A novel PCR-based marker for identifying Ns chromosomes in wheat-Psathyrostachys huashanica Keng derivative lines

Psathyrostachys huashanica Keng is an endangered species that is endemic to China, which provides an important gene pool for wheat improvement. We developed a quick and reliable PCR-based diagnostic assay to accurately and efficiently detect P. huashanica DNA sequences from introgression lines, which was based on a species-specific marker derived from genomic DNA. The 900-bp PCR-amplified band used as a P. huashanica-specific RAPD marker was tested with 21 different plant species and was converted into a sequence-characterized amplified region (SCAR) marker by cloning and sequencing the selected fragments (pHs11). This SCAR marker, which was designated as RHS-23, could clearly distinguish the presence of P. huashanica DNA repetitive sequences in wheat-P. huashanica derivative lines. The specif icity of the marker was validated using 21 different plant species and a complete set of wheatP. huashanica disomic addition lines (1Ns – 7Ns, 2n = 44 = 22II). This specific sequence targeted the Ns genome of P. huashanica and it was present in all the seven P. huashanica chromosomes. Therefore, this SCAR marker is specific for P. huashanica chromosomes and may be used in the identification of alien repetitive sequences in large gene pools. This diagnostic PCR assay for screening the target genetic material may play a key role in marker-assisted selective breeding programs. Additional key words: addition lines; marker-assisted selection; repetitive sequences; RAPD; SCAR. * Corresponding author: cxh2089@126.com; wj2105@163.com Received: 24-03-13. Accepted: 15-10-13. This work has one Supplementary Figure that does not appear in the printed article but that accompanies the paper online. Abbreviations used: AFLP (amplified fragment length polymorphism); EST (expressed sequence tag); GISH (genomic in situ hybridization); MAS (marker-assisted selection); RAPD (random amplified polymorphic DNA); SCAR (sequence-characterized amplified region); SSR (simple sequence repeat); STS (sequence tagged site). Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA) Spanish Journal of Agricultural Research 2013 11(4): 1094-1100 Available online at www.inia.es/sjar ISSN: 1695-971-X http://dx.doi.org/10.5424/sjar/2013114-4245 eISSN: 2171-9292


Introduction
The genome of Psathyrostachys huashanica Keng (2n = 2x = 14, NsNs) contains many desirable characteristics, such as resistance to biotic and abiotic stresses, which make it suitable for wheat improvement (Kang et al., 2009).Several P. huashanica genes have already been introgressed successfully into the wheat (Triticum aestivum L.) genome (Zhao et al., 2010;Du et al., 2013a,b).In the 1990s, we generated (Chen et al., 1991(Chen et al., , 1996) ) the F 1 hybrid H881 (2n = 28, ABDNs) and a derived heptaploid H8911 (2n = 49, AABBDDNs) of common wheat cv.7182 and P. huashanica (GenBank Acc.No. 0503383) via embryo culture, two backcrosses, and selfing.As a result, we generated a large and complex range of wheat-P.huashanica offspring that carried different chromosome number or structure variants, i.e., chromosome additions, deletions, and rearrangements.However, a major problem was the selection of recombinant plants derived from the large interbred population.After several years of screening and identification, a complete set of wheat-P.huashanica disomic addition lines (1Ns -7Ns, 2n = 44 = 22II) was developed.This was a repetitive and difficult work.Although cytogenetic techniques proved to be very useful tools for alien identification, they had limitations because of the small population number, the complexity of the experimental techniques, and envi-A novel PCR-based marker for identifying Ns chromosomes in wheat-Psathyrostachys huashanica Keng derivative lines ronmental effects.Therefore, an alternative approach was needed urgently to facilitate the rapid and accurate identification of alien chromatin from incorporated lines, which is essential before they can be utilized.
The choice of a DNA marker depends on the specific scale and the aims of identification.Some molecular markers techniques have already been applied successfully to identifying P. huashanica chromatin, i.e., EST-SSR and EST-STS (Du et al., 2013a,b), as well as for mapping P. huashanica resistance genes, i.e., SSR (Li et al., 2012) and AFLP (Cao et al., 2008).In particular, random amplified polymorphic DNA (RAPD) markers are simple and cost-effective, while they require no previous sequence information and can be conducted using a small sample as the template DNA (Masojé et al., 2001).Several RAPD markers have been developed for the effective screening of wheat lines containing P. huashanica chromatin (Kang et al., 2008), However, the short primers and low annealing temperatures mean RAPD markers are extremely sensitive to the reaction conditions, none of which are suitable for large-scale selection because of their low reproducibility and specificity (Goulão et al., 2001;Quian et al., 2001).To overcome these problems, longer primers have been developed from RAPD fragment sequences to generate sequence-characterized amplified region (SCAR) markers, thereby allowing the amplification of a target DNA fragment by PCR.
SCAR markers are generally simpler to use and more suited to high-throughput applications than RAPD markers (Chowdhury et al., 2001;Bautista et al., 2002).It has been used to develop molecular markers for genes that confer resistance to scald (Genger et al., 2003), barley yellow dwarf (Zhang et al., 2004), leaf rust (Gupta et al., 2006), dwarf bunt (Gao et al., 2010), and common bunt (Zhang et al., 2012), as well as for specific genomes including Secale africanum Stapf.(Jia et al., 2009), Agropyron cristatum (L.) Gaertn.(Wu et al., 2010), Thinopyrum elongatum (Host) D.R. Dewey (Xu et al., 2012), and Thinopyrum intermedium (Host) Barkworth and Dewey (Hu et al., 2012).Thus, SCAR markers are the most practical method for screening large number of samples in a time-efficient and labor-saving manner because they are accurate, easy to use, and cost efficient (Kasai et al., 2000).In this study, we isolated a new repetitive DNA sequence from P. huashanica by RAPD analysis and converted it into a stable Ns-chromosome-specific SCAR marker based on a comparative analysis.The SCAR marker was used to authenticate large-scale populations of wheat-P.huashanica derivatives and to detect Ns chromosomes in wheat-P.huashanica introgression lines.This diagnostic marker was designed to facilitate the tracing of P. huashanica genome sequences in a wheat background, which will improve the efficiency of targeting genetic material; it is suitable for high-throughput diagnosis.

