小麦2B染色体新SSR标记开发
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摘要
为开发用于小麦育种的新SSR分子标记,利用普通小麦中国春2B染色体25 Mb的DNA序列进行了SSR筛选,在检测到的2 852个SSR中80.40%是二核苷酸重复。二、三、四、五核苷酸重复序列分别有6,30,17,1种类型。二核苷酸中GA/CT、AG/TC、AT/TA数量最多,分别占SSR总数的20.44%,19.00%,17.15%;三核苷酸中CTT/GAA的数量最多,占SSR总数的2.56%;四、五核苷酸重复出现的频率都较低。进一步分析SSR的重复次数,发现二核苷酸重复次数总体较高,重复次数≥10的SSR数量有327个,占所有二核苷酸序列的14.26%,其中,AG/TC、AT/TA的重复次数≥10的SSR数量分别有98,97个。三核苷酸的重复次数总体偏低,重复次数≥8的SSR数量仅有62个,占所有三核苷酸序列的11.55%,其中TTC/AAG的重复次数≥8的SSR数量最多,有12个。四核苷酸中只有ACAT/TGTA、TAGA/ATCT的重复次数分别达到了16,14次,其他重复基元的重复次数主要为5～7次。根据筛选到的SSR位点共设计合成了135对SSR引物,发现有101对(74.81%)引物在小麦品系Cf5019-21和Cf5240-41扩增出清晰的DNA条带,17对引物在二者间表现出差异扩增。
In order to develop novel SSR markers for wheat breeding,simple sequence repeat(SSR)was identified from a 25 Mb region according to the Chinese Spring 2 B chromosome reference sequence. In 2 852 SSR loci identified,80.40% belonged to dinucleotides repeat.Six,thirty,seventeen,one kinds of SSR repeats were found in dinucleotide,trinucleotide,tetranucleotide,pentanucleotide repeats,respectively. For dinucleotides,GA/CT,AG/TC,AT/TA had the highest frequencies,occupying 20.44%,19.00%,17.15% of all SSR loci identified,respectively; for trinucleotides,CTT/GAA had the highest frequency,occupying 2.56% of all SSR loci identified; whereas the frequencies of tetranucleotide and pentanucleotide repeats were relatively low. Furthermore,the replication numbers were higher in dinucleotide than others,and 327 SSR loci which occupied 14.26% of all dinucleotides loci identified were with ≥10 replications. The numbers of SSR loci with ≥10 replications for AG/TC and AT/TA reached 98 and 97,respectively. The replication numbers in trinucleotide were relatively low,and 62 SSR loci which occupied 11.55% of all trinucleotides loci identified were with ≥8 replications. The number of TTC/AAG with ≥8 replications was the biggest,reached 12. For tetranucleotide,the numbers in ACAT/TGTA and TAGA/ATCT reached 16 and 14,respectively,and the replication numbers in other repeats of tetranucleotide varied between 5 and 7. Based on the found SSR loci,135 SSR primer pairs were designed and synthesized. Of these,101(74.81%)SSR primer pairs amplified clear products in wheat lines Cf5019-21 and Cf5240-41,and 17 SSR primer pairs showed polymorphism between them.
In order to develop novel SSR markers for wheat breeding,simple sequence repeat(SSR)was identified from a 25 Mb region according to the Chinese Spring 2 B chromosome reference sequence. In 2 852 SSR loci identified,80.40% belonged to dinucleotides repeat.Six,thirty,seventeen,one kinds of SSR repeats were found in dinucleotide,trinucleotide,tetranucleotide,pentanucleotide repeats,respectively. For dinucleotides,GA/CT,AG/TC,AT/TA had the highest frequencies,occupying 20.44%,19.00%,17.15% of all SSR loci identified,respectively; for trinucleotides,CTT/GAA had the highest frequency,occupying 2.56% of all SSR loci identified; whereas the frequencies of tetranucleotide and pentanucleotide repeats were relatively low. Furthermore,the replication numbers were higher in dinucleotide than others,and 327 SSR loci which occupied 14.26% of all dinucleotides loci identified were with ≥10 replications. The numbers of SSR loci with ≥10 replications for AG/TC and AT/TA reached 98 and 97,respectively. The replication numbers in trinucleotide were relatively low,and 62 SSR loci which occupied 11.55% of all trinucleotides loci identified were with ≥8 replications. The number of TTC/AAG with ≥8 replications was the biggest,reached 12. For tetranucleotide,the numbers in ACAT/TGTA and TAGA/ATCT reached 16 and 14,respectively,and the replication numbers in other repeats of tetranucleotide varied between 5 and 7. Based on the found SSR loci,135 SSR primer pairs were designed and synthesized. Of these,101(74.81%)SSR primer pairs amplified clear products in wheat lines Cf5019-21 and Cf5240-41,and 17 SSR primer pairs showed polymorphism between them.
