Introduction

PCR-based molecular markers have been developed and mapped onto chromosomes in the Triticeae tribe (Harper and Cande 2000). Chromosomal alterations are informative not only for the mapping of genes from specific chromosomal regions but also for structural and functional analyses of the genetic relationships between homoeologous chromosomes of wheat and its relatives (Kojima et al. 2000). Thus far, chromosomal aberrations of wheat and its relatives, barley and rye, have been produced (Endo and Gill 1996; Joshi et al. 2011; Friebe et al. 2000; Tsuchida et al. 2008), and these unique resources have provided excellent tools for estimating the actual physical chromosomal locations of molecular markers and genes, thus facilitating the utility of genetic linkage maps for map-based gene cloning (Harper and Cande 2000; Qi et al. 2003).

The V genome of Dasypyrum villosum (L.) Candargy (Dv), also known as Haynaldia villosa (L.) Schur, an allogamous and annual wild diploid relative of common wheat (Gradzielewska 2006), contains genes conferring various disease resistances (Bizzarri et al. 2009; Xu et al. 2009; Qi et al. 2011; Chen et al. 1995; Yildirim et al. 2000; Li et al. 2002; Zhang et al. 2005a, b; Zhang et al. 2016a, b), increasing seed protein content (Montebove et al. 1987; De Pace et al. 2001; Zhang et al. 2014), increasing grains per spike (Zhang et al. 2015), and enhancing salt tolerance and zinc efficiency (Zhong and Dvorak 1995; Schlegel et al. 1998). D. villosum is therefore recognized as a potentially useful resource for enhancing wheat’s genetic diversity. Several D. villosum accessions have been successfully crossed with tetraploid or hexaploid wheat (reviewed by De Pace et al. 2011) with the goal of transferring the alien genes from the V genome into wheat, but researchers rarely progressed further than the induction of wheat-D. villosum translocation lines. Three sets of wheat-D. villosum disomic addition (DA) lines have been developed by Sears (1953), Chen and Liu (1986), and Lukaszewski (1988) using different D. villosum accessions. Based on the addition lines that Lukaszewski developed, Liu et al. (2011) further developed a set of compensating Triticum aestivum-D. villosum Robertsonian translocation lines in a Chinese Spring genetic background. In addition, the wheat-D. villosum lines developed by Chen and Liu (1986) were used to further produce the V chromosome alterations. Specific potential advantages associated with this V genome have been identified and include powdery mildew resistance genes Pm21 and Pm55, a photoperiod response gene Ppd-V1, a cereal nematode resistance gene CreV, and the wheat yellow mosaic virus resistance gene Wss1, all of which have been physically mapped into special bins associated with DNA markers (Chen et al. 2013; Zhao et al. 2013; Zhang et al. 2015, 2016a, b).

The integration of DNA markers into chromosome engineering strategies facilitates the recovery of small alien segments and the physical tagging of genes. Thus far, 103 expressed sequence tag (EST)-sequence-tagged site (STS) and eight SSR markers specific to V chromosomes have been screened, and some of them have been physically mapped onto the chromosome arms 1VS, 2VS, 4VS, 5VS, 6VS, and 6VL (Liu et al. 2004; Zhang et al. 2006; Cao et al. 2006; Wang et al. 2007; Cao et al. 2009a; Qi et al. 2011; Zhao et al. 2013; Chen et al. 2013; Zhang et al. 2014, 2015, 2016a, b). Integration of more physical DNA markers along with complete coverage of the V genome is expected to result in the construction of more precise maps of the D. villosum chromosomes. Therefore, we attempted to develop more V chromosome-specific molecular markers and structural aberrations that mostly focused on 1VL, 2VL, 3VS, 3VL, 4VL, 5VL, 7VS, and 7VL of D. villosum in the present study and to physically map the DNA markers into V chromosome bins using a series of wheat-D. villosum genetic stocks. These wheat-D. villosum translocations, together with V chromosome-specific molecular markers and a V genome physical map, would be useful for comparative genomics and alien gene tagging.

