Development of Thinopyrum ponticum-specific molecular markers and FISH probes based on SLAF-seq technology
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Based on SLAF-seq, 67 Thinopyrum ponticum-specific markers and eight Th. ponticum-specific FISH probes were developed, and these markers and probes could be used for detection of alien chromatin in a wheat background.
Decaploid Thinopyrum ponticum (2n = 10x = 70) is a valuable gene reservoir for wheat improvement. Identification of Th. ponticum introgression would facilitate its transfer into diverse wheat genetic backgrounds and its practical utilization in wheat improvement. Based on specific-locus-amplified fragment sequencing (SLAF-seq) technology, 67 new Th. ponticum-specific molecular markers and eight Th. ponticum-specific fluorescence in situ hybridization (FISH) probes have been developed from a tiny wheat—Th. ponticum translocation line. These newly developed molecular markers allowed the detection of Th. ponticum DNA in a variety of materials specifically and steadily at high throughput. According to the hybridization signal pattern, the eight Th. ponticum-specific probes could be divided into two groups. The first group including five dispersed repetitive sequence probes could identify Th. ponticum chromatin more sensitively and accurately than genomic in situ hybridization (GISH). Whereas the second group having three tandem repetitive sequence probes enabled the discrimination of Th. ponticum chromosomes together with another clone pAs1 in wheat–Th. ponticum partial amphiploid Xiaoyan 68.
KeywordsFISH GISH Molecular markers SLAF-seq Thinopyrum ponticum Triticum aestivum
Genome in situ hybridization
Fluorescence in situ hybridization
Wheat cv. Chinese spring
Specific-locus-amplified fragment sequencing
Decaploid tall wheatgrass [Thinopyrum ponticum (Podp.) Barkworth & D. R. Dewey, 2n = 10x = 70, syn. Agropyron elongatum (Host) P. Beauv., Elytrigia pontica (Podp.) Holub, Lophopyrum ponticum (Podp.) Á Löve] is an important forage crop (Li and Wang 2009) and a valuable gene donor for wheat (Triticum aestivum, 2n = 6x = 42) improvement because of its biotic and abiotic stress tolerance and high crossability with various Triticum species (Shannon 1978; Sharma et al. 1989). To transfer its advantageous traits into wheat, distant hybridization has been performed between wheat and Th. ponticum (Yang et al. 2006; Jauhar et al. 2009; Lin et al. 2009). Rapid and accurate characterization of tall wheatgrass chromatin would clearly improve the efficiency of applying elite genes in wheat breeding. Nowadays, cytological methods and molecular markers are widely used to distinguish and follow alien chromosomes or chromosome segments during crossing and selection (Schwarzacher et al. 1992; Kruppa et al. 2016; Sibikeev et al. 2017).
A conventional cytological method, genomic in situ hybridization (GISH), can be used to recognize related species chromosomes in a wheat background (Han et al. 2003; Sepsi et al. 2008; Wang et al. 2011). Li et al. (2003) characterized the chromosome constitution of the wheat—Th. ponticum partial amphiploid line and its derivatives by GISH. Zheng et al. (2006b) identified eight T. aestivum–Th. ponticum translocations and estimated the size of Th. ponticum chromosome segments by GISH. However, GISH is rather time-consuming and its hybridization specificity is influenced by the ratio of probe DNA to blocking DNA. Another cytological method, fluorescence in situ hybridization (FISH) with labeled cloned DNA containing a species-specific sequence, can also paint alien chromosome segments (Zheng et al. 2006a; Linc et al. 2012). Kong et al. (1999) developed a C-genome-specific repetitive sequence pAeca212 from Aegilops caudata so as to separate Ae. caudate chromosome and translocation segments from wheat chromosome. FISH localized the dispersed repetitive DNA sequence pHvNAU62 to six of seven Haynaldia villosa chromosome pairs in telomeric or sub-telomeric regions (Li et al. 1995). Using the probes developed from Thinopyrum elongatum, chromosome painting of Thinopyrum chromosomes was successfully performed (Bournival et al. 1994). However, up to now, only a few probes developed from Thinopyrum have been reported (Bai et al. 2002).
