Fengbo Xue，Jun Guo，Changying Guan，Hongwei Wang，Anfei Li，Lingrang Kong*

State Key Laboratory of Crop Biology，Shandong Key Laboratory of Crop Biology，College of Agronomy，Shandong Agricultural University，Tai'an 271018，China

Molecular mapping of the hybrid necrosis gene NetJingY176 in Aegilops tauschii using microsatellite markers

Fengbo Xue，Jun Guo，Changying Guan，Hongwei Wang，Anfei Li，Lingrang Kong*

State Key Laboratory of Crop Biology，Shandong Key Laboratory of Crop Biology，College of Agronomy，Shandong Agricultural University，Tai'an 271018，China

A R T I C L E I N F O

Article history:

Received 3 April 2015

Received in revised form

13 May 2015

Accepted 1 June 2015

Available online 6 June 2015

Aegilops tauschii Coss

Hybrid necrosis

NetJingY176

Molecular marker

The rich genetic variation preserved in collections of Aegilops tauschii can be readily exploited to improve common wheat using synthetic hexaploid wheat lines.However，hybrid necrosis，which is characterized by progressive death of leaves or plants，has been observed in certain interspecific crosses between tetraploid wheat and Ae.tauschii.The aim of this study was to construct a fine genetic map of a gene（temporarily named NetJingY176）conferring hybrid necrosis in Ae.tauschii accession Jing Y176.A triploid F1population derived from distant hybridization between Ae.tauschii and tetraploid wheat was used to map the gene with microsatellite markers.The newly developed markers XsdauK539 and XsdauK561 co-segregated with NetJingY176 on chromosome arm 2DS.The tightly linked markers developed in this study were used to genotype 91 Ae.tauschii accessions.The marker genotype analysis suggested that 49.45%of the Ae.tauschii accessions carry NetJingY176.Interestingly，hybrid necrosis genotypes tended to appear more commonly in Ae.tauschii ssp.tauschii than in Ae.tauschii ssp.strangulata.

?2015 Crop Science Society of China and Institute of Crop Science，CAAS.Production and hosting by Elsevier B.V.This is an open access article under the CC BY-NC-ND license（http://creativecommons.org/licenses/by-nc-nd/4.0/）.

1.Introduction

Commonwheat（Triticum aestivum L.，2n=6x=42，AABBDD）isa naturallyformedallohexaploid species；tetraploid wheat（Triticum turgidum L.，2n=4x=28，AABB）provided the A and B genomes and the diploid goatgrass species Aegilops tauschii Coss.（2n=2x=DD）provided the D genome approximately 8000 years ago［1，2］.As the source of the D genome of common wheat，Ae.tauschii shows abundant genetic variation useful for wheat improvement［3-9］.Ae.tauschii has historically been classified into two subspecies，ssp.tauschii and ssp.strangulata［3，10］.Elongated cylindrical spikelets are a prominent typical characteristic of Ae.tauschii ssp.tauschii，whereas quadrate spikelets are a prominent typical characteristic of Ae.tauschii ssp.strangulata［3，10］.The subspecies tauschii is distributed from eastern Turkey to China and Pakistan，whereas the subspecies strangulata is distributed only in the southeastern Caspian coastal region and Transcaucasia［3-5，33］.

The rich natural variation in Ae.tauschii accessions，which remains largely unexplored，can be used for the improvement of wheat breeding through the production of synthetic hexaploid wheats［11-15］.However，abnormal phenotypeshave been observed in many F1triploid hybrids between tetraploid wheat and Ae.tauschii［16-20］.Four abnormal phenotypes have been reported:type II and type III hybrid necrosis，hybrid chlorosis，and severe growth abortion［21］. Hybrid plants showing any of the four types of hybrid necrosis rarely produce selfed seeds［22］.Thus，in the process of transferring genes from related species to cultivated wheat，hybrid necrosis has been a serious obstacle［23，24］.