Materials and methods

Plant material and genetic stocks

T. aestivum cv. Chinese Spring (CS), T. durum cv. ZY1286 (AABB), D. villosum GP005 (VV, originally introduced from the Cambridge Botanical Garden, UK), T. durum cv. ZY1286-D. villosum (GP005) amphiploid (Tritpyrum, AABBVV), and other T. aestivum-D. villosum genetic stocks listed in Table 1 were employed to screen the molecular markers specific to V genome chromosomes. All materials were maintained at the Cytogenetic Institution, Nanjing Agricultural University, China (CINAU). Sets of compensating CS nullisomic tetrasomic (NT) lines (Sears 1966) and ditelosomic (DT) lines (Sears and Sears 1978) were used for assigning the homoeoloci of DNA markers to individual chromosomes on the A, B, and D genomes. The NT and DT lines were kindly provided by Prof B. S. Gill of Kansas State University, Kansas, USA.

Table 1 Wheat-D. villosum genetic stocks used in this study
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Wheat-D. villosum translocation development

The flowering spikes of the T. durum-D. villosum amphiploid (AABBVV) were irradiated by 60CO γ-rays at a dose of 1200 Rad to induce the V chromosomal aberrations. Mature, fresh pollen was harvested within 2 days after irradiation from irradiated spikes and was applied to stigmas of emasculated florets of CS plants. Subsequent progenies were backcrossed using CS as recurrent parent. The plants with moderately or fully fertile spikes in the BC2F1 and BC2F2 generations were screened using genomic in situ hybridization (GISH) and V chromosome-specific molecular markers.

Cytogenetic analysis

The protocol used for chromosome in situ hybridization was according to Chen et al. (1995). Genomic in situ hybridization (GISH) was performed using total genomic DNA of D. villosum and labeling with fluorescein-12-dUTP as a probe to detect the chromosomal aberrations. Fluorescence in situ hybridization (FISH) was performed using the oligonucleotide probes pSc119.2, pAs1 and (GAA)10 labeled with digoxigenin-11-dUTP (Roche Diagnostics GmbH, Germany) following the procedures described by Mukai et al. (1993) and Zhang et al. (2004). After hybridization, signals were examined with an Olympus BX60 epifluorescence microscope (Olympus Co., Tokyo, Japan). GISH/FISH images were captured with a SPOT Cooled Color Digital Camera (Diagnostic Instruments, Sterling Heights, MI, USA).

D. villosum-specific EST-STS marker development and analysis

Previously reported molecular markers specific to D. villosum, including 93 STS markers and seven SSR markers, were used to identify the novel translocations and to construct the physical maps (Table S1). To develop more DNA markers specific to chromosomes 3V, 4V, 5V, and 7V of D. villosum, 543 STS primers based on the wheat expressed sequence tags (ESTs) were mapped onto the group 3, 4, 5, and 7 chromosomes of CS, respectively, without paralogous sequences in different homologous groups (http://wheat.pw.usda.gov/cgi-bin/westsql/map_locus.cgi); the primers were designed using the software Primer 3 (http://frodo.wi.mit.edu). In addition, 582 intron-flanking primers based on the wheat Unigene sequences (http://www.ncbi.nlm.nih.gov/unigene) and compared with the Brachypodium genome sequences were designed using Conserved Primers 2.0 software (Frank et al. 2009). The wheat high-molecular-weight glutenin subunit (HMW-GS) gene-specific primer P1/P5 (Pang et al. 2009) and photoperiod response (Ppd) gene-specific primer XHvF11 (Turner et al. 2005) were also mapped onto V chromosomes. All the primers listed in Table S1 were synthesized by Invitrogen Company (Shanghai, China).

Genomic DNA was isolated from young leaves according to instructions accompanying the DNAsecure Plant Kit (Tiangen Biotech Co., Ltd., Beijing, China). The polymerase chain reaction (PCR) amplifications were conducted in a 25-μL reaction mixture containing 1× Taq DNA polymerase buffer, 0.8 mmol/L MgCl2, 0.8 mmol/L dNTPs, 0.5 μL (10 μM) of each primer, 2 U of DNA polymerase, and 30–50 ng of genomic DNA as a template. The samples were denaturated at 94 °C for 5 min and subjected to 33 cycles of 30 s of denaturation at 94 °C, 53–60 °C (depending on the specific primers) for 40 s and 2 min of elongation at 72 °C, with a final extension at 72 °C for 8 min. The PCR products were analyzed on 10% non-denaturing polyacrylamide gels with a 39:1 ratio of acrylamide/bisacrylamide.

