CN116042889B - A Molecular Marker Linked to the Major QTL Locus of Flowering Stage in Brassica napus and Its Application - Google Patents
A Molecular Marker Linked to the Major QTL Locus of Flowering Stage in Brassica napus and Its Application Download PDFInfo
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Abstract
本发明公开了一种与甘蓝型油菜开花期主效QTL位点连锁的分子标记及其应用,属于植物分子遗传育种领域。该分子标记为InDel分子标记,其核苷酸序列如SEQ ID NO:3所示。本发明通过检测甘蓝型油菜中该分子标记的扩增产物,发现携带该InDel标记的株系为晚开花株系,未携带该InDel标记的株系为早开花株系。本发明为甘蓝型油菜早花早熟育种改良提供了优良的分子标记,通过分子标记辅助选择选育早花材料进而可培育出早花、早熟油菜,具有准确性好、效率高、性价比高的优点。本发明将有助于油菜开花时间相关基因的克隆、开花时间相关基因功能标记的开发。
The invention discloses a molecular marker linked with a main effect QTL locus of flowering stage of Brassica napus and an application thereof, belonging to the field of plant molecular genetics and breeding. The molecular marker is an InDel molecular marker, and its nucleotide sequence is shown in SEQ ID NO:3. The invention detects the amplified product of the molecular marker in Brassica napus, and finds that the line carrying the InDel marker is a late flowering line, and the line not carrying the InDel marker is an early flowering line. The invention provides excellent molecular markers for the improvement of early-flowering and early-maturing breeding of Brassica napus, and can breed early-flowering and early-maturing rapeseed through molecular marker-assisted selection and breeding of early-flowering rapeseed, which has the advantages of good accuracy, high efficiency, and high cost performance . The invention will contribute to the cloning of genes related to flowering time and the development of functional markers of genes related to flowering time in rapeseed.
Description
技术领域technical field
本发明涉及植物分子遗传育种领域,特别是涉及一种与甘蓝型油菜开花期主效QTL位点连锁的分子标记及其应用。The invention relates to the field of plant molecular genetics and breeding, in particular to a molecular marker linked to a major QTL locus for flowering stage of Brassica napus and its application.
背景技术Background technique
开花是高等植物生长过程中的重要转折点,表示植物由营养生长向生殖生长转变。在外界环境因素和植物内源物质共同作用下,植物从幼苗阶段生长发育到成株期并经诱导而开花,这在植物的一生中是最为重要的阶段。植物不同于动物,植物只有不断改变自身来适应外界环境,才能更好地生存;这就是达尔文《进化论》的中心思想:物竞天择,适者生存。作物由营养生长向生殖生长转变的时间决定着作物能否适时开花,而作物能否适时开花最终决定着作物的产量和品质。甘蓝型油菜是我国重要的油料作物之一,根据其开花是否需要春化将甘蓝型油菜分为冬油菜、半冬性油菜和春油菜三种生态型。深入研究甘蓝型油菜开花调控机理将有助于育种家培育出能够适应国内不同生态环境的油菜优良品种,对于确保高产与稳产具有重要意义。Flowering is an important turning point in the growth process of higher plants, which represents the transformation of plants from vegetative growth to reproductive growth. Under the joint action of external environmental factors and plant endogenous substances, plants grow from the seedling stage to the adult stage and are induced to flower, which is the most important stage in the life of a plant. Plants are different from animals. Only by constantly changing themselves to adapt to the external environment can plants survive better; this is the central idea of Darwin's "Evolution": natural selection, survival of the fittest. The timing of crop transition from vegetative growth to reproductive growth determines whether the crop can bloom in a timely manner, and whether the crop can bloom in a timely manner ultimately determines the yield and quality of the crop. Brassica napus is one of the important oil crops in my country. According to whether it needs vernalization for flowering, Brassica napus can be divided into three ecological types: winter rape, semi-winter rape and spring rape. An in-depth study of the flowering regulation mechanism of Brassica napus will help breeders to breed excellent varieties of rapeseed that can adapt to different ecological environments in China, which is of great significance for ensuring high and stable yields.
针对甘蓝型油菜开花基因的研究,目前常用的有两种克隆开花基因的策略:一种是参照模式植物拟南芥中开花基因的信息,在甘蓝型油菜中直接进行同源序列法分离,得到各个拷贝之后,在材料中进行开花基因的表达量分析(Tadege et al 2001;Zou et al2012;Calderwood et al2021)。第二种策略是利用不同群体从正向遗传学的角度来定位甘蓝型油菜开花期QTL。其中很多研究是基于双亲的连锁作图分析,基于该策略,研究人员找到了开花调控中比较重要的基因如FLC、FT基因在甘蓝型油菜中的不同拷贝,并且发现了这些拷贝在功能上有强弱的分化(Raman et al 2013;代书桃2015;Chen et al 2018;Tudoret al 2020;Xu et al 2021)。此外,近年来大量研究是基于自然群体的关联作图分析,通过自然群体发现了很多SNP标记位于开花基因区域,这些SNP标记后期可以很好的用于分子育种中(Raman et al 2016;Xu et al 2016;Li et al 2018;Lu et al 2019;Vollrath etal 2021)。For the research on flowering genes of Brassica napus, there are currently two commonly used strategies for cloning flowering genes: one is to directly isolate the homologous sequence method in Brassica napus with reference to the information of flowering genes in the model plant Arabidopsis, and obtain After each copy, the expression levels of flowering genes were analyzed in the materials (Tadege et al 2001; Zou et al2012; Calderwood et al2021). The second strategy is to use different populations to locate QTLs for anthesis in Brassica napus from the perspective of forward genetics. Many of these studies are based on linkage mapping analysis of parents. Based on this strategy, researchers have found different copies of genes important in flowering regulation, such as FLC and FT genes in Brassica napus, and found that these copies have functional differences. Strong and weak differentiation (Raman et al 2013; Dai Shutao 2015; Chen et al 2018; Tudor et al 2020; Xu et al 2021). In addition, a large number of studies in recent years have been based on association mapping analysis of natural populations. Many SNP markers have been found in the flowering gene region through natural populations. These SNP markers can be well used in molecular breeding later (Raman et al 2016; Xu et al 2016; Xu et al. al 2016; Li et al 2018; Lu et al 2019; Vollrath et al 2021).
为了丰富甘蓝型油菜开花期的研究,本发明拟通过构建DH群体及其高密度遗传图谱,定位一个与甘蓝型油菜开花期主效QTL位点紧密连锁的分子标记,为甘蓝型油菜早花早熟育种改良提供新的思路。In order to enrich the research on the flowering period of Brassica napus, the present invention intends to locate a molecular marker closely linked to the main QTL locus of flowering stage of Brassica napus by constructing the DH population and its high-density genetic map, which is early flowering and early maturation in Brassica napus. Breeding improvement provides new ideas.