Type II hybrid necrosis，which is characterized by tiller number increase and culm length decrease under low-temperature conditions，is one of the abnormal phenotypes observed in hybrids between tetraploid wheat and Ae. tauschii［22］.This type of necrosis is controlled by Net1 and Net2，two complementary genes located on the A or B genome and the D genome，respectively［18］.Net2 is located on the short arm of chromosome 2D，while the chromosomal location of Net1 remains uncertain［14］.However，molecular markers closely linked to the two genes have not yet been identified. The tetraploid durum wheat（T.turgidum ssp.durum）cultivar Langdon has been extensively used as the AB genome source in creating synthetic hexaploid wheats［25］and carries Net1［26］.

Simple sequence repeat（SSR）markers，also known as microsatellite markers，show highly polymorphic polymerase chain reaction（PCR）products in many plant species.In most cases，SSR markers are chromosome-specific and codominant. For this reason，they have been used in many areas of genetic analysis；for example，constructing genetic linkage maps［27-29］，detecting genetic diversity［30］，and mapping genes of interest［31，32］.Diverse breeding materials can be genotypically distinguished for phylogenetic studies by analysis of a small number of SSR markers［33，34］.A genome sequence of Ae.tauschii based on a whole-genome shotgun strategy has been published［35］.In addition，a physical map of Ae.tauschii covering 4 Gb has been developed［36］.The 1.72-Gb genome sequence and physicalmap of Ae.tauschii establish a foundation for developing SSR markers for fine mapping and map-based cloning oftargetgenes in Ae.tauschii.

The aim of this study was to construct a fine genetic map of NetJingY176 and to determine whether or not Aegilops accessions carry NetJingY176，using newly developed closely linked markers.

2.Materials and methods

2.1.Plant materials Twelve Ae.tauschii accessions（CIae 9，CIae 17，CIae 25，PI 268210，PI 276985，PI 369527，PI 428564，PI 511375，Jing Y176，Jing Y199，Jing Y215，and Jing Y225）were used as pollen parents for crossing with the durum wheat cultivar Langdon（genome formula AABB）.Tightly linked markers developed in this study were used for the identification of NetJingY176 in 91 Aegilops accessions，including 49 Ae.tauschii ssp.tauschii and 42 Ae.tauschii ssp.strangulata accessions（Table S1）.

To determine the chromosome location of NetJingY176，a triploid F1mapping population was developed.First an F1was obtained by crossing Ae.tauschii accession Jing Y225（showing a normal wild-type（WT）phenotype when crossed with Langdon）with Ae.tauschii accession Jing Y176（showing hybrid necrosis when crossed with Langdon）.Ae.tauschii accessions Jing Y225 and Jing Y176 are classified as subspecies strangulata and tauschii，respectively（Fig.1-a）.Second，a mapping population with 199 triploid F1individuals was developed by crossing Langdon with the F1（Jing Y225/Jing Y176）using embryo rescue.The triploid F1carried the same AB genomes from Langdon as the female parent and a recombinant D genome derived from the hybrid of Jing Y176 and Jing Y225 as the male parents.After vernalization for 20 days at 4°C，the triploid F1individuals to be used for phenotypic and genetic studies were planted in the greenhouse at Shandong Agricultural University，Tai'an，Shandong province，China.

2.2.Trypan blue staining

Each plant was grown at room temperature in the greenhouse for 3 weeks before staining.Leaves were harvested and placed in phosphate buffer solution（PBS），which was then mixed in equal volumes with 0.4%trypan blue dye solution.After incubation for 2 min in a boiling water bath，the leaves were cooled overnight.They were then bleached in 1.25 g mL-1chloral hydrate for 3 d and examined under an inverted microscope［37］.Leaves stained with dye were considered dead.

2.3.DNA extraction and analysis of microsatellite markers Genomic DNA was isolated from young leaves by the CTAB（Cetyltrimethyl Ammonium Bromide）method as described by Guo et al.［38］.

