中间偃麦草第6同源群特异STS标记开发
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摘要
中间偃麦草(2n=6x=42,JJJ~sJ~sStSt)是小麦遗传改良的重要野生资源之一,因其基因组尚未完成测序,造成目前已报道的特异分子标记数量较少,不能满足小麦生产和研究领域内对杂交材料中外源片段或外源基因鉴定的需求。本研究利用中间偃麦草GBS芯片探针数据组装了5877409条Contig序列,筛选后获得5452条与小麦基因组相似性低于80%的、具有染色体位置信息的非冗余序列,据此开发2019个序列标签位点(sequence-tagged site,STS)分子标记,在中间偃麦草第1至第7同源群中的分布依次为250、215、323、253、323、253和402;利用5株中间偃麦草和5份小麦农家种的DNA从253个第6同源群(G6)标记中进一步筛选出160个中间偃麦草特异标记,其中"+/-"型特异标记共有53个,分布在G6-Chr1(32个)、G6-Chr2(13个)和G6-Chr3(8个)染色体上;接着利用拟鹅观草(2n=2x=14,StSt)、百萨偃麦草(2n=2x=14,J~bJ~b)、二倍体长穗偃麦草(2n=2x=14,J~eJ~e)以及中国春-二倍体长穗偃麦草6J~e代换系推断G6-Chr2为6St染色体;最后利用6St上开发的13个"+/-"型标记,从分子水平对小麦-偃麦草代换系F881(6St/6D)中的外源6St染色体进行了验证。研究结果将为中间偃麦草染色体或染色体片段的鉴定提供较为方便和经济的检测手段。
Thinopyrum intermedium(2n=6x=42, JJJ~sJ~sStSt) is one of the important wild resources for wheat genetic improvement. However, as the Th. intermedium genome has not yet been sequenced, the numbers of reported Thinopyrum-specific molecular markers are insufficient for the purpose of identification of the small aline fragments or genes in the offspring of wheat-Th. intermedium hybrids in wheat breeding research. In this study, a total of 5877409 Contigs(overlapping DNA sequences) were obtained by assembling the probe sequences of a Th. intermedium genotyping-by-sequencing chip downloaded from a database. Through informatics analysis, 5452 non-redundant Contigs with chromosome location information and a similarity less than 80% with the wheat genome were screened out, and 2019 sequence-tagged site(STS) molecular markers were developed. The distribution numbers of markers from Thinopyrum homologous groups 1(G1) to G7 were 250, 215, 323, 253, 323, 253 and 402, respectively. Then, 160 Thinopyrum-G6 specific markers were selected based on amplification of five Th. intermedium plants and five wheat landraces, 53 of which were of "+/-" specificity and were distributed on the three G6 chromosomes: G6-Chr1(32), G6-Chr2(13) and G6-Chr3(8). Next, G6-Chr2 was deduced to be the 6 St chromosome by using three progenitor species of Th. intermedium, including Th. elongatum(2n=2x=14, J~eJ~e), Th. bessarabicum(2n=2x=14, J~bJ~b) and Pseudoroegneiria strigosa(2n=2x=14, StSt), as well as 6 J~e substitution lines of Chinese spring-Th. elongatum. Finally, the 13 "+/-" specific markers developed that were located on Thinopyrum-6 St were used to verify the exogenous chromosome in wheat-Thinopyrum substitution line F881-6 St/6 D at the molecular level. These results will provide a more convenient and economical method for the identification of chromosomes or chromosome fragments of Th. intermedium.
Thinopyrum intermedium(2n=6x=42, JJJ~sJ~sStSt) is one of the important wild resources for wheat genetic improvement. However, as the Th. intermedium genome has not yet been sequenced, the numbers of reported Thinopyrum-specific molecular markers are insufficient for the purpose of identification of the small aline fragments or genes in the offspring of wheat-Th. intermedium hybrids in wheat breeding research. In this study, a total of 5877409 Contigs(overlapping DNA sequences) were obtained by assembling the probe sequences of a Th. intermedium genotyping-by-sequencing chip downloaded from a database. Through informatics analysis, 5452 non-redundant Contigs with chromosome location information and a similarity less than 80% with the wheat genome were screened out, and 2019 sequence-tagged site(STS) molecular markers were developed. The distribution numbers of markers from Thinopyrum homologous groups 1(G1) to G7 were 250, 215, 323, 253, 323, 253 and 402, respectively. Then, 160 Thinopyrum-G6 specific markers were selected based on amplification of five Th. intermedium plants and five wheat landraces, 53 of which were of "+/-" specificity and were distributed on the three G6 chromosomes: G6-Chr1(32), G6-Chr2(13) and G6-Chr3(8). Next, G6-Chr2 was deduced to be the 6 St chromosome by using three progenitor species of Th. intermedium, including Th. elongatum(2n=2x=14, J~eJ~e), Th. bessarabicum(2n=2x=14, J~bJ~b) and Pseudoroegneiria strigosa(2n=2x=14, StSt), as well as 6 J~e substitution lines of Chinese spring-Th. elongatum. Finally, the 13 "+/-" specific markers developed that were located on Thinopyrum-6 St were used to verify the exogenous chromosome in wheat-Thinopyrum substitution line F881-6 St/6 D at the molecular level. These results will provide a more convenient and economical method for the identification of chromosomes or chromosome fragments of Th. intermedium.