发明内容Contents of the invention
本发明的目的是提供一种与甘蓝型油菜开花期主效QTL位点连锁的分子标记及其应用,以解决上述现有技术存在的问题,本发明为甘蓝型油菜早花早熟育种改良提供了优良的分子标记,通过该分子标记辅助选择选育早花材料进而可培育出早花、早熟油菜,具有准确性好、效率高、性价比高的优点。The purpose of the present invention is to provide a molecular marker linked with the main effect QTL site of Brassica napus flowering stage and its application, so as to solve the problems in the above-mentioned prior art. Excellent molecular markers, through the molecular marker-assisted selection and breeding of early-flowering materials, early-flowering and early-maturing rapeseed can be bred, which has the advantages of good accuracy, high efficiency, and high cost performance.
本发明采用简化基因组测序的方法,针对双亲及178份DH材料进行基因组测序,然后通过基于SNP的Bin标记分析,进行该群体遗传图谱的构建。结合三年六个重复的开花期表型数据,采用WinQTLcart2.5软件的复合区间作图标准模型(model 6)进行三年六重复开花期QTL的检测,进一步确定主效QTL的稳定性。本发明发现了位于染色体C02上的一个主效开花时间QTL,命名为cqDTF-C02,该QTL是一个环境稳定的QTL。本发明预测了该QTL的候选基因,并为cqDTF-C02基因位点开发了紧密连锁的插入缺失(InDel)标记。这项研究的结果增强了对开花时间分子调控机理的理解,并提供了可用于油菜开花时间性状育种的分子标记。The present invention adopts a simplified genome sequencing method to carry out genome sequencing on parents and 178 DH materials, and then constructs the population genetic map through SNP-based Bin marker analysis. Combined with the phenotypic data of the three-year and six-repetition flowering period, the standard model of compound interval mapping (model 6) of WinQTLcart2.5 software was used to detect the QTLs of the three-year and six-replicate flowering period, and further determine the stability of the main QTL. The present invention discovers a main flowering time QTL located on chromosome C02, which is named cqDTF-C02, and the QTL is an environmentally stable QTL. The present invention predicts the candidate gene of the QTL, and develops a closely linked insertion-deletion (InDel) marker for the cqDTF-C02 gene locus. The results of this study enhance the understanding of the molecular regulation mechanism of flowering time and provide molecular markers that can be used in breeding for flowering time traits in rapeseed.
为实现上述目的,本发明提供了如下方案:To achieve the above object, the present invention provides the following scheme:
本发明提供一种甘蓝型油菜开花时间主效QTL位点的分子标记,所述分子标记为InDel分子标记,其核苷酸序列如SEQ ID NO:3所示。The present invention provides a molecular marker of the major QTL locus of flowering time in Brassica napus, the molecular marker is an InDel molecular marker, and its nucleotide sequence is shown in SEQ ID NO:3.
本发明还提供一种扩增上述的分子标记的引物组,所述引物组的核苷酸序列如SEQ ID NO:1-2所示。The present invention also provides a primer set for amplifying the above-mentioned molecular marker, the nucleotide sequence of the primer set is shown in SEQ ID NO: 1-2.
本发明还提供一种含有上述引物组的试剂盒。The present invention also provides a kit containing the above primer set.
本发明还提供一种根据上述的分子标记或上述的引物组或上述的试剂盒在甘蓝型油菜育种中的应用。The present invention also provides an application of the above-mentioned molecular marker or the above-mentioned primer set or the above-mentioned kit in the breeding of Brassica napus.
本发明还提供一种根据上述的分子标记或上述的引物组或上述的试剂盒在筛选甘蓝型油菜早花品种中的应用。The present invention also provides an application of the above-mentioned molecular marker or the above-mentioned primer set or the above-mentioned kit in screening early flowering varieties of Brassica napus.
本发明还提供一种筛选甘蓝型油菜早花品种的方法,以待测甘蓝型油菜样品的基因组DNA为模板,利用上述的引物组对模板进行PCR扩增,将扩增产物进行电泳检测。The present invention also provides a method for screening early-flowering varieties of Brassica napus, using the genomic DNA of the Brassica napus sample to be tested as a template, using the above primer set to perform PCR amplification on the template, and performing electrophoresis detection on the amplified product.
进一步地,若电泳检测结果显示出200bp的电泳条带,则样品为甘蓝型油菜晚花品种,若无200bp的电泳条带,则样品为甘蓝型油菜早花品种。Further, if the electrophoresis detection result shows a 200bp electrophoresis band, the sample is a late-flowering Brassica napus variety, and if there is no 200bp electrophoresis band, the sample is an early-flowering Brassica napus variety.
进一步地,所述PCR扩增的体系为:DNA模板2μL、2×Tap PCR Mix 5μL、上下游引物共1μL和ddH2O 2μL。Further, the PCR amplification system is: 2 μL of DNA template, 5 μL of 2×Tap PCR Mix, 1 μL of upstream and downstream primers, and 2 μL of ddH 2 O.
进一步地,所述PCR扩增的程序为:94℃预变性3min;94℃变性30s,60℃退火30s,72℃延伸45s,共35个循环;72℃延伸10min。Further, the PCR amplification procedure is: pre-denaturation at 94°C for 3 minutes; denaturation at 94°C for 30 s, annealing at 60°C for 30 s, extension at 72°C for 45 s, a total of 35 cycles; extension at 72°C for 10 min.
本发明公开了以下技术效果:The invention discloses the following technical effects:
本发明通过构建DH群体及其高密度遗传图谱,成功定位了一个甘蓝型油菜主效开花QTL位点cqDTF-C02,利用cqDTF-C02区段本身的序列设计了紧密连锁的分子标记:InDel标记C2-5,经实验验证,携带该InDel标记的株系为晚开花株系,未携带该InDel标记的株系为早开花株系。本发明为甘蓝型油菜早花早熟育种改良提供了优良的分子标记,通过分子标记辅助选择选育早花材料进而可培育出早花、早熟油菜,具有准确性好、效率高、性价比高的优点。本发明将有助于油菜开花时间相关基因的克隆、开花时间相关基因功能标记的开发。By constructing the DH population and its high-density genetic map, the present invention successfully located a major flowering QTL site cqDTF-C02 in Brassica napus, and designed a closely linked molecular marker using the sequence of the cqDTF-C02 segment itself: InDel marker C2 -5. It has been verified by experiments that the line carrying the InDel marker is a late flowering line, and the line not carrying the InDel marker is an early flowering line. The invention provides excellent molecular markers for the improvement of early flowering and early maturity breeding of Brassica napus, through molecular marker-assisted selection and breeding of early flowering materials, early flowering and early maturity rapeseed can be bred, and has the advantages of good accuracy, high efficiency, and high cost performance . The invention will contribute to the cloning of genes related to flowering time and the development of functional markers of genes related to flowering time in rapeseed.
