作物品种分子标记鉴定及指纹图谱构建研究
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摘要
种子是农业生产中最基本的生产资料。快速、准确、高效的作物品种鉴定及指纹图谱构建方法研究及其应用,可以满足农业生产中不同条件下品种鉴定的需要,对保证农业生产中种子质量具有重要的意义。选择适宜的分子标记及其配套检测技术,建立一套简便、经济、准确而高效的作物品种鉴定和指纹图谱构建技术体系,是打击假冒伪劣种子、保护作物遗传育种家、种子生产经营企业和农民的合法权益、规范我国种子市场和提高我国种子产业国际市场竞争力的迫切需要。本研究以我国主要农作物水稻、棉花、小麦、玉米及烟草为研究对象,研究其品种分子标记鉴定及指纹图谱构建方法，主要结果如下
1.利用均匀设计方法对水稻、棉花、烟草、小麦和玉米的ISSR-PCR反应体系分别进行优化,得到每种作物各自适宜的扩增结果稳定的ISSR-PCR反应体系,并对24个水稻品种、12个烟草品种、10个棉花品种、8个小麦品种和18个玉米品种分别进行品种鉴定。结果显示,PCR反应体系中Taq酶Mg2-、dNTPs及引物浓度或用量的差异均会不同程度影响ISSR-PCR扩增效果；不同作物各自适宜的ISSR-PCR反应体系不同;2条ISSR引物(UBC900和UBC825)组合可以成功鉴别24个水稻品种,3条ISSR引物(UBC848, UBC841和UBC830)组合可以将12个烟草品种鉴别出来,ISSR引物UBC849可以成功鉴别出8个近亲小麦品种,3条ISSR引物(UBC807,UBC811和UBC820)组合可以将10个棉花品种鉴别出来、3条引物(UBC807, UBC811和UBC822)组合可以成功鉴别出18个玉米品种。本试验研究结果表明,均匀设计是一种有效的ISSR-PCR反应体系优化方法,ISSR可以用于室内水稻、棉花、烟草、小麦和玉米品种的快速鉴定,是一种很有潜力的作物品种鉴定和指纹图谱构建技术。
2.构建了利用琼脂糖凝胶电泳检测SSR和ISSR分子标记进行作物品种鉴定和指纹图谱构建的新方法。该方法包括以下步骤：用改进的CTAB法提取种子或幼苗DNA;以提取的DNA为模板进行SSR或ISSR-PCR扩增；琼脂糖凝胶电泳检测(GelRed染色)PCR扩增产物,并在全自动凝胶成像系统紫外光下拍照：对电泳图谱进行数据分析。采用新方法构建的DNA指纹图谱,即以品种编号、扩增谱带带型编号、每条谱带的编号及“0”“1”阵列为基本信息,分别制得每种作物所有参试品种特定单一SSR或ISSR引物扩增所得的DNA指纹图谱,根据指纹差异进行品种鉴定。该方法与以往指纹图谱构建方法相比,检索更加方便、快捷、更适合农作物品种快速鉴定的要求。
3.利用多态性信息含量(PIC)、平均位点信息含量(Ibav)、引物分辨能力(R)、标记指数(MI)、引物鉴别力(D)和极限引物鉴别力(DL)作为评价指标,比较了SSR和ISSR两种分子标记在水稻品种鉴定中的效率。结果显示,SSR分子标记的PIC(0.33)和Ibav(0.47)比ISSR分子标记(0.28和0.41)略高,而ISSR分子标记的D值(0.642)和DL值(0.636)比SSR分子标记(0.614和0.606)略高。但是,ISSR分子标记的R值和MI(2.41和1.63)均比SSR(1.03和0.72)高1倍以上。因此,根据本试验结果推测ISSR较SSR分子标记具有更高的水稻品种鉴定效率。但是,多次重复试验表明,ISSR适用于水稻品种快速鉴别,但该技术还需要进一步改进以提高其稳定性。SSR比ISSR扩增结果更稳定,且SSR扩增产物更适用于现代分子标记自动化检测技术。因此,选择SSR作为理想分子标记用于本论文作物品种分子标记鉴定和指纹图谱构建方法的深入研究。
4.利用SSR分子标记技术成功构建浙江省48个主栽水稻品种的DNA指纹图谱。18对多态性SSR引物在48个水稻品种中共扩增出42条电泳谱带,其中41(97.6%)条是多态性谱带,其中每对引物检测出多态性谱带的数目从1条(RM210)到4条(RM16)不等,平均每对引物2.28条。每对引物扩增的谱带带型数目最少为2种,最多5种,平均每对引物2.88种。根据每对引物的扩增图谱,分别构建18对引物对48个水稻品种的DNA指纹图谱,根据指纹图谱鉴定结果,48个水稻品种中的40个品种可以被——鉴别出来。根据指纹图谱对8组近亲品种进行品种鉴别,除了秀水114和秀水09两个品种无法鉴别外,其他7组近亲品种都可以——鉴别出来。18对SSR引物的UPGMA聚类结果分为两组：第一组包含34个籼稻品种,第二组则聚集了14个粳稻品种。其中,引物RM249和RM250可以分别将粳稻和籼稻品种鉴别出来。研究结果表明,SSR分子标记和琼脂糖凝胶电泳(GelRed染色)检测相结合的方法可以用于常规室内便捷、经济的水稻品种鉴定以及粳稻和籼稻种质间的鉴别。
5. TP-M13-SSR技术是基于毛细管电泳荧光检测的高通量低成本SSR扩增产物检测分析技术。利用TP-M13-SSR技术构建130个陆地棉品种的DNA指纹图谱。以来自10个国家的130个陆地棉品种为材料,利用TP-M13-SSR毛细管电泳荧光检测方法对初步筛选出的在陆地棉种质资源遗传多样性分析中具有多态性的100对SSR引物进行分析,其中60组TP-M13-SSR引物具有稳定、清晰且多态性的扩增片段,扩增片段大小在102bp到388bp之间。根据毛细管电泳荧光检测分析结果,选用其中50组扩增稳定且多态性丰富的TP-M13-SSR引物用于130个陆地棉品种的DNA指纹图谱构建。50组TP-M13-SSR引物共扩增出368条核苷酸片段,平均每组引物7.36条。根据每组引物扩增的核苷酸片段数目和长度信息,利用品种鉴定系统软件V1.0构建130个陆地棉品种的DNA指纹图谱数据库。根据构建的DNA指纹数据,可以将130个品种鉴别出来。该数据库可以用于SSR指纹数据的查询及品种鉴定中待测品种与标准样品的指纹比对。研究结果表明,TP-M13-SSR毛细管电泳荧光检测方法适用于陆地棉品种标准DNA指纹图谱构建和品种鉴定,且更适合于大量样品指纹图谱构建。TP-M13-SSR毛细管电泳荧光检测方法与传统凝胶电泳方法和作物品种DNA分子标记指纹图谱数据库相结合,应该是作物品种分子标记快速、准确鉴定的有效方法之一
