地黄SCoT分子标记体系的建立和指纹图谱的构建
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摘要
该研究采用L_(25)(5~6)正交设计和单因素两种方法,对影响地黄SCoT-PCR反应的5个因素(模板DNA浓度,引物浓度,ddH_2O和Mix的用量以及退火温度)进行了优化。结果表明:优化后的反应体系总体积为25μL,含有8μL ddH_2O,1μL模板DNA(80 ng·μL~(-1)),1μL引物(8μmol·L~(-1))和15μL Mix,退火温度为45℃。运用30份地黄种质材料,对优化的SCoT-PCR正交体系进行多次重复验证,获得了多态性丰富、条带清晰的扩增图谱,证明该反应体系稳定可靠。利用该体系对32条SCoT引物进行两次筛选,得到14个扩增产物清晰、重复性好且多态性条带相对较高的引物。利用SCoT_4等5条引物构建了上述地黄2个种共30份种质的SCoT指纹图谱。利用这5个SCoT引物指纹图谱可将7个地黄常用栽培品种区分开。这表明SCoT分子标记体系适用于地黄主要品种亲缘关系及遗传多样性的研究,所构建的指纹图谱也为地黄常见的7个栽培品种的区分提供参考依据。
We used L_(25)( 5~6) orthogonal design and single factor method to optimize the five factors( template DNA concentration,primer concentration,dd H_2 O,Mix amount and annealing temperature) that affect the SCo T-PCR reaction of Rehmannia glutinosa. The results were as follows: The reaction system was a total volume of 25 μL containing 8 μL of dd H_2 O,1 μL of template DNA( 80 ng·μL~(-1)),1 μL of primer( 8 μmol·L~(-1)) and 15 μL of Mix,and an annealing temperature of 45 ℃. The optimized SCo T-PCR orthogonal system was repeatedly verified by using 30 parts of Rehmannia germplasm materials,and the amplified spectrum with rich polymorphism and clear bands was obtained,which proves that the reaction system is stable and reliable. Using this system,32 SCo T primers were screened twice,and 14 primers with clear,reproducible and relatively high polymorphic bands were obtained. Finally,SCo T fingerprints of 30 species of the above two species of Rehmannia were constructed by using five primers such as SCo T_4. Using these five SCo T primer fingerprints,seven common cultivars of Rehmannia glutinosa can be distinguished. The study indicates that the SCo T molecular marker system is suitable for the study of genetic relationship and genetic diversity of the main varieties of Rehmannia glutinosa. The fingerprints constructed also provide reference for the differentiation of seven cultivars commonly found in Rehmannia glutinosa.
We used L_(25)( 5~6) orthogonal design and single factor method to optimize the five factors( template DNA concentration,primer concentration,dd H_2 O,Mix amount and annealing temperature) that affect the SCo T-PCR reaction of Rehmannia glutinosa. The results were as follows: The reaction system was a total volume of 25 μL containing 8 μL of dd H_2 O,1 μL of template DNA( 80 ng·μL~(-1)),1 μL of primer( 8 μmol·L~(-1)) and 15 μL of Mix,and an annealing temperature of 45 ℃. The optimized SCo T-PCR orthogonal system was repeatedly verified by using 30 parts of Rehmannia germplasm materials,and the amplified spectrum with rich polymorphism and clear bands was obtained,which proves that the reaction system is stable and reliable. Using this system,32 SCo T primers were screened twice,and 14 primers with clear,reproducible and relatively high polymorphic bands were obtained. Finally,SCo T fingerprints of 30 species of the above two species of Rehmannia were constructed by using five primers such as SCo T_4. Using these five SCo T primer fingerprints,seven common cultivars of Rehmannia glutinosa can be distinguished. The study indicates that the SCo T molecular marker system is suitable for the study of genetic relationship and genetic diversity of the main varieties of Rehmannia glutinosa. The fingerprints constructed also provide reference for the differentiation of seven cultivars commonly found in Rehmannia glutinosa.