附图说明Description of drawings
为了更清楚地说明本发明实施例或现有技术中的技术方案,下面将对实施例中所需要使用的附图作简单地介绍,显而易见地,下面描述中的附图仅仅是本发明的一些实施例,对于本领域普通技术人员来讲,在不付出创造性劳动的前提下,还可以根据这些附图获得其他的附图。In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the accompanying drawings required in the embodiments. Obviously, the accompanying drawings in the following description are only some of the present invention. Embodiments, for those of ordinary skill in the art, other drawings can also be obtained based on these drawings without any creative effort.
图1为本发明构建DH群体、表型鉴定、图谱构建、QTL定位、主效QTL候选基因鉴定以及主效QTL区间分子标记开发鉴定应用的技术流程图;Fig. 1 is a technical flow chart of the present invention for constructing DH populations, phenotypic identification, map construction, QTL mapping, main QTL candidate gene identification, and main QTL interval molecular marker development and identification application;
图2为甘蓝型油菜早花亲本158A和晚花亲本SGDH284的花期表型图;A是冬油菜环境2019年10月21日播种后于2020年03月21日拍摄田间表型图片,B是冬油菜环境2019年10月21日播种后于2020年03月28日拍摄田间表型图片;Figure 2 is the florescence phenotype of Brassica napus early-flowering parent 158A and late-flowering parent SGDH284; A is the field phenotype picture taken on March 21, 2020 after sowing in winter rapeseed environment on October 21, 2019, and B is winter rape Field phenotype pictures were taken on March 28, 2020 after sowing rapeseed environment on October 21, 2019;
图3为甘蓝型油菜158A-SGDH群体2018-2019年度一个重复、2019-2020年度三个重复和2020-2021年度两个重复的开花期表型频率分布图;Figure 3 is a diagram showing the frequency distribution of flowering stage phenotypes of Brassica napus 158A-SGDH population in 2018-2019, three repetitions in 2019-2020 and two repetitions in 2020-2021;
图4为三年六个重复开花期表型的相关性分析;Fig. 4 is the correlation analysis of six repeated flowering period phenotypes in three years;
图5为部分样品基因组DNA提取质量检测胶图;M表示DL15000 DNA Marker,1-18为部分样品基因组DNA;Fig. 5 is a part of the sample genomic DNA extraction quality detection gel map; M indicates DL15000 DNA Marker, 1-18 is part of the sample genomic DNA;
图6为简化测序获得的SNP在染色体上的分布;Figure 6 is the distribution of SNPs obtained by simplified sequencing on chromosomes;
图7为C02的连锁群标记信息;Figure 7 is the linkage group marker information of C02;
图8为甘蓝型油菜C02连锁群开花期QTL区间候选基因的鉴定;上曲线代表在三种环境中六个重复中鉴定的QTL,下曲线代表相同颜色的QTL的加性效应,主效应QTL cqDTF-C02显示在置信区间下,与开花时间相关的候选基因显示在主效QTL cqDTF-C02下;Figure 8 shows the identification of candidate genes in the flowering period QTL interval of Brassica napus C02 linkage group; the upper curve represents the QTL identified in six replicates in three environments, the lower curve represents the additive effect of QTL of the same color, the main effect QTL cqDTF -C02 is shown under the confidence interval, and candidate genes associated with flowering time are shown under the main QTL cqDTF-C02;
图9为通过cqDTF-C02基因位点特异性InDel标记C2-5分析的10个极早开花家系和10个极晚开花家系的小群体以及双亲158A(P1)和SGDH284(P2)的带型图;Figure 9 is a band diagram of 10 very early flowering families and 10 very late flowering families and the band patterns of the parents 158A (P1) and SGDH284 (P2) analyzed by cqDTF-C02 locus-specific InDel marker C2-5 ;
图10为通过cqDTF-C02基因位点特异性InDel标记C2-5分析的158A-SGDH群体A03-A47以及双亲158A(P1)和SGDH284(P2)的带型图;Figure 10 is a band diagram of the 158A-SGDH population A03-A47 analyzed by the cqDTF-C02 locus-specific InDel marker C2-5 and the parents 158A (P1) and SGDH284 (P2);
图11为在cqDTF-C02基因位点使用特异性InDel标记C2-5分析158A-SGDH群体的六个重复的开花时间,标记基因型结合表型进行方差分析的结果。Figure 11 shows the analysis of flowering time of six replicates of the 158A-SGDH population using the specific InDel marker C2-5 at the cqDTF-C02 gene locus, and the results of variance analysis of marker genotype combined with phenotype.
具体实施方式Detailed ways
现详细说明本发明的多种示例性实施方式,该详细说明不应认为是对本发明的限制,而应理解为是对本发明的某些方面、特性和实施方案的更详细的描述。Various exemplary embodiments of the present invention will now be described in detail. The detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features and embodiments of the present invention.
应理解本发明中所述的术语仅仅是为描述特别的实施方式,并非用于限制本发明。另外,对于本发明中的数值范围,应理解为还具体公开了该范围的上限和下限之间的每个中间值。在任何陈述值或陈述范围内的中间值以及任何其他陈述值或在所述范围内的中间值之间的每个较小的范围也包括在本发明内。这些较小范围的上限和下限可独立地包括或排除在范围内。It should be understood that the terminology described in the present invention is only used to describe specific embodiments, and is not used to limit the present invention. In addition, regarding the numerical ranges in the present invention, it should be understood that each intermediate value between the upper limit and the lower limit of the range is also specifically disclosed. Each smaller range between any stated value or intervening value in a stated range and any other stated value or intervening value in a stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded from the range.
除非另有说明,否则本文使用的所有技术和科学术语具有本发明所述领域的常规技术人员通常理解的相同含义。虽然本发明仅描述了优选的方法和材料,但是在本发明的实施或测试中也可以使用与本文所述相似或等同的任何方法和材料。本说明书中提到的所有文献通过引用并入,用以公开和描述与所述文献相关的方法和/或材料。在与任何并入的文献冲突时,以本说明书的内容为准。Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only the preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and/or materials in connection with which the documents are described. In case of conflict with any incorporated document, the contents of this specification control.
在不背离本发明的范围或精神的情况下,可对本发明说明书的具体实施方式做多种改进和变化,这对本领域技术人员而言是显而易见的。由本发明的说明书得到的其他实施方式对技术人员而言是显而易见的。本发明说明书和实施例仅是示例性的。It will be apparent to those skilled in the art that various modifications and changes can be made in the specific embodiments of the present invention described herein without departing from the scope or spirit of the present invention. Other embodiments will be apparent to the skilled person from the description of the present invention. The description and examples of the invention are illustrative only.
关于本文中所使用的“包含”、“包括”、“具有”、“含有”等等,均为开放性的用语,即意指包含但不限于。As used herein, "comprising", "comprising", "having", "comprising" and so on are all open terms, meaning including but not limited to.