6.高分辨率熔解曲线分析是基于实时荧光定量PCR技术发展起来的新型基因分型技术。利用SSR高分辨率熔解曲线分析检测方法,成功构建了浙江省3个主栽水稻杂交种及其亲本的DNA指纹图谱。研究结果表明,SSR高分辨率熔解曲线分析检测方法适用于水稻品种高效鉴定,具有高通量、高灵敏度、自动化、快速、直观等特点,满足大小不同规模样品快速、准确鉴别的要求。
7.比较了三种基于SSR分子标记的指纹图谱构建方法,即琼脂糖凝胶电泳检测、TP-M13-SSR毛细管电泳荧光检测和SSR高分辨率熔解曲线分析检测的优劣,提出高效农作物品种鉴定和指纹图谱构建的策略。首先,利用高浓度琼脂糖凝胶电泳检测方法,以小样本品种为测试目标,检测引物扩增片段的大小和数量及其扩增效果,筛选出扩增结果清晰、稳定且具有多态性的引1物;然后,用TP-M13-SSR毛细管电泳荧光检测方法构建大量品种的标准DNA指纹图谱；最后,以标准DNA指纹图谱为参考进行作物品种鉴定。扩增片段大小相差大于10bp的引物,可以选用高浓度琼脂糖凝胶电泳检测方法；检测扩增片段大小相差较小的引物,若待测品种数目很大,选用高通量的TP-M13-SSR毛细管电泳荧光检测或者SSR高分辨率熔解曲线分析的方法,如果只有少量品种,可优先选用SSR高分辨率熔解曲线分析的方法。
Seed was the most basic means of agricultural production. Fast, accurate and high efficient crop cultivar identification and DNA fingerprinting methods and their application were of great importance in seed quality control. Combining suitable molecular markers with detection techniques to invent simple, low cost, accurate and high efficient technical system for cultivar identification and fingerprinting was urgent need to crack down on counterfeit and shoddy seeds, protect the legitimate rights and interests of the breeder, seed company and farmers, standardize seed market and improve the international competitiveness of Chinese seed industry. In present study, rice, cotton, wheat, maize and tobacco were selected to study the cultivar identification and fingerprinting methods. The achieved results were as follows:
1. Uniform design was applied to optimize ISSR-PCR reaction system for rice. cotton, tobacco, wheat and maize, and the optimal ISSR-PCR reaction systems for each crop were established, respectively.24rice cultivars.12tobacco cultivars.10cotton cultivars.8wheat cultivars and18maize cultivars were identified using the optimal PCR reaction systems, respectively. The results showed that variations of Mg2-, dNTPs. primer, and Taq polymerase concentrations changed the fingerprinting patterns; each crop had different optimal ISSR-PCR reaction system. Combination of2ISSR primers (UBC900and UBC825) was able to identify24rice cultivars.3ISSR primers (UBC848. UBC841and UBC830) were able to identify12tobacco cultivars. Combination of3ISSR primers (UBC807. UBC811and UBC820) could identify10cotton cultivars. ISSR primer UBC849was able to discriminate8close-related wheat cultivars. A group of3ISSR primers (UBC807. UBC811and UBC822) were able to identify18maize cultivars. The results showed that uniform design was an efficient method for optimization of ISSR-PCR reaction systems. ISSR molecular markers could be used in rapid cultivar identification of rice, cotton, tobacco, wheat and maize, which might be a potential technique for crop cultivar identification and fingerprinting.
