山楂种质资源的分子鉴定及创新研究
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摘要
山楂属(Crataegus spp．)植物是重要的果树种质资源。本研究以原产于中国的山楂种质资源为试材，探索建立并优化山楂的RAPD分析体系和ISSR分析体系，在此基础上，以在国家果树种质沈阳山楂圃保存的96份山楂属植物资源为试材，利用RAPD标记和ISSR标记对中国山楂属植物的遗传多样性进行了分析。同时进行了山楂离体再生体系建立和四倍体诱导的研究。主要结果如下：
1．改进的CTAB法(提取缓冲液中含3％的CTAB)和QIAGEN试剂盒法是适宜的山楂总DNA提取方法。改进的CTAB法提取的山楂总DNA的产率较低，但是纯度较高，OD_(260)／OD_(280)比值在1.638～1.889之间；QIAGEN试剂盒法提取的山楂总DNA的产率和纯度均较高，供试8个样品的总DNA产率均高于100μg／g，OD_(260)／OD_(280)比值在1.758～1.850之间。以改进的CTAB法和QIAGEN试剂盒法提取的山楂总DNA为模板进行RAPD和ISSR扩增，能扩增出多条清晰的谱带，多态性、稳定性、重复性良好。以SDS法提取的山楂总DNA为模板进行RAPD和ISSR扩增，未能扩增出谱带。
2．对循环次数、反应体系中Mg~(2+)浓度、DNA模板质量、Taq DNA聚合酶的来源和浓度等多个影响RAPD反应结果的因子进行了研究，首次建立了优化的山楂属植物RAPD分析体系，即：PCR反应体积为20μl，内含1×PCR反应缓冲液(Promega)，2.0mmol／LMgCl_2，0.2 mmol／LdNTP(Promega)，0.3μmol／L引物，0.5 U Taq DNA聚合酶(Tiangen)，50 ng改进的CTAB法或QIAGEN试剂盒法提取的山楂总DNA；扩增程序为94℃30 s；94℃30 s，40℃2min，72℃3min，45循环，72℃7min。根据谱带的多少、多态性、清晰性，从146个RAPD引物中筛选出30个适宜于山楂属植物遗传分析的引物。
3．对退火温度、循环次数、反应体系中Mg~(2+)浓度、DNA模板质量、Taq酶的来源和浓度等影响ISSR反应结果的因子进行了研究，首次建立了优化的山楂属植物ISSR分析体系，即：PCR反应体积为20μl，内含1×PCR反应缓冲液(Promega)，3.0 mmol／LMgCl_2，0.2 mmol／L dNTP，0.3μmol／L引物，0.5 U Taq DNA聚合酶(Tiangen)，50 ng改进的CTAB法或QIAGEN试剂盒法提取的总DNA；扩增程序为94℃3 min；94℃30s，退火温度(通过温度梯度PCR筛选出的适宜温度)1 min，72℃2 min，38循环，72℃7 min。根据谱带的多少、多念性、清晰性，从36个ISSR引物中筛选出13个适宜于山楂属植物遗传分析的引物，其中有8个是基于(GA／CT) n或(AG／TC) n重复序列的在3’端加锚1～2个碱基的ISSR引物。
4．回收了1个山楂RAPD多态性谱带和16个ISSR多态性谱带，然后对回收的谱带进行克隆和测序，共获得了9个不同的山楂基因组序列。基于解析的山楂序列设计特异引物，成功地将1个RAPD标记和5个ISSR标记转化成SCAR标记。
5．首次利用分子标记技术对山楂属植物资源进行遗传研究，表明山楂属植物存在较高的遗传多样性。用12个RAPD引物在96份山楂属植物资源上共检测到136个多态性位点，不同山楂种质资源之间的相异系数在0～0.73之间，UPGMA法聚类分析结果显示不同山楂种质资源之间距离系数在0～0.58之间；用13个ISSR引物在96份山楂属植物资源上共检测到130个多态性位点，不同山楂种质资源之间的相异系数在0～0.80之间，UPGMA法聚类分析结果显示不同山楂种质资源之间距离系数在0～0.65之间。
6．分别绘制了基于RAPD标记和基于ISSR标记的山楂属植物分子系统进化树。基于RAPD标记的山楂属植物聚类结果与基于ISSR标记的聚类结果基本一致。但是与ISSR标记分析结果相比，基于RAPD标记的分类结果更接近于根据形态学特征进行的山楂传统植物学分类结果，RAPD标记更适合于山楂属植物的种内、种间的遗传变异分析。
7．RAPD和ISSR标记的分析结果均表明：在植物学分类上的伏山楂(C．brettschneideri Schneid．)属于山楂(C．pinnatifida Bge．)的一个变种或亚种，而不是一个独立的新种；从辽宁省盖州市熊岳镇引入的表现为黑皮绿肉的‘绿肉山楂’资源属于绿肉山楂(C．chlorosarca Maxim．)；而‘彰武山里红’是一个新种。
8．全面地进行了山楂胚、胚轴和子叶离体再生的研究，结果表明：①山楂未成熟子叶、成熟子叶和子叶叶片的再生不定芽能力明显不同，其中子叶叶片具有较高的再生能力，其不定芽再生频率是成熟子叶的6倍以上，在附加BA和TDZ各1.0 mg／L的MS培养基上，秋金星子叶叶片的不定芽再生频率达到33.3％；②山楂的胚轴分化出不定芽，而胚根只形成了愈伤组织，未能分化不定芽；③花后40 d的山楂幼胚离体培养的成苗率很低，供试的15份资源的幼胚的成苗率都小于20％，而成熟胚离体培养的成苗率较高，在50％以上。
9．用0.5％秋水仙碱＋1％二甲基亚砜的混合溶液处理隆化粉肉的成熟胚48 h，然后在SC＋IBA 0.1 mg／L＋TDZ 1.0 mg／L培养基上诱导芽分化。染色体数目观察结果表明，67个检测植株中有3个是四倍体，变异率达4.5％。