本发明利用的双亲材料158A和SGDH284,是前期利用小孢子培养技术,分别通过对生产中推广的半冬性材料中油9988和欧洲高含油量品种冬性材料Sollux进行小孢子培养并加倍获得的DH纯系(Ke et al 2020)。这两份纯系材料在安徽地区秋播至入冬前均表现耐寒稳健生长,年后158A相比SGDH284表现出快速生长,快速抽薹,提前8天左右开花的特点。利用二者作为亲本,杂交得到的F1世代,通过其花药小孢子培养,得到一个包含178份材料的DH群体,本研究将其命名为158A-SGDH。图1为本发明构建DH群体、表型鉴定、图谱构建、QTL定位、主效QTL候选基因鉴定以及主效QTL区间分子标记开发鉴定应用的技术流程图。The parental materials 158A and SGDH284 used in the present invention are DH obtained by microspore culture and doubling of the semi-winter material Zhongyou 9988 and the European high-oil content variety Sollux respectively, using the microspore culture technology in the early stage. Pure line (Ke et al 2020). These two pure-line materials showed cold-resistant and steady growth from autumn sowing to winter in Anhui area. Compared with SGDH284, the latter 158A showed rapid growth, rapid bolting, and flowering about 8 days earlier. Using the two as parents, the F1 generation obtained by crossing was cultivated through its anther microspores, and a DH population containing 178 materials was obtained, which was named 158A-SGDH in this study. Figure 1 is a technical flow chart of the present invention for constructing DH populations, phenotypic identification, map construction, QTL mapping, main QTL candidate gene identification, and main QTL interval molecular marker development and identification applications.
实施例1 158A-SGDH群体的开花期表型分析Example 1 Phenotype analysis of flowering period of 158A-SGDH population
双亲材料早花亲本158A和晚花亲本SGDH284在半冬性油菜环境下(安徽凤阳)的花期表型差异水平达到极显著(表1,图2,图2中A是在冬油菜环境2019年10月21日播种后于2020年03月21日拍摄田间表型图片,此时早花亲本158A已经开花;B是在冬油菜环境2019年10月21日播种后于2020年03月28日拍摄田间表型图片,此时晚花亲本SGDH284开始开花;标尺=10cm。)。基于二者作为双亲构建158A-SGDH群体之后,在冬油菜环境下:我们于2018年10月18日将该群体种植了一个重复(FY18.1);并在2019年03-04月份调查该群体的开花期表型(表1,图3)。接着我们于2019年10月21日将该群体种植了三个重复(FY19.1、FY19.2、FY19.3);然后2020年03-04月份调查该群体的开花期表型(表1,图3)。本研究于2020年10月15日继续种植了该群体两个重复(FY20.1、FY20.2),并于2021年03-04月份调查该群体材料的开花期表型(表1,图3)。因此,通过三年的表型鉴定,共收集到了6个重复的开花期表型数据(表1,图3)。从表型分布图可看出,158A-SGDH群体三年6重复均呈现正态分布的趋势,表明开花期是多基因控制的数量性状。而且和2018-2019年度相比,2019-2020年度以及2020-2021年度158A-SGDH群体开花期明显提前(图3,横坐标是开花时间(天),纵坐标是密度(当天开花的单株数占总数的百分比))。Parental materials, the early-flowering parent 158A and the late-flowering parent SGDH284 have extremely significant differences in flowering phenotypes in the semi-winter rapeseed environment (Fengyang, Anhui) (Table 1, Figure 2, A in Figure 2 is in the winter rapeseed environment in 2019 The field phenotype pictures were taken on March 21, 2020 after sowing on October 21, when the early-flowering parent 158A had bloomed; B was taken on March 28, 2020 after sowing on October 21, 2019 in the winter rapeseed environment Field phenotype picture, when the late-flowering parent SGDH284 begins to flower; scale bar = 10 cm.). After constructing the 158A-SGDH population based on the two as parents, in the winter rapeseed environment: we planted a duplicate (FY18.1) of the population on October 18, 2018; and investigated the population in March-April 2019 The anthesis phenotype of the (Table 1, Figure 3). Then we planted three replicates (FY19.1, FY19.2, FY19.3) of this group on October 21, 2019; image 3). In this study, two replicates (FY20.1, FY20.2) of this group were planted on October 15, 2020, and the flowering period phenotypes of the materials of this group were investigated from March to April 2021 (Table 1, Figure 3 ). Therefore, through three years of phenotypic identification, a total of six replicate flowering phenotype data were collected (Table 1, Figure 3). It can be seen from the phenotype distribution map that the 6 replicates of the 158A-SGDH population in three years showed a normal distribution trend, indicating that flowering period is a quantitative trait controlled by polygenes. Moreover, compared with 2018-2019, the flowering period of 158A-SGDH populations in 2019-2020 and 2020-2021 is significantly earlier (Figure 3, the abscissa is the flowering time (days), and the ordinate is the density (the number of individual plants that flowered on the day accounted for % of the total)).
表1 158A-SGDH群体及其亲本开花时间(天)的表型变异Table 1 Phenotypic variation of flowering time (days) in 158A-SGDH population and its parents
通过t检验显着性水平。***p<0.001。a平均值±SEM,SEM表示平均值的标准误差。Significance level by t test. ***p<0.001. a Mean ± SEM, SEM represents the standard error of the mean.
为了判断2018-2019年度与2019-2020年度、2020-2021年度的158A-SGDH群体开花期整体偏移情况是否会对后面的开花期QTL检测有影响。我们分析了以上三个年度158A-SGDH群体6个重复开花期的相关性。发现6个重复间开花期的相关性均达到极显著相关水平(图4)。说明各重复间的开花期表型可以用于下一步的开花期QTL检测。In order to judge whether the overall deviation of flowering period of 158A-SGDH population in 2018-2019, 2019-2020 and 2020-2021 will affect the subsequent QTL detection of flowering period. We analyzed the correlation of the six repeated flowering periods of the above three annual 158A-SGDH populations. It was found that the correlation of flowering period among the 6 replicates all reached extremely significant correlation level (Fig. 4). It shows that the flowering period phenotype among the replicates can be used for the next step of flowering period QTL detection.
实施例2 158A-SGDH群体的遗传图谱构建Example 2 Genetic map construction of 158A-SGDH population
将158A-SGDH群体共计178份材料样品及双亲样品送往上海派森诺生物科技股份有限公司进行简化基因组测序,经检测各样品提取的基因组DNA质量较好,均符合测序要求(图5)。经过简化基因组测序总共生成了360.87Gb的数据。其中Q20质量平均分98.69%以上,GC含量约39.09%。DH群体样品测不少于1G的数据量,每个株系的平均测序深度为1.70倍。双亲158A和SGDH284分别获得5,064,588,459bp和5,362,611,753bp的序列,测序测序深度分别为3.67倍和4.19倍。A total of 178 material samples and parental samples of the 158A-SGDH population were sent to Shanghai Passino Biotechnology Co., Ltd. for simplified genome sequencing. The quality of the genomic DNA extracted from each sample was tested to be good and all met the sequencing requirements (Figure 5). A total of 360.87Gb of data was generated after simplified genome sequencing. Among them, the average quality score of Q20 is over 98.69%, and the GC content is about 39.09%. The amount of data measured by the DH population sample is not less than 1G, and the average sequencing depth of each strain is 1.70 times. The sequences of parents 158A and SGDH284 were 5,064,588,459bp and 5,362,611,753bp respectively, and the sequencing depths were 3.67 times and 4.19 times respectively.
