BACKGROUND: Haynaldia villosa (L.) Schur (syn. Dasypyrum villosum L. Candargy, 2n = 14, genome VV) is the tertiary gene pool of wheat, and thus a potential resource of genes for wheat improvement. Among other, wheat yellow mosaic (WYM) resistance gene Wss1 and a take-all resistance gene were identified on the short arm of chromosome 4 V (4VS) of H. villosa. We had obtained introgressions on 4VS chromosome arm, with the objective of utilizing the target genes. However, monitoring these introgressions has been a daunting task because of inadequate knowledge as to H.villosa genome, as reflected by the lack of specific markers. RESULTS: This study aims to develop 4VS-specific markers by combination of chromosome sorting and next-generation sequencing. The short arm of chromosome 4VS of H.villosa was flow-sorted using a FACSVantage SE flow cytometer and sorter, and then sequenced by Illumina sequencing. The sequence of H. villosa 4VS was assembled by the software Hecate, and then was compared with the sequence assemblies of wheat chromosome arms 4AL, 4BS and 4DS and Ae. tauschii 4DS, with the objectives of identifying exon-exon junctions and localizing introns on chromosome 4VS of H. villosa. The intron length polymorphisms suitable for designing H. villosa primers were evaluated with criteria. Consequently, we designed a total of 359 intron targeting (IT) markers, among which 232 (64.62%) markers were specific for tracing the 4VS chromatin in the wheat background. CONCLUSION: The combination of chromosome sorting and next-generation sequencing to develop specific IT markers for 4VS of H. villosa has high success rate and specificity, thus being applicable for the development of chromosome-specific markers for alien chromatin in wheat breeding.

Institute of Experimental Botany Centre of the Haná Region for Biotechnological and Agricultural Research Šlechtitelů 31 CZ 783671 Olomouc Czech Republic

State Key Laboratory of Crop Genetics and Germplasm Enhancement Cytogenetics Institute Nanjing Agricultural University JCIC MCP Nanjing 210095 China

Zobrazit více v PubMed

Colmer TD, Flowers TJ, Munns R. Use of wild relatives to improve salt tolerance in wheat. J Exp Bot. 2006;57(5):1059–1078. doi: 10.1093/jxb/erj124. PubMed DOI

Friebe B, Jiang J, Raupp WJ, McIntosh RA, Gill BS. Characterization of wheat-alien translocations conferring resistance to diseases and pests: Current status. Euphytica. 1996;91(1):59–87.3. doi: 10.1007/BF00035277. DOI

Triebe B, Mukai Y, Dhaliwal HS, Martin TJ, Gill BS. Identification of alien chromatin specifying resistance to wheat streak mosaic and greenbug in wheat germ plasm by C-banding and insitu hybridization. Theor Appl Genet. 1991;81(3):381–89. PubMed

Lukaszewski AJ, Lapinski B, Rybka K. Limitations of in situ hybridization with total genomic DNA in routine screening for alien introgressions in wheat. Cytogenet Genome Res. 2005;109(1–3):373–77. PubMed

Kimura M. Rare variant alleles in the light of the neutral theory. Mol Biol Evol. 1983;1(1):84–93. PubMed

Poczai P, Varga I, Laos M, Cseh A, Bell N, Valkonen JP, et al. Advances in plant gene-targeted and functional markers: a review. Plant Methods. 2013;9(1):6.7. doi: 10.1186/1746-4811-9-6. PubMed DOI PMC

Ishikawa G, Yonemaru J, Saito M, Nakamura T. PCR-based landmark unique gene (PLUG) markers effectively assign homoeologous wheat genes to A, B and D genomes. BMC genomics. 2007;8:135. PubMed PMC

Ishikawa G, Nakamura T, Ashida T, Saito M, Nasuda S, Endo TR, et al. Localization of anchor loci representing five hundred annotated rice genes to wheat chromosomes using PLUG markers. Theor Appl Genet. 2009;118(3):499–514. doi: 10.1007/s00122-008-0916-y. PubMed DOI

Poczai P, Cernak I, Gorji AM, Nagy S, Taller J, Polgar Z. Development of intron targeting (IT) markers for potato and cross-species amplification in Solanum nigrum (Solanaceae) Am J Bot. 2010;97(12):e142–e145. doi: 10.3732/ajb.1000360. PubMed DOI