2. New method for crop variety identification and fingerprinting based on SSR and ISSR molecular markerswas constructed. The new method included the following steps:extracting DNA of seedlings with improved CTAB method; SSR and ISSR PCR amplification with the genomic DNA as the template; detecting PCR products by agarose gel electrophoresis (staining with GelRed) and take pictures in the automated gel imaging system under UV light:analyzing the data of electrophoresis bands. DNA fingerprinting constructed by the new method was based on cultivar number, type of amplified bands, band number and'0'and'1'array as the basic information. DNA fingerprinting was prepared for each tested cultivars based on each SSR or ISSR primers. The method was prior to the previous methods, which was more convenient, faster and more suitable for the rapid identification of crop cultivar.
3. Comparing the discriminating efficiency between SSR and ISSR markers on rice fingerprinting and cultivar identification based on PIC (polymorphic information content), Ibav (average band informativeness), R (Resolving Power). MI (Marker Index), D (Discriminating Power Calculated) and D□(Discriminating Power Estimated). The results showed that average PIC and Ibav values of SSR (0.33,0.47) were higher than ISSR (0.28.0.41). respectively, and the average D, DL, R and MI values of ISSR (0.642,0.636,2.41.1.63) were higher compared with SSR (0.614,0.606,1.03.0.72), respectively. It was indicated that both SSR and ISSR molecular markers were suitable for rapid rice cultivar identification, and ISSR might have a higher discriminating efficiency in rice cultivar identification compared with SSR marker system. Combination of SSR and ISSR markers might be a capable strategy in fingerprinting and cultivar identification of rice in further study. However, the stability of ISSR technique might be improved in further study, and SSR molecular markers were much steadier and more suitable for automatic analysis. So, SSR molecular markers were selected as the ideal marker for further study of crop cultivar identification and fingerprinting in present paper.
