CN109722488B - SCAR molecular marker linked with root knot nematode resistant gene Me3 and application thereof - Google Patents
SCAR molecular marker linked with root knot nematode resistant gene Me3 and application thereof Download PDFInfo
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Abstract
本发明公开了一种与抗根结线虫基因Me3连锁的SCAR分子标记及其应用,该与抗根结线虫基因Me3连锁的SCAR分子标记含有SEQ ID No:5所示序列;扩增与抗根结线虫基因Me3连锁的SCAR分子标记的引物对的引物1为SEQ ID No:6所示序列,引物2为SEQ ID No:7所示序列。本发明所提供的SCAR分子标记可用于分子标记辅助育种,具有特异性强,稳定性高的特点,利用该SCAR分子标记鉴定辣椒是否抗根结线虫病,对底物模板质量的要求不高,且该SCAR分子标记为共显性标记,准确度高,适用性广。
The invention discloses a SCAR molecular marker linked with the root knot nematode gene Me3 and an application thereof. The SCAR molecular marker linked with the root knot nematode gene Me3 contains the sequence shown in SEQ ID No: 5; The primer 1 of the primer pair for the Me3-linked SCAR molecular marker of the K. elegans gene is the sequence shown in SEQ ID No: 6, and the primer 2 is the sequence shown in SEQ ID No: 7. The SCAR molecular marker provided by the present invention can be used for molecular marker-assisted breeding, and has the characteristics of strong specificity and high stability. Using the SCAR molecular marker to identify whether pepper is resistant to root-knot nematode disease does not require high quality of the substrate template. And the SCAR molecular marker is a co-dominant marker with high accuracy and wide applicability.
Description
技术领域technical field
本发明属于分子生物学的技术领域,涉及一种分子标记,具体涉及一种与抗根结线虫基因Me3连锁的SCAR分子标记,还涉及扩增该分子标记的引物对,及该分子标记或引物对在辣椒辅助育种或选育抗根结线虫病辣椒品种中的应用。The invention belongs to the technical field of molecular biology, and relates to a molecular marker, in particular to a SCAR molecular marker linked to the root-knot nematode gene Me3, a primer pair for amplifying the molecular marker, and the molecular marker or primer The application of the pair in the auxiliary breeding of peppers or the selection of pepper varieties resistant to root knot nematode disease.
背景技术Background technique
根结线虫(Root-knot nematode)广泛分布于世界各地的辣椒种植区,每年危害5500多种作物并造成严重的经济损失,是一种毁灭性的土传病害。危害最大的四种根结线虫为南方根结线虫(Meloidogyne.Incognita),花生根结线虫(M.arenaria),爪哇根结线虫(M.javanica)以及北方根结线虫(M.hapla),其中,南方根结线虫寄主最为广泛,可侵染1700种作物。目前生产上防治根结线虫的方法主要是化学防治,但许多化学杀线虫药剂会破坏环境安全和威胁公众健康。Root-knot nematode (Root-knot nematode) is widely distributed in pepper-growing areas all over the world. It damages more than 5,500 crops and causes serious economic losses every year. It is a devastating soil-borne disease. The four most harmful root-knot nematodes are Meloidogyne. Incognita, peanut root-knot nematode (M. arenaria), Java root-knot nematode (M.javanica) and northern root-knot nematode (M.hapla). , the most extensive host of root-knot nematode, can infect 1700 kinds of crops. At present, the main method for controlling root-knot nematodes in production is chemical control, but many chemical nematicides will damage environmental safety and threaten public health.