Plant materials
We used genome samples of 21 different species (Table 1) for RAPD polymorphic analysis, including common wheat cv.7182 (AABBDD, 2n = 6x = 42) and P. huashanica Keng (NsNs, 2n = 2x = 14).These species were provided partly by the Center for Crop Germplasm Resources Research (CGRR) at the Institute of Crop Science, CAAS, Beijing, China.We also used a complete set of wheat-P.huashanica disomic addition lines (1Ns -7Ns, 2n = 44=22II) that carried different P. huashanica chromosomes, i.e., each line included all 42 wheat chromosomes and a pair of alien P. huashanica chromosomes, as shown in Table 2.These specimens were deposited at the Shaanxi Key Laboratory of Genetic Engineering for Plant Breeding, College of Agronomy, Northwest A&F University, Shaanxi, China.

Cloning and sequencing of the RAPD product
The putative marker OPL05 amplified (≈900 bp) by the random primer (ACGCAGGCAC, annealing temperature 34°C) was excised from 1% agarose gel, then purified and extracted using a TaKaRa Agarose Gel DNA Purification Kit (Takara, Japan).The selected DNA fragments were ligated into the pMD19-T vector and transformed into Escherichia coli DH5α-competent cells by heat shock transformation.Positive colonies were determined by blue/white screening.The white colonies were picked from LB-ampicillin plates and the recombinant DNA was extracted using a plasmid kit for each overnight cultured colony.Both ends of each DNA insert were sequenced by Sangon Biotech (Shanghai, China).The DNA sequence was submitted to GenBank (Acc.No. HR614210).Homology searches were performed in the GenBank nonredundant database using BLASTn and BLASTx via the National Center for Biotechnology Information (NCBI) website.

Detection of P. huashanica genome sequences in addition lines
The corresponding SCAR marker primer pair of P. huashanica was designed based on the sequence of the RAPD marker.This was evaluated as a tool to test the validity of the molecular marker in a complete set of disomic addition lines (1Ns -7Ns, 2n = 44 = 22 II) and their parents, common wheat cv.7182 and P. huashanica.The PCR reactions, electrophoresis, and imaging were performed as reported previously.

Identification of a RAPD marker for P. huashanica
Two-hundred primers with arbitrary sequences were screened and 30 primers produced distinct, reproducible amplification profiles with all of the screened DNA samples (data not shown).Of these, the primer OPL05 consistently amplified a single, intense band of approximately 900-bp that was specific to P. huashanica, but absent from the other 20 species (Fig. 1).This band (pHs11) was selected as a putative P. huashanicaspecific marker and used to develop the SCAR marker.

Sequence analysis
The cloned DNA fragments of interest were sequenced and the length of pHs11 was found to be 900-bp (Fig. 2; Suppl.Fig. 1[pdf]).A BLAST search showed that the pHs11 sequence did not have homology with any sequence deposited in public databases.