引文
[1] Wang H G, Liu S B, Qi Z J, et al. Application studies of Elytrigia intermedium in hereditary improvement of wheat. Journal of Shandong Agricultural University (Natural Science), 2000, 31(3): 333-336. 王洪刚, 刘树兵, 亓增军, 等. 中间偃麦草在小麦遗传改良中的应用研究. 山东农业大学学报(自然科学版), 2000, 31(3): 333-336.
[2] Peto F H. Hybridization of Triticum and Agropyron. II. cytology of the male parents and F1 generation. Canadian Journal of Research, 1936, 14(5): 203-214.
[3] Matsumura K. Cell genetics and breeding in wheat. Tokyo: Yokendo Company Limited, 1950: 64-72. 松村清二. 小麦细胞遗传育种. 东京: 养贤堂株式会社, 1950: 64-72.
[4] Stebbins G L, Pun F T. Artificial and natural hybrids in the gramineae, tribe hordeae. VI. chromosome pairing in Secale cereale×Agropyron intermedium and the problem of genome homologies in the Triticinae. Genetics, 1953, 38(6): 600-608.
[5] Dewey D R. The genomics system of classification as a guide to intergeneric hybridization within perennial Triticeae//Gene manipulation in plant improvement. Boston, MA: Springer, 1984: 209-279.
[6] Piao Z S. The studies on the chromosomal morphology and banding pattern in Agropyron intermedium. Acta Genetica Sinica, 1982, 9(5): 350-356. 朴真三. 天兰冰草染色体形态和带型的研究. 遗传学报, 1982, 9(5): 350-356.
[7] Chen Q, Conner R L, Laroche A, et al. Genome analysis of Thinopyrum intermedium and Thinopyrum ponticum using genomic in situ hybridization. Genome, 1998, 41(4): 580-586.
[8] Tang S, Li Z, Jia X, et al. Genomic in situ hybridization (GISH) analyses of Thinopyrum intermedium, its partial amphiploid Zhong 5, and disease-resistant derivatives in wheat. Theoretical and Applied Genetics, 2000, 100(3/4): 344-352.
[9] Ji W Q, Xue X Z, Wang Q Y, et al. GISH analysis of Thinopyrum intermedium. Acta Botanica Boreali-Occidentalia Sinica, 2001, 21(3): 401-405. 吉万全, 薛秀庄, 王秋英, 等. 中间偃麦草的GISH分析. 西北植物学报, 2001, 21(3): 401-405.
[10] Wang R R, Larson S R, Jensen K B, et al. Genome evolution of intermediate wheatgrass as revealed by EST-SSR markers developed from its three progenitor diploid species. Genome, 2015, 58(2): 63-70.
[11] Linc G, Gaál E, Molnár I, et al. Molecular cytogenetic (FISH) and genome analysis of diploid wheatgrasses and their phylogenetic relationship. PLoS One, 2017, 12(3): e0173623.
[12] Chen Q. Detection of alien chromatin introgression from Thinopyrum into wheat using S genomic DNA as a probe-A landmark approach for Thinopyrum genome research. Cytogenetic and Genome Research, 2005, 109: 350-359.
[13] Lü W D, Xü P B, Pu X. Summary of the situation for applying genetic resources from Elytrigia in Triticum aestivum breeding. Acta Prataculturae Sinica, 2007, 16(6): 136-140. 吕伟东, 徐鹏彬, 蒲训. 偃麦草属种质资源在普通小麦育种中的应用现状简介. 草业学报, 2007, 16(6): 136-140.