Wheat microsatellite markers（Xcfa，Xcfd，Xgdm，Xgwm，Xbarc，Xpsp，and Xwmc）［27-29，38］on chromosomes 1D-7D based on previously published maps were chosen to analyze polymorphism between the two Ae.tauschii parents and the linkage relationships of NetJingY176 in 10 plants showing normal phenotype（WT）and 10 plants showing hybrid necrosis using bulk segregant analysis（BSA）.Wheat SSR markers on chromosome 2D were then selected for gene mapping. The sequences of the selected primers were obtained from GrainGenes 2.0（http://www.wheat.pw.usda.gov/）.

The PCR reaction mixtures of 15 μL contained 10 mmol L-1Tris-HCl，pH 8.3，50 mmol L-1KCl，1.5 mmol L-1MgCl2，0.2 mmol L-1dNTPs，25 ng of each primer，50-100 ng of genomic DNA，and 0.75 U of Taq DNA polymerase.DNA amplification was performed by denaturing template DNA at 94°C for 5 min；followed by 35 cycles of 30 s at 94°C，30 s at 50-60°C（depending on the annealing temperature of each primer），and 30 s at 72°C，and 10 min at 72°C as the final step. The PCR products were mixed with 6 μL of 6×loading buffer and separated by 8%nondenaturing polyacrylamide gels（39:1 acrylamide:bisacrylamide）by electrophoresis at 120 V for 3 h. Gels were visualized by silver staining as described by Guo et al.［38］.A genetic linkage map was constructed using JoinMap 4.0［39］with a minimum LOD threshold of 2.0 for grouping order.

2.4.Development of new SSR markers closely linked to NetJingY176

Scaffolds of the Ae.tauschii 2D genome sequence［35，36］surrounding the necrosis gene NetJingY176 were obtainedand used for marker development.The software SSR Finder（http://www.fresnostate.edu/ssrfinder/）was used to identify SSR regions in the scaffolds and sequences flanking the SSR region were selected to design SSR primers using Primer3 Input Version 0.4.0（http://bioinfo.ut.ee/primer3-0.4.0/）under general settings.These primers were used to screen for polymorphisms between the two Ae.tauschii parents.Primer sequences of newly developed SSR markers are listed in Table S2.The primers were then used to screen for polymorphism between Ae.tauschii accessions Jing Y176 and Jing Y225. Marker analyses were performed as described above.

Fig.1-Phenotypic characterization of hybrid necrosis in an F1triploid individual.（a）Panicle traits of Aegilops tauschii accessions Jing Y225 and Jing Y176，tetraploid wheat cultivar Langdon，and a synthetic hexaploid wheat line（Langdon/Jing Y225）.Ae.tauschii accessions Jing Y225 and Jing Y176 are classified as the subspecies strangulata and tauschii，respectively.（b）Phenotypic characterization of leaves by staining with trypan blue.Untreated leaves are on the left and leaves stained with trypan blue are on the right.（c）Triploid F1individuals in a constant-temperature incubator showed no obvious symptoms.（d）Phenotypic characterization of plants.I:Triploid F1individual with the wild-type phenotype at the seedling stage；II: Triploid F1individual with the hybrid necrosis phenotype at the seedling stage；III:Triploid F1individuals with the wild-type phenotype at the heading stage；IV:Triploid F1individuals with the hybrid necrosis phenotype stopped growing at the tillering stage and then died.

3.Results

3.1.Phenotypic characterization of necrotic wheat—Ae. tauschii triploid hybrids showing necrosis

Four of 12 F1hybrids from crosses between tetraploid wheat Langdon and four Ae.tauschii accessions（Jing Y176，PI 276985，PI 428564，and PI 511375）clearly showed an identical necrotic phenotype:dwarfing and markedly increased tiller number. The remaining hybrids showed normal growth.

Compared to WT，the phenotypic progression of hybrids with necrosis was divided into three stages based on plant height and leaf senescence（Fig.1）.In the first stage，no obvious necrotic symptoms were observed in a constant-temperature incubator at 4°C for one month.In the second stage，after vernalization，triploid F1individuals were planted in the greenhouse at24°C/16°C（day/night）for one month.Compared to WT individuals，hybrids with necrosis showed dwarfing and dramatically increased tiller numbers.Incomplete leaf expansion and stem tip necrosis were also observed at the tillering stage.In the third stage，the leaves of hybrids with necrosis gradually turned yellowish and the meristem gradually died，while the WT individuals produced selfed seeds.