国内外研究和生产实践证明,选育抗病品种是防治和减轻根结线虫危害的最经济、安全和有效的防治方法。现在被公认的抗线虫材料有遵辣1号、Yolo Wonder、PM687等。目前报道的抗线虫基因有马铃薯Gpa2基因,番茄Mi-1基因和辣椒Me3基因等。其中,对高温抗性稳定的辣椒抗线虫基因Me3基因被定位在辣椒9号染色体上,该染色体还含有多个抗线虫基因位点,如Me4、Mech2、Me7及Me1基因等。早前,多个与Me3基因连锁较近的AFLP标记、SSCP标记及SSR标记已被开发。许小艳等人开发出SSCP_B322和EPMS658两个分子标记,与Me3基因的遗传距离分别为0.56cM和1.33cM。Domestic and foreign research and production practice have proved that breeding disease-resistant varieties is the most economical, safe and effective control method to control and reduce the damage of root-knot nematodes. Now recognized anti-nematode materials are Zunla No. 1, Yolo Wonder, PM687, etc. Currently reported nematode resistance genes include potato Gpa2 gene, tomato Mi-1 gene and pepper Me3 gene. Among them, the pepper nematode resistance gene Me3, which is stable to high temperature resistance, is located on
随着现代生物技术的迅猛发展,分子标记辅助选择育种成为育种工作的重要辅助手段。该技术是利用与决定特定性状基因紧密连锁的分子标记,通过对标记进行检测来确定基因是否存在,从而大大加速育种进程,节约大量成本,显著缩短了育种周期。因此,开发有效的分子标记对于新品种的选育具有重要意义。With the rapid development of modern biotechnology, molecular marker-assisted selective breeding has become an important auxiliary means of breeding. This technology uses molecular markers closely linked to genes that determine specific traits, and determines whether the genes exist by detecting the markers, thereby greatly accelerating the breeding process, saving a lot of costs, and significantly shortening the breeding cycle. Therefore, the development of effective molecular markers is of great significance for the selection and breeding of new varieties.
发明内容SUMMARY OF THE INVENTION
为解决以上技术问题,本发明提供了一种与抗根结线虫基因Me3连锁的SCAR分子标记及其应用。In order to solve the above technical problems, the present invention provides a SCAR molecular marker linked with the root-knot nematode gene Me3 and its application.
为了实现上述目的,本发明提供如下技术方案:In order to achieve the above object, the present invention provides the following technical solutions:
本发明提供了一种与抗根结线虫基因Me3连锁的SCAR分子标记,所述分子标记含有SEQ ID No:5所示序列。The present invention provides a SCAR molecular marker linked with the root-knot nematode gene Me3, and the molecular marker contains the sequence shown in SEQ ID No:5.
本发明还提供了扩增与抗根结线虫基因Me3连锁的SCAR分子标记的引物对,所述引物对的引物1为SEQ ID No:6所示序列,引物2为SEQ ID No:7所示序列。The present invention also provides a primer pair for amplifying the SCAR molecular marker linked to the root-knot nematode gene Me3, the
本发明还提供了一种与抗根结线虫基因Me3连锁的SCAR分子标记,所述分子标记是由所述的引物对,以抗根结线虫病辣椒的基因组DNA为模板经PCR扩增得到的。The present invention also provides a SCAR molecular marker linked with the root-knot nematode gene Me3, the molecular marker is obtained by using the primer pair and the genome DNA of the root-knot nematode disease-resistant pepper as a template through PCR amplification .
进一步的,所述抗根结线虫病辣椒为辣椒HDA149。Further, the root-knot nematode-resistant pepper is pepper HDA149.
进一步的,所述分子标记为SEQ ID No:5所示序列。Further, the molecular marker is the sequence shown in SEQ ID No:5.
进一步的,所述分子标记与抗根结线虫基因Me3的遗传距离为0.5cM。Further, the genetic distance between the molecular marker and the root knot nematode resistance gene Me3 is 0.5cM.
本发明还提供了一种利用所述的分子标记鉴定待测辣椒是否抗根结线虫病的方法,该方法包括以下步骤:The present invention also provides a method for identifying whether the pepper to be tested is resistant to root-knot nematode disease using the molecular marker, the method comprising the following steps:
(1)以SEQ ID No:6、SEQ ID No:7所示序列为上下游引物,待测辣椒的DNA为模板进行扩增,得到扩增产物;(1) take the sequence shown in SEQ ID No:6 and SEQ ID No:7 as the upstream and downstream primers, and the DNA of the pepper to be tested is amplified as a template to obtain an amplification product;
(2)判断扩增产物是否存在上述分子标记。(2) Determine whether the amplification product has the above-mentioned molecular marker.