Physical mapping of the specific molecular markers into V chromosome bins

For physical mapping of the V chromosome-specific molecular markers, 30 wheat-D. villosum translocations with different breakpoints and two deletions previously reported (Table 1) were used. Markers were first allocated to their respective arms using wheat-D. villosum whole-arm translocations or ditelosomic additions, and the markers were then assigned to arm bins according to whether or not the expected PCR-amplified band was present when the genomic DNA from a particular cytogenetic stock was used as a template. Tests with each marker were repeated twice.

Results

Identification of STS markers specific to V genome chromosomes

Of 1125 STS-containing wheat ESTs, 197 (17.5%) produced PCR amplicons using DNA from D. villosum, indicating a high rate of transferability between wheat and D. villosum. These markers were subsequently used to detect the polymorphisms among the D. villosum (GP005) and T. durum cv. ZY1286 plants, the T. durum cv. ZY1286-D. villosum (GP005) amphiploid, the CS and CS-D. villosum 1V to 7V addition lines, and the wheat-D. villosum ditelosomic addition and whole-arm translocation lines. Seventy-two primers produced stable and clear polymorphic bands that could be allocated to individual V chromosomes or to chromosome arms, and the remaining 125 exhibited no polymorphism between wheat and D. villosum (Table S1). Of the 72 markers, 14 were distributed on 1VL and one was on 2VL. Six markers were assigned on 3VS, and seven were on 3VL. One marker was on 4VS, and seven were assigned to 4VL. Seventeen 5V-specific markers were all located on chromosome arm of 5VL, and three 6V-specific markers were all on 6VS. Nine of the 16 7V-specific markers were on 7VS and the remaining seven were on 7VL.

Development of V chromosome structural aberrations

Twenty V chromosomal aberrations mostly found on 1VL, 2VL, 3VS, 3VL, 4VL, 5VL, 7VS, and 7VL were detected in the progenies following the irradiated pollen treatment. Four aberrations, 4V-4, 7V-1, 7V-2, and 7V-3, were centric; aberrations 2V-7, 3V-2, 4V-5, 4V-6, 6V-10, 7V-5, and 7V-7 were large-segment and the remaining aberrations, 1V-9, 1V-10, 1V-11, 2V-5, 2V-6, 4V-7, 5V-8, 7V-4, and 7V-6, were small-segment translocations (Fig. 1a). Based on the chromosome-specific molecular markers amplified in the structural aberrations and the V chromosome pSc119.2/pAs1 FISH patterns described by Zhang et al. (2013), the translocation breakpoint of 1V-9 on 1VS contained a terminal segment covering approximately 50% of the physical length of arm, and translocation segments of 1V-10 and 1V-11 constituted approximately 50 and 25% of the 1VL terminal segment, respectively. The breakpoint of 2V-5 retained approximately 30% of the terminal region of 2VS, and the 2V-6 translocation segment covered approximately 25% of the terminal region of 2VL. Line 2V-7 consisted of an entire 2VS arm and approximately 50% of the 2VL arm. The line 3V-2 was a T2BS-3VL.3VS(del) translocation confirmed by FISH of (GAA)10 (Fig. 1b) and pSc119.2 (Fig. 1c), and the breakpoints were located approximately 30% of 3VS and 70% of 3VL away from the centromere, respectively. The whole-arm translocation 4V-4 was a T5DL.4VL translocation based on the results of the pAs1 FISH patterns (Fig. 1d) and the 5D-marker analysis (Fig. S2). The breakpoints of 4V-5 and 4V-6 were located approximately 45% of 4VL and 78% of 4VS away from the centromere, respectively, and the 4V-7 translocated segment covered approximately 35% of the terminal region of 4VL. The translocation breakpoint of 5V-8 was located on 5VL covering approximately 30% of the terminal physical length of arm, and 6V-10 was located approximately 70% of 6VS away from the centromere. Of the seven 7V structural aberrations, the centric translocation 7V-1 was T7AL.7VS, confirmed by FISH results using pSc119.2 (Fig. 1e) and the molecular marker CINAU44 (Fig. S2). Another two centric fusion lines, 7V-2 and 7V-3, were T4BL.7VS and T4BS.7VL, respectively, according to the pSc119.2 patterns (Fig. 2f, g). The breakpoint of 7V-4 was located in approximately 20% of the 7VS terminal region, 7V-5 consisted of the entire 7VS arm and approximately 55% of the proximal portion of 7VL, 7V-6 carried approximately 35% of the terminal region of 7VL, and 7V-7 consisted of the entire 7VL arm and approximately 35% of the proximal portion of 7VS.