4. DNA fingerprinting of the48main rice cultivars in Zhejiang province were constructed based on SSR molecular markers. A total of42bands were produced with the18polymorphic SSR primer pairs,41(97.6%) of which were polymorphic. The number of polymorphic bands detected by each SSR primer pair ranged from1(RM210) to4(RM16), with an average of2.28bands per primer pair. The number of band patterns produced by each primer pair ranged from2to5, with an average of2.88. DNA fingerprinting of48rice cultivars based on each primer pair were constructed, which were able to identify40out of the48rice cultivars.7of the8groups of close-related cultivars were able to be identified based on the fingerprinting. UPGMA clustering results based on18polymorphic SSR primer pairs divided the48rice cultivars into two groups: the first group consists of34indica rice cultivars, and the second group gathered14japonica rice cultivars. And the primer pairs RM249and RM250could identify japonica from indica rice cultivars. The results showed that combination of SSR molecular markers and agarose gel electrophoresis (staining with GelRed) detection method could be used for convenient, economy rice cultivar identification, and japonica and indica germplasm identification.
5. TP-M13-SSR technique was a high throughput and low cost analysis method of SSR product based on fluorescence detection of capillary electrophoresis. DNA fingerprinting of130upland cotton were constructed with TP-M13-SSR method.130cotton cultivars from10countries were selected as the test materials to analyze the100SSR primer pairs which showed good polymorphic among upland cotton in previous study. Among the100groups of TP-M13-SSR primers,60sets of primers showed steady, clear and polymorphic amplification results, and the length of produced fragments ranged from102bp to388bp.50sets of steady and polymorphic TP-M13-SSR primers were selected for fingerprinting of130cotton cultivars. A total of368DNA fragments were produced with the50sets of primers, with an average of7.36. DNA fingerprinting database of130cotton cultivars were constructed using the cultivar identification system software (V1.0) based on the number and length information of the nucleotide fragment produced.130cotton cultivars were able to be identified based on the fingerprinting. The fingerprinting database could be used for fingerprinting data query and match between tested material and standard cultivar. The results showed that TP-M13-SSR method was suitable for standard fingerprinting and cultivar identification of upland cotton, especially for a large number of samples. Combination of TP-M13-SSR technique, conventional gel electrophoresis. and DNA fingerprinting database of crop cultivars might be an efficient strategy for rapid and accurate crop cultivar identification based on molecular markers.
6. High resolution melting curve analysis (HRM) was a new genotyping method developed based on real-time fluorescence quantitative PCR technology, and its principle was that the melting temperatures of nucleotide fragments were different base on their different base composition or sequence. SSR molecular markers combining high resolution melting curve analysis method were used to construct the DNA fingerprinting of3main cultivated rice hybrids and their parents in Zhejiang province. The results showed that the method based on combination of SSR molecular markers with high resolution melting curve analysis (SSR-HRM) was suitable for rice cultivar identification and had the characteristics of high throughput, high sensitivity, automatic, fast, and intuitive.
7. The advantages and disadvantages of agarose gel electrophoresis, TP-M13-SSR capillary fluorescence detection and SSR high resolution melting curve analysis were compared. A suitable strategy for crop cultivar identification and fingerprinting was proposed. Firstly, high concentration agarose gel electrophoresis was used to detect the sizes, numbers and amplification effect of the produced fragments to screen out SSR primers with clear, stable and polymorphic bands. Secondly, standard DNA fingerprinting was constructed by the TP-M13-SSR capillary electrophoresis with fluorescence detection method. Finally, the fingerprinting was used for cultivar identification. If the difference among fragment sizes of the primer was greater than10bp. high concentration of agarose gel electrophoresis detection method could be selected. If the difference among fragment sizes was smaller than10bp. capillary electrophoresis with fluorescence detection or high resolution melting curve analysis should be selected if there were a large number of tested cultivars. or high resolution melting curve analysis should be given priority with only a small number of samples.
