DR736025),were targeted to develop 2B-specific PCR markers(Tables 1–2,Fig.2).

Fig.1-EW appearance on Langdon and LDNDIC521-2B.Only the spike(S),peduncle(P),and leaf sheath(LS)are shown.

Seven wheat ESTs,including BE444541,BE498111,BE498396,BQ788707,CD893659,CD927782,and CD938589,were targeted to develop PCR markers from their genomic sequences.BE444541 and BE498396 were previously mapped to deletion bin 6S in the group 2 consensus map[30].BQ788707,CD893659,and CD927782,which were reported to co-segregate with the Iw1 gene,are equivalent to JIC011/CJ876545,JIC009/BF474014,and JIC010,respectively [20,23].BE498111 and CD938589 are orthologs of the genes in rice BAC clones OSJNBb0003A12(AL731620) and OSJNBa0095E20 (AL731627) and show microcolinearity to the Iw2 gene on wheat chromosome 2DS [21].In total,10 EST/SNP based PCR markers were developed,including three dominant markers in Langdon,one dominant marker in LDNDIC521-2B,two InDel markers,and four CAPS/dCAPS markers(Table 1).The 2B locations of some of the new markers were demonstrated by testing the“Chinese Spring” nulli-tetrasomic(NT)lines(Fig.2).

Table 1-PCR markers used for linkage analysis; upper,SSR markers; lower,gene-based markers developed from ESTs/SNPs.

3.3.PI 481521 carries a dominant Iw1 gene on chromosome 2BS

Using the F2population of Langdon/LDNDIC521-2B,we constructed a 2B linkage map containing 29 SSR and EST/SNP-based PCR markers spanning ca.79.9 cM (Fig.3).In this map,Xwmc272,Xwmc592,and Xcfa2278 are located near the centromere [32].As the Iw allele from PI 481521 displayed dominance for non-glaucousness,the EW phenotypes of F2plants and F3lines were sufficient to conclude the Iw genotype in F2plants.We mapped the Iw gene of PI 481521 to the distal region of chromosome 2BS (Fig.3).In the current map,Xbarc35,BQ788707,CD893659,CD927782,and CD938589 were completely linked to the Iw locus.In other reports,CD893659/BF474014/JIC009,CD927782/JIC010,and BQ788707/CJ876545/JIC011 (hereafter designated as CD893659,CD927782,and BQ788707) were shown to co-segregate with the Iw1 and Iw2 loci [20,23].Most likely,the dominant Iw locus of PI 481521 is Iw1.

The 2B linkage map of Langdon/LDNDIC521-2Bwas in agreement with the “Shamrock”/“Shango” map [22]; six SSR markers aligned in the same order on chromosome 2BS;the non-glaucous allele was mapped within the Xgwm614–Xwmc264 interval (Fig.3-A,B).Using EST-based markers,we then investigated the syntenic relationship of the Triticeae homologous group 2,and the Iw1 and Iw2 regions were highly conserved among the 2BS,2DS,and 2HS chromosomes(Fig.3-B,C,D,E).Seven ESTs were mapped in the same order on 2BS and 2DS (Fig.3-D,E).However,data for other ESTs implied chromosome inversions between 2BS and 2HS,such as the chromosome blocks MC43775–MC37223,MC1559514–MC36937,and MC1580487–MC135036 on the barley physical map(Fig.3-B,C).

Table 2-SNPs on the chromosome 2B polymorphic between Langdon and PI 481521.

Fig.2-Chromosome 2B specificities of newly developed PCR markers.PCR were performed on Langdon(AABB),“DV92”(T.monococcum,AmAm),“AS75”(Ae.tauschii,DD),and the“Chinese Spring” nulli-tetrasomic(NT)lines.BQ788707,a dominant marker in LDNDIC521-2B,is not detected in Langdon.

