Introduction

Common wheat (Triticum aestivum L.) is an allohexaploid species with an AABBDD genome, derived from interspecific crossing between cultivated tetraploid wheat (Triticum turgidum L., genome constitution AABB) and Aegilops tauschii Coss. (DD) (Matsuoka 2011). The allohexaploid speciation process can be artificially reproduced through interspecific crosses and the following formation of synthetic wheat hexaploids (Kihara and Lilienfeld 1949; Mujeeb-Kazi et al. 1996). The tetraploid wheat cultivar Langdon (Ldn) is an efficient female parent for production of synthetic wheat hexaploids (Matsuoka and Nasuda 2004), whereas many accessions of Ae. tauschii show abnormal growth in artificial hybrids (2n = 3x = 21, ABD genome) crossed into Ldn (Matsuoka et al. 2007; Mizuno et al. 2010). The growth abnormalities in ABD triploids have been proposed to occur due to epistatic interaction between the tetraploid AB and the Ae. tauschii D genomes (Nishikawa 1960, 1962). Thus, postzygotic reproductive barriers between tetraploid wheat and Ae. tauschii inhibit the normal production of synthetic wheat hexaploids, and make it difficult to use agriculturally important genes in the Ae. tauschii population for wheat breeding through wheat synthetics. Therefore, fine mapping of the causal genes for the growth abnormalities in ABD triploids is important for efficient introgression of Ae. tauschii alleles into the common wheat genome.

The growth abnormalities in the ABD triploids between Ldn and Ae. tauschii include hybrid chlorosis, severe growth abortion (SGA), and two types of hybrid necrosis (type II and type III) (Mizuno et al. 2010). Abnormal chloroplasts form in the mesophyll cells before the leaves yellow in the triploid plants showing hybrid chlorosis (Nakano et al. 2015). SGA is always lethal, because SGA-exhibiting ABD triploids cease development and growth after expansion of the second or third leaves (Nishikawa 1960; Hatano et al. 2012). Cell death occurs gradually beginning with older tissues in hybrid lines showing type III necrosis, whereas type II necrosis lines show a necrotic phenotype under low temperature conditions (Nishikawa 1960; Mizuno et al. 2011). In addition to necrotic symptoms, low temperature represses mitotic cell division in the shoot apices of hybrid plants displaying type II necrosis (Mizuno et al. 2011). Out of these growth abnormalities, type II necrosis, is induced in most of the Ae. tauschii accessions examined previously (Mizuno et al. 2010). The Ae. tauschii accessions resulting in type II necrosis were mainly from the eastern regions of the native range (≥60° longitude), predominantly from Afghanistan and Pakistan (Mizuno et al. 2010). The early-flowering accessions spread mainly in the eastern habitats of Ae. tauschii (Matsuoka et al. 2008), and the spike and spikelet morphology in the eastern habitats clearly differs from that in the western habitats (Matsuoka et al. 2009; Takumi et al. 2009). Since the birthplace of common wheat has been supposed to be restricted to a narrow distribution range within the western habitats (Tsunewaki 1966; Dvorak et al. 1998; Wang et al. 2013; Nishijima et al. 2014), the eastern habitat accessions that provide agriculturally useful genes are not represented in the D genome of common wheat (Kajimura et al. 2011; Jones et al. 2013). Thus, type II necrosis should be overcome to comprehensively use the eastern habitat accessions of Ae. tauschii for wheat breeding.

Dislike type I necrosis observed in crosses between common wheat cultivars, type II necrosis has not been reported in any intraspecific cross of the Ae. tauschii accessions. Causal genes for type I necrosis, Ne1 and Ne2, were respectively located on chromosome arms of 5BL and 2BS (Tsunewaki 1960; Chu et al. 2006). Ne2 was assumed to be a wheat leaf rust resistance gene Lr13 (Zhang et al. 2016). The D-genome causal gene, Net2, for type II necrosis in ABD triploids and wheat synthetics was assigned to the short arm of chromosome 2D (Mizuno et al. 2011), whereas homoeologous relation between Ne2 and Net2 is still unknown. Another Net gene, Net1, complementary to Net2, is proposed to be in the AB genome of tetraploid wheat, but no information on its chromosome location has been reported (Takumi and Mizuno 2011). At least one wild tetraploid wheat accession was reported to show no hybrid necrotic phenotype in the ABD triploid with Net2 (Takumi and Mizuno 2011). In hybrid necrosis of higher plants, most causal genes that have been isolated to date are related to disease resistance (Bomblies et al. 2007; Alcázar et al. 2009, 2010; Yamamoto et al. 2010). Plants with type II necrosis exhibit a distinct phenotype from hybrid plants showing other types of hybrid necrosis, including marked growth suppression induced by low temperature (Mizuno et al. 2011). Arrest of cell division at the shoot apical meristem seems to occur prior to the autoimmune response in type II necrosis as well as in SGA (Mizuno et al. 2011; Hatano et al. 2012). In addition, tiller number is dramatically increased at normal temperatures in type II necrosis, although plants are significantly shorter (Takumi and Mizuno 2011). Due to these characteristics, it is still unknown which complementary genes are associated with the initial step in expression of type II necrosis.

