SUN Hao-jie, SONG Jing-jing, XlAO Jin, XU Tao, WEl Xing, YUAN Chun-xia, CAO Ai-zhong, XlNG Liping, WANG Hai-yan, WANG Xiu-e

State Key Laboratory of Crop Genetics and Germplasm Enhancement, Ministry of Science and Technology/Cytogenetics Institute,Nanjing Agricultural University/Jiangsu Collaborative Innovation Center for Modern Crop Production (JCIC-MCP), Nanjing 210095,P.R.China

Abstract Expressed sequence tags-derived polymerase chain reaction (EST-PCR) molecular markers specific for alien chromosomes can be used to not only monitor the introgressed alien chromatin in wheat background, but also provide the evidence of the syntenic relationship between homoeologous chromosomes. In the present study, in order to develop high density and evenly distributed molecular markers specific for chromosome 6VL of Dasypyrum villosum, 297 primer pairs were designed based on the expressed sequence tags (EST) sequences, which were previously mapped in different bins of the long arms of wheat homoeologous 6AL, 6BL, and 6DL. By using the Triticum aestivum, D. villosum, T. durum-D. villosum amphiploid,and T. aestivum-D. villosum alien chromosome lines involving chromosome 6V, it was found that 32 (10.77%) primers could amplify specific bands for chromosome 6V, and 31 could be allocated to chromosome arm 6VL. These 6VL specific markers provided efficient tools for the characterization of structural variation involving the chromosome 6VL in common wheat background as well as for the selection of useful genes located on 6VL in breeding programs.

Keywords: Dasypyrum villosum, molecular marker, common wheat, alien chromosome line

1. lntroduction

Bread wheat (Triticum aestivumL.) is a major staple food crop in the world. The amount of genetic variation of cultivated wheat is relatively low under modern agricultural systems. Wild relatives of wheat provide a vast source of genetic variation that can be transferred into wheat through introgression and subsequently used in breeding of superior adapted wheat varieties (Rileyet al. 1968; Blancoet al. 1991;Bankset al. 1995; Crastaet al. 2000; Chenet al. 2005). These species exhibit huge diversity in phenotype and adaptation to a wide range of environments and favorable traits such as resistances and tolerances to biotic and abiotic stresses.Integrated techniques have been established for alien gene transfer, including the production of small alien introgressions by the induction of chromosome structural changes and homoeologous chromosome pairing. However, it is still difficult to identify the introgressed chromatin efficiently in a large breeding population.

Dasypyrum villosum(L.) (Candargy, Dv; 2n=14, VV)Schur (syn.Haynaldia villosa(L.) Schur), is the tertiary gene pool of wheat, carrying several resistance genes to wheat diseases, including powdery mildew, eyespot, takeall, and wheat spindle streak mosaic virus (WSSMV) (Chen and Liu 1982; Blancoet al. 1987; Chenet al. 1995; Zhanget al. 2005; Gradzielewska 2006; Liet al. 2011). Both the stem rust resistance geneSr52and cereal cyst nematode resistance geneCreVwere all located in the long arm of 6V chromosome (Qiet al. 2011; Zhanget al. 2012). The chromosome 6V also carries loci having potential positive effects on salt tolerance (Zhong and Dvorak 1995).Considering the agronomic importance of 6VL chromosome,recently, many small segmental translocations involving 6VL chromosome ofD.villosumhave been inducedviairradiation (Bieet al. 2007) and Chinese Spring (CS)ph1bsystem. However, identifying alien chromatin in wheat background is difficult because of the shortage of genetic and molecular mapping information ofD.villosum.Therefore, the development of high density and evenly distributed molecular markers on chromosome 6VL ofD.villosumis highly significant.

Expressed sequence tags-derived polymerase chain reaction (EST-PCR) markers, deriving from expressed gene sequences, have higher conservatism but variation in some extent among species or between genera. ESTPCR is a novel and rich-in-source functional marker system, which could be generated simply by direct primer design based on EST sequences. Unlike other PCR-based markers, such as random amplification polymorphism DNA(RAPD), amplified fragment length polymorphism (AFLP),and simple sequence repeat (SSR) that often target noncoding regions, EST-PCR markers directly target coding regions, and may directly represent the phenotype-related genes, making EST-PCR markers more convenient for screening desirable breeding materials (Schubertet al.2001; Hagraset al. 2005; Luet al. 2006; Ayala-Navarreteet al. 2007; Shen and Ohm 2007; Ayala-Navarreteet al.2009). Furthermore, EST-PCR markers are more likely transportable across taxonomic boundaries (Rowlandet al. 2003; Sargentet al. 2007; Wanget al. 2010). This transportability is especially valuable for development of molecular markers specific for certain chromosome of some species which whole genome sequences are not yet available.