With regard to molecular markers, developing species-specific markers for the identification of alien chromosomes is of great significance in breeding programs. In recent years, several Th. ponticum species-specific molecular markers have been developed by random-amplified polymorphic (RAPD), expressed sequence tag (EST), cleaved amplification polymorphism sequence (CAPS), sequence-tagged sites (STS), and simple sequence repeats (SSR) (Liu and Kolmer 1998; Yu et al. 2001; Santra et al. 2006; Li et al. 2007; Wang et al. 2010, 2015). Nevertheless, the existing markers are extremely inadequate to meet the needs in Th. ponticum chromatin detection. Fortunately, since next-generation sequencing (NGS) technology became available, it is now possible to achieve dense marker coverage without a reference genome (Davey et al. 2011). As one method using NGS technology, specific-locus-amplified fragment sequencing (SLAF-seq) provides a high-throughput, high-accuracy, and low-cost tool for developing an ample number of markers of Triticale species (Chen et al. 2013a, b; Li et al. 2016). In fact, 518 specific fragments on the 7E chromosome of Th. elongatum were successfully obtained by SLAF-seq, and depended on 135 randomly selected fragments, 89 specific molecular markers of Th. elongatum were developed, with efficiency of up to 65.9% (Chen et al. 2013a). However, to the best of our knowledge, the development of specific molecular markers of decaploid Th. ponticum based on SLAF-seq technology has yet to be reported.
In this study, SLAF-seq technology was applied to a small wheat—Th. ponticum translocation line, EA, which carried a TTh-4DS·4DL translocation. By comparison of genome-wide SLAFs between wheat and Thinopyrum species, we selected all of the EA-specific sequences for the development of new PCR-based Th. ponticum-specific molecular markers and FISH probes, which make it more convenient to discriminate Th. ponticum chromatin from wheat chromosome.
Materials and methods
Plant materials and DNA extraction
Chinese spring (CS) (2n = 42), wheat cultivated variety Xiaoyan 81 (2n = 42), Shaan 229 (2n = 42), wheat–Th. ponticum partial amphiploid Xiaoyan 68 (2n = 56), Th. ponticum (2n = 70), wheat–Th. ponticum translocation line EA (2n = 42) (Xiaoyan 81/4/Xiaoyan 81/3/Xiaoyan 81//Shaan 229/Xiaoyan 68), the 61 individuals from F2 population of wheat cv. Xiaoyan 60 and EA (2n = 42) were maintained by our laboratory. Seeds of Th. elongatum (2n = 14, EeEe), Thinopyrum bessarabicum (2n = 14, EbEb) and Pseudoroegneria strigosa (2n = 14, StSt) were obtained from the US National Plant Germplasm System (NPGS). Seeds of Haynaldia villosa (2n = 14, VV) and Hordeum vulgare (2n = 14, HH) were kindly provided by Prof. Yonghong Zhou (Sichuan Agricultural University), Agropyrum cristatum (2n = 14, PP) by Dr. Jinpeng Zhang (Chinese Academy of Agricultural Sciences), Secale cereale (2n = 14, RR) by Prof. Diaoguo An (Institute of Genetics and Developmental Biology, Chinese Academy of Sciences). Total genomic DNA was extracted from the fresh young leaves, using a cetyl-trimethyl-ammonium bromide (CTAB) method (Gill et al. 1991), and adjusted to a final concentration of 150 ng/µl.
Specific-locus amplified fragment sequencing
Genomic DNA samples of EA, Th. ponticum, and CS were subjected to SLAF-seq with some modifications (Sun et al. 2013), performed by the Beijing Biomarker Technologies Corporation (Beijing, China). All paired-end reads (160 bp per read) generated from SLAF-seq raw reads were clustered according to sequence similarity. Sequences with over 90% identity were grouped into one SLAF locus, as described (Sun et al. 2013).
Sequence comparison and Th. ponticum-specific fragment acquisition
To get the Th. ponticum-specific SLAFs, we filtered the SLAFs by a specificity comparison with some modifications (Chen et al. 2013a). First, the good quality sequences of EA were compared with the CS sequences on http://www.ncbi.nlm.nih.gov and http://www.cerealsdb.uk.net, and then, they were compared with the CS sequences acquired by SLAF-seq in this study. Finally, the comparison was made between the sequences of EA and Th. ponticum, and the specific sequences of Th. ponticum were acquired.