The cell death response was observed in the leaves of the triploid hybrids.To further confirm this phenomenon，trypan blue was used to stain the necrotic leaves.No staining was observed in the leaves of WT individuals.In contrast，the leaves of hybrid necrotic plants showed extensive cell death（Fig.1-b）.

3.2.Genetic analysis of the hybrid necrosis phenomenon

A triploid F1mapping population was produced to map the gene for hybrid necrosis.In total，199 triploid F1individualssegregated as 110 WT and 89 hybrid necrosis plants，fitting a 1:1 segregation ratio（χ2=1.114，P=0.29）.This result indicates that the gene controlling hybrid necrosis on the D genome is a single genetic locus.Thus，the phenotypic segregation of WT and hybrid necrosis in the triploid F1population can be used to confirm the genotypes of Ae.tauschii accessions Jing Y176 and Jing Y225 at the NetJingY176 locus.

3.3.Marker development and molecular mapping of the gene NetJingY176 NetJingY176 on the D genome controlled hybrid necrosis in intraspecific crosses of tetraploid wheat and Ae.tauschii.To map the gene，140 D-genome SSR markers were chosen to analyze polymorphism between the two Ae.tauschii parents and their linkage relationships of NetJingY176 using BSA.Five markers on chromosome 2D were linked to NetJingY176.Then，64 SSR markers that were previously mapped to chromosome 2D were investigated for polymorphism between Ae.tauschii accessions Jing Y225 and Jing Y176.Of these，only 16 identified polymorphic fragments were linked to NetJingY176 in the population，suggesting that NetJingY176 was located on the short arm of chromosome 2D.Markers Xgwm102 and Xgwm515 were located on each side of NetJingY176 at genetic distances of 4.5 and 3.8 cM，respectively（Fig.2）.

Based on the genome sequence and the physical map of Ae.tauschii，131 SSR markers were developed.Twenty-nine markers that were closely linked to NetJingY176 were assigned to chromosome arm 2DS（Fig.2）and 10 of them i.e.，XsdauK547，XsdauK554，XsdauK555，XsdauK558，XsdauK548，XsdauK534，XsdauK536，XsdauK541，XsdauK549，and XsdauK552 developed on the basis of Ae.tauschii scaffold sequences［35］were anchored on the chromosome 2D physical map constructed by Luo et al.［36］.Of these markers，XsdauK539 and XsdauK561 co-segregated with NetJingY176 on chromosome 2DS and were designed from the same scaffold.The corresponding relationships between scaffold numbers and the new developed markers are presented in Table S2.

Comparative analysis was performed to assess the collinearity among Ae.tauschii 2D chromosome regions containing NetJingY176 and Brachypodium（http://www.Brachypodium.org/），Oryza（http://rice.plantbiology.msu.edu/），and Sorghum（http:// www.plantgdb.org/SbGDB/cgi-bin/blastGDB.pl）.The results indicated that this syntenic region between XsdauK552 and XsdauK549 corresponds to Brachypodium chromosome 1（Bradi1g19627 and Bradi1g19570），Oryza chromosome 7（Os07g45160 and Os07g45280）and Sorghum chromosome 2（Sb02g041248 and Sb02g041320）（Fig.2）.Detailed comparative analyses revealed that only one gene，Bradi1g19620 in Brachypodium，corresponding to Os07g45170 in Oryza and Sb02g041250 in Sorghum，is conserved.Thus，very lower levels of genomic collinearity were observed among Brachypodium，Oryza，and Sorghum in the Ae.tauschii chromosome region containing NetJingY176.