[14] Li H J, Wang X M. Thinopyrum ponticum and Th. intermedium: The promising source of resistance to fungal and viral diseases of wheat. Journal of Genetics and Genomics, 2009, 36(9): 557-565.
[15] Cui Z F, Lin Z S, Xin Z Y, et al. Identification of wheat-Thinopyrum intermedium telosomic lines resistant to barley yellow dwarf virus by GISH and STS markers converted from RFLP. Acta Agronomica Sinica, 2006, 32(12): 1855-1859. 崔志富, 林志珊, 辛志勇, 等. 应用 GISH 与 STS 标记鉴定小麦-中间偃麦草抗黄矮病端体系. 作物学报, 2006, 32(12): 1855-1859.
[16] Zhan H X, Zhang X J, Li G R, et al. Molecular characterization of a new wheat-Thinopyrum intermedium translocation line with resistance to powdery mildew and stripe rust. International Journal of Molecular Sciences, 2015, 16(1): 2162-2173.
[17] Salina E A, Adonina I G, Badaeva E D, et al. A Thinopyrum intermedium chromosome in bread wheat cultivars as a source of genes conferring resistance to fungal diseases. Euphytica, 2015, 204(1): 91-101.
[18] Bao Y G, Wu X, Zhang C, et al. Chromosomal consititutions and reactions to powdery mildew and stripe rust of four novel wheat-Thinopyrum intermedium partial amphiploids. Journal of Genetics and Genomics, 2014, 41(12): 663-666.
[19] Bao Y G, Li X, Liu S, et al. Molecular cytogenetic characterization of a new wheat-Thinopyrum intermedium partial amphiploid resistant to powdery mildew and stripe rust. Cytogenetic and Genome Research, 2009, 126(4): 390-395.
[20] Li J B, Qiao L Y, Li X, et al. Molecular mapping of powdery mildew resistance gene PmCH7124 in a putative wheat-Thinopyrum intermedium introgression line. Acta Agronomica Sinica, 2015, 41(1): 49-56. 李建波, 乔麟轶, 李欣, 等. 小麦-中间偃麦草渗入系抗白粉病基因PmCH7124的分子定位. 作物学报, 2015, 41(1): 49-56.
[21] Qi X L, Li X F, He F, et al. Cytogenetic and molecular identification of a new wheat-Thinopyrum intermedium addition line with resistance to powdery mildew. Cereal Research Communications, 2015, 43(3): 353-363.
[22] Li J B, Lang T, Li B, et al. Introduction of Thinopyrum intermedium ssp. trichophorum chromosomes to wheat by trigeneric hybridization involving Triticum, Secale and Thinopyrum genera. Planta, 2017, 245(6): 1121-1135.
[23] Pu J, Wang Q, Shen Y F, et al. Physical mapping of chromosome 4J of Thinopyrum bessarabicum using gamma radiation-induced aberrations. Theoretical and Applied Genetics, 2015, 128(7): 1319-1328.
[24] Kantarski T, Larson S, Zhang X F, et al. Development of the first consensus genetic map of intermediate wheatgrass (Thinopyrum intermedium) using genotyping-by-sequencing. Theoretical and Applied Genetics, 2016, 130(1): 1-14.
[25] Tsitsin N V. Remote hybridization as a method of creating new species and varieties of plants. Euphytica, 1965, 14: 326-330.
[26] Kuraparthy V, Sood S, Chhuneja P, et al. A cryptic wheat-Aegilops triuncialis translocation with leaf rust resistance gene Lr58. Crop Science, 2007, 47(5): 1995-2003.
[27] Kuraparthy V, Chhuneja P, Dhaliwal H S, et al. Characterization and mapping of cryptic alien introgression from Aegilops geniculata with new leaf rust and stripe rust resistance genes Lr57 and Yr40 in wheat. Theoretical and Applied Genetics, 2007, 114(8): 1379-1389.
[28] Wang L M, Li X F, Liu S B, et al. Studies on the transferability of common wheat (T. aestivum) microsatellites (SSR) markers used in Thinopyrum intermedium. Acta Agriculturae Boreali-Sinica, 2007, 22(6): 50-52.王黎明, 李兴锋, 刘树兵, 等. 小麦微卫星标记在中间偃麦草中通用性研究. 华北农学报, 2007, 22(6): 50-52.
[29] Hegarty M J, Hiscock S J. Hybrid speciation in plants: New insights from molecular studies. New Phytologist, 2005, 165(2): 411-423.