进一步的,步骤(2)中,如果扩增产物大小为153bp,则判定待测辣椒为抗根结线虫病植株;如果扩增产物不含有该片段,则判定待测辣椒为感根结线虫病植株。Further, in step (2), if the size of the amplified product is 153bp, it is determined that the pepper to be tested is a root-knot nematode-resistant plant; if the amplified product does not contain the fragment, it is determined that the pepper to be tested is susceptible to root-knot nematode disease. plant.
本发明又提供了所述的分子标记或所述的引物对在辣椒辅助育种中的应用。The present invention further provides the application of the molecular marker or the primer pair in pepper-assisted breeding.
本发明又提供了所述的分子标记或所述的引物对在选育抗根结线虫病辣椒品种中的应用。The present invention further provides the application of the molecular marker or the primer pair in the selection and breeding of root-knot nematode resistant pepper varieties.
本发明的有益效果是:The beneficial effects of the present invention are:
本发明所提供的分子标记SCAR可用于分子标记辅助育种,具有特异性强,稳定性高的特点,利用该分子标记SCAR鉴定辣椒是否抗根结线虫病,对底物模板质量的要求不高,且该SCAR分子标记为共显性标记,准确度高,适用性广。本发明为辣椒抗根结线虫基因Me3的精细定位及克隆提供了重要的遗传学信息,可进一步通过染色体步移法精细定位基因,从而克隆该基因。本发明的分子标记可应用于辣椒育种工作,通过降低南方根结线虫对辣椒植株的侵染,从而减轻南方根结线虫造成的经济损失,降低生产成本以及农药施用量。The molecular marker SCAR provided by the invention can be used for molecular marker-assisted breeding, and has the characteristics of strong specificity and high stability. Using the molecular marker SCAR to identify whether pepper is resistant to root-knot nematode disease does not require high quality of the substrate template. And the SCAR molecular marker is a co-dominant marker with high accuracy and wide applicability. The invention provides important genetic information for the fine mapping and cloning of the root-knot nematode gene Me3 in pepper, and the gene can be further finely positioned by the chromosome walking method, so as to clone the gene. The molecular marker of the invention can be applied to pepper breeding, and by reducing the infection of pepper plants by M. incognita, the economic loss caused by M. incognita can be reduced, and the production cost and the amount of pesticide application can be reduced.
附图说明Description of drawings
图1为辣椒抗根结线虫基因Me3与分子标记的连锁遗传图谱;Fig. 1 is the linked genetic map of pepper root knot nematode resistance gene Me3 and molecular markers;
图2为本发明分子标记在双亲以及F2群体中随机挑选的抗病和感病单株中的PCR扩增结果;其中,M:Maker;1:抗病亲本HDA149;2:感病亲本8214;3-10:F2群体单株;Figure 2 shows the PCR amplification results of the molecular markers of the present invention in the disease-resistant and susceptible individual plants randomly selected from the parents and the F2 population; wherein, M: Maker; 1: the resistant parent HDA149; 2: the susceptible parent 8214; 3-10: F2 population single plant;
图3为本发明分子标记在父本HDA149、母本茄门以及F2群体中随机挑选的单株的PCR扩增结果;其中,M:Maker;1:抗病亲本HDA149;2:感病亲本茄门;3-10:F2群体单株;Fig. 3 is the PCR amplification result of the molecular markers of the present invention randomly selected from the male parent HDA149, the female parent Solanum and the F2 population; wherein, M: Maker; 1: disease-resistant parent HDA149; 2: susceptible parent Solanum door; 3-10: F2 population individual plant;
图4为本发明待测辣椒植株的PCR扩增结果;其中,M:Maker;H:抗病亲本HDA1491;1-8:待测辣椒植株。Figure 4 shows the PCR amplification results of the pepper plants to be tested in the present invention; wherein, M: Maker; H: disease-resistant parent HDA1491; 1-8: pepper plants to be tested.