4.Discussion

Wild emmer is the progenitor of cultivated durum and the A and B genome donors of common wheat via cultivated emmer.As a largely untapped genetic reservoir,wild emmer represents a significant resource for wheat improvement[38].Wild emmer PI 481521,collected from Israel in 1983,was used to produce 14 chromosome substitution lines (LDNDIC521) in which single chromosome pairs of Langdon are replaced by the corresponding homologous pairs from PI 481521 [26].Studies on PI 481521 and the LDNDIC521substitutions demonstrated that PI 481521 possesses genes affecting disease resistance[39],kernel characteristics and protein molecular weight distribution [40],the glutenin subunits and gliadins[26].

In wheat,the EW phenotype is a significant morphological character.Glaucousness prevails in cultivated wheat,but non-glaucousness is common in wild germplasms,including wheat donors such as wild emmer and Ae.tauschii [41].EW phenotype is determined by four dominant genes,including the wax production genes(W1 and W2)and the wax inhibitors(Iw1 and Iw2).The inhibitors pair act epistatically on the wax producing genes[19].The absence of both dominant W genes or the presence of either dominant Iw gene leads to a non-glaucous phenotype.Both Iw1 and Iw2 were precisely mapped in wheat materials originating from wild emmer and Ae.tauschii[20,23].

Fig.3-Comparative maps of the two homologous groups containing the Iw gene.A:2B linkage map of the Shamrock/Shango population[22];B: 2B linkage map of the Langdon/LDNDIC521-2B population(current study);C:2H physical map of barley“Morex”(MC = morex_contig;http://barleyflc.dna.affrc.go.jp/bexdb/blast.html)[42]; D:2B linkage map of the WE74/Xuezao population[20];E:2DS linkage map of the ITMI population[20].Previously,BE498358(WE6)was mapped ca.1.4 cM proximal to the Iw2 gene[21];an approximate location of BE498358 is shown on the ITMI map.Of markers co-segregating with the Iw loci,only the 2B orthologs are included.For comparison with other studies,CD893659,CD927782,and BQ788707 are equivalent to JIC009/BF474014,JIC010 and JIC011/CJ876545,respectively[20,23].

Wild emmer accession PI 481521 and the LDNDIC521-2Bsubstitution lines are both non-glaucous.In contrast,the durum wheat cultivar Langdon is glaucous.Using the wheat 90K iSelect array,we first identified 345 SNPs polymorphic between LDN and its 2B substitution line LDNDIC521-2B.Using the wheat 90K consensus map as a reference,257 SNPs were ordered along the chromosome(Table 2).In conjunction with SSR and EST-based markers,the non-glaucous trait of PI 481521 was mapped to the distal region of the chromosome.In the current study,the Langdon/LDNDIC521-2Bpopulation contained only 85 F2plants,which consequently led to a low mapping resolution such that BQ788707,CD893659,CD927782,CD938589,and Xbarc35 co-segregated with the Iw.With population sizes of 2111 in the Shango/Shamrock population[23]and 4949 in the“Xuezao”/“WE74”population[20],BQ788707,CD893659,and CD927782 remained linked to the Iw1 locus.In addition,BQ788707 and CD893659 also co-segregated with the Iw2 locus in 1161 recombinant inbred lines(RIL)from“W7984”/“Opata M85” [20].However,only CD893659 cosegregated with the Iw1 gene among 850 F2plants in Langdon/ “TTD140” [23].Apparently,Iw1 and Iw2 are orthologs on 2BS and 2DS[20],and the dominant Iw locus of PI 481521 is assumed to be the Iw1 locus,although an allelism test should be made to determine whether PI 481521 carries the Iw1 allele.In barley,MLOC_20994 and MLOC_6767 are in close proximity to MLOC_77461 (an ortholog of CD893659),and are potential candidates for the Iw1 gene[23].In the future,large Langdon/LDNDIC521-2Bpopulations(e.g.5000 F2plants)should be obtained to test for linkage among MLOC_20994,MLOC_6767,and the Iw1 gene.If MLOC_20994 and MLOC_6767 show recombination with the Iw1 locus,map-based cloning of the Iw1 gene could be performed on large F2populations of the Langdon/LDNDIC521-2Bcross.