3.4.Identification of Ae.tauschii accessions putatively carrying NetJingY176 using linked SSR markers

First，eight Ae.tauschii accessions showing the WT phenotype and four Ae.tauschii accessions showing hybrid necrosis phenotypes confirmed by hybridization were used to validate the efficacy of NetJingY176-linked markers XsdauK536，XsdauK549，XsdauK555，and XsdauK560 for the identification of Ae.tauschii accessions putatively carrying NetJingY176.The marker assay indicated that the marker genotypes were in agreement with the phenotypes in all 12 Ae.tauschii accessions tested，so the markers closely linked to NetJingY176 are useful for identifying the necrosis gene NetJingY176 in Ae.tauschii.Accordingly，the 91 Ae.tauschii accessions were screened with the linked SSR markers to detect the presence of NetJingY176 gene，and the amplification bands for both WT and hybrid necrosis genotypes are shown in Fig.3.Among the 91 Ae.tauschii accessions screened with markers XsdauK536，XsdauK549，XsdauK555，and XsdauK560，45 showed hybrid necrosis marker genotypes and the remainder showed WT marker genotypes.The Ae.tauschii accessions with clear genotypes identified by SSR markers included 49 ssp.tauschii accessions and 42 ssp.strangulata accessions.Interestingly，of the 42 ssp.strangulata accessions，30 showed WT marker genotypes and the rest showed necrosis marker genotypes. However，among the 49 ssp.tauschii accessions，only 16 showed WT marker genotypes and the other 33 showed hybrid necrosis marker genotypes（Fig.4）.

Fig.2-Comparative genetic linkage map of NetJingY176 and its syntenic genomic regions on the chromosome 2D physical map，Brachypodium chromosome 1，rice chromosome 7，and sorghum chromosome 2，respectively.The units of these maps are cM，cM，10 kb，kb，and 100 kb，respectively.

4.Discussion

Hybrid necrosis in wheat was first described in the 1940s［40］. Genes Ne1 and Ne2 are two dominant complementary genes that，when occurring together in a hybrid wheat，confer hybrid necrosis［41-44］.Ne1 lies on the proximal half of chromosome arm 5BL，whereas Ne2 lies on the distal half of 2BS［45］.The necrosis of wheat triploid F1hybrids was first mentioned by Nishikawa［16-18］.The tetraploid cultivar Langdon is an effective AB genome source for producing synthetic hexaploid wheat［12］.Large numbers of synthetic hexaploid wheat lines can be created with different D genomes from various Ae. tauschii accessions［46，47］.In synthetic wheat production，there are four types of hybrid abnormality［20，21］.Type II necrosis shows unique features，such as low temperature-induced necrotic symptoms and growth repression［22］.In this study，hybrid necrotic plants clearly exhibited a necrotic phenotype: dwarf plants and markedly increased tiller number after vernalization.This result suggests that the hybrid necrosis phenotype oftriploid F1hybrids in the presentstudy was that of type II necrosis.

Net2 is located on chromosome arm 2DS［14］.The molecular mapping performed in this study showed that gene NetJingY176 from Ae.tauschii accession Jing Y176 co-segregated with the newly developed microsatellite markers XsdauK539 and XsdauK561 on chromosome arm 2DS.Comparison of the physical map of chromosome 2D［28］with the genetic map of 2D developed in this study shows that NetJingY176 is physically located on the proximal half of the short arm.Two SSR markersXgwm102 and Xgwm515 that flank NetJingY176 are located in deletion bins 2DS-1-0.33-0.47 and 2DS-5-0.47-1.00，respectively. This result suggests that NetJingY176 is likely to be identical to Net2.Thus，Ae.tauschii accession JingY176 or other accessions carrying Net should not be used as parents when mapping populations for genetic research are created with synthetic hexaploid wheat lines.The closely linked microsatellite markers could be effective for identifying the genotypes of parental lines for NetJingY176 or for using marker-assisted selection to remove the hybrid necrosis gene.

Fig.4-Distribution of the gene NetJingY176 in Ae.tauschii ssp.tauschii and Ae.tauschii ssp.strangulata.The Ae.tauschii accessions comprised 49 ssp.tauschii and 42 ssp.strangulata accessions.Based on markers，49.45%of the Ae.tauschii accessions showed hybrid necrosis genotypes，and 50.55% showed WT genotypes.Among 91 Ae.tauschii with clear genotypes，28.57%of the ssp.strangulata accessions showed hybrid necrosis genotypes，whereas 67.35%of the ssp. tauschii accessions showed hybrid necrosis genotypes.