1.利用均匀设计方法对水稻、棉花、烟草、小麦和玉米的ISSR-PCR反应体系分别进行优化,得到每种作物各自适宜的扩增结果稳定的ISSR-PCR反应体系,并对24个水稻品种、12个烟草品种、10个棉花品种、8个小麦品种和18个玉米品种分别进行品种鉴定。结果显示,PCR反应体系中Taq酶Mg2-、dNTPs及引物浓度或用量的差异均会不同程度影响ISSR-PCR扩增效果；不同作物各自适宜的ISSR-PCR反应体系不同;2条ISSR引物(UBC900和UBC825)组合可以成功鉴别24个水稻品种,3条ISSR引物(UBC848, UBC841和UBC830)组合可以将12个烟草品种鉴别出来,ISSR引物UBC849可以成功鉴别出8个近亲小麦品种,3条ISSR引物(UBC807,UBC811和UBC820)组合可以将10个棉花品种鉴别出来、3条引物(UBC807, UBC811和UBC822)组合可以成功鉴别出18个玉米品种。本试验研究结果表明,均匀设计是一种有效的ISSR-PCR反应体系优化方法,ISSR可以用于室内水稻、棉花、烟草、小麦和玉米品种的快速鉴定,是一种很有潜力的作物品种鉴定和指纹图谱构建技术。
2.构建了利用琼脂糖凝胶电泳检测SSR和ISSR分子标记进行作物品种鉴定和指纹图谱构建的新方法。该方法包括以下步骤：用改进的CTAB法提取种子或幼苗DNA;以提取的DNA为模板进行SSR或ISSR-PCR扩增；琼脂糖凝胶电泳检测(GelRed染色)PCR扩增产物,并在全自动凝胶成像系统紫外光下拍照：对电泳图谱进行数据分析。采用新方法构建的DNA指纹图谱,即以品种编号、扩增谱带带型编号、每条谱带的编号及“0”“1”阵列为基本信息,分别制得每种作物所有参试品种特定单一SSR或ISSR引物扩增所得的DNA指纹图谱,根据指纹差异进行品种鉴定。该方法与以往指纹图谱构建方法相比,检索更加方便、快捷、更适合农作物品种快速鉴定的要求。
3.利用多态性信息含量(PIC)、平均位点信息含量(Ibav)、引物分辨能力(R)、标记指数(MI)、引物鉴别力(D)和极限引物鉴别力(DL)作为评价指标,比较了SSR和ISSR两种分子标记在水稻品种鉴定中的效率。结果显示,SSR分子标记的PIC(0.33)和Ibav(0.47)比ISSR分子标记(0.28和0.41)略高,而ISSR分子标记的D值(0.642)和DL值(0.636)比SSR分子标记(0.614和0.606)略高。但是,ISSR分子标记的R值和MI(2.41和1.63)均比SSR(1.03和0.72)高1倍以上。因此,根据本试验结果推测ISSR较SSR分子标记具有更高的水稻品种鉴定效率。但是,多次重复试验表明,ISSR适用于水稻品种快速鉴别,但该技术还需要进一步改进以提高其稳定性。SSR比ISSR扩增结果更稳定,且SSR扩增产物更适用于现代分子标记自动化检测技术。因此,选择SSR作为理想分子标记用于本论文作物品种分子标记鉴定和指纹图谱构建方法的深入研究。
4.利用SSR分子标记技术成功构建浙江省48个主栽水稻品种的DNA指纹图谱。18对多态性SSR引物在48个水稻品种中共扩增出42条电泳谱带,其中41(97.6%)条是多态性谱带,其中每对引物检测出多态性谱带的数目从1条(RM210)到4条(RM16)不等,平均每对引物2.28条。每对引物扩增的谱带带型数目最少为2种,最多5种,平均每对引物2.88种。根据每对引物的扩增图谱,分别构建18对引物对48个水稻品种的DNA指纹图谱,根据指纹图谱鉴定结果,48个水稻品种中的40个品种可以被——鉴别出来。根据指纹图谱对8组近亲品种进行品种鉴别,除了秀水114和秀水09两个品种无法鉴别外,其他7组近亲品种都可以——鉴别出来。18对SSR引物的UPGMA聚类结果分为两组：第一组包含34个籼稻品种,第二组则聚集了14个粳稻品种。其中,引物RM249和RM250可以分别将粳稻和籼稻品种鉴别出来。研究结果表明,SSR分子标记和琼脂糖凝胶电泳(GelRed染色)检测相结合的方法可以用于常规室内便捷、经济的水稻品种鉴定以及粳稻和籼稻种质间的鉴别。
5. TP-M13-SSR技术是基于毛细管电泳荧光检测的高通量低成本SSR扩增产物检测分析技术。利用TP-M13-SSR技术构建130个陆地棉品种的DNA指纹图谱。以来自10个国家的130个陆地棉品种为材料,利用TP-M13-SSR毛细管电泳荧光检测方法对初步筛选出的在陆地棉种质资源遗传多样性分析中具有多态性的100对SSR引物进行分析,其中60组TP-M13-SSR引物具有稳定、清晰且多态性的扩增片段,扩增片段大小在102bp到388bp之间。根据毛细管电泳荧光检测分析结果,选用其中50组扩增稳定且多态性丰富的TP-M13-SSR引物用于130个陆地棉品种的DNA指纹图谱构建。50组TP-M13-SSR引物共扩增出368条核苷酸片段,平均每组引物7.36条。根据每组引物扩增的核苷酸片段数目和长度信息,利用品种鉴定系统软件V1.0构建130个陆地棉品种的DNA指纹图谱数据库。根据构建的DNA指纹数据,可以将130个品种鉴别出来。该数据库可以用于SSR指纹数据的查询及品种鉴定中待测品种与标准样品的指纹比对。研究结果表明,TP-M13-SSR毛细管电泳荧光检测方法适用于陆地棉品种标准DNA指纹图谱构建和品种鉴定,且更适合于大量样品指纹图谱构建。TP-M13-SSR毛细管电泳荧光检测方法与传统凝胶电泳方法和作物品种DNA分子标记指纹图谱数据库相结合,应该是作物品种分子标记快速、准确鉴定的有效方法之一