Validation of the SCAR primer RHS23
A pair of specific SCAR primers, RHS23, was designed based on the nucleotide sequence of pHs11 to test its specificity and efficiency.The primer pair was designed to produce the full-length RAPD fragment.In some cases, the SCAR was shorter than the initial RAPD fragments because the reverse primer was in-Psathyrostachys huashanica SCAR marker 1097  ternal to the RAPD fragments so annealing temperature was optimized.The PCR primer pair was further validated using 21 different plant species.The PCR amplification product with primer pair RHS23 had high specificity and it clearly distinguished the expected 900-bp specific band present in the P. huashanica genome, which was absent from the ABD, A, AB, AG, AAG, C, C, D, E, H, K, M, PPP, B'C', AtG, R, S, SY, SYW, and St genomes.This conf irmed that the RHS23 marker was a novel, Ns-genome-specific DNA marker for P. huashanica, and the species specificity of the marker.

SCAR marker for identifying P. huashanica genome sequences
The practical applicability of the marker was tested by detecting the presence of P. huashanica in wheat-P.huashanica addition lines.The primer pair designed to produce the genome-specific SCAR marker was used to amplify the genomic DNA of a complete set of wheat-P.huashanica disomic addition lines (1Ns -7Ns, 2n = 44 = 22II) and their parents, common wheat cv.7182 and P. huashanica, to determine the chromosomal locations of the SCAR marker sequence.This showed that RHS23 produced a very intense marker at 900-bp (see Fig. 3), which was present in P. huashanica and in all the seven wheat-P.huashanica addition lines (1Ns to 7Ns), whereas it was absent from the maternal parent (the common wheat cv.7182).Being the marker present on all the seven P. huashanica chromosomes, it could be used as a marker-assisted selection (MAS) tool for tracking P. huashanica chromatin.

Discussion
Although genetic diversity in wheat was reduced during its domestication, some diversity can be restored by introgression from its progenitors or from more distant wild relatives (Dubcovsky & Dvorak, 2007).The introgression of genetic material from wild relatives or distantly related species into wheat germplasm is a classical and effective approach for broadening the genetic basis of this crop (Hernández et al., 1999).In previous studies, we successfully transferred the Ns genome of P. huashanica into wheat using wide hybridization methods and we generated a complete set of wheat-P.huashanica disomic addition lines (1Ns -7Ns, 2n = 44 = 22II).However, a rapid and accurate method for identifying P. huashanica chromosomes and chro-   fragments in derived lines is required before they can be utilized effectively (Wu et al., 2010).Molecular markers, particularly genome-specific markers, are useful for identifying the genome constitution of unknown species and they also provide efficient tools for confirming the transfer of target alien genes to wheat (Wang et al., 2010 ).
In the present study, we developed a SCAR marker known as RHS23 by directly sequencing RAPD products and it was verified by wide screening of a complete set of wheat-P.huashanica disomic addition lines (1Ns -7Ns, 2n = 44 = 22II) that carried different P. huashanica chromosomes.The result demonstrated that this novel SCAR marker is suitable for distinguishing P. huashanica genome in a wheat background.This is the first report of the development of a SCAR marker for the detection of P. huashanica in wheat-P.huashanica derivative lines.
Marker-assisted selection (MAS) has great potential for increasing the efficiency of the breeding process by increasing the number of traits that can be selected in one population and by improving the precision of genotype selection (Wang et al., 2012).A SCAR marker with high reproducibility and reliability can be used easily in a MAS program and this is an attractive technique for indentifying exogenous chromatin from introgression lines.To ensure that the Ns-chromosomespecific marker delivered consistent performance with homologous groups, the complete set of wheat-P.huashanica disomic addition lines with different Ns chromosomes were used to test the SCAR marker.All homologous groups of P. huashanica chromosomes could be located in the addition lines, which show that our primer pair can amplify the Ns chromosome-specific sequence in all the different homologous groups.The development of a SCAR marker from specif ic repetitive DNA bands provides a practical tool for determining introgression, which supports MAS in wheat.Our results suggest that this SCAR marker could be used to discriminate derivative lines from wheat-P.huashanica addition lines and for detecting alien P. huashanica DNA fragments in wheat.Further diagnostic markers for P. huashanica chromatin are also required for the fine mapping of genes and for future genomic studies.
In summary, this PCR-based assay can be rapidly applied to monitor the presence of P. huashanica in a wheat background during the early stages of development.Thus, breeders could obtain early information about the presence of P. huashanica chromatin, which may contribute to making decisions about the retention or elimination of lines.This rapid and efficient method has high specificity and reproducibility so it could be used for screening the targeted genetic material.

Figure 1 .
Figure 1.RAPD patterns of 21 different plant species (a) and (b) generated using the primer OPL05 900 .The arrow indicates the species-specific diagnostic band of P. huashanica.The full species names are listed inTable 1.

Table 1 .
List of 21 different plant species used to screen the P. huashanica-specific RAPD marker

Table 2 .
The genetic constitution of the complete set of whe-