Here, we report fine mapping of Net2 as a step toward map-based cloning of the type II necrosis-causative gene in future studies. Recently, a draft genome sequence of Ae. tauschii from a whole-genome shotgun strategy was made available, which anchored 1.72 Gb of the 4.36 Gb genome to physical chromosomes (Jia et al. 2013), and a physical map of the Ae. tauschii genome that covers 4 Gb is available (Luo et al. 2013). In addition, barley genome information helps to efficiently assign wheat genomic and transcript sequences in silico to the barley physical map (Iehisa et al. 2012, 2014). Thus, the scaffold sequence data of the Ae. tauschii genome are quite useful for fine mapping of loci assigned to the D genome, such as Iw2 and Hch1 on 2DS and 7DS, respectively (Nishijima et al. 2014; Hirao et al. 2015). To make a fine map of the Net2 region on 2DS, we developed Net2-linked markers using Ae. tauschii and barley genome information, and compared the chromosomal structure of the Net2 region with the barley orthologous region based on the marker order. Moreover, to verify the usefulness of the newly developed markers around Net2, we analysed the genetic association of the markers closely linked to Net2 using Ae. tauschii accessions that had been crossed to Ldn for phenotypic evaluation in the ABD triploids (Mizuno et al. 2010).

Materials and methods

Plant materials

Two synthetic hexaploid wheat lines generated through interspecific crosses of tetraploid wheat accession Ldn with two Ae. tauschii accessions, KU-2025 and KU-2075 (Mizuno et al. 2010), were used in this study. A synthetic hexaploid, Ldn/KU-2025, revealed type II necrosis (Mizuno et al. 2010), and another synthetic line, Ldn/KU-2075, showed normal growth (wild type; WT). As reported in Mizuno et al. (2010), the type II necrosis line produced small seeds when maintained at the normal growth temperature. An F2 mapping population was produced from crosses between Ldn/KU-2025 and Ldn/KU-2075. Seeds of the Ldn/KU-2025//Ldn/KU-2075 population were sown in November 2012, resulting in 188 F2 individuals, which were grown in the experimental field of Kobe University. Additionally, F3 and F4 individuals derived from F2 and F3 plants heterogeneous for the Net2 chromosomal region were respectively used for fine mapping. In the 2013–2014 season, 400 F3 individuals of the Ldn/KU-2025//Ldn/KU-2075 population, and in the 2014–2015 season, 200 F4 individuals were grown to increase the size of the mapping population.

Out of the Ae. tauschii core collection with 206 accessions, 83 were used in this study (Table S1), and their passport data, including geographical coordinates, were provided in our previous report (Matsuoka et al. 2008, 2009). Phenotypes of the 83 accessions in their ABD triploids were previously evaluated, and these accessions showed normal or type II necrosis (Mizuno et al. 2010). Previously, the 206 Ae. tauschii accessions were grouped into three lineages, TauL1, TauL2, and TauL3, based on DArT marker genotyping analysis (Matsuoka et al. 2013). The genotyping information on the 206 accessions was used for the 83 Ae. tauschii accessions for the genetic association study.