Hawthorn (Crataegus spp.) is one of important germplasm resources of fruit trees. This research took hawthorn germplasm resources which are originally from China as the materials and RAPD system and ISSR system of hawthorn were established and optimized. Base on that, genetic diversity of the genus Crataegus was analyzed by RAPD markers and ISSR markers with the 96 accessions of the genus Crataegus growing at the National Hawthorn Germplasm Repository in Shenyang. Besides these, the in vitro regeneration system of hawthorn was established and the tetraploid plants were inducted. The main results were as follows:
1. The modified CTAB method (3% CTAB in the extraction buffer) and the QIAGEN Plant DNeasy Kit were the suitable protocols for the extraction of total DNA of hawthorn. The production of total DNA was lower with the modified CTAB method, but the purity was relatively high and OD_(260)/OD_(280) value was between 1.638 and 1.889. Both the production and the purity of total DNA was high with the QIAGEN Plant DNeasy Kit, since total DNA of the 8 samples tested in this research were higher than 100μg/g and OD_(260)/OD_(280) value were between 1.758 and 1.850. RAPD and ISSR amplifications were carried on using total DNA of hawthorn with the modified CTAB method and the QIAGEN Plant DNeasy Kit, and several clear bands with better polymorphism, stability and repeatability could be obtained. Meanwhile, RAPD and ISSR amplifications were carried on using total DNA of hawthorn with the SDS method, but no band was obtained.
2. A number of factors affected the amplification results of RAPD were investigated such as cycle numbers, Mg~(2+) concentration, quality of DNA template and kinds and concentration of Taq DNA polymerase etc. The optimized RAPD analysis system of the genus Crataegus was established for the first time, i.e., reaction mixture contained I×PCR buffer (Promega), 2.0 mM MgCl_2, 0.2 mM dNTPs (Promega), 0.3μM primer, 0.5 U Taq enzyme (Tiangen) and 50 ng total DNA of hawthorn extracted with the modified CTAB method or the QIAGEN Plant DNeasy Kit in a total volume of 20μl. The cycling conditions consisted of an initial denaturation step at 94℃for 30 s, followed by 45 cycles at 94℃for 30 s, 40℃for 2 min and 72℃for 3 min, and then a final elongation step at 72℃for 7 min. Thirty primers screened out from 146 RAPD primers were suitable for the genetic analysis of the genus Crataegus based on the number, polymorphism and clarity of the bands.