158A-SGDH群体的遗传图谱是经过简化基因组测序,通过分析筛选一共得到946690个SNPs,以200Kb为窗口统计SNP的数目,每条染色体上的SNP分布情况见图6(横坐标为物理位置(Mb),纵坐标为连锁群)。The genetic map of the 158A-SGDH population was simplified genome sequencing, and a total of 946,690 SNPs were obtained through analysis and screening. The number of SNPs was counted with a window of 200Kb. The distribution of SNPs on each chromosome is shown in Figure 6 (the abscissa is the physical position (Mb ), the ordinate is the linkage group).
为了构建遗传图谱,对得到946690个SNP进一步过滤,主要过滤条件如下:In order to construct the genetic map, further filter the obtained 946690 SNPs, the main filtering conditions are as follows:
(1)亲本基因型:保留在两个亲本中纯合并且亲本间不一致的位点;(1) Parental genotype: the sites that are homozygous in the two parents and inconsistent between the parents are retained;
(2)测序深度:保留子代的测序深度>2的位点;(2) Sequencing depth: retain the sites where the sequencing depth of the progeny is >2;
(3)缺失率:保留子代的缺失率<0.5的位点;(3) Deletion rate: retain the loci with a deletion rate < 0.5 in the progeny;
最终筛选出13395个bin marker,转换基因型格式,整理成的MSTmap软件的输入文件格式。利用软件MSTmap进行连锁群的分群,并绘制连锁群,最终共有2777个bin marker被锚定到19条连锁群中,连锁群的总长度为3268.01cM,标记平均间距为1.18cM。Finally, 13,395 bin markers were selected, converted into genotype format, and organized into the input file format of MSTmap software. The software MSTmap was used to group the linkage groups and draw the linkage groups. In the end, a total of 2777 bin markers were anchored to 19 linkage groups. The total length of the linkage groups was 3268.01cM, and the average distance between markers was 1.18cM.
实施例3 158A-SGDH群体的开花期QTL位点定位Example 3 Mapping of QTL loci for anthesis in 158A-SGDH population
利用WinQTLCart 2.5软件的复合区间作图的标准模型(model 6)进行QTL分析。开花期的表型数据为178个品系的表型值,三年内重复6次。通过QTL分析,共检测到56个开花期的QTL(表2)。单个QTL解释的表型变异比例为2.70%~32.04%。鉴定出的QTLs分布在12个连锁群中,不同实验中QTLs的置信区间(CIs)重叠。在56个QTL中,44个可以通过元分析整合成12个可重复的共有QTL,分别命名为cqDTF-A02、cqDTF-A04、cqDTF-A06-1、cqDTF-A06-2、cqDTF-A07、cqDTF-A09、cqDTF-C02、cqDTF-C04-1、cqDTF-C04-2、cqDTF-C05、cqDTF-C06和cqDTF-C09(表2)。在这些共有的QTL中,cqDTF-C02在所有六个重复中检测到;cqDTF-C06也在六个重复中检测到,其中有五个重复检测到位于共同的区间,有一次重复位于3.38-28.99cM区间;cqDTF-A04、cqDTF-A07和cqDTF-C09在所有五个重复中检测到。在三个重复中检测到四个QTL(cqDTF-A02、cqDTF-A06-1、cqDTF-A06-2和cqDTF-C05),在三个重复中检测到cqDTF-A09、cqDTF-C04-1和cqDTF-C04-2两个重复(表2)。The standard model (model 6) of composite interval mapping in WinQTLCart 2.5 software was used for QTL analysis. The phenotypic data of flowering period are the phenotypic values of 178 lines, repeated 6 times in three years. Through QTL analysis, a total of 56 QTLs for anthesis were detected (Table 2). The proportion of phenotypic variation explained by a single QTL ranged from 2.70% to 32.04%. The identified QTLs were distributed in 12 linkage groups, and the confidence intervals (CIs) of QTLs in different experiments overlapped. Among the 56 QTLs, 44 could be integrated into 12 reproducible consensus QTLs through meta-analysis, named cqDTF-A02, cqDTF-A04, cqDTF-A06-1, cqDTF-A06-2, cqDTF-A07, cqDTF - A09, cqDTF-C02, cqDTF-C04-1, cqDTF-C04-2, cqDTF-C05, cqDTF-C06 and cqDTF-C09 (Table 2). Among these shared QTLs, cqDTF-C02 was detected in all six replicates; cqDTF-C06 was also detected in six replicates, five of which were detected in the common interval, and one replicate was located at 3.38-28.99 cM interval; cqDTF-A04, cqDTF-A07 and cqDTF-C09 were detected in all five replicates. Four QTLs (cqDTF-A02, cqDTF-A06-1, cqDTF-A06-2, and cqDTF-C05) were detected in three replicates, and cqDTF-A09, cqDTF-C04-1, and cqDTF were detected in three replicates - Two replicates of C04-2 (Table 2).
表2 158A-SGDH群体开花期QTL检测Table 2 Detection of QTLs for anthesis in 158A-SGDH population
a QTL解释的表型变异的比例;b+和-表示加性效应的方向。 a Proportion of phenotypic variation explained by QTL; b + and - indicate direction of additive effect.
在开花期检测到两个稳定的主效QTL(cqDTF-C02和cqDTF-C06)。cqDTF-C02基因位点位于C02连锁群上(图7),解释了13.54-32.04%的开花期表型变异(表2),cqDTF-C06解释了6.07-16.13%的表型变异(表2)。元分析结果显示,cqDTF-C02位点在遗传连锁图谱中的峰值为34.02cM,CI为30.07-37.97cM,两侧分别标记为chrC02_bin6298和chrC02_bin6308;标记之间的物理距离为1,885,589到2,918,506bp(图7)。Two stable major QTLs (cqDTF-C02 and cqDTF-C06) were detected in anthesis. The cqDTF-C02 gene locus is located on the C02 linkage group (Fig. 7), explaining 13.54-32.04% of the phenotypic variation in flowering period (Table 2), and cqDTF-C06 explaining 6.07-16.13% of the phenotypic variation (Table 2) . The results of meta-analysis showed that the cqDTF-C02 locus had a peak value of 34.02cM in the genetic linkage map, CI was 30.07-37.97cM, and the two sides were marked as chrC02_bin6298 and chrC02_bin6308 respectively; the physical distance between the markers was 1,885,589 to 2,918,506 bp (Fig. 7).