Development, verification, and detection of Th. ponticum-specific molecular markers
According to these Th. ponticum-specific sequences, PCR primers (forward and reverse primer) were designed for the amplification of EA, Th. ponticum, Xiaoyan 68, Xiaoyan 81, Shaan 229, and CS. All PCR primers were synthesized by Shanghai Sangon Biotechnology Co., Ltd. (Shanghai, China). The amplified products were examined by 2% agarose electrophoresis. Those markers amplified the specific bands in EA, Xiaoyan 68 and Th. ponticum, but not in Xiaoyan 81, Shaan 229 and CS were identified as the Th. ponticum-specific molecular markers. Then, the stability, repeatability, and specificity of these markers were detected in the F2 individuals of Xiaoyan 60 and EA and in several wheat-related species.
The PCR system final volume was set to 20 µl containing 1 µl of template DNA (150 ng/µl), 17 µl of 1.1 × Taq Mix (Tsingke Biological Technology, Beijing, China), and 1 µl of each primer (10 µM). The PCR reactions were performed as follows: denaturation at 94 °C for 3 min, followed by 30 cycles of 94 °C for 30 s, appropriate anneal temperature (48–60 °C) for 30 s, 72 °C for 30 s, and a final extension at 72 °C for 5 min.
Seeds were germinated on moistened filter paper in petri dishes at 23 °C for 48 h. Actively growing roots of 1–2 cm length were removed from seedlings and pretreated in N2O at 10 atm pressure for 2 h to accumulate metaphases, then fixed in 90% acetic acid and stored in 70% v/v ethanol. Chromosome spread preparation was carried out following the procedures of Zheng et al. (2014). Good slides were stored at 4 °C until use.
Fluorescence in situ hybridization
For cytological characterization, FISH was performed using published protocols with some modification (Zheng et al. 2014). All Th. ponticum-specific sequences were amplified by PCR from Th. ponticum genomic DNA and labelled with fluorescein-12-dUTP (green) (Life Technologies Corporation, Eugene, OR, USA) or Texas-red-5-dCTP (red) (PerkinElmer Life Sciences, Boston, MA, USA) served as probes. After hybridization, the slides were washed in 2 × SSC and mounted in Vectashield mounting medium with 4, 6-diamindino-2-phenyl-indole (DAPI) (Vector Laboratories, Youngstown, OH, USA) (Zheng et al. 2014). All cells with good hybridization signals were captured by Olympus DP80 CCD camera attached to an Olympus BX53 and analyzed using program CellSens Standard 1.12 (Olympus Corporation, Tokyo, Japan).
Sequential genomic and multicolor fluorescence in situ hybridization (sequential GISH and mc-FISH)
Procedures for sequential GISH and mc-FISH were according to Zheng et al. (2015) with some modification. Briefly, Th. ponticum genomic DNA was labelled with Texas-red-5-dCTP (red) and served as probe, and wheat DNA was used as a block. The ratio of probe to block was 1:250. Mc-FISH was carried out after GISH analysis using two probes, pAs1 labeled with Texas-red-5-dCTP (red) and Th. ponticum-specific sequence labelled with fluorescein-12-dUTP (green). Two probes were mixed 1:1 before the hybridization. After the hybridization, the slides were counterstained with DAPI. The image acquisition and analysis were described as those in FISH process.
Molecular marker development for Th. ponticum
A total of 96,565, 121,256, and 108,782 effective SLAFs were obtained for CS, Th. ponticum, and EA by high-throughput sequencing, respectively. After comparison with the CS fragments and sequences in the online databases at http://www.ncbi.nlm.nih.gov and http://www.cerealsdb.Uk.net, 2035 EA fragments with homologies less than 50% of CS were acquired. By sequence alignment with the Th. ponticum fragments obtained by SLAF-seq, 171 EA fragments whose homology with Th. ponticum was greater than 50% were selected, which were identified as the Th. ponticum-specific fragments.