3. A number of factors affected the results of ISSR were investigated such as annealing temperature, cycle numbers, Mg~(2+) concentration, quality of DNA template and kinds and concentration of Taq DNA polymerase etc. The optimized ISSR analysis system of the genus Crataegus was established for the first time, i.e., reaction mixture contained 1×PCR buffer (Promega), 3.0 mM MgCl_2, 0.2 mM dNTPs, 0.3μM primer, 0.5 U Taq enzyme (Tiangen) and 50 ng total DNA of hawthorn extracted with the modified CTAB method or the QIAGEN Plant DNeasy Kit in a total volume of 20μl. The cycling conditions consisted of an initial denaturation step at 94℃for 3 min, followed by 38 cycles at 94℃for 30s, suitable annealing temperature (screened out by temperature gradient PCR) for 1 min and 72℃for 2 min, and then a final elongation step at 72℃for 7 min. Thirteen primers, in which 8 primers were added 1～2 anchor bases in 3' end based on the repeated sequence of (GA/CT)_n or (AG/TC)_n, screened out from 36 ISSR primers were suitable for the genetic analysis of the genus Crataegus based on the number, polymorphism and clarity of the bands.
4. After 1 polymorphism band of RAPD and 16 polymorphism bands of ISSR were recovered, cloned and sequenced, 9 different genome sequences of hawthorn were obtained. Based on the analysis of genome sequences of hawthorn, specific primers were designed and SCAR conversions of one RAPD marker and five ISSR markers were done successful.
5. It is the first time to analyze the genetic diversity of Crataegus germplasms with molecular marker techniques and the results indicated that the genus Crataegus had abundance genetic diversity. One hundred and thirty-six polymorphism loci were detected from the 96 accessions of the genus Crataegus with 12 RAPD primers. The dissimilarity coefficients of different germplasms of hawthorn were between 0 and 0.73, and dendrogram with UPGMA method displayed that distance coefficients of different germplasm resources of hawthorn were between 0 and 0.58; 130 polymorphism loci were detected from the 96 accessions of the genus Crataegus with 13 RAPD primers. The dissimilarity coefficients of different germplasms of hawthorn were between 0 and 0.80, and dendrogram with UPGMA method displayed that distance coefficients of different germplasm resources of hawthorn were between 0 and 0.65.
6. Dendrograms of the genus Crataegus were drawn separately based on the RAPD marker and the ISSR marker. Dendrogram based on the RAPD marker was nearly the same as that based on the ISSR marker. But compared with the analysis result of ISSR marker, the classification result based on the RAPD marker was more closed to traditional botany classification result which was mainly based on the morphological characteristics, therefore, RAPD marker was more suitable to study on the genetic diversity of intraspecies and interspecies of the genus Crataegus.
7. Both the results of RAPD marker and ISSR marker indicated that C. brettschneideri Schneid. was a variety or a subspecies of C. pinnatifida Bge., but not a new independent species. Lvroushazha, a hawthorn resource introduced from Xiongyue Town, Gaizhou City, Liaoning Province, with black seed case and green flesh belonged to C. chlorosarca Maxim; Moreover, the hawthorn resource Zhangwushanlihong was a new species.
8. In vitro regeneration from embryo, hypocotyl and cotyledon explants of hawthorn was investigated comprehensively and the results were as follows:①The regeneration abilities of immature cotyledon, mature cotyledon and cotyledon leaf were obviously different, especially the regeneration ability of cotyledon leaves was higher and regeneration percentage of its adventitious bud was six times higher than that of mature cotyledon. On MS medium supplemented with 1.0 mg/L BA and 1.0 mg/L TDZ, the percentage of bud regeneration from cotyledon leaves of cultivar 'Qiujinxing' was 33.3%.②Adventitious bud regenerated from hypocotyl of hawthorn and on radicel no adventitious bud but only callus formed.③The percentage of seedling forming in vitro from immature embryo of hawthorn harvested after 40 d of flowering was very low and it was lower than 20% for 15 accessions tested in this research. But the percentage of seedling forming in vitro from mature embryo was relatively high—more than 50%.