Marker development and genotyping

In our previous studies, we performed deep-sequencing analyses of the leaf and spike transcriptomes of two Ae. tauschii accessions, PI476874 and IG47182, that represent the two major lineages, and more than 16,000 high-confidence single nucleotide polymorphisms (SNPs) were found in 5808 contigs (Iehisa et al. 2012, 2014). The SNP information between PI476874 and IG47182 were applied to detect SNPs between Ldn/KU-2025 and Ldn/KU-2075. PI476874 and KU-2025 belong to the same lineage TauL1, and IG47182 and KU-2075 are grouped to TauL2 (Matsuoka et al. 2013). Contig sequences with the SNPs were searched with the BlastN algorithm against the Ae. tauschii and barley genomes (International Barley Genome Sequencing Consortium (IBSC) et al. 2012; Jia et al. 2013). 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 Net2 chromosomal region, all the genes contained in each scaffold were searched with BlastN against the barley genomic sequence using the parameters described above. Scaffolds containing at least one gene aligning to the putatively corresponding region of chromosome 2HS (between 52.784 and 74.898 Mb) were considered candidates for marker development. Scaffolds without genes were anchored based on respective results from BlastN searches against the barley genome. First, high-confidence SNPs (Iehisa et al. 2012, 2014) plotted on barley 2HS were used for marker development to refine the Net2 region. Next, SciRoKo version 3.4 software (Kofler et al. 2007) was used with the search mode setting “mismatched; fixed penalty” to identify additional simple sequence repeat (SSR) markers in sequence data of candidate scaffolds. Additional SNPs were identified in the Net2 regions by sequencing approximately 700 bp of amplified DNA of Ae. tauschii accessions KU-2025 and KU-2075. The nucleotide sequences were determined using an Applied Biosystems 3730xl DNA Analyzer (Applied Biosystems, Foster City, CA, USA), and SNPs were found through sequence alignment using GENETYX-MAC version 12.00 software (Whitehead Institute for Biomedical Research, Cambridge, MA, USA). The identified SNPs were converted to cleaved amplified polymorphic sequence (CAPS) markers. The primer sequences for newly developed markers and any relevant restriction enzymes are listed in Table S2.

Total DNA was extracted from leaves of the parental strains and individuals of the mapping population. For SSR genotyping, 40 cycles of PCR were performed using 2× Quick Taq HS DyeMix (TOYOBO, Osaka, Japan) and the following conditions: 10 s at 94°C, 30 s at the annealing temperature, and 30 s at 68 °C. For CAPS markers, the PCR annealing temperature was 58 °C. PCR products and their digests were resolved in 2 % agarose or 13 % non-denaturing polyacrylamide gels and visualized under UV light after staining with ethidium bromide.

Construction of the 2DS linkage map

For the initial mapping of Net2, 188 F2 plants were used in the Ldn/KU-2025//Ldn/KU-2075 population. Then, 400 F3 and 200 F4 individuals from the Ldn/KU-2025//Ldn/KU-2075 population were added for fine mapping of Net2 with tightly linked molecular markers. The MAPMAKER/EXP version 3.0b package was used for genetic mapping (Lander et al. 1987). The threshold for log-likelihood (LOD) scores was set at 3.0, and genetic distances were calculated with the Kosambi function (Kosambi 1944).

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

Nucleotide sequences and annotation information for the selected Ae. tauschii scaffolds were analysed with reference to the Ae. tauschii draft genome data, which were published by Jia et al. (2013). Reference sequences from Brachypodium (The International Brachypodium Initiative 2010) 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 type II necrosis

The Q+K method was conducted using a mixed linear model function in TASSEL ver 5.0 software (Bradbury et al. 2007) 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 (Pritchard et al. 2000) was used with the setting k = 2 to predict the population structure of the Ae. tauschii accessions (Matsuoka et al. 2013). The Q-matrix of population membership probabilities served as covariates in the mixed linear model. Kinship (K) was calculated in TASSEL based on the genotyping information of the 169 DArT markers for the 83 Ae. tauschii accessions (Matsuoka et al. 2013). We performed F statistics and calculated the P values for the F test, and the threshold value was set as 1E−3 for a significant association. We omitted the target markers from the association analysis when their minor allele frequencies were less than 0.05.