Fig. 1
figure 1

The GISH/FISH patterns of the wheat-D. villosum translocations. GISH used Dasypyrum villosum genomic DNA labeled with digoxigenin-11-dUTP as a probe and D. villosum chromatin fluoresced with a yellowish-green color. Dual-color FISH used pSc119.2, pAs1 or (GAA)10 labeled with digoxigenin-11-dUTP (red) and total genomic DNA of D. villosum labeled with fluorescein-12-dUTP (green) as probes, and chromosomes were counterstained with DAPI (blue). a GISH patterns of the 20 wheat-D. villosum translocated chromosomes developed in the present study. b Dual-color GISH/GAA-FISH patterns of 3V-2. Signal patterns present on the short arm of the translocated chromosome confirmed the assignment to 3VS. c Dual-color GISH/pSc119.2-FISH patterns of 3V-2. B-genome chromosomes colored by the pSc119.2 probe (red) are marked, and the patterns show the translocated chromosome in 3V-2 is T2BS-3VL.3VS(del). d Dual-color GISH/pAs1-FISH patterns of 4V-4. GISH used D. villosum DNA as a probe (green), and D-genome chromosomes colored by the pAs1 probe (red) are marked, indicating that the translocated chromosome in 4V-4 is T5DL.4VL. e, f, g Dual-color pSc119.2-FISH patterns of 7V-1, 7V-2, and 7V-3. The patterns suggest that the translocations in 7V-1, 7V-2, and 7V-3 are TW.7VS, T4BL.7VS, and T4BS.7VL, respectively (color figure online)

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Fig. 2
figure 2

The rough physical map of Dasypyrum villosum. 174 V genome-specific molecular markers are integrated into 37 bins covering 14 V chromosome arms. The alien genes (red) are also mapped into the specific bins that refer to the previous reports of Zhang et al. (2014, 2015, 2016a, 2016b), Zhao et al. (2013), and Chen et al. (2013)

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Distribution of molecular marker loci in V chromosome bins

The DNA markers, including the 72 EST-STS primer pairs developed in this study and the 102 previously reported markers, were allocated to V chromosome bins (Fig. 2) using 50 translocations and two deletions (Table 1). Physical maps of 1VS, 2VS, 5VS, and 6VL are the same as those of earlier reports (Zhang et al. 2014, 2015, 2016a, b). However, when comparing the map with the 4VS physical map reported by Zhao et al. (2013), a novel STS marker, CINAU202, was physically positioned onto 4VS FL0.78-1.00, which is associated with the wheat yellow mosaic virus resistance gene, Wss1. In addition, three new STS markers, 6EST802, 6EST928, and 6EST970, were mapped onto 6VS FL0.45-0.58, which is associated with Pm21 when compared with the 6VS physical map constructed by Chen et al. (2013). The remaining physical maps of 1VL, 2VL, 3VS, 3VL, 4VL, 5VL, 7VS, and 7VL were constructed in the present study.

Of the 174 V chromosome-specific markers, 114 have been allocated to special bins of wheat chromosomes (Qi et al. 2004), and the remaining 60 STS markers were mapped onto the CS chromosomes using NT and DT genetic stocks in the present study. Among the 60 STS markers, 56 markers provided homoeoloci that can be assigned onto the chromosome arms of CS. Two markers, CINAU17 and CINAU18 (mapping to 6 VS), only had single locus in D. villosum and none in CS. Another two markers, 6EST245 and 6EST267 (also mapping to 6VS), produced a single band in CS, but their homoeoloci lacked polymorphism. Most EST-STS homoeoloci were allocated to homoeologous chromosomes of CS and D. villosum, with the exception of the 4VL-specific markers CINAU206, CINAU207, CINAU208, and CINAU209, which mapped onto group 7 chromosomes of CS.