具体实施方式Detailed ways
下面将通过具体实施例对本发明进行详细的描述。提供这些实施例是为了能够更透彻地理解本发明,并且能够将本发明的范围完整的传达给本领域的技术人员。The present invention will be described in detail below through specific embodiments. These embodiments are provided so that the present invention can be more thoroughly understood, and will fully convey the scope of the present invention to those skilled in the art.
下述实施例中所用的材料、试剂等,如无特殊说明,均可从商业途径得到。The materials, reagents, etc. used in the following examples can be obtained from commercial sources unless otherwise specified.
下述实施例中所用的辣椒感根结线虫病品种8214为母本(P1),辣椒抗根结线虫病品种HDA149作为父本(P2),利用两个亲本配置得到F1和F2群体。The pepper root-knot nematode cultivar 8214 used in the following examples was the female parent (P1), and the pepper root-knot nematode disease-resistant variety HDA149 was used as the male parent (P2).
下述实施例中含有Me3基因的抗根结线虫病辣椒品种HDA149、不含Me3基因的感根结线虫病辣椒品种8214和茄门均来自于中国农业科学院蔬菜花卉研究所。In the following examples, the root-knot nematode-resistant pepper variety HDA149 containing the Me3 gene, the root-knot nematode-susceptible pepper variety 8214 and the eggplant without the Me3 gene were all from the Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences.
下述实施例中病原线虫为南方根结线虫,来自中国农业科学院蔬菜花卉研究所。The pathogenic nematodes in the following examples are root knot nematodes, from the Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences.
下述实施例中的南方根结线虫二龄幼虫的制备方法:从感病辣椒茄门的根部挑取新鲜的卵块,质量浓度0.9%的NaClO溶液表面消毒后,用无菌水清洗两遍。将卵块放入无菌水中,于28℃培养箱中,黑暗条件下孵化并收集南方根结线虫二龄幼虫,将收集到的二龄幼虫悬浮液浓度调节至600条/mL,在24h内进行接种。The preparation method of the second instar larvae of R. incognita in the following examples: pick fresh egg masses from the root of the susceptible capsicum solanum, 0.9% mass concentration of NaClO solution surface disinfection, rinse with sterile water twice. Put the egg mass into sterile water, incubate at 28°C in the dark, incubate and collect the second-instar larvae of R. incognita, adjust the concentration of the collected second-instar larvae suspension to 600/mL, and carry out the process within 24 hours. inoculation.
实施例1Example 1
一、苗期辣椒的抗病性分析1. Analysis of disease resistance of pepper in seedling stage
1、苗期辣椒的抗性鉴定1. Resistance identification of pepper in seedling stage
采用辣椒抗南方根结线虫病品种HDA149,辣椒感南方根结线虫病8214以及利用两亲本配制得到F2群体进行抗性鉴定,以茄门及其F2代群体(HDA149为父本,茄门为母本)为对照。具体步骤如下:待辣椒苗长出4片真叶时,在临近根系的土壤上打两个1.5cm深的孔,将南方根结线虫二龄幼虫分别接种到两个孔内,每株接种南方根结线虫600条,接种时注意摇匀线虫悬浮液。接种后的辣椒在25-28℃的温室培养,期间光照16h,黑暗8h,正常管理。接种线虫60d后,取出辣椒根系,轻轻抖下根表的土壤,用清水小心冲洗干净,统计并记录每个单株的根结数及卵块数。Using pepper varieties HDA149 resistant to M. incognita, pepper susceptible to M. incognita 8214, and F2 populations prepared from two parents for resistance identification, Agaricula and its F2 generation population (HDA149 as the male parent and Agaricus as the female) This) is the control. The specific steps are as follows: when the pepper seedlings grow 4 true leaves, punch two 1.5cm deep holes in the soil near the root system, inoculate the second instar larvae of M. incognita into the two holes respectively, and inoculate each plant with southern 600 root-knot nematodes, pay attention to shaking the nematode suspension when inoculating. The inoculated peppers were cultured in a greenhouse at 25-28°C with 16 hours of light and 8 hours of darkness during normal management. 60 days after inoculation with nematodes, take out the pepper root system, shake off the soil on the root surface gently, rinse it carefully with clean water, and count and record the number of root knots and egg masses per plant.