9. The mature embryo of Longhuafenrou was disposed by the mixed solution of 0.5% colchicine and 1% dimethyl sulfoxide for 48 h and transferred to SC medium supplemented with 0.1 mg/L IBA and 1.0 mg/L TDZ to induce bud differentiation. The result of chromosome number indicated that 3 out of 67 tested plants were tetraploid and the variation rate reached 4.5%.
1．改进的CTAB法(提取缓冲液中含3％的CTAB)和QIAGEN试剂盒法是适宜的山楂总DNA提取方法。改进的CTAB法提取的山楂总DNA的产率较低，但是纯度较高，OD_(260)／OD_(280)比值在1.638～1.889之间；QIAGEN试剂盒法提取的山楂总DNA的产率和纯度均较高，供试8个样品的总DNA产率均高于100μg／g，OD_(260)／OD_(280)比值在1.758～1.850之间。以改进的CTAB法和QIAGEN试剂盒法提取的山楂总DNA为模板进行RAPD和ISSR扩增，能扩增出多条清晰的谱带，多态性、稳定性、重复性良好。以SDS法提取的山楂总DNA为模板进行RAPD和ISSR扩增，未能扩增出谱带。
2．对循环次数、反应体系中Mg~(2+)浓度、DNA模板质量、Taq DNA聚合酶的来源和浓度等多个影响RAPD反应结果的因子进行了研究，首次建立了优化的山楂属植物RAPD分析体系，即：PCR反应体积为20μl，内含1×PCR反应缓冲液(Promega)，2.0mmol／LMgCl_2，0.2 mmol／LdNTP(Promega)，0.3μmol／L引物，0.5 U Taq DNA聚合酶(Tiangen)，50 ng改进的CTAB法或QIAGEN试剂盒法提取的山楂总DNA；扩增程序为94℃30 s；94℃30 s，40℃2min，72℃3min，45循环，72℃7min。根据谱带的多少、多态性、清晰性，从146个RAPD引物中筛选出30个适宜于山楂属植物遗传分析的引物。
3．对退火温度、循环次数、反应体系中Mg~(2+)浓度、DNA模板质量、Taq酶的来源和浓度等影响ISSR反应结果的因子进行了研究，首次建立了优化的山楂属植物ISSR分析体系，即：PCR反应体积为20μl，内含1×PCR反应缓冲液(Promega)，3.0 mmol／LMgCl_2，0.2 mmol／L dNTP，0.3μmol／L引物，0.5 U Taq DNA聚合酶(Tiangen)，50 ng改进的CTAB法或QIAGEN试剂盒法提取的总DNA；扩增程序为94℃3 min；94℃30s，退火温度(通过温度梯度PCR筛选出的适宜温度)1 min，72℃2 min，38循环，72℃7 min。根据谱带的多少、多念性、清晰性，从36个ISSR引物中筛选出13个适宜于山楂属植物遗传分析的引物，其中有8个是基于(GA／CT) n或(AG／TC) n重复序列的在3’端加锚1～2个碱基的ISSR引物。
4．回收了1个山楂RAPD多态性谱带和16个ISSR多态性谱带，然后对回收的谱带进行克隆和测序，共获得了9个不同的山楂基因组序列。基于解析的山楂序列设计特异引物，成功地将1个RAPD标记和5个ISSR标记转化成SCAR标记。
5．首次利用分子标记技术对山楂属植物资源进行遗传研究，表明山楂属植物存在较高的遗传多样性。用12个RAPD引物在96份山楂属植物资源上共检测到136个多态性位点，不同山楂种质资源之间的相异系数在0～0.73之间，UPGMA法聚类分析结果显示不同山楂种质资源之间距离系数在0～0.58之间；用13个ISSR引物在96份山楂属植物资源上共检测到130个多态性位点，不同山楂种质资源之间的相异系数在0～0.80之间，UPGMA法聚类分析结果显示不同山楂种质资源之间距离系数在0～0.65之间。
6．分别绘制了基于RAPD标记和基于ISSR标记的山楂属植物分子系统进化树。基于RAPD标记的山楂属植物聚类结果与基于ISSR标记的聚类结果基本一致。但是与ISSR标记分析结果相比，基于RAPD标记的分类结果更接近于根据形态学特征进行的山楂传统植物学分类结果，RAPD标记更适合于山楂属植物的种内、种间的遗传变异分析。
7．RAPD和ISSR标记的分析结果均表明：在植物学分类上的伏山楂(C．brettschneideri Schneid．)属于山楂(C．pinnatifida Bge．)的一个变种或亚种，而不是一个独立的新种；从辽宁省盖州市熊岳镇引入的表现为黑皮绿肉的‘绿肉山楂’资源属于绿肉山楂(C．chlorosarca Maxim．)；而‘彰武山里红’是一个新种。
8．全面地进行了山楂胚、胚轴和子叶离体再生的研究，结果表明：①山楂未成熟子叶、成熟子叶和子叶叶片的再生不定芽能力明显不同，其中子叶叶片具有较高的再生能力，其不定芽再生频率是成熟子叶的6倍以上，在附加BA和TDZ各1.0 mg／L的MS培养基上，秋金星子叶叶片的不定芽再生频率达到33.3％；②山楂的胚轴分化出不定芽，而胚根只形成了愈伤组织，未能分化不定芽；③花后40 d的山楂幼胚离体培养的成苗率很低，供试的15份资源的幼胚的成苗率都小于20％，而成熟胚离体培养的成苗率较高，在50％以上。
9．用0.5％秋水仙碱＋1％二甲基亚砜的混合溶液处理隆化粉肉的成熟胚48 h，然后在SC＋IBA 0.1 mg／L＋TDZ 1.0 mg／L培养基上诱导芽分化。染色体数目观察结果表明，67个检测植株中有3个是四倍体，变异率达4.5％。