In this study, high density and evenly-distributed ESTPCR based markers specific for the chromosome arm 6VL were developed by exploiting the information of the mapped wheat ESTs. These newly developed markers will facilitate the identification of various structural variations involving 6VL and the selection of 6VL-carrying target genes in wheat breeding programs.

2. Materials and methods

2.1. Plant materials

The plant materials used in this study includedT.durum-D.villosumamphiploid (AABBVV), a complete set ofT.aestivum cv. Chinese Spring (CS)-D.villosumdisomic addition lines (DA1V-7V),T.aestivum-D.villosumdisomic substitution line (DS6V(6A)),T.aestivum-D.villosumtranslocation lines T6AS·6VL and T6AL·6VS.These cytogenetic materials were all developed by the Cytogenetics Institute, Nanjing Agricultural University(CINAU, hereafter). TheD.villosum(accession no. 91C43)was introduced from Cambridge Botanical Garden, UK.To identify and locate markers specific for wheat group 6 chromosomes andD.villosumchromosome 6V, the CS nulli/tetrasomic lines N6AT6B, N6BT6A, and N6DT6A were kindly provided by Dr. Bikram S. Gill (Wheat Genetics and Genomics Resource Center, Kansas State University), were also used in this study.

2.2. DNA extraction

Genomic DNA was extracted from 2 g fresh leaves at 3-leaf stage with SDS-phenol-chloroform method described by Devos and Gale (1992) and purified for further elimination of RNA, amylase, and other unwanted components. The concentration of DNA was calculated by comparison with microplate reader (M200, TECan, Switzerland). Each DNA sample was finally stored at –20°C until use.

2.3. Primer design

ESTs (200 bp) with no paralogous gene on other homologous groups in each bin of chromosomes 6A, 6B,and 6D were selected. According to the published wheat EST sequences cytogenetically mapped to specific deletion bins, a total of 297 EST-PCR primer pairs were designed using the online software Primer 3 V0.4.0 (http://frodo.wi.mit.edu/primer3/).

2.4. PCR amplification and products visualization

PCR amplification was conducted in a 10-μL reaction containing 40 ng genomic DNA, 2 μmol L–1each of the primer pairs, 2.5 mmol L–1each dNTPs, 2.5 mmol L–1MgCl2, 1×PCR buffer (10 mmol L–1Tris-HCl, pH 8.5, 50 mmol L–1KCl),and 0.5 UTaqDNA polymerase with a PTC-200 thermal cycler (Bio-Rad, Hercules, CA, USA). The samples were denaturated at 94°C for 3 min and subjected to 32 cycles of the following: 30 s of denaturation at 94°C, 45 s at 50–58°C,according to the different primers, and a 70-s elongation at 72°C. A final cycle with an extension of 10 min at 72°C was applied to complete the reactions. The PCR products were separated in 8% non-denaturing poly-acrylamide gels(Acr:Bis=19:1 or 39:1) and visualized with silver staining(Bassam and Gresshoff 2007).

3. Results

3.1. Amplification patterns with different EST-PCR primer pairs

A total of 297 primer pairs were mined based on the EST sequences located in different bins of chromosomes 6A, 6B,and 6D. The genomic DNA of CS,D.villosum,T.durum-D.villosumamphiploid,T.durum, andT.aestivum-D.villosumdisomic substitution line DS6V(6A) were used as templates for amplification. The results indicated that 31 primer pairs had no amplification products both in wheat andD.villosumand the majority of the primers (216,72.73%) resulted in successful amplicon in both wheat andD.villosum. In addition, 50 primer pairs only amplified products from the CS template. Among the 216 primer pairs,only 32 primer pairs (10.77%) amplified specific polymorphic fragments which were common inD.villosum,T.durum-D.villosumamphiploid, and DS6V(6A), indicating they were specific for chromosome 6V.