Gene deletion or insertion，which plays an important role in adaptation to stress and environment changes for plant species，is very common in the grass family［48，49］.In this study，comparative analysis was performed to investigate the collinearity among Brachypodium，Oryza，and Sorghum and the Aegilops chromosome 2D region containing NetJingY176.The results showed very low levels of genomic collinearity among these species in this Aegilops chromosome region containing NetJingY176.The low genomic collinearity could be explained by the better survival in unstable environments of species with gene deletions or insertions than of their ancestors［50，51］.

Fig.3-Amplification bands of the wild type and hybrid necrotic plants with SSR markers.（a）Marker XsdauK560；（b）Marker XsdauK549.M:The size of an amplified fragment was estimated with a GeneRulerExpress DNA ladder；1:Jing Y225；2:Jing Y176；3-12:amplification bands of the wild type（WT）；13-22:amplification bands of hybrid necrosis.

Determining the genetic and evolutionary relationships between the D genome of common wheat and the Ae.tauschii genome is crucial for understanding the development of common wheat.The Ae.tauschii genotypes associated with the origin of common wheat are confined to a narrow distribution range relative to the distribution range of the species，suggesting that Ae.tauschii harbors abundant genetic diversity that is not represented in common wheat［52］.In fact，only a few Ae.tauschii lineages were involved in the origin and development of common wheat.In addition，the wheat D genome is more closely related to that of Ae.tauschii ssp.strangulata than to that of Ae.tauschii ssp. tauschii［31］.In this study，91 Ae.tauschii accessions were used to detect the NetJingY176 gene.Based on the screening of SSR markers，49.45%of the Ae.tauschii accessions showed necrosis marker genotypes and 50.55%of the Ae.tauschii accessions showed WT marker genotypes.However，among 91 Ae.tauschii accessions with genotypes revealed by the associated markers，28.57% of the ssp.strangulata but 67.35%of the ssp.tauschii accessions showed necrosis marker genotypes（Fig.4）.Hybrid necrosis marker genotypes tended to appear more commonly in ssp.tauschii than in ssp.strangulata.

The molecular mechanisms of hybrid necrosis in synthetic hexaploid wheat are largely unknown.The mapping performed in this study has shown the location of the gene NetJingY176 in the Ae.tauschii genome and opens the way for further elucidating the structure，products，and function of the hybrid necrosis genes.The distribution of NetJingY176 locus in the various Ae.tauschii accessions may provide a guide for parental selection in use of Ae.tauschii germplasm collections for wheat germplasm enhancement and mapping population development.

5.Conclusion

We developed a triploid F1population by interspecific crosses between tetraploid wheat and Ae.tauschii to map the hybrid necrosis gene NetJingY176.The developed microsatellite markers XsdauK539 and XsdauK561 co-segregated with NetJingY176 on chromosome arm 2DS.The tightly linked markers developed in this study are useful for identifying Ae.tauschii genotypes carrying NetJingY176 and for cloning this gene from the Ae. tauschii genome.Genotyping analysis using SSR markers closely linked to NetJingY176 revealed that 49.45%of 91 Ae.tauschii accessions potentially carry NetJingY176.Hybrid necrosis genotypes tended to occur more frequently in Ae.tauschii ssp.tauschii than in Ae.tauschii ssp.strangulata.

Acknowledgments

This research was financially supported by the NationalNatural Science Foundation of China（grant numbers 31171553 and 31471488），the National Basic Research Program of China（2014CB138100），the National High Technology Research and Development Program of China （2011AA100102 and 2012AA101105）， Shandong Seed Engineering Project（2015-2019）and the Program of Introducing International Super Agricultural Science and Technology（948 program，2013-S19）.

Supplementary material

Supplementary material to this article can be found online at http://dx.doi.org/10.1016/j.cj.2015.05.003.

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*Corresponding author.Tel.:+86 538 8249278.

E-mail address:lkong@sdau.edu.cn（L.Kong）.

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