以上结果表明cqDTF-C02在三年6个重复中均能够稳定的检测到,因此,该位点是一个稳定存在的开花期主效QTL位点。The above results indicated that cqDTF-C02 could be detected stably in 6 replicates in three years. Therefore, this locus was a stable major QTL locus for anthesis.
实施例4主效开花时间QTL cqDTF-C02区域的候选基因预测Example 4 Candidate Gene Prediction of Main Effect Flowering Time QTL cqDTF-C02 Region
根据甘蓝型油菜ZS11参考基因组(http://yanglab.hzau.edu.cn/BnIR),预测1.03-Mb cqDTF-C02区域有195个基因。基于甘蓝型油菜和拟南芥目标区域的微共线性,我们检测到四个可能与开花时间相关的基因:BnaC02G0032100ZS(AT5G08370/AGAL2)、BnaC02G0038900ZS(AT5G10130/DFC)、BnaC02G0039100ZS(AT5G10140/FLC)、和BnaC02G0046300ZS(AT5G11530/EMF1)(图8,上曲线代表在三种环境中六个重复中鉴定的QTL,下曲线代表相同颜色的QTL的加性效应,主效应QTL cqDTF-C02显示在置信区间下;与开花时间相关的候选基因显示在主效QTL cqDTF-C02下)。Ke等人(2020)对欧洲油菜158A和SGDH284苗期叶片进行了转录组测序。本研究利用该文献得到的到大量基因的表达信息,对幼苗期亲本158A和SGDH284的转录组数据进行分析,揭示了该QTL区域中的四个开花相关基因(BnaC02G0032100ZS、BnaC02G0038900ZS、BnaC02G0039100ZS和BnaC02G0046300ZS)。BnaC02G0032100ZS基因在158A和SGDH284中的FPKM值分别为71.96和74.88。BnaC02G0038900ZS、BnaC02G0039100ZS、BnaC02G0046300ZS的FPKM(fragments perkilobase transcript per million reads)值在158A母本中均小于5,然而这些基因的FPKM值在SGDH284亲本中更高(分别为20.64、277.51和31.31)(表3)。According to the Brassica napus ZS11 reference genome (http://yanglab.hzau.edu.cn/BnIR), 195 genes were predicted in the 1.03-Mb cqDTF-C02 region. Based on the microcollinearity of target regions in Brassica napus and Arabidopsis, we detected four genes that may be associated with flowering time: BnaC02G0032100ZS (AT5G08370/AGAL2), BnaC02G0038900ZS (AT5G10130/DFC), BnaC02G0039100ZS (AT5G10140/FLC ), and BnaC02G0046300ZS (AT5G11530/EMF1) (Fig. 8, the upper curve represents the QTL identified in six replicates in three environments, the lower curve represents the additive effect of the QTL of the same color, the main effect QTL cqDTF-C02 is shown under the confidence interval; Candidate genes associated with flowering time are shown under the main QTL cqDTF-C02). Ke et al. (2020) performed transcriptome sequencing of Brassica napus 158A and SGDH284 seedling leaves. In this study, the transcriptome data of parents 158A and SGDH284 at the seedling stage were analyzed using the expression information of a large number of genes obtained from this literature, revealing four flowering-related genes (BnaC02G0032100ZS, BnaC02G0038900ZS, BnaC02G0039100ZS and BnaC02G0046300ZS) in this QTL region. The FPKM values of BnaC02G0032100ZS gene in 158A and SGDH284 were 71.96 and 74.88, respectively. The FPKM (fragments perkilobase transcript per million reads) values of BnaC02G0038900ZS, BnaC02G0039100ZS, and BnaC02G0046300ZS were all less than 5 in the 158A female parent, but the FPKM values of these genes were higher in the SGDH284 parent (20.64, 277.51 and 31.31) (Table 3) .
表3亲本158A和SGDH284苗期主效QTLcqDTF-C02开花时间相关基因的FPKM值Table 3 FPKM values of genes related to flowering time of main QTLcqDTF-C02 in seedling stage of parents 158A and SGDH284
实施例5主效开花时间QTL cqDTF-C02区域的InDel标记开发及应用Example 5 Development and application of InDel markers in the main flowering time QTL cqDTF-C02 region
基于158A和SGDH284的Illumina测序数据,使用ZS11参考基因组开发了cqDTF-C02的InDel标记。我们为cqDTF-C02基因位点开发了九个InDel标记,其中一个具有亲本多态性和强条带模式的InDel标记是C2-5。扩增该标记的引物序列为C2-5L:5’-CGTGTCAAGTCTGCATTGTTGT-3’(SEQ ID NO:1);C2-5R:5’-TTCCTGCCTTATCCATCCCA-3’(SEQID NO:2)。我们用C2-5标记分析一个小群体包含10个极早开花株系、10个极晚开花株系以及分析双亲158A(P1)和SGDH284(P2),试验如下:Based on the Illumina sequencing data of 158A and SGDH284, an InDel marker for cqDTF-C02 was developed using the ZS11 reference genome. We developed nine InDel markers for the cqDTF-C02 locus, and one of the InDel markers with parental polymorphism and strong banding pattern was C2-5. The primer sequence for amplifying the marker is C2-5L: 5'-CGTGTCAAGTCTGCATTGTTGT-3' (SEQ ID NO: 1); C2-5R: 5'-TTCCTGCCTTATCCATCCCA-3' (SEQ ID NO: 2). We used the C2-5 marker to analyze a small population containing 10 very early-flowering lines, 10 very late-flowering lines and analyze the parents 158A (P1) and SGDH284 (P2), the experiment is as follows:
1检测样品的DNA提取1 DNA extraction of test samples
采用CTAB法进行DNA的提取,试剂及步骤如下(参考Doyle et al;1987):The CTAB method was used to extract DNA, and the reagents and steps were as follows (refer to Doyle et al; 1987):
1.1试剂的配制1.1 Preparation of reagents
(1)Tris-HCl(1.0M/L,pH8.0):60.58g Tris-Base和21mL浓HCl加dd H2O定容至500mL。(1) Tris-HCl (1.0M/L, pH8.0): 60.58g Tris-Base and 21mL of concentrated HCl, add dd H 2 O to make up to 500mL.
(2)EDTA (0.5M/L,pH8.0):186g EDTA和25g NaOH(颗粒)加dd H2O定容至1L。(2) EDTA (0.5M/L, pH8.0): 186g EDTA and 25g NaOH (granule) add dd H 2 O to make up to 1L.
(3)2%CTAB:81.9g NaCl、100mL 1.0M/L Tris-HCl(pH8.0)、40mL 0.5M/L EDTA(pH8.0)、20g CTAB,加ddH2O定容至1L,灭菌后即可用于DNA的提取。(3) 2% CTAB: 81.9g NaCl, 100mL 1.0M/L Tris-HCl (pH8.0), 40mL 0.5M/L EDTA (pH8.0), 20g CTAB, add ddH 2 O to 1L, extinguish The bacteria can be used for DNA extraction.