引文
CHEN DX,LI LY,PENG R,et al.,2009.SRAP study on genetic relationship and genetic diversity of three cultivation types of Scrophularia sinensis[J].Chin J Mat Med,34(2):138-142.[陈大霞,李隆云,彭锐,等,2009.玄参3种栽培类型遗传关系和遗传多样性的SRAP研究[J].中国中药杂志,34(2):138-142.]
FENG FJ,LI MJ,GU L,et al.,2015.Development of EST-SSR markers and establishment of amplification system for Rehmannia glutinosa[J].Guangdong Agric Sci,42(10):120-126.[冯法节,李明杰,古力,等,2015.地黄EST-SSR标记的开发及扩增体系的建立[J].广东农业科学,42(10):120-126.]
GUO J,YU X,YIN H,et al.,2016.Phylogenetic relationships of Thinopyrum and Triticum species revealed by SCo T and CDDP markers[J].Plant Syst Evol,302(9):1301-1309.
JIANG YQ,LI Y,LI WJ,et al.,2014.Application of SCo Tmarkers on genetic diversity analysis of Loofah Mill.[J].J SAgric,45(12):2117-2122.[蒋雅琴,黎炎,李文嘉,等,2014.SCo T分子标记技术在丝瓜上的应用[J].南方农业学报,45(12):2117-2122.]
LI PR,JIANG JF,CHEN SM,et al.,2013.Application of SCo T molecular marker technology in genetic diversity analysis of Chrysanthemum[J].J Hortic,40(10):2015-2025.[李丕睿,蒋甲福,陈素梅,等,2013.菊属植物SCo T分子标记技术在遗传多样性分析中的应用[J].园艺学报,40(10):2015-2025.]
LIU CY,YUAN C,LI XT,et al.,2015.Development of microsatellites from Rehmannia glutinosa transcriptome and evaluation of genetic diversity among Rehmannia species[J].Biochem Syst Ecol,59:177-182.
LONG ZJ,FAN LZ,XU G,et al.,2015.Progress in the application of SCo T molecular markers in plant research[J].J Plant Gen Resour,16(2):336-343.[龙治坚,范理璋,徐刚,等,2015.SCo T分子标记在植物研究中的应用进展[J].植物遗传资源学报,16(2):336-343.]
MAHJBI A,BARAKET G,OUESLATI A,et al.,2015.Start Codon Targeted(SCo T)markers provide new insights into the genetic diversity analysis and characterization of Tunisian Citrus species[J].Biochem Syst Ecol,61:390-398.
MARIAPPAN M,THIRUPPATHI SK,MANDALI VR,2016.Organogenesis and evaluation of genetic homogeneity through SCo T and ISSR markers in Helicteres isora L.,a medicinally important tree[J].Safr J Bot,106:204-210.
WANG WS,2016.Screening and identification of DNA barcode sequences of Rehmannia glutinosa[D].Xinxiang:Henan Normal University.[王婉珅,2016.地黄DNA条形码序列的筛选与鉴定研究[D].新乡:河南师范大学.]
XU LT,2015.Identification of wheat-intermediate ryegrass germplasm line[D].Tai'an:Shandong Agricultral University.[徐林涛,2015.小麦-中间偃麦草种质系的鉴定[D].泰安:山东农业大学.]
XIA Z,WANG LJ,HUANG Y,et al.,2016.Identification of DNA barcodes of Rehmannia and the origin of Rehmannia cultivation[J].Chin Trad Herb Drugs,47(4):648-654.[夏至,王璐静,黄勇,等,2016.地黄属植物DNA条形码鉴定及地黄栽培起源研究[J].中草药,47(4):648-654.]
ZHOU YQ,YAO HL,ZHOU CE,et al.,2010.Research progress of Rehmannia breeding[J].Guihaia,30(3):373-376.[周延清,姚换灵,周春娥,等,2010.地黄育种研究进展[J].广西植物,30(3):373-376.]
ZHOU YQ,WANG WS,WANG XN,et al.,2015.Progress in DNA molecular markers and gene functions of Rehmannia glutinosa[J].Chin J Plant Sci,50(5):665-672.[周延清,王婉珅,王向楠,等,2015.地黄DNA分子标记与基因功能研究进展[J].植物学报,50(5):665-672.]