6.高分辨率熔解曲线分析是基于实时荧光定量PCR技术发展起来的新型基因分型技术。利用SSR高分辨率熔解曲线分析检测方法,成功构建了浙江省3个主栽水稻杂交种及其亲本的DNA指纹图谱。研究结果表明,SSR高分辨率熔解曲线分析检测方法适用于水稻品种高效鉴定,具有高通量、高灵敏度、自动化、快速、直观等特点,满足大小不同规模样品快速、准确鉴别的要求。
7.比较了三种基于SSR分子标记的指纹图谱构建方法,即琼脂糖凝胶电泳检测、TP-M13-SSR毛细管电泳荧光检测和SSR高分辨率熔解曲线分析检测的优劣,提出高效农作物品种鉴定和指纹图谱构建的策略。首先,利用高浓度琼脂糖凝胶电泳检测方法,以小样本品种为测试目标,检测引物扩增片段的大小和数量及其扩增效果,筛选出扩增结果清晰、稳定且具有多态性的引1物;然后,用TP-M13-SSR毛细管电泳荧光检测方法构建大量品种的标准DNA指纹图谱；最后,以标准DNA指纹图谱为参考进行作物品种鉴定。扩增片段大小相差大于10bp的引物,可以选用高浓度琼脂糖凝胶电泳检测方法；检测扩增片段大小相差较小的引物,若待测品种数目很大,选用高通量的TP-M13-SSR毛细管电泳荧光检测或者SSR高分辨率熔解曲线分析的方法,如果只有少量品种,可优先选用SSR高分辨率熔解曲线分析的方法。
Seed was the most basic means of agricultural production. Fast, accurate and high efficient crop cultivar identification and DNA fingerprinting methods and their application were of great importance in seed quality control. Combining suitable molecular markers with detection techniques to invent simple, low cost, accurate and high efficient technical system for cultivar identification and fingerprinting was urgent need to crack down on counterfeit and shoddy seeds, protect the legitimate rights and interests of the breeder, seed company and farmers, standardize seed market and improve the international competitiveness of Chinese seed industry. In present study, rice, cotton, wheat, maize and tobacco were selected to study the cultivar identification and fingerprinting methods. The achieved results were as follows:
1. Uniform design was applied to optimize ISSR-PCR reaction system for rice. cotton, tobacco, wheat and maize, and the optimal ISSR-PCR reaction systems for each crop were established, respectively.24rice cultivars.12tobacco cultivars.10cotton cultivars.8wheat cultivars and18maize cultivars were identified using the optimal PCR reaction systems, respectively. The results showed that variations of Mg2-, dNTPs. primer, and Taq polymerase concentrations changed the fingerprinting patterns; each crop had different optimal ISSR-PCR reaction system. Combination of2ISSR primers (UBC900and UBC825) was able to identify24rice cultivars.3ISSR primers (UBC848. UBC841and UBC830) were able to identify12tobacco cultivars. Combination of3ISSR primers (UBC807. UBC811and UBC820) could identify10cotton cultivars. ISSR primer UBC849was able to discriminate8close-related wheat cultivars. A group of3ISSR primers (UBC807. UBC811and UBC822) were able to identify18maize cultivars. The results showed that uniform design was an efficient method for optimization of ISSR-PCR reaction systems. ISSR molecular markers could be used in rapid cultivar identification of rice, cotton, tobacco, wheat and maize, which might be a potential technique for crop cultivar identification and fingerprinting.