Results and discussion

Fine mapping of Net2 to 2DS

In the winter season, Net2-homozygous plants exhibited a severe growth defect and generally died in the Ldn/KU-2025//Ldn/KU-2075 F2 population (Fig. 1). Net2-heterozygous plants frequently showed an intermediate phenotype of defective and delayed growth and weak necrotic symptoms. In spring, with an increase in the growth temperature, the tiller numbers increased in the Net2-heterozygous plants. Although the heading and flowering times were remarkably delayed compared with the net2-homozygous plants, selfed seeds were obtained in the Net2-heterozygous plants as previously reported (Mizuno et al. 2011). Based on phenotypic observation, 188 F2 individuals segregated into 130 plants with the necrotic phenotype and 58 WT plants, fitting a 3:1 ratio (χ2 = 3.433, P = 0.064). Thus, type II necrosis is controlled by a single dominant allele of the Net2 locus in the Ldn/KU-2075//Ldn/KU-2025 population. Effects of Net2 on gamete formation and seed fertility were still unknown. The 3:1 segregation suggests no direct effects of Net2 on the seed production-related traits.

Fig. 1
figure 1

Phenotypes of segregating individuals in the Ldn/KU-2025//Ldn/KU-2075 F2 population. Net2- and Net2-homozygous plants respectively showed WT and severe necrotic symptoms. Net2/net2 heterozygous plants sometimes showed an intermediate phenotype, with growth inhibition and weak necrotic symptoms

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In our previous studies, Net2 was assigned to 2DS, and it mapped within the 8.2-cM region between an SSR (gwm102) and a SNP (HRM-10) marker (Mizuno et al. 2011; Matsuda et al. 2012). According to the available physical map information on the Ae. tauschii genome, 16,876 scaffolds that constitute 1.49 Gb from the draft Ae. tauschii genome sequence have been anchored to the linkage map of Ae. tauschii (Jia et al. 2013; Luo et al. 2013). The nucleotide sequences of cDNAs corresponding to the Net2-linked SNP markers were used as queries to select carrier scaffolds from the Ae tauschii sequences. We selected the Ae tauschii scaffolds near the Net2-linked marker-carrying scaffolds based on the chromosomal synteny between the wheat and barley genomes (Mayer et al. 2011; IBSC et al. 2012). In all, 55 Ae tauschii scaffolds were assigned in silico to an area showing synteny to the Net2 region on the physical map of barley chromosome 2H (Fig. 2). Using the 2DS linkage map of Ae. tauschii (Luo et al. 2013), nine Ae tauschii scaffolds were in silico mapped to the corresponding region on the 2DS map. Nucleotide sequences of the selected scaffolds were used to design CAPS or SSR markers for each scaffold, and the markers were mapped in the F2 population after their polymorphisms were confirmed between the parental synthetic hexaploids Ldn/KU-2025 and Ldn/KU-2075. In the selected scaffolds, 510 SSR and 99 SNP markers were designed, and polymorphisms between the parental lines were found in 20 SSR and eight SNP markers.

Fig. 2
figure 2

Comparison of Net2 linkage map, which contains Ae. tauschii scaffolds, with the Ae. tauschii linkage map and the barley physical map. The Ae. tauschii scaffolds were assigned to regions of the barley physical map of chromosome 2H (IBSC et al. 2012). A previously reported Ae. tauschii linkage map (Luo et al. 2013) is represented with the mapped scaffolds

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Using 24 of the polymorphic markers, 20 of the selected Ae tauschii scaffolds were anchored to the Net2 chromosomal region (Fig. 2; Table S2). No polymorphisms between Ldn/KU-2025 and Ldn/KU-2075 were found in the remaining scaffolds. In the Ldn/KU-2025//Ldn/KU-2075 population with 188 F2 individuals, the Net2 locus was mapped within a 0.6 cM interval between the linked marker scaf5-2, derived from scaffold 38996, and scaf13, from scaffold 15574. Next, the Net2 locus was mapped together with 11 co-dominant markers in 788 individuals of the Ldn/KU-2025//Ldn/KU-2075 population to construct the Net2 fine map (Fig. 3). The 788 individuals were segregated into 602 plants with the necrotic phenotype and 186 WT plants, fitting a 3:1 ratio (χ2 = 0.819, P = 0.365). On the fine map, Net2 was located within the 1.6 cM interval between scaf5-2 and scaf13. There were nine recombinants among the 788 individuals for the markers. In the Ae. tauschii linkage map (Luo et al. 2013), the scaffolds 38996 and 15574 co-localized to the 59.445-cM position of 2DS (Fig. 2). These observations suggest that Net2 is localized near scaffolds 38996 and 15574 or within one of them on 2DS. The scaffolds 38996 and 15574 were respectively 130.4 and 88.1 kb in length (Fig. 3), whereas the gap length between them is unknown. Construction of a bacterial artificial chromosome contig covering the Net2 region will be required for further studies toward map-based cloning of Net2.