(4)5M/L NH4AC:385.4g NH4AC加dd H2O定容至1L。(4) 5M/L NH 4 AC: add 385.4g NH 4 AC and add dd H 2 O to make up to 1L.
(5)76%Ethanol(含10mM NH4AC):760mL无水乙醇和2mL 5M/L NH4AC,加dd H2O定容至1L。(5) 76% Ethanol (containing 10mM NH4AC): 760mL absolute ethanol and 2mL 5M/L NH4AC , add ddH2O to make up to 1L.
(6)3M/L NaAc(pH5.2):246.09g NaAc加dd H2O和HAC定容至1L,并用HAC调pH值至5.2。(6) 3M/L NaAc (pH5.2): 246.09g NaAc, add dd H 2 O and HAC to make the volume to 1L, and adjust the pH value to 5.2 with HAC.
(7)24:1:吸取22mL异戊醇加到500mL三氯甲烷中混匀。(7) 24:1: Add 22mL of isoamyl alcohol to 500mL of chloroform and mix well.
1.2CTAB法提取DNA步骤1.2 CTAB method to extract DNA steps
1)用写好编号的2mL离心管取少量幼嫩叶片(室内发芽的幼苗取子叶)放在-20℃冰柜冷冻备用;1) Use a numbered 2mL centrifuge tube to take a small amount of young leaves (cotyledons from indoor germinated seedlings) and freeze them in a -20°C freezer for later use;
2)磨样:2) Grinding sample:
磨样机法:取出离心管后置于冰上,打开盖子加入干净钢珠,同时加入100μLCTAB,盖好后置于磨样机适配器中(28times/s,30s),磨碎后取出离心管,打开盖子,倒出钢珠,再加入300μL CTAB。(磨样机使用时严格按照使用说明,注意平衡对称)Grinding machine method: take out the centrifuge tube and put it on ice, open the cover and add clean steel balls, and add 100μLCTAB at the same time, cover it and put it in the sample grinding machine adapter (28times/s, 30s), after grinding, take out the centrifuge tube, open the cover, Pour off the steel beads and add 300 μL CTAB. (When using the prototype mill, strictly follow the instructions for use, pay attention to balance and symmetry)
3)将离心管置于离心盒板上,在55~60℃的水浴锅中水浴50~60min,每10分钟轻轻摇晃一次,水浴完后放置于通风厨中室温冷却(大约30~60min);3) Place the centrifuge tube on the centrifuge box plate, bathe in a water bath at 55-60°C for 50-60 minutes, shake gently every 10 minutes, and place it in a fume hood to cool at room temperature after the water bath (about 30-60 minutes) ;
4)加入等体积(400μL)的24:1溶液于管中,轻轻摇晃10分钟后,12000rpm离心10min;4) Add an equal volume (400 μL) of 24:1 solution to the tube, shake gently for 10 minutes, and then centrifuge at 12,000 rpm for 10 minutes;
5)吸取上清夜(200μL)转至与原编号相同的1.5mL离心管(预先加入上清液1/10体积的3M/L NaAc),加入两倍体积冰冻的无水乙醇(-20℃冰箱过夜),静置20~30min;5) Transfer the supernatant (200 μL) to a 1.5 mL centrifuge tube with the same number as the original (pre-add 1/10 volume of 3M/L NaAc of the supernatant), add twice the volume of frozen absolute ethanol (-20 °C refrigerator Overnight), let stand for 20-30min;
6)若DNA团状物较大则可直接用枪头挑出DNA,倒出乙醇,若DNA量少则盖好盖子,8000rpm离心2min后开盖子倒酒精;6) If the DNA clumps are large, you can directly pick out the DNA with a pipette tip and pour out the ethanol. If the amount of DNA is small, cover the lid, centrifuge at 8000rpm for 2 minutes, open the lid and pour alcohol;
7)随后加入76%乙醇洗涤沉淀(可过夜),偶尔转动,重复1~2次;7) Then add 76% ethanol to wash the precipitate (overnight), rotate occasionally, repeat 1-2 times;
8)轻轻倒出酒精,将DNA放置于管底,室温下吹干,加入TE或ddH2O溶解,37℃恒温箱中放置1h后摇匀即可。长期保存请置于-20℃冰柜。8) Gently pour out the alcohol, place the DNA at the bottom of the tube, blow dry at room temperature, add TE or ddH 2 O to dissolve, place in a 37°C incubator for 1 hour, and then shake well. For long-term storage, please store in a -20°C freezer.
1.3扩增体系及程序:1.3 Amplification system and procedures:
PCR反应体系(10μL体系)如下:The PCR reaction system (10 μL system) is as follows:
1.4扩增程序:1.4 Amplification procedure:
第一步:预变性94℃3分钟;Step 1: Pre-denaturation at 94°C for 3 minutes;
第二步:变性94℃30秒;Step 2: Denaturation at 94°C for 30 seconds;
第三步:退火60℃30秒;The third step: annealing at 60°C for 30 seconds;
第四步:延伸72℃45秒;Step 4: extend at 72°C for 45 seconds;
第五步:回到第二步进行35个循环;Step 5: Go back to Step 2 for 35 cycles;
第六步:72℃10分钟;Step 6: 72°C for 10 minutes;
第七步:4℃5分钟。Step 7: 5 minutes at 4°C.
1.5电泳检测方法:1.5 Electrophoresis detection method:
聚丙烯酰胺凝胶电泳检测(6%PAGE)Polyacrylamide gel electrophoresis detection (6% PAGE)
1.5.1试剂配制1.5.1 Reagent preparation
A.5×TBE配置如下:Tris-base 107.8g、EDTA 7.44g、硼酸55.0g,用超纯水定容至2L。A. 5×TBE configuration is as follows: Tris-base 107.8g, EDTA 7.44g, boric acid 55.0g, dilute to 2L with ultrapure water.
B.6%PAGE配置如下:5×TBE 200mL、丙烯酰胺114g、甲叉丙烯酰胺6g,用dd H2O定容至2L,双层滤纸过滤备用。B. The configuration of 6% PAGE is as follows: 5×TBE 200mL, acrylamide 114g, methylene acrylamide 6g, dilute to 2L with dd H 2 O, filter with double-layer filter paper for later use.
C.10%过硫酸氨(AP)的配制:过硫酸氨10克,加超纯水定容至100mL。C. Preparation of 10% ammonium persulfate (AP): add 10 grams of ammonium persulfate to 100 mL with ultrapure water.
D.银染液D. Silver stain
1.5g硝酸银溶于1.5L dH2O。1.5g of silver nitrate was dissolved in 1.5L of dH 2 O.
E.显影液E. Developer
0.6g碳酸钠和30g氢氧化钠溶于1.5L dH2O中,显影时在通风橱中加入6mL甲醛(37%)混匀即可使用。Dissolve 0.6g of sodium carbonate and 30g of sodium hydroxide in 1.5L of dH 2 O, add 6mL of formaldehyde (37%) in a fume hood and mix well before developing.