2. New method for crop variety identification and fingerprinting based on SSR and ISSR molecular markerswas constructed. The new method included the following steps:extracting DNA of seedlings with improved CTAB method; SSR and ISSR PCR amplification with the genomic DNA as the template; detecting PCR products by agarose gel electrophoresis (staining with GelRed) and take pictures in the automated gel imaging system under UV light:analyzing the data of electrophoresis bands. DNA fingerprinting constructed by the new method was based on cultivar number, type of amplified bands, band number and'0'and'1'array as the basic information. DNA fingerprinting was prepared for each tested cultivars based on each SSR or ISSR primers. The method was prior to the previous methods, which was more convenient, faster and more suitable for the rapid identification of crop cultivar.
3. Comparing the discriminating efficiency between SSR and ISSR markers on rice fingerprinting and cultivar identification based on PIC (polymorphic information content), Ibav (average band informativeness), R (Resolving Power). MI (Marker Index), D (Discriminating Power Calculated) and D□(Discriminating Power Estimated). The results showed that average PIC and Ibav values of SSR (0.33,0.47) were higher than ISSR (0.28.0.41). respectively, and the average D, DL, R and MI values of ISSR (0.642,0.636,2.41.1.63) were higher compared with SSR (0.614,0.606,1.03.0.72), respectively. It was indicated that both SSR and ISSR molecular markers were suitable for rapid rice cultivar identification, and ISSR might have a higher discriminating efficiency in rice cultivar identification compared with SSR marker system. Combination of SSR and ISSR markers might be a capable strategy in fingerprinting and cultivar identification of rice in further study. However, the stability of ISSR technique might be improved in further study, and SSR molecular markers were much steadier and more suitable for automatic analysis. So, SSR molecular markers were selected as the ideal marker for further study of crop cultivar identification and fingerprinting in present paper.