Li J, Endo TR, Saito M, Ishikawa G, Nakamura T, Nasuda S. Homoeologous relationship of rye chromosome arms as detected with wheat PLUG markers. Chromosoma. 2013;122(6):555–564. doi: 10.1007/s00412-013-0428-7. PubMed DOI

Hu LJ, Liu C, Zeng ZX, Li GR, Song XJ, Yang ZJ. Genomic rearrangement between wheat and Thinopyrum elongatum revealed by mapped functional molecular markers. Genes Genom. 2012;34(1):67–75. doi: 10.1007/s13258-011-0153-7. DOI

Zhan HX, Li GR, Chang ZJ, Zhang XJ, Li X, Yang ZJ. Molecular identification of a new wheat-Thinopyrum intermedium cryptic translocation line for resistance to powdery mildew. IJBBB. 2013;4(3):376–378.

Dolezel J, Vrana J, Safar J, Bartos J, Kubalakova M, Simkova H. Chromosomes in the flow to simplify genome analysis. Funct Integr Genomic. 2012;12(3):397–416. doi: 10.1007/s10142-012-0293-0. PubMed DOI PMC

Tiwari VK, Wang SC, Sehgal S, Vrana J, Friebe B, Kubalakova M, et al. SNP Discovery for mapping alien introgressions in wheat. BMC Genomics. 2014;15. PubMed PMC

Molnar I, Kubalakova M, Simkova H, Cseh A, Molnar-Lang M, Dolezel J. Chromosome isolation by flow sorting in Aegilops umbellulata and Ae. comosa and their allotetraploid hybrids Ae. biuncialis and Ae. geniculata. Plos One. 2011;6(11):e27708. PubMed PMC

Molnar I, Vrana J, Farkas A, Kubalakova M, Cseh A, Molnar-Lang M, et al. Flow sorting of C-genome chromosomes from wild relatives of wheat Aegilops markgrafii, Ae. triuncialis and Ae. cylindrica, and their molecular organization. Ann Bot. 2015;116(2):189–200. doi: 10.1093/aob/mcv073. PubMed DOI PMC

Molnar I, Kubalakova M, Simkova H, Farkas A, Cseh A, Megyeri M, et al. Flow cytometric chromosome sorting from diploid progenitors of bread wheat, T. urartu, Ae. speltoides and Ae. tauschii. Theor Appl Genet. 2014;127(5):1091–1104. doi: 10.1007/s00122-014-2282-2. PubMed DOI

Molnar I, Simkova H, Leverington-Waite M, Goram R, Cseh A, Vrana J, et al. Syntenic relationships between the U and M genomes of Aegilops, wheat and the model species Brachypodium and rice as revealed by COS markers. Plos One. 2013;8(8):e70844. PubMed PMC

Gradzielewska A. The genus Dasypyrum - Part 2. Dasypyrum villosum - a wild species used in wheat improvement. Euphytica. 2006;152(3):441–454. doi: 10.1007/s10681-006-9245-x. DOI

Zhang QP, Li Q, Wang X, Wang HY, Lang SP, Wang YN, et al. Development and characterization of a Triticum aestivum-Haynaldia villosa translocation line T4VS · 4DL conferring resistance to wheat spindle streak mosaic virus. Euphytica. 2005;145(3):317–320. doi: 10.1007/s10681-005-1743-8. DOI

Zhao RH, Wang HY, Xiao J, Bie T, Cheng SH, Jia Q, et al. Induction of 4VS chromosome recombinants using the CS ph1b mutant and mapping of the wheat yellow mosaic virus resistance gene from Haynaldia villosa. Theor Appl Genet. 2013;126(12):2921–2930. doi: 10.1007/s00122-013-2181-y. PubMed DOI

Vrana J, Kubalakova M, Simkova H, Cihalikova J, Lysak MA, Dolezel J. Flow sorting of mitotic chromosomes in common wheat (Triticum aestivum L.) Genetics. 2000;156(4):2033–2041. PubMed PMC

Kubalakova M, Valarik M, Bartos J, Vrana J, Cihalikova J, Molnar-Lang M, et al. Analysis and sorting of rye (Secale cereale L.) chromosomes using flow cytometry. Genome. 2003;46(5):893–905. doi: 10.1139/g03-054. PubMed DOI