引文
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[13] Cho Y G,Ishii T,Temnykh S,Chen X,Lipovich L,McCouch S R,Park W D,Ayres N.Diversity of microsatellites derived from genomic libraries and GenBank sequences in rice(Oryza sativa L.)[J].Theoretical and Applied Genetics,2000,100(5):713-722.doi:10.1007/s001220051343.
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[15] 李立新,司守霞,魏安智,刘玉林,冯世静,杨途熙.基于花椒转录组序列SSR分子标记开发及花椒种质鉴定[J].华北农学报,2017,32(5):69-77.doi:10.7668/hbnxb.2017.05.011.Li L X,Si S X,Wei A Z,Liu Y L,Feng S J,Yang T X.Study on development of SSR molecular markers based on transcriptome sequencing and germplasm identification in germplasm[J].Acta Agriculturae Boreali-Sinica,2017,32(5):69-77.
[16] Li Q,Wan J M.SSRHunter:Development of a local searching software for SSR sites[J].Hereditas,2005,27(5):808-810.doi:10.3321/j.issn:0253-9772.2005.05.023.
[17] 陈军方,任正隆,高丽锋,贾继增.从小麦EST序列中开发新的SSR引物[J].作物学报,2005,31(2):154-158.doi:10.3321/j.issn:0496-3490.2005.02.004.Chen J F,Ren Z L,Gao L F,Jia J Z.Developing new SSR markers from EST of wheat[J].Acta Agronomica Sinica,2005,31(2):154-158.
[18] Li L,Wang J,Guo Y,Jiang F,Xu Y,Wang Y,Pan H,Han G,Li R,Li S.Development of SSR markers from ESTs of gramineous species and their chromosome location on wheat[J].Progress in Natural Science,2008,18(12):1485-1490.doi:10.1016/j.pnsc.2008.05.012.
[19] 刘泽涛,苑少华,杨迪,王玉昆,张立平,赵昌平,刘丽华,李宏博,庞斌双.小麦穗部组织EST-SSR标记引物开发及其在小麦遗传多样性分析中的应用[J].麦类作物学报,2013,33(6):1093-1099.doi:10.7606/j.issn.1009-1041.2013.06.005.Liu Z T,Yuan S H,Yang D,Wang Y K,Zhang L P,Zhao C P,Liu L H,Li H B,Pang B S.Development of SSR markers derived from EST of wheat spikes and their use in genetic diversity analysis of wheat[J].Journal of Triticeae Crops,2013,33(6):1093-1099.
[20] Pestsova E,Ganal M W,R der M S.Isolation and mapping of microsatellite markers specific for the D genome of bread wheat[J].Genome,2000,43(4):689-697.doi:10.1139/g00-042.
[2] 朱振东,贾继增.小麦SSR标记的发展及应用[J].遗传,2003,25(3):355-360.doi:10.16288/j.yczz.2003.03.026.Zhu Z D,Jia J Z.Microsatellite marker development and applications in wheat genetics and breeding[J].Hereditas,2003,25(3):355-360.doi:10.16288/j.yczz.2003.03.026.
[3] Rasheed A,Hao Y,Xia X,Khan A,Xu Y,Varshney R K,He Z.Crop breeding chips and genotyping platforms:Progress,challenges,and perspectives[J].Molecular Plant,2017,10(8):1047-1064.doi:10.1016/j.molp.2017.06.008.
[4] Liu J,Luo W,Qin N,Ding P,Zhang H,Yang C,Mu Y,Tang H,Liu Y,Li W,Jiang Q,Chen G,Wei Y,Zheng Y,Liu C,Lan X,Ma J.A 55 K SNP array-based genetic map and its utilization in QTL mapping for productive tiller number in common wheat[J].Theoretical and Applied Genetics,2018,131(11):2439-2450.doi:10.1007/s00122-018-3164-9.
[5] Sun C,Zhang F,Yan X,Zhang X,Dong Z,Cui D,Chen F.Genome-wide association study for 13 agronomic traits reveals distribution of superior alleles in bread wheat from the Yellow and Huai Valley of China[J].Plant Biotechnology Journal,2017,15(8):953-969.doi:10.1111/pbi.12690.
[6] Cui F,Zhang N,Fan X L,Zhang W,Zhao C H,Yang L J,Pan R Q,Chen M,Han J,Zhao X Q,Ji J,Tong Y P,Zhang H X,Jia J Z,Zhao G Y,Li J M.Utilization of a Wheat 660K SNP array-derived high-density genetic map for high-resolution mapping of a major QTL for kernel number[J].Scientific Reports,2017,7(1):3788.doi:10.1038/s41598-017-04028-6.
[7] Guyomarc′h H,Sourdille P,Charmet G,Edwards K J,Bernard M.Characterisation of polymorphic microsatellite markers from Aegilops tauschii and transferability to the D-genome of bread wheat[J].Theoretical and Applied Genetics,2002,104(6):1164-1172.doi:10.1007/s00122-001-0827-7.