Hawthorn (Crataegus spp.) is one of important germplasm resources of fruit trees. This research took hawthorn germplasm resources which are originally from China as the materials and RAPD system and ISSR system of hawthorn were established and optimized. Base on that, genetic diversity of the genus Crataegus was analyzed by RAPD markers and ISSR markers with the 96 accessions of the genus Crataegus growing at the National Hawthorn Germplasm Repository in Shenyang. Besides these, the in vitro regeneration system of hawthorn was established and the tetraploid plants were inducted. The main results were as follows:
1. The modified CTAB method (3% CTAB in the extraction buffer) and the QIAGEN Plant DNeasy Kit were the suitable protocols for the extraction of total DNA of hawthorn. The production of total DNA was lower with the modified CTAB method, but the purity was relatively high and OD_(260)/OD_(280) value was between 1.638 and 1.889. Both the production and the purity of total DNA was high with the QIAGEN Plant DNeasy Kit, since total DNA of the 8 samples tested in this research were higher than 100μg/g and OD_(260)/OD_(280) value were between 1.758 and 1.850. RAPD and ISSR amplifications were carried on using total DNA of hawthorn with the modified CTAB method and the QIAGEN Plant DNeasy Kit, and several clear bands with better polymorphism, stability and repeatability could be obtained. Meanwhile, RAPD and ISSR amplifications were carried on using total DNA of hawthorn with the SDS method, but no band was obtained.
2. A number of factors affected the amplification results of RAPD were investigated such as cycle numbers, Mg~(2+) concentration, quality of DNA template and kinds and concentration of Taq DNA polymerase etc. The optimized RAPD analysis system of the genus Crataegus was established for the first time, i.e., reaction mixture contained I×PCR buffer (Promega), 2.0 mM MgCl_2, 0.2 mM dNTPs (Promega), 0.3μM primer, 0.5 U Taq enzyme (Tiangen) and 50 ng total DNA of hawthorn extracted with the modified CTAB method or the QIAGEN Plant DNeasy Kit in a total volume of 20μl. The cycling conditions consisted of an initial denaturation step at 94℃for 30 s, followed by 45 cycles at 94℃for 30 s, 40℃for 2 min and 72℃for 3 min, and then a final elongation step at 72℃for 7 min. Thirty primers screened out from 146 RAPD primers were suitable for the genetic analysis of the genus Crataegus based on the number, polymorphism and clarity of the bands.