Kubalakova M, Vrana J, Cihalikova J, Simkova H, Dolezel J. Flow karyotyping and chromosome sorting in bread wheat (Triticum aestivum L.) Theor Appl Genet. 2002;104(8):1362–1372. doi: 10.1007/s00122-002-0888-2. PubMed DOI

Simkova H, Svensson JT, Condamine P, Hribova E, Suchankova P, Bhat PR, et al. Coupling amplified DNA from flow-sorted chromosomes to high-density SNP mapping in barley. BMC Genomics. 2008;9. PubMed PMC

Mayer KFX, Rogers J, Dolezel J, Pozniak C, Eversole K, Feuillet C, et al. A chromosome-based draft sequence of the hexaploid bread wheat (Triticum aestivum) genome. Science. 2014; 345(6194):1251788. PubMed

Jia JZ, Zhao SC, Kong XY, Li YR, Zhao GY, He WM, et al. Aegilops tauschii draft genome sequence reveals a gene repertoire for wheat adaptation. Nature. 2013;496(7443):91–95. doi: 10.1038/nature12028. PubMed DOI

Sharp PJ, Chao S, Desai S, Gale MD. The isolation, characterization and application in the Triticeae of a set of wheat RFLP probes identifying each homoeologous chromosome arm. Theor Appl Genet. 1989;78(3):342–348. doi: 10.1007/BF00265294. PubMed DOI

Devos KM, Atkinson MD, Chinoy CN, Liu CJ, Gale MD. RFLP-based genetic map of the homoeologous group 3 chromosomes of wheat and rye. Theor Appl Genet. 1992;83(8):931–939. doi: 10.1007/BF00232953. PubMed DOI

Bassam BJ, Gresshoff PM. Silver staining DNA in polyacrylamide gels. Nat Protoc. 2007;2(11):2649–2654. doi: 10.1038/nprot.2007.330. PubMed DOI

Mullan DJ, Platteter A, Teakle NL, Appels R, Colmer TD, Anderson JM, et al. EST-derived SSR markers from defined regions of the wheat genome to identify Lophopyrum elongatum specific loci. Genome. 2005;48(5):811–822. doi: 10.1139/g05-040. PubMed DOI

Zhang W, Gao AL, Zhou B, Chen PD. Screening and applying wheat microsatellite markers to trace individual Haynaldia villosa chromosomes. Acta Genet Sin. 2006;33(3):236–243. doi: 10.1016/S0379-4172(06)60046-2. PubMed DOI

Liu SB, Tang ZH, You MS, Li BY, Song JM, Liu GT. Development and application of a genome specific PCR marker for Haynaldia villosa. Acta Genet Sin. 2003;30(4):350–356. PubMed

Qi LL, Friebe B, Zhang P, Gill BS. Homoeologous recombination, chromosome engineering and crop improvement. Chromosome Res. 2007;15(1):3–19. doi: 10.1007/s10577-006-1108-8. PubMed DOI

Zhao RH, Wang HY, Jia Q, Xiao J, Yuan CX, Zhang YJ, et al. Development of EST-PCR markers for the chromosome 4 V of Haynaldia villosa and their application in Identification of 4 V chromosome structural aberrants. J Integr Agr. 2014;13(2):282–289. doi: 10.1016/S2095-3119(13)60359-7. DOI

Dolezel J, Vrana J, Capal P, Kubalakova M, Buresova V, Simkova H. Advances in plant chromosome genomics. Biotechnol Adv. 2014;32(1):122–136. doi: 10.1016/j.biotechadv.2013.12.011. PubMed DOI

Miftahudin RK, Ma XF, Mahmoud AA, Layton J, Milla MAR, et al. Analysis of expressed sequence tag loci on wheat chromosome group 4. Genetics. 2004;168(2):651–663. doi: 10.1534/genetics.104.034827. PubMed DOI PMC

Flow Cytometric Analysis and Sorting of Plant Chromosomes

Long-range assembly of sequences helps to unravel the genome structure and small variation of the wheat-Haynaldia villosa translocated chromosome 6VS.6AL

Chromosome analysis and sorting

Sequencing flow-sorted short arm of Haynaldia villosa chromosome 4V provides insights into its molecular structure and virtual gene order