1)凝胶制备1) Gel preparation
A.长玻璃使用前要用洗洁精洗干净后晾干备用。A. Before using the long glass, wash it with detergent and dry it for later use.
B.用无水乙醇分别擦拭长、短玻璃后,晾至乙醇挥发;B. After wiping the long and short glasses with absolute ethanol, let the ethanol volatilize;
C.将两玻璃用封条装好,放到电泳槽中,拧动螺帽调节松紧度,并用琼脂进行封边处理。C. Install the two glasses with sealing strips, put them in the electrophoresis tank, twist the nuts to adjust the tightness, and seal the edges with agar.
E.在烧杯中倒入30mL左右6%PAGE,再分别加入300μL AP及30μL TEMED,快速搅拌均匀;E. Pour about 30mL of 6% PAGE into the beaker, then add 300μL AP and 30μL TEMED respectively, and stir quickly;
F.干净利落地将PAGE胶灌入两玻璃间,适当轻敲玻璃以防产生气泡。然后将干净的梳子背部插入两玻璃间。一般20-30min后即可电泳。F. Cleanly pour PAGE glue into the gap between the two glasses, and tap the glass properly to prevent air bubbles. Then insert the back of the clean comb between the two glasses. Generally, electrophoresis can be performed after 20-30min.
2)电泳2) Electrophoresis
电泳前将玻璃板上的碎胶用移液枪吸取缓冲液冲洗干净,小心取下梳子后架上电泳槽进行预电泳。预电泳结束,在玻璃中插入点样用梳子进行点样。以功率80W电泳至需要时间。Before electrophoresis, wash the broken gel on the glass plate with a pipette gun to absorb the buffer solution, carefully remove the comb and put it on the electrophoresis tank for pre-electrophoresis. After the pre-electrophoresis is completed, insert the spotting comb into the glass and spot the spotting. It takes time to electrophoresis with a power of 80W.
3)显影3) Development
A.取下长玻璃,胶面向上,用1L ddH2O快速漂洗(10秒钟左右);A. Take off the long glass, with the glue side up, rinse it quickly with 1L ddH 2 O (about 10 seconds);
B.银染8-10分钟(其中银染液为0.5gAgNO3溶于于1L dH2O中);B. Silver staining for 8-10 minutes (the silver staining solution is 0.5gAgNO 3 dissolved in 1L dH 2 O);
C.取出凝胶,轻轻甩脱附在凝胶表面的染色液,用1L dH2O漂洗(40-60秒钟左右);C. Take out the gel, gently shake off the staining solution attached to the surface of the gel, and rinse with 1L dH 2 O (about 40-60 seconds);
D.取出凝胶,轻轻甩干,在显影液(0.5L dH2O,9.5gNaOH,1mL甲醛)中显影至满意效果;D. Take out the gel, dry it gently, and develop it in a developer solution (0.5L dH 2 O, 9.5gNaOH, 1mL formaldehyde) to a satisfactory effect;
E.显影完毕将凝胶放入漂洗盆略作浸泡以去除表面的显影液;E. After developing, put the gel into the rinsing tub for a while to soak to remove the developer on the surface;
F.取出凝胶,在专用灯箱上读带拍照。F. Take out the gel, read the tape on the special light box and take pictures.
结果如图9,实验结果显示该InDel标记C2-5与甘蓝型油菜的开花时间有关。The results are shown in Figure 9. The experimental results show that the InDel marker C2-5 is related to the flowering time of Brassica napus.
通过测序发现,10个极晚开花株系的InDel标记C2-5序列的扩增片段长度为200bp,序列如SEQ ID NO:3所示:It was found by sequencing that the length of the amplified fragment of the InDel-marked C2-5 sequence of the 10 extremely late flowering lines was 200bp, and the sequence is shown in SEQ ID NO: 3:
CGTGTCAAGTCTGCATTGTTGTGCTTTGCTAATGCTAGTTCCATTGAGGAAAGGTATTAATATACAAATCTTTGAATACAAATCTTTTCAAATCTTTGAATACAAATCTTTTAGTTTCAAAGTTAAAAATTTTAGGAGTACATGTATTCAACCATGCATTTATGCTTGCTTGTAGGATTATGGGATGGATAAGGCAGGAA。CGTGTCAAAGTCTGCATTGTTGTGCTTTGCTAATGCTAGTTCCATTGAGGAAAGGTATTAATATACAAATCTTTGAATACAAATCTTTTCAAATCTTTGAATACAAATCTTTTAGTTTCAAAGTTAAAATTTTAGGAGTACATGTATTCAACCATGCATTTATGCTTGCTTGTAGGATTATGGGATGGATAAGGCAGGAA.
因此,为了验证该标记的应用情况,本研究使用InDel标记C2-5在cqDTF-C02基因位点分析了158A-SGDH群体的六个重复的开花时间。图10展示的是利用C2-5标记检测鉴定158A-SGDH群体家系编号为A03到A47以及双亲158A(P1)和SGDH284(P2)的检测结果。检测完158A-SGDH群体共计178份材料之后,通过标记基因型结合三年6个重复的开花期表型进行方差分析,结果如图11(图中“-”表示没有cqDTF-C02基因位点的家系,“+”表示具有cqDTF-C02基因位点的家系;开花时间统计的为平均值±s.e.m。***表示P<0.001),表明携带该InDel标记的株系和没有该InDel标记的品系的开花时间差异水平达到极显著,携带该InDel标记的株系为晚开花株系,未携带该InDel标记的株系为早开花株系。Therefore, in order to verify the application of this marker, this study analyzed the flowering time of six replicates of the 158A-SGDH population at the cqDTF-C02 locus using the InDel marker C2-5. Figure 10 shows the detection results of the identification of 158A-SGDH population families numbered A03 to A47 and their parents 158A (P1) and SGDH284 (P2) using C2-5 marker detection. After detecting a total of 178 materials from the 158A-SGDH population, ANOVA was performed by combining the marker genotypes with the flowering phenotypes of 6 repetitions in three years. The results are shown in Figure 11 ("-" in the figure indicates that there is no cqDTF-C02 gene locus Families, "+" means the family with the cqDTF-C02 gene locus; the flowering time statistics are the mean ± s.e.m. *** means P<0.001), indicating the lines carrying the InDel marker and the lines without the InDel marker The level of difference in flowering time reached extremely significant, the lines carrying the InDel marker were late flowering lines, and the lines not carrying the InDel marker were early flowering lines.
以上所述的实施例仅是对本发明的优选方式进行描述,并非对本发明的范围进行限定,在不脱离本发明设计精神的前提下,本领域普通技术人员对本发明的技术方案做出的各种变形和改进,均应落入本发明权利要求书确定的保护范围内。The above-mentioned embodiments are only to describe the preferred mode of the present invention, and are not intended to limit the scope of the present invention. Variations and improvements should fall within the scope of protection defined by the claims of the present invention.
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