3. A number of factors affected the results of ISSR were investigated such as annealing temperature, cycle numbers, Mg~(2+) concentration, quality of DNA template and kinds and concentration of Taq DNA polymerase etc. The optimized ISSR analysis system of the genus Crataegus was established for the first time, i.e., reaction mixture contained 1×PCR buffer (Promega), 3.0 mM MgCl_2, 0.2 mM dNTPs, 0.3μM primer, 0.5 U Taq enzyme (Tiangen) and 50 ng total DNA of hawthorn extracted with the modified CTAB method or the QIAGEN Plant DNeasy Kit in a total volume of 20μl. The cycling conditions consisted of an initial denaturation step at 94℃for 3 min, followed by 38 cycles at 94℃for 30s, suitable annealing temperature (screened out by temperature gradient PCR) for 1 min and 72℃for 2 min, and then a final elongation step at 72℃for 7 min. Thirteen primers, in which 8 primers were added 1～2 anchor bases in 3' end based on the repeated sequence of (GA/CT)_n or (AG/TC)_n, screened out from 36 ISSR primers were suitable for the genetic analysis of the genus Crataegus based on the number, polymorphism and clarity of the bands.
4. After 1 polymorphism band of RAPD and 16 polymorphism bands of ISSR were recovered, cloned and sequenced, 9 different genome sequences of hawthorn were obtained. Based on the analysis of genome sequences of hawthorn, specific primers were designed and SCAR conversions of one RAPD marker and five ISSR markers were done successful.
5. It is the first time to analyze the genetic diversity of Crataegus germplasms with molecular marker techniques and the results indicated that the genus Crataegus had abundance genetic diversity. One hundred and thirty-six polymorphism loci were detected from the 96 accessions of the genus Crataegus with 12 RAPD primers. The dissimilarity coefficients of different germplasms of hawthorn were between 0 and 0.73, and dendrogram with UPGMA method displayed that distance coefficients of different germplasm resources of hawthorn were between 0 and 0.58; 130 polymorphism loci were detected from the 96 accessions of the genus Crataegus with 13 RAPD primers. The dissimilarity coefficients of different germplasms of hawthorn were between 0 and 0.80, and dendrogram with UPGMA method displayed that distance coefficients of different germplasm resources of hawthorn were between 0 and 0.65.
6. Dendrograms of the genus Crataegus were drawn separately based on the RAPD marker and the ISSR marker. Dendrogram based on the RAPD marker was nearly the same as that based on the ISSR marker. But compared with the analysis result of ISSR marker, the classification result based on the RAPD marker was more closed to traditional botany classification result which was mainly based on the morphological characteristics, therefore, RAPD marker was more suitable to study on the genetic diversity of intraspecies and interspecies of the genus Crataegus.
7. Both the results of RAPD marker and ISSR marker indicated that C. brettschneideri Schneid. was a variety or a subspecies of C. pinnatifida Bge., but not a new independent species. Lvroushazha, a hawthorn resource introduced from Xiongyue Town, Gaizhou City, Liaoning Province, with black seed case and green flesh belonged to C. chlorosarca Maxim; Moreover, the hawthorn resource Zhangwushanlihong was a new species.
8. In vitro regeneration from embryo, hypocotyl and cotyledon explants of hawthorn was investigated comprehensively and the results were as follows:①The regeneration abilities of immature cotyledon, mature cotyledon and cotyledon leaf were obviously different, especially the regeneration ability of cotyledon leaves was higher and regeneration percentage of its adventitious bud was six times higher than that of mature cotyledon. On MS medium supplemented with 1.0 mg/L BA and 1.0 mg/L TDZ, the percentage of bud regeneration from cotyledon leaves of cultivar 'Qiujinxing' was 33.3%.②Adventitious bud regenerated from hypocotyl of hawthorn and on radicel no adventitious bud but only callus formed.③The percentage of seedling forming in vitro from immature embryo of hawthorn harvested after 40 d of flowering was very low and it was lower than 20% for 15 accessions tested in this research. But the percentage of seedling forming in vitro from mature embryo was relatively high—more than 50%.
9. The mature embryo of Longhuafenrou was disposed by the mixed solution of 0.5% colchicine and 1% dimethyl sulfoxide for 48 h and transferred to SC medium supplemented with 0.1 mg/L IBA and 1.0 mg/L TDZ to induce bud differentiation. The result of chromosome number indicated that 3 out of 67 tested plants were tetraploid and the variation rate reached 4.5%.
引文
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