Utility verification of Th. ponticum-specific markers
FISH probes exploitation of Th. ponticum
SLAF sequences of the Th. ponticum-specific probes
SLAF-seq sequences (5′–3′) of the special probe
These two Th. ponticum-specific probes, pThp3.93 and pThp3.96, were cloned and sequenced (Table S3), and then compared with common wheat sequences in http://www.ncbi.nlm.nih.gov, http://www.cerealsdb.uk.net and https://wheat.pw.usda.gov. The lengths of the probes pThp3.93 and pThp3.96 were 469 and 591-bp, with GC content 49.68 and 54.31%, respectively. BLAST search revealed that the 16–455 bp nucleotide sequence of pThp3.93 had 83% sequence identity to an LTR retrotransposon of Aegilops tauschii (CM008370.1). BLAST search revealed that the 3–412 bp nucleotide sequence of pThp3.96 had 86% sequence identity to an intron of gene (LOC109786650) of Ae. tauschii (NW_017930880.1) (Table S4).
When mixing pThp3.96 with another repetitive DNA clone pAs1 as mc-FISH probes (Fig. 4c), 12 Th. ponticum chromosomes and two wheat—Th. ponticum translocation chromosomes could be clearly distinguished from each other (Fig. 4f, g). Chromosome 1 was shown to be the longest chromosome with an intense pThp3.96 signal on its short arm. Neither chromosome 2 nor chromosome 3 had any pThp3.96 site; however, chromosome 2 was a metacentric chromosome, while chromosome 3 was a sub-metacentric one. Chromosome 2 had strong pAs1 bands on its short arm, while chromosome 3 had these on its long arm. Chromosomes 4 and 6 carried pThp3.96 signals at both ends. The pAs1 signals were more intense on chromosome 4 than on chromosome 6. Chromosome 5 had pThp3.96 sites distributed on its long arm. Chromosome 7 was clearly the most easily identifiable chromosome, because it showed pThp3.96 band on the Th. ponticum chromosome segment and distinctive pAs1 signals on the wheat chromosome segment.
Detection and application of Th. ponticum-specific FISH probes
Compared with the GISH pattern of total Th. ponticum genomic DNA as a probe (Fig. 5a), the FISH pattern of pThp3.93 in the translocation line EA did more clearly show the two alien chromosome segments without unspecific hybridization on the wheat chromatin (Fig. 5b). Moreover, the pThp3.93 signals extended to all chromosomes in decaploid Th. ponticum (Fig. 6a), as well as in the three progenitor diploid species (Fig. 7a−c), with stronger hybridization towards the distal ends on both short and long arms.
As for pThp3.96, the hybridization band was strong on the alien chromosome segments in EA (Fig. 5c). In decaploid Th. ponticum, there were 22 chromosomes having pThp3.96 signals on both chromosome ends, 29 chromosomes having strong or faint pThp3.96 signals on their short or long arm ends, and 19 chromosomes having no pThp3.96 bands at all (Fig. 6b). In three progenitor diploid species, there was one pair of chromosomes in each of Th. elongatum and Th. bessarabicum showing hybridization signals at the terminals of their short arms (Fig. 7d, e). Significantly, the hybridization signals in the two arms differed in Th. elongatum: the signal was intense at one site, while it was weak at the other. In contrast, in Th. bessarabicum, both signals were faint. However, no hybridization site of pTh3.96 was found in the chromosomes of Ps. strigosa (Fig. 7f).
SLAF-seq, an efficient next-generation sequencing (NGS)-based method and high-resolution strategy of large-scale genotyping, has been applied successfully in the development of large numbers of molecular markers in various species (Jia et al. 2016; Luo et al. 2016). Compared with the conventional methods, such as RFLP and AFLP, SLAF-seq technology possesses a remarkable advantage in developing large numbers of highly accurate molecular markers with less sequencing. In a previous study (Chen et al. 2013a), 89 specific molecular markers of diploid Th. elongatum were developed, which notably were from the whole length of chromosome 7E. In our study, 67 specific molecular markers were developed from a tiny Th. ponticum chromosome segment. The obtained findings proved that the SLAF-seq technology is efficient for developing alien-specific molecular markers in a wheat background. The markers were also shown to be suitable for tracking chromosome fragments in advanced backcross derivatives from the distance hybridization between wheat and its wild relatives.