[30] Chen S Y, Ma X, Zhang X Q, et al. Interspecific relationships between hexaploid species in the Triticeae tribe with St, H and Y genomes. Acta Prataculturae Sinica, 2018, 27(9): 142-151.陈仕勇, 马啸, 张新全, 等. 基于SSR标记的小麦族St、H、Y基因组六倍体物种遗传变异及种间亲缘关系研究. 草业学报, 2018, 27(9): 142-151.
[31] Hu L J, Li G R, Zeng Z X, et al. Molecular characterization of a wheat-Thinopyrum ponticum partial amphiploid and its derived substitution line for resistance to stripe rust. Journal of Applied Genetics, 2011, 52(3): 279-285.
[2] Peto F H. Hybridization of Triticum and Agropyron. II. cytology of the male parents and F1 generation. Canadian Journal of Research, 1936, 14(5): 203-214.
[3] Matsumura K. Cell genetics and breeding in wheat. Tokyo: Yokendo Company Limited, 1950: 64-72. 松村清二. 小麦细胞遗传育种. 东京: 养贤堂株式会社, 1950: 64-72.
[4] Stebbins G L, Pun F T. Artificial and natural hybrids in the gramineae, tribe hordeae. VI. chromosome pairing in Secale cereale×Agropyron intermedium and the problem of genome homologies in the Triticinae. Genetics, 1953, 38(6): 600-608.
[5] Dewey D R. The genomics system of classification as a guide to intergeneric hybridization within perennial Triticeae//Gene manipulation in plant improvement. Boston, MA: Springer, 1984: 209-279.
[6] Piao Z S. The studies on the chromosomal morphology and banding pattern in Agropyron intermedium. Acta Genetica Sinica, 1982, 9(5): 350-356. 朴真三. 天兰冰草染色体形态和带型的研究. 遗传学报, 1982, 9(5): 350-356.
[7] Chen Q, Conner R L, Laroche A, et al. Genome analysis of Thinopyrum intermedium and Thinopyrum ponticum using genomic in situ hybridization. Genome, 1998, 41(4): 580-586.
[8] Tang S, Li Z, Jia X, et al. Genomic in situ hybridization (GISH) analyses of Thinopyrum intermedium, its partial amphiploid Zhong 5, and disease-resistant derivatives in wheat. Theoretical and Applied Genetics, 2000, 100(3/4): 344-352.
[9] Ji W Q, Xue X Z, Wang Q Y, et al. GISH analysis of Thinopyrum intermedium. Acta Botanica Boreali-Occidentalia Sinica, 2001, 21(3): 401-405. 吉万全, 薛秀庄, 王秋英, 等. 中间偃麦草的GISH分析. 西北植物学报, 2001, 21(3): 401-405.
[10] Wang R R, Larson S R, Jensen K B, et al. Genome evolution of intermediate wheatgrass as revealed by EST-SSR markers developed from its three progenitor diploid species. Genome, 2015, 58(2): 63-70.
[11] Linc G, Gaál E, Molnár I, et al. Molecular cytogenetic (FISH) and genome analysis of diploid wheatgrasses and their phylogenetic relationship. PLoS One, 2017, 12(3): e0173623.
[12] Chen Q. Detection of alien chromatin introgression from Thinopyrum into wheat using S genomic DNA as a probe-A landmark approach for Thinopyrum genome research. Cytogenetic and Genome Research, 2005, 109: 350-359.
[13] Lü W D, Xü P B, Pu X. Summary of the situation for applying genetic resources from Elytrigia in Triticum aestivum breeding. Acta Prataculturae Sinica, 2007, 16(6): 136-140. 吕伟东, 徐鹏彬, 蒲训. 偃麦草属种质资源在普通小麦育种中的应用现状简介. 草业学报, 2007, 16(6): 136-140.
[14] Li H J, Wang X M. Thinopyrum ponticum and Th. intermedium: The promising source of resistance to fungal and viral diseases of wheat. Journal of Genetics and Genomics, 2009, 36(9): 557-565.
[15] Cui Z F, Lin Z S, Xin Z Y, et al. Identification of wheat-Thinopyrum intermedium telosomic lines resistant to barley yellow dwarf virus by GISH and STS markers converted from RFLP. Acta Agronomica Sinica, 2006, 32(12): 1855-1859. 崔志富, 林志珊, 辛志勇, 等. 应用 GISH 与 STS 标记鉴定小麦-中间偃麦草抗黄矮病端体系. 作物学报, 2006, 32(12): 1855-1859.