[8] R?der M S,Korzun V,Wendehake K,Plaschke J,Tixier M H,Leroy P,Ganal M W.A microsatellite map of wheat[J].Genetics,1998,149(4):2007-2023.doi:10.0000/PMID9691054.
[9] Nicot N,Chiquet V,Gandon B,Amilhat L,Legeai F,Leroy P,Bernard M,Sourdille P.Study of simple sequence repeat(SSR)markers from wheat expressed sequence tags(ESTs)[J].Theoretical and Applied Genetics,2004,109(4):800-805.doi:10.1007/s00122-004-1685-x.
[10] Gao L F,Jing R L,Huo N X,Li Y,Li X P,Zhou R H,Chang X P,Tang J F,Ma Z Y,Jia J Z.One hundred and one new microsatellite loci derived from ESTs(EST-SSRs)in bread wheat[J].Theoretical and Applied Genetics,2004,108(7):1392-1400.doi:10.1007/s00122-003-1554-z.
[11] Chen H M,Li L Z,Wei X Y,Li S S,Lei T D,Hu H Z,Wang H G,Zhang X S.Development,chromosome location and genetic mapping of EST-SSR markers in wheat[J].Chinese Science Bulletin,2005,50(20):2328-2336.doi:10.1360/982005-379.
[12] 潘海涛,汪俊君,王盈盈,齐照良,李斯深.小麦EST-SSR标记的开发和遗传作图[J].中国农业科学,2010,43(3):452-461.doi:10.3864/j.issn.0578-1752.2010.03.002.Pan H T,Wang J J,Wang Y Y,Qi Z L,Li S S.Development and mapping of EST-SSR markers in wheat[J].Scientia Agricultura Sinica,2010,43(3):452-461.
[13] Cho Y G,Ishii T,Temnykh S,Chen X,Lipovich L,McCouch S R,Park W D,Ayres N.Diversity of microsatellites derived from genomic libraries and GenBank sequences in rice(Oryza sativa L.)[J].Theoretical and Applied Genetics,2000,100(5):713-722.doi:10.1007/s001220051343.
[14] Kantety R V,La Rota M,Matthews D E,Sorrells M E.Data mining for simple sequence repeats in expressed sequence tags from barley,maize,rice,sorghum and wheat[J].Plant Molecular Biology,2002,48(5-6):501-510.doi:10.1023/A:1014875206165.
[15] 李立新,司守霞,魏安智,刘玉林,冯世静,杨途熙.基于花椒转录组序列SSR分子标记开发及花椒种质鉴定[J].华北农学报,2017,32(5):69-77.doi:10.7668/hbnxb.2017.05.011.Li L X,Si S X,Wei A Z,Liu Y L,Feng S J,Yang T X.Study on development of SSR molecular markers based on transcriptome sequencing and germplasm identification in germplasm[J].Acta Agriculturae Boreali-Sinica,2017,32(5):69-77.
[16] Li Q,Wan J M.SSRHunter:Development of a local searching software for SSR sites[J].Hereditas,2005,27(5):808-810.doi:10.3321/j.issn:0253-9772.2005.05.023.
[17] 陈军方,任正隆,高丽锋,贾继增.从小麦EST序列中开发新的SSR引物[J].作物学报,2005,31(2):154-158.doi:10.3321/j.issn:0496-3490.2005.02.004.Chen J F,Ren Z L,Gao L F,Jia J Z.Developing new SSR markers from EST of wheat[J].Acta Agronomica Sinica,2005,31(2):154-158.
[18] Li L,Wang J,Guo Y,Jiang F,Xu Y,Wang Y,Pan H,Han G,Li R,Li S.Development of SSR markers from ESTs of gramineous species and their chromosome location on wheat[J].Progress in Natural Science,2008,18(12):1485-1490.doi:10.1016/j.pnsc.2008.05.012.
[19] 刘泽涛,苑少华,杨迪,王玉昆,张立平,赵昌平,刘丽华,李宏博,庞斌双.小麦穗部组织EST-SSR标记引物开发及其在小麦遗传多样性分析中的应用[J].麦类作物学报,2013,33(6):1093-1099.doi:10.7606/j.issn.1009-1041.2013.06.005.Liu Z T,Yuan S H,Yang D,Wang Y K,Zhang L P,Zhao C P,Liu L H,Li H B,Pang B S.Development of SSR markers derived from EST of wheat spikes and their use in genetic diversity analysis of wheat[J].Journal of Triticeae Crops,2013,33(6):1093-1099.
[20] Pestsova E,Ganal M W,R der M S.Isolation and mapping of microsatellite markers specific for the D genome of bread wheat[J].Genome,2000,43(4):689-697.doi:10.1139/g00-042.