Discussion

Several chromosome engineering approaches have been used to produce alien introgressions from D. villosum into common wheat. These include pairing and recombination of alien chromosomes with their wheat homoeologues (Qi et al. 2011; Liu et al. 2011; Li et al. 2011; Zhao et al. 2013), random chromosome breakage by irradiation or gametocidal chromosomes (Chen et al. 2002; Chen et al. 2008; Bie et al. 2007; Cao et al. 2009b), and even chromosome aberrations occurring in tissue culture (Li et al. 2000; Li et al. 2005). Introgressions via chromosome fragmentation irradiation are effective; however, the chromosome breakpoints are random; therefore, these types of introgressions usually are unsuitable for agriculture but still are useful genetic stocks to physically map if they are stable in the wheat background. The 20 wheat-D. villosum translocations developed in the present study may be stable in the wheat background because these plants have homozygous translocated chromosomes with moderate or full spike fertility after two generations of backcrossing using CS as a recurrent parent. Of the four novel whole-arm translocations, T5DL.4VL, T4BL.7VS, and T4BS.7VL were not compensating, while translocation T7AL.7VS was compensating, which may be useful for wheat breeding. Yildirim et al. (1998) mapped the eyespot (Pseudocercosporella herpotrichoides) resistance gene onto chromosome 4VL, and Liu et al. (1989) located the water-soluble endosperm protein gene Wsp-1 onto chromosome 7V. Therefore, these new whole-arm translocation lines will be promising donors for the production of useful small-fragment translocations to aid in using alien genes for wheat breeding.

An abundance of EST sequence polymorphisms exist between wheat and the homoeologous chromosomes belonging to closely related species (Zhang et al. 2005b). Deletion-based physical mapping using chromosome structural changes enabling the physical allocation of EST-PCR markers has been used in wheat (Qi et al. 2004), rye (Lukaszewski et al. 2004) and barley (Harper and Cande 2000). Using 52V chromosome alterations, we integrated 174 markers, including 167 EST-STS and seven SSR markers, into 37 bins of the V chromosomes and provided the first rough physical map of D. villosum in the present study (Fig. 2). The homoeoloci of most markers were distributed on the chromosome arms of the V genome and the A, B, and D genomes, revealing a high degree of homoeology between the genomes of D. villosum and wheat (Table S1). However, homoeoloci of wheat group 7 markers CINAU206, CINAU207, CINAU208, and CINAU209 were specific to 4VL of D. villosum, suggesting that D. villosum chromosome 4V showed some affinity to both wheat homoeologous groups 4 and 7. The physical mapping of EST-STS markers in V chromosome bins will help in further elucidating the relationships between the wheat and D. villosum genomes, making them a valuable source for comparative genomic research.

Physical mapping of DNA markers in V chromosome bins also facilitates the linkage between those EST sequences and alien genes. On the terminal end of chromosome arm 1VS, there are genes at complex loci coding for high-molecular-weight glutenins (Glu-V1) and prolamins (Gli-V1) and low-molecular-weight (LMW) polymeric prolamin proteins (Glu-V3) detected by SDS-PAGE (Zhang et al. 2014). However, the homoeoloci of the HMW-GS gene-specific marker P1/P5 were present on 1VL, 1BL, and 1DL (Fig. S1), indicating that an HMW-GS pseudogene orthologous to the Glu-A1, Glu-B1, and Glu-D1 loci of hexaploid wheat may be present on 1VL. Three wheat group 6 EST-STS markers, 6EST802, 6EST928, and 6EST970, were added to the 6VS FL0.45-0.58, where a powdery mildew resistance gene Pm21 has been physically mapped (Chen et al. 2013). These markers provide the reference sequences for the assignment of alien candidate genes through the microcollinearity between these regions of D. villosum and the homoeologous chromosome sequences of wheat.