Glaucousness in common wheat is controlled by two pairs of wax producing gene(W loci)and wax production inhibitors(Iw loci);W1 and Iw1 are located on chromosome 2BS,and the W2 and Iw2 are located on chromosome 2DS.At least one dominant W allele is required for a glaucous phenotype,and only one dominant Iw allele is capable to prevent glaucousness [19].The glaucous parent Langdon must have genotype W1iw1,but whether non-glaucous PI 481521 has genotype W1Iw1 or w1Iw1 remains to be answered.Because the W1 locus is ca.2 cM proximal to the Iw1 locus [19],the EW phenotype on F2and F3plants of a cross between W1iw1 and W1Iw1(or w1Iw1)is primarily controlled by the Iw1 locus.If a dominant W1 allele is present in PI 481521 the Iw1 genotype can be precisely predicted using the F2phenotype in conjunction with the F3segregation pattern of glaucousness.The non-glaucous phenotype is solely caused by the dominant Iw1 gene.At the same time,all lines heterozygous for the Iw1 locus should segregate in an approximate ratio of one glaucous to three non-glaucous plants.The current study indeed displayed this segregation pattern.

If a recessive (w1) gene is present in PI 481521,the EW phenotype of the F2plants is again controlled by the Iw1 locus resulting from non-recombinant gametes at W1 and Iw1.Given a 2 cM genetic distance between W1 and Iw1,ca.96.04%of F2plants will be derived from non-recombinant gametes.Of the remaining 3.96% F2plants derived from recombinant gametes,about one quarter of them(ca.0.98%of all F2plants)will segregate at the W1 locus in homozygous iw1 background(W1w1iw1iw1),resulting in an approximate segregation ratio of three glaucous to one non-glaucous plant in F3lines.In the current study,only 82 F3lines were investigated,which allows only a 55.41% chance to containing at least one F3line segregating 3:1 for glaucousness versus non-glaucous plants P = 1 │(82 | 0)(0.98%) ↑0 (1 │0.98%) ↑82] with at least 468 F3lines being required to ensure a 99% possibility of recovering at least one such segregant.Therefore,a large F2population and progeny testing are necessary to determine whether a recessive(w1)gene is present in PI 481521.

Wild emmer has been widely studied with the objective of improving cultivated wheat [38].For example,the UK bread-making variety Shamrock is characterized by its viridescent appearance derived from the non-glaucous wild emmer[22].As for wild emmer PI 481521,a complete set of 14 chromosome substitution lines (LDNDIC521) were generated in the cultivated durum wheat Langdon [26].The LDNDIC521lines have been made publically accessible by the U.S.scientists with the USDA-ARS,and these are becoming important genetic resources for wheat improvement.

5.Conclusions

The non-glaucous phenotype of wild emmer accession PI 481521 and LDNDIC521-2Bis controlled by the dominant Iw1 allele on the chromosome 2B.In total,371 polymorphic markers,including 345 SNPs,19 SSRs,and 7 EST-based markers,were assigned to the chromosome 2B.All SSRs and 10 SNP/EST-based markers were used to construct a 79.9 cM linkage map,spanning the short arm of chromosome 2B.The Iw1 gene was mapped within the Xgwm614–BE498111 interval in the distal region of 2BS,and five markers(BQ788707,CD893659,CD927782,CD938589,and Xbarc35)cosegregated with the EW phenotype in the current mapping population.

This study was supported by the Natural Science Foundation of Shandong Province,China(JQ201107),the National Natural Science Foundation of China (31110103917),and the Cooperative Innovation Center of Efficient Production with High Annual Yield of Wheat and Corn,Shandong Province,China.We thank Professor G.F.Marais for providing the DIC479 seeds and Dr.Mingcheng Luo for the critical reading of the manuscript.

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