FISH analysis with repetitive DNA probes, which can identify alien introgression in a wheat genetic background, would facilitate gene transfer from Thinopyrum to wheat (Gonzalez-Garcia et al. 2011). For instance, Yao et al. (2016) isolated seven Th. ponticum genome-specific repetitive sequences, which could discriminate chromosomes of Th. ponticum and Th. intermedium from wheat chromosomes without wheat DNA block. In the present study, five Th. ponticum-specific sequence probes, namely, pThp2.31, pThp2.77, pThp2.83, pThp3.93, and pThp5.84, produced a dispersed pattern on all Th. ponticum chromosomes and chromosome segments. Although there was no need to add blocking DNA, nonspecific hybridization was not obvious on the wheat chromosomes. Therefore, FISH analysis with dispersed repetitive DNA probes is more convenient, efficient and accurate than GISH. Three tandem repetitive sequence, probes, namely pThp3.81, pThp3.96, and pThp5.121, showed hybridization sites at the majority of Th. ponticum chromosome arms, and thus, they offered a good probability of distinguishing some different Th. ponticum chromosomes with other repetitive DNA clones, such as pAs1, in this study. Although, owing to the limited number of tandem repetitive sequences, it is difficult to achieve the accurate identification of every chromosome of Th. ponticum, FISH with repetitive DNA has become a useful tool in detecting alien chromatin.
The complex genomic composition of Th. ponticum has been investigated for decades and various hypotheses have been proposed (Dewey 1984; Muramatsu 1990; Wang et al. 1991). Zhang et al. (1996a, b) proposed that the formula of the Th. ponticum genome was EeEbExStSt, whereas Chen et al. (1998) concluded that the haploid genomic constitution of Th. ponticum was JJJJsJs. The previous studies also indicated that Th. elongatum (EeEe), Th. bessarabicum (EbEb), and Pseudoroegneria (StSt) were probably the donor species of Th. ponticum (Muramatsu 1990; Wang et al. 1991). However, our specificity analysis of newly developed Th. ponticum-specific markers showed that the amplification frequencies of the markers in these three putative diploid progenitors are far higher than in other wheat-related species. In addition, all chromosomes of Th. elongatum, Th. bessarabicum, and Ps. strigosa were dyed by the Th. ponticum-specific probe pThp3.93. Moreover, only two chromosomes had pThp3.96 signals on the short arm end in Th. elongatum and Th. bessarabicum. In contrast, in decaploid Th. ponticum, there were 51 chromosomes showing pThp3.96 signals. Among these, 22 chromosomes had pThp3.96 signals on both ends. The above FISH analysis results showed that the distribution and number of repetitive DNA sequence had altered a lot. The most likely explanation for these finding is that diploid progenitor DNA sequences had changed as a result of polyploidization (Mcintyre et al. 1988). Our results provide further evidence that Th. ponticum originated from diploid Th. elongatum, Th. bessarabicum, and Pseudoroegneria and these three genomes were modified during hybridization and polyploidization.
In conclusion, based on SLAF-seq, 67 Th. ponticum-specific markers and eight Th. ponticum-specific FISH probes were successfully developed. These markers and probes could be used not only for sensitive and accurate detection of alien chromosomes and chromosome segments in a wheat background, but also for chromosome fingerprinting in partial amphiploid Xiaoyan 68 as well as for understanding the genomic composition of Th. ponticum.
Author contribution statement
QZ, ZSL, and LQL conceived and designed the research. LQL, QLL, and WT performed the research. LQL and QZ wrote the manuscript. WT, BL, HWL, and YWL participated in the preparation of the reagents and materials in this study. All authors read and approved the manuscript.
We would like to thank Prof. Yonghong Zhou from Sichuan Agricultural University, Prof. Diaoguo An from Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, and Dr. Jinpeng Zhang from Chinese Academy of Agricultural Sciences, for kindly providing seeds of some of the accessions used in this study. This project was supported by the National Key Research and Development Program of China (2016YFD0102000) and the Strategic Priority Research Program of the Chinese Academy of Sciences (No. XDA08030105).
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