Fig. 3
figure 3

Comparison of Ae. tauschii linakge map of the Net2 region with 788 individuals, the scaffolds, and Brachypodium chromosomes. The nine Ae. tauschii scaffolds, shown with gene and contig numbers, were putatively anchored to the Net2 region. Positions of the Net2-linked markers on the nine scaffolds are shown by the connected lines. The regions orthologous to Net2 on Brachypodium chromosomes with gene models are represented, and orthologous relationships are indicated by the connected lines

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Chromosomal synteny in the Net2 region among related species

The markers mapping near Net2 in the 188 F2 individuals covered the 57.568–59.649 cM chromosomal region in the Ae. tauschii genome (Fig. 2). This Net2 chromosomal region corresponded to the 52.784–74.898 Mb region on 2HS of the barley genome. Scaffold 38996 was located at the 74.898 Mb position, whereas scaffold 15574 was at the 70.506 Mb site in the Net2-corresponding chromosomal region. The order of the Net2-linked markers on the Ae. tauschii 2DS map was not necessarily conserved in the corresponding region of the barley 2HS physical map (Fig. 2). These observations indicate that complicated rearrangements occurred in the chromosomal region containing Net2 during the divergence between barley and Ae. tauschii. Similar rearrangements such as inversions have been reported in the chromosomal regions for Iw2, a wax inhibitor on 2DS, and Hch1, controlling hybrid chlorosis on 7DS, between the barley and Ae. tauschii genomes (Nishijima et al. 2014; Hirao et al. 2015). Chromosomal synteny was generally well conserved on each chromosome arm between the barley and Ae. tauschii genomes, as previously reported (Mayer et al. 2011). However, a lot of minor rearrangements likely accumulated locally after divergence of the diploid genomes in the tribe Triticeae.

The 11 markers used for the Net2 fine mapping were derived from the nine Ae. tauschii scaffolds. Each of the scaffolds was 30.8–130.4 kb and contained one to eight protein-coding genes (Jia et al. 2013; Luo et al. 2013). Thirty genes, including 24 with an AEGTA number and six contigs, appeared on the nine scaffolds (Fig. 3). Scaffolds 38996 and 15574 contained eight and two genes, respectively. The Ae. tauschii scaffolds that included protein-coding genes were used as queries to search the Brachypodium genomic information by a BlastN search. Putative orthologs of the Ae. tauschii genes from the nine scaffolds were mainly assigned to chromosome 1 of Brachypodium (Fig. 3). One gene in scaffold 38996 was orthologous to genes assigned to chromosome 2, and some paralogous copies to AEGTA15710 in scaffold 74122 were distributed to chromosomes 1–5. With these exceptions, the Net2 region on 2DS showed chromosomal synteny to the broad region of Brachypodium chromosome 1. In spite of some small inversions, translocations and duplications, the gene order was generally conserved during divergence between Brachypodium and Ae. tauschii.

Hybrid necrosis is widely known to be triggered by autoimmune responses due to epistatic interaction of disease resistance-related genes in higher plants (Bomblies et al. 2007; Alcázar et al. 2009, 2010; Yamamoto et al. 2010). Of the 18 Ae. tauschii genes in the four scaffolds near Net2, 10 (8 AEGTAs and 2 contigs) had obvious orthologs in the Brachypodium genome (Table 1). However, three AEGTA genes were putatively orthologous to Brachypodium genes encoding GDSL-like lipase protein or ARM repeat protein in the Net2-linked scaffolds 38996 and 15774, and thus no apparent NB-LRR-type resistance genes were present. In addition to autoimmune responses, arrest of cell division has been observed at shoot apical meristems with the type II necrosis phenotype under low temperature (Mizuno et al. 2011). Suppression of cell cycle-related gene expression inhibits plant growth and induces necrotic cell death in Arabidopsis (Wang and Liu 2006; Lin et al. 2007). Therefore, Net2 might be related to the cell cycle and cell division rather than disease resistance. To clarify the initial gene interaction resulting in type II necrosis, the molecular nature of Net2 should first be elucidated.