[16] Zhan H X, Zhang X J, Li G R, et al. Molecular characterization of a new wheat-Thinopyrum intermedium translocation line with resistance to powdery mildew and stripe rust. International Journal of Molecular Sciences, 2015, 16(1): 2162-2173.
[17] Salina E A, Adonina I G, Badaeva E D, et al. A Thinopyrum intermedium chromosome in bread wheat cultivars as a source of genes conferring resistance to fungal diseases. Euphytica, 2015, 204(1): 91-101.
[18] Bao Y G, Wu X, Zhang C, et al. Chromosomal consititutions and reactions to powdery mildew and stripe rust of four novel wheat-Thinopyrum intermedium partial amphiploids. Journal of Genetics and Genomics, 2014, 41(12): 663-666.
[19] Bao Y G, Li X, Liu S, et al. Molecular cytogenetic characterization of a new wheat-Thinopyrum intermedium partial amphiploid resistant to powdery mildew and stripe rust. Cytogenetic and Genome Research, 2009, 126(4): 390-395.
[20] Li J B, Qiao L Y, Li X, et al. Molecular mapping of powdery mildew resistance gene PmCH7124 in a putative wheat-Thinopyrum intermedium introgression line. Acta Agronomica Sinica, 2015, 41(1): 49-56. 李建波, 乔麟轶, 李欣, 等. 小麦-中间偃麦草渗入系抗白粉病基因PmCH7124的分子定位. 作物学报, 2015, 41(1): 49-56.
[21] Qi X L, Li X F, He F, et al. Cytogenetic and molecular identification of a new wheat-Thinopyrum intermedium addition line with resistance to powdery mildew. Cereal Research Communications, 2015, 43(3): 353-363.
[22] Li J B, Lang T, Li B, et al. Introduction of Thinopyrum intermedium ssp. trichophorum chromosomes to wheat by trigeneric hybridization involving Triticum, Secale and Thinopyrum genera. Planta, 2017, 245(6): 1121-1135.
[23] Pu J, Wang Q, Shen Y F, et al. Physical mapping of chromosome 4J of Thinopyrum bessarabicum using gamma radiation-induced aberrations. Theoretical and Applied Genetics, 2015, 128(7): 1319-1328.
[24] Kantarski T, Larson S, Zhang X F, et al. Development of the first consensus genetic map of intermediate wheatgrass (Thinopyrum intermedium) using genotyping-by-sequencing. Theoretical and Applied Genetics, 2016, 130(1): 1-14.
[25] Tsitsin N V. Remote hybridization as a method of creating new species and varieties of plants. Euphytica, 1965, 14: 326-330.
[26] Kuraparthy V, Sood S, Chhuneja P, et al. A cryptic wheat-Aegilops triuncialis translocation with leaf rust resistance gene Lr58. Crop Science, 2007, 47(5): 1995-2003.
[27] Kuraparthy V, Chhuneja P, Dhaliwal H S, et al. Characterization and mapping of cryptic alien introgression from Aegilops geniculata with new leaf rust and stripe rust resistance genes Lr57 and Yr40 in wheat. Theoretical and Applied Genetics, 2007, 114(8): 1379-1389.
[28] Wang L M, Li X F, Liu S B, et al. Studies on the transferability of common wheat (T. aestivum) microsatellites (SSR) markers used in Thinopyrum intermedium. Acta Agriculturae Boreali-Sinica, 2007, 22(6): 50-52.王黎明, 李兴锋, 刘树兵, 等. 小麦微卫星标记在中间偃麦草中通用性研究. 华北农学报, 2007, 22(6): 50-52.
[29] Hegarty M J, Hiscock S J. Hybrid speciation in plants: New insights from molecular studies. New Phytologist, 2005, 165(2): 411-423.
[30] Chen S Y, Ma X, Zhang X Q, et al. Interspecific relationships between hexaploid species in the Triticeae tribe with St, H and Y genomes. Acta Prataculturae Sinica, 2018, 27(9): 142-151.陈仕勇, 马啸, 张新全, 等. 基于SSR标记的小麦族St、H、Y基因组六倍体物种遗传变异及种间亲缘关系研究. 草业学报, 2018, 27(9): 142-151.
[31] Hu L J, Li G R, Zeng Z X, et al. Molecular characterization of a wheat-Thinopyrum ponticum partial amphiploid and its derived substitution line for resistance to stripe rust. Journal of Applied Genetics, 2011, 52(3): 279-285.