FENG FJ,LI MJ,GU L,et al.,2015.Development of EST-SSR markers and establishment of amplification system for Rehmannia glutinosa[J].Guangdong Agric Sci,42(10):120-126.[冯法节,李明杰,古力,等,2015.地黄EST-SSR标记的开发及扩增体系的建立[J].广东农业科学,42(10):120-126.]
GUO J,YU X,YIN H,et al.,2016.Phylogenetic relationships of Thinopyrum and Triticum species revealed by SCo T and CDDP markers[J].Plant Syst Evol,302(9):1301-1309.
JIANG YQ,LI Y,LI WJ,et al.,2014.Application of SCo Tmarkers on genetic diversity analysis of Loofah Mill.[J].J SAgric,45(12):2117-2122.[蒋雅琴,黎炎,李文嘉,等,2014.SCo T分子标记技术在丝瓜上的应用[J].南方农业学报,45(12):2117-2122.]
LI PR,JIANG JF,CHEN SM,et al.,2013.Application of SCo T molecular marker technology in genetic diversity analysis of Chrysanthemum[J].J Hortic,40(10):2015-2025.[李丕睿,蒋甲福,陈素梅,等,2013.菊属植物SCo T分子标记技术在遗传多样性分析中的应用[J].园艺学报,40(10):2015-2025.]
LIU CY,YUAN C,LI XT,et al.,2015.Development of microsatellites from Rehmannia glutinosa transcriptome and evaluation of genetic diversity among Rehmannia species[J].Biochem Syst Ecol,59:177-182.
LONG ZJ,FAN LZ,XU G,et al.,2015.Progress in the application of SCo T molecular markers in plant research[J].J Plant Gen Resour,16(2):336-343.[龙治坚,范理璋,徐刚,等,2015.SCo T分子标记在植物研究中的应用进展[J].植物遗传资源学报,16(2):336-343.]
MAHJBI A,BARAKET G,OUESLATI A,et al.,2015.Start Codon Targeted(SCo T)markers provide new insights into the genetic diversity analysis and characterization of Tunisian Citrus species[J].Biochem Syst Ecol,61:390-398.
MARIAPPAN M,THIRUPPATHI SK,MANDALI VR,2016.Organogenesis and evaluation of genetic homogeneity through SCo T and ISSR markers in Helicteres isora L.,a medicinally important tree[J].Safr J Bot,106:204-210.
WANG WS,2016.Screening and identification of DNA barcode sequences of Rehmannia glutinosa[D].Xinxiang:Henan Normal University.[王婉珅,2016.地黄DNA条形码序列的筛选与鉴定研究[D].新乡:河南师范大学.]
XU LT,2015.Identification of wheat-intermediate ryegrass germplasm line[D].Tai'an:Shandong Agricultral University.[徐林涛,2015.小麦-中间偃麦草种质系的鉴定[D].泰安:山东农业大学.]
XIA Z,WANG LJ,HUANG Y,et al.,2016.Identification of DNA barcodes of Rehmannia and the origin of Rehmannia cultivation[J].Chin Trad Herb Drugs,47(4):648-654.[夏至,王璐静,黄勇,等,2016.地黄属植物DNA条形码鉴定及地黄栽培起源研究[J].中草药,47(4):648-654.]
ZHOU YQ,YAO HL,ZHOU CE,et al.,2010.Research progress of Rehmannia breeding[J].Guihaia,30(3):373-376.[周延清,姚换灵,周春娥,等,2010.地黄育种研究进展[J].广西植物,30(3):373-376.]
ZHOU YQ,WANG WS,WANG XN,et al.,2015.Progress in DNA molecular markers and gene functions of Rehmannia glutinosa[J].Chin J Plant Sci,50(5):665-672.[周延清,王婉珅,王向楠,等,2015.地黄DNA分子标记与基因功能研究进展[J].植物学报,50(5):665-672.]