Table 1 Colinearity between Ae. tauschii and Brachypodium in the syntenic genomic regions around Net2
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Genetic association of the Net2-linked markers with type II necrosis

To determine the genetic association between developed markers and type II necrosis, 12 Net2-linked PCR markers including 11 SSRs and one CAPS were used to genotype the 83 Ae. tauschii accessions (Table S1). Of the 83 accessions, 22 showed type II necrosis in ABD triploid hybrids after crossing to Ldn, and 61 produced WT synthetic hexaploids. Most of the accessions showing type II necrosis belong to the TauL1b sublineage, dispersed in eastern habitats in the Ae. tauschii population (Matsuoka et al. 2013; Table S1). The restricted distribution suggests that the Net2 dominant allele might be derived from one mutation in the original net2 recessive allele. All 12 markers exhibited just two apparent alleles, the KU-2075 type and KU-2025 type. No obvious marker in which the genotype completely corresponded to the WT/type II necrosis phenotypes was found in the examined 12 markers (Table 2).

Table 2 Association between Net2-linked marker genotypes and WT versus type II necrosis phenotypes in 83 accessions of Ae. tauschii
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Out of the 12 Net2-linked markers, three SSRs, scaf8, scaf5-1 and scaf5-2 and one CAPS marker, scaf13, were significantly (P < 1E−3) associated with the WT/type II necrosis phenotypic difference (Fig. 4). Three associated markers, scaf5-1, scaf5-2 and scaf13, were the most closely linked to Net2, whereas the scaf8 marker was slightly further from the three markers and Net2. At the scaf5-2 marker with the lowest P value, six of the 61 WT accessions showed the KU-2025-type allele and seven of the 22 type II necrosis accessions carried the KU-2075-type allele. Therefore, the chromosomal region around scaffolds 38996 and 15574 were highly associated with the WT/type II necrosis phenotypic difference, meaning that Net2 should be localized in this region. These results suggest that the three PCR markers derived from the two scaffolds will be efficient for selection of Net2 non-carrier individuals from the Ae. tauschii population and its intraspecific progeny. The eastern habitat accessions of Ae. tauschii provide useful genes for breeding of the D genome of common wheat (Kajimura et al. 2011; Jones et al. 2013), and many of them contain Net2 (Mizuno et al. 2010). Thus, these markers could help the introgression of agriculturally important genes from Ae. tauschii to common wheat.

Fig. 4
figure 4

Trait associations with marker polymorphisms at the Net2 locus. The horizontal axis represents the position of each examined marker in the linkage map (N = 188). The −log(P value) values were calculated based on a mixed linear model in TASSEL ver. 4.0 software. The threshold for significance (P < 1E−3) is indicated by the dashed line

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Conclusion

Net2, the causal gene for low-temperature induced hybrid necrosis, inhibits efficient use of Ae. tauschii for the D-genome breeding of common wheat. In the present study, the Net2 locus, the D-genome causal gene for type II necrosis, was mapped within the 0.6-cM region on 2DS of synthetic hexaploid wheat. The most closely linked markers also showed highly significant association with the WT/type II necrosis phenotype in Ae. tauschii. Although local chromosomal rearrangements were observed in the region corresponding to Net2 in comparison of the barley/Brachypodium and Ae. tauschii genomes, our results provide useful information toward map-based cloning of Net2. On the other hand, the number of scaffolds assigned to the Net2 region was higher in the barley physical map than in the Ae. tauschii linkage map. In the draft genome sequence of Ae. tauschii, many scaffolds have failed to be positioned to chromosomes (Jia et al. 2013; Luo et al. 2013; Iehisa et al. 2012, 2014). In addition, the lowest P values in the association study for Net2 was much higher than the values for Iw2 on 2DS (Nishijima et al. 2014). These observations imply the possible existence of additional scaffolds that failed in assignments to the Net2 region in this study. Updated information for the diploid and polyploid wheat genome sequences using assemblies with NRGene’s DeNovoMAGIC software (https://wheat-urgi.versailles.inra.fr) could be useful for the additional detection of scaffolds around Net2. A recent report showed that bulked segregant analysis using next generation sequencing technology is effective in identifying molecular markers tightly linked to target loci (Borrill et al. 2015). Thus, RNA sequencing-based bulked segregant analysis could be efficient for discovery of additional unanchored Ae. tauschii scaffolds in the Net2 region.