3.2. Marker specificity and arm allocation on chromosome 6V

To further map these specific markers to specific arms of 6V,the 32 EST-PCR markers were amplified using the genomic DNA of wheat-D.villosumaddition/substitution lines, the T6AS·6VL and T6AL·6VS translocation lines as templates.When a primer pair generated a distinct PCR product shown as polymorphic band inD.villosum,T.durum-D.villosumamphiploid, DA6V, DS6V(6A), and T6AS·6VL(or T6AL·6VS) translocation line, but not inT.durumand CS, it was consider as a specific marker for chromosome arm 6VL (or 6VS). By this, we assigned 31 specific markers to 6VL, and 1 specific marker to 6VS (Fig. 1 and Table 1).

3.3. Marker assisted selection of chromosome 6VL

The background of the translocation line T6AS·6VL used in this experiment was homozygousph1bph1b. In order to induce homoeologous meiotic recombination between the chromosome arm 6VL and wheat homoeologous group 6 chromosomes, the homozygous translocation line T6AS·6VL was crossed to the homozygous CSph1bmutant.The F1plants were self-crossed and the derived 103 F2progenies were screened by genomicin situhybridization(GISH) and PCR using 7 6VL specific EST-PCR markers.

Fig. 1 Electrophoresis patterns of 6V-specific expressed sequence tags-derived polymerase chain reaction (EST-PCR markers.Each pattern represents markers as follows: 6VL-specific markers 6EST387 (A), 6VL-specific markers 6EST431 (B), 6VL-specific markers 6EST541 (C), 6VL-specific markers 6EST387 (D), 6VL-specific markers 6EST431 (E), and 6VL-specific markers 6EST541(F). Arrows show 6V-specific fragments. M, DNA ladder; lanes 1–16, 1, Dasypyrum villosum; 2, Triticum durum-D. villosum amphiploid; 3, T. durum; 4, Chinese Spring (CS); 5–11, DA 1V–7V; 12, T6AL·6VS translocation line; 13, T6AS·6VL translocation line; 14, N6AT6B; 15, N6BT6A; 16, N6DT6A.

Table 1 Molecular markers specific to 6VL chromosome of Dasypyrum villosum based on PCR1)

We found that in most cases, 6VL (48.54%, 50 plants)was detected by at least 1 of the 7 specific markers, however,none of the specific markers was present in 53 plants(51.46%), indicating 6VL was not transmitted. Combined with results from molecular-assisted selection (MAS)(Table 2) and GISH (Fig. 2), 4 different groups of plants were identified in the F2population. Group 1: all the 6VL specific markers could not amplify the specific fragments, and GISH could not detect any alien chromatin from 6VL, indicating that the 6VL might fail to transmit (Fig. 2-A). Group 2: all the 6VL specific markers could amplify their specific fragments,indicating only the long arm of 6VL was present in these lines. GISH confirmed that in these plants 1 or a pair of 6VL chromosomes was present (Fig. 2-B and C). Group 3: only 4 or 3 of the 6VL specific markers could amplify the specific bands, and GISH further identified they were small segment terminal translocations (SSTTs) involving 6VL(Fig. 2-D and E). Group 4: only 3 of the 6VL specific markers could amplify the specific bands, and GISH identified they were large segment intercalary translocations involving 6VL(Fig. 2-F). In total, 4 translocation lines were identified. The consistency of the results from marker analysis and GISH further proved that these chromosome arm specific markers can be used in the tracing of alien chromosome fragments and in the determination of chromosome constitutions of large population derived from wide hybridization.

4. Discussion

4.1. The utilization of EST-PCR markers in tracing the alien chromosome in wheat background

Wild relatives consist of vast valuable gene pool for wheat improvement. Alien introgressions have been developed,however, the selection of alien chromatin in wheat background by phenotype analysis, biochemical markers such as proteins and isozymes, and cytogenetics analysis has been low efficient and low throughput. Phenotype analysis is sometimes of low efficiency, especially where environmental effects are large as in many diseases. The disadvantage of biochemical markers is the relatively low level of polymorphism at loci identified by isozymes and proteins and these are now largely replaced by genome wide molecular markers. Chromosome banding makes chromosome identification fast, reliable, and economical.However, chromosome banding is uninformative if the alien chromosome segments are deficient in diagnosticbands. Banding polymorphism in different wheat genotypes sometimes also confuses the identification of the alien chromosome segments.In situhybridization (ISH) has been used to directly localize genes or DNA sequences on chromosomes in cytological preparations. However,GISH has the limitation of low throughput. DNA markers can detect small amounts of alien chromatin that cannot be recognized cytogenetically, are highly efficient thus are highly recommended for the identification of the introgressed fragments. But the numbers of molecular markers are very low in particular for the tertiary gene pool species of wheat due to the shortage of genetic and molecular mapping information of the wild species. The identification of alien chromatin in the wheat background was based on only limited number of markers, useful smaller introgressions may have escaped detection. This hampers the identification of lines with small fragment introgression carrying the traits of interest.