4. DNA fingerprinting of the48main rice cultivars in Zhejiang province were constructed based on SSR molecular markers. A total of42bands were produced with the18polymorphic SSR primer pairs,41(97.6%) of which were polymorphic. The number of polymorphic bands detected by each SSR primer pair ranged from1(RM210) to4(RM16), with an average of2.28bands per primer pair. The number of band patterns produced by each primer pair ranged from2to5, with an average of2.88. DNA fingerprinting of48rice cultivars based on each primer pair were constructed, which were able to identify40out of the48rice cultivars.7of the8groups of close-related cultivars were able to be identified based on the fingerprinting. UPGMA clustering results based on18polymorphic SSR primer pairs divided the48rice cultivars into two groups: the first group consists of34indica rice cultivars, and the second group gathered14japonica rice cultivars. And the primer pairs RM249and RM250could identify japonica from indica rice cultivars. The results showed that combination of SSR molecular markers and agarose gel electrophoresis (staining with GelRed) detection method could be used for convenient, economy rice cultivar identification, and japonica and indica germplasm identification.
5. TP-M13-SSR technique was a high throughput and low cost analysis method of SSR product based on fluorescence detection of capillary electrophoresis. DNA fingerprinting of130upland cotton were constructed with TP-M13-SSR method.130cotton cultivars from10countries were selected as the test materials to analyze the100SSR primer pairs which showed good polymorphic among upland cotton in previous study. Among the100groups of TP-M13-SSR primers,60sets of primers showed steady, clear and polymorphic amplification results, and the length of produced fragments ranged from102bp to388bp.50sets of steady and polymorphic TP-M13-SSR primers were selected for fingerprinting of130cotton cultivars. A total of368DNA fragments were produced with the50sets of primers, with an average of7.36. DNA fingerprinting database of130cotton cultivars were constructed using the cultivar identification system software (V1.0) based on the number and length information of the nucleotide fragment produced.130cotton cultivars were able to be identified based on the fingerprinting. The fingerprinting database could be used for fingerprinting data query and match between tested material and standard cultivar. The results showed that TP-M13-SSR method was suitable for standard fingerprinting and cultivar identification of upland cotton, especially for a large number of samples. Combination of TP-M13-SSR technique, conventional gel electrophoresis. and DNA fingerprinting database of crop cultivars might be an efficient strategy for rapid and accurate crop cultivar identification based on molecular markers.
6. High resolution melting curve analysis (HRM) was a new genotyping method developed based on real-time fluorescence quantitative PCR technology, and its principle was that the melting temperatures of nucleotide fragments were different base on their different base composition or sequence. SSR molecular markers combining high resolution melting curve analysis method were used to construct the DNA fingerprinting of3main cultivated rice hybrids and their parents in Zhejiang province. The results showed that the method based on combination of SSR molecular markers with high resolution melting curve analysis (SSR-HRM) was suitable for rice cultivar identification and had the characteristics of high throughput, high sensitivity, automatic, fast, and intuitive.
7. The advantages and disadvantages of agarose gel electrophoresis, TP-M13-SSR capillary fluorescence detection and SSR high resolution melting curve analysis were compared. A suitable strategy for crop cultivar identification and fingerprinting was proposed. Firstly, high concentration agarose gel electrophoresis was used to detect the sizes, numbers and amplification effect of the produced fragments to screen out SSR primers with clear, stable and polymorphic bands. Secondly, standard DNA fingerprinting was constructed by the TP-M13-SSR capillary electrophoresis with fluorescence detection method. Finally, the fingerprinting was used for cultivar identification. If the difference among fragment sizes of the primer was greater than10bp. high concentration of agarose gel electrophoresis detection method could be selected. If the difference among fragment sizes was smaller than10bp. capillary electrophoresis with fluorescence detection or high resolution melting curve analysis should be selected if there were a large number of tested cultivars. or high resolution melting curve analysis should be given priority with only a small number of samples.
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