Table 2 Six types of chromosome structural variations observed in the F2 population identified by developed expressed sequence tags-derived polymerase chain reaction (EST-PCR) markers1)

4.2. Comparative analysis of 6VL with wheat homoeologous group 6 chromosomes

Fig. 2 Genomic in situ hybridization (GISH) pattern of chromosomes at mitotic metaphase on root tip cells of plants from F2 progeny of the translocation line T6AS·6VL×CS ph1b.Total genomic DNA of Dasypyrum villosum was labeled with fluorescein-12-dUTP, visualized with green fluorescence; wheat chromosomes were counterstained with propidium iodide and fluoresced red. A, no alien chromosome. B, heterozygous 6VL translocation line. C, homozygous 6VL translocation line. D, a small segment terminal translocation (SSTT) line. E, an SSTT line. F, large segment intercalary translocations (LSIT) line.

In recent years, excessive accumulation of molecular markers and DNA sequences have made the comparative genomics study among grass family possible. The colinearity of genetic markers is well conserved among different grass genomes (Gale and Devos 1998). It was found by comparative mapping that there were extensive colinearity in different species and this colinearity is increased between closely related species (Feuillet and Keller 2002; Feuilletet al. 2003; Bossoliniet al. 2007).Large amount of molecular markers have been allocated to chromosomes or chromosome fragments belong to the same homoeologous groups. Among them, many markers such as EST-based ones, were found to be polymorphic between the alien chromatin and its corresponding wheat chromatin.EST-PCR has been used to identify alien chromosomes and chromosome segments (Qiet al. 2007,2008; Liuet al.2011; Niuet al. 2011). In the present study, we designed 297 wheat EST derived primer pairs according to the EST sequences allocated in different bins of chromosomes 6AL,6BL, and 6DL (Qiet al. 2004). Most of EST-PCR molecular markers could be used to amplify in both wheat andD.villosum, of which, 31 were mapped to the chromosome 6VL. By RFLP analysis, Qiet al. (1999) found that theD.villosumchromosome 6V was homoeologous to wheat group 6 confirmed their homoeologous relationship using wheat microsatellite markers. Zhaoet al. (2014) confirmed their homoeologous relationship using wheat EST-PCR markers. This confirmed thatD.villosumandT.aestivumhad close phylogenetic relationship and suggested that their collinear regions were highly conserved and reinforced the homoeologous relationship betweenD.villosumand wheat chromosomes (Qiet al. 1999; Zhanget al. 2006). However,only one marker showed complex syntenic relationships between the wheat andD.villosumgenomes. Marker 6EST325 located on the wheat 6L arm was assigned to the 6VS arm. From this syntenic relationship of the marker,we deduced chromosomal rearrangements that most likely occurred during the karyotype evolution ofD.villosum.

5. Conclusion

The 31 specific EST-PCR markers for 6VL were developed in the present study. The colinearity of the EST-PCR markers is well conserved between 6VL and wheat homoeologous group 6 chromosomes. These markers will be helpful for the accurate characterization of structural variation involving 6VL and for the selection of alien useful genes in breeding programs.

Acknowledgements

This research was supported by the National Key Research and Development Program of China (2016YFD0102001),the National Natural Science Foundation of China(31571653, 31771782, 31201204), the Technology Support Program of Jiangsu Province, China (BE2013439), the Fundamental Research Funds for the Central Universities,China (KYZ201403), the Jiangsu Agricultural Science and Technology Innovation Fund, China (CX151001),the Program of Introducing Talents of Discipline to Universities, China (B08025), the Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions, China (PAPD), and the Six Talent Peaks Project in Jiangsu Province, China.