本实验所采用方法是Djian-Caporalino等人使用的抗性鉴定方法,若植株卵块数≤5,则记为抗病单株;若植株卵块数>5。则记为感病单株;若植株卵块数<5,但根结数≥6,记为抗性不确定单株。The method used in this experiment is the resistance identification method used by Djian-Caporalino et al. If the number of egg masses in a plant is less than or equal to 5, it is recorded as a disease-resistant single plant; if the number of egg masses in a plant is >5. If the number of egg masses is less than 5, but the number of root knots is greater than or equal to 6, it is recorded as a single plant with uncertain resistance.
2、苗期辣椒的抗性鉴定结果2. Results of resistance identification of pepper in seedling stage
苗期辣椒的抗性鉴定结果表明:辣椒抗南方根结线虫病品种HDA149的所有单株未发病,辣椒感南方根结线虫病8214均表现为高度感病;对照材料茄门均表现为高度感病;F1代群体的所有单株均表现为抗病;F2代群体中抗病植株有2148株,感病植株有594株,经χ2检验符合3R:1S的分离比例(p=0.05)。以上结果表明,HDA149对南方根结线虫无毒品种的抗性是由一对单显性基因控制的。The results of resistance identification of pepper at seedling stage showed that all individual plants of pepper resistant to M. incognita disease HDA149 did not develop disease, while pepper-susceptible M. incognita 8214 showed high susceptibility; the control material Solanum spp. All individual plants in the F1 generation population were resistant to disease; there were 2148 resistant plants and 594 susceptible plants in the F2 generation population, which were in line with the 3R:1S segregation ratio (p=0.05) by χ 2 test. The above results indicated that the resistance of HDA149 to avirulent cultivars of M. incognita was controlled by a pair of single-dominant genes.
二、分子标记SCAR537的获得2. Acquisition of molecular marker SCAR537
采用CTAB法分别提取感病品种和抗病品种的基因组DNA,并采用混合分组分析法(Bulked Segregation Analysis,BSA法)进行分子标记的筛选。具体步骤如下:The genomic DNAs of susceptible and resistant varieties were extracted by CTAB method, and molecular markers were screened by Bulked Segregation Analysis (BSA). Specific steps are as follows:
1、抗感池的获得1. Obtaining the anti-inductive pool
在F2分离群体中随机选取30株极端抗病植株和30株极端感病植株,分别提取DNA,然后将30株抗病植株的DNA等量混合,得到抗病池;将30株感病植株的DNA等量混合,得到感病池。30 extreme disease-resistant plants and 30 extremely susceptible plants were randomly selected from the F2 isolated population, DNA was extracted respectively, and then the DNA of the 30 disease-resistant plants was mixed in equal amounts to obtain a disease-resistant pool; Equal amounts of DNA were mixed to obtain a susceptible pool.
2、分子标记Indel537的开发2. Development of molecular marker Indel537
通过测序,抗病池得到2664759945条序列,基因组覆盖度为90.8%,感病池得到2688580724条序列,基因组覆盖度为91.6%。以遵辣1号基因组为参考基因组,分别计算两个混池中各SNPs位点的频率及两个混池中对应SNPs频率差值(即delta值),最后以平均delta值(1MB为窗口,20Kb为步长)为纵坐标,染色体位置为横坐标作图,发现在9号染色体上的1522400bp和7862507bp之间出现了SNP不平衡的状况,说明抗病池中的单株在9号染色体上的该区间内含有与抗病相关的片段,感病池中的单株在同一区域含有与感病相关的片段。Through sequencing, the resistant pool obtained 2664759945 sequences with a genome coverage of 90.8%, and the susceptible pool obtained 2688580724 sequences with a genome coverage of 91.6%. Taking the Zunla No. 1 genome as the reference genome, the frequency of each SNPs site in the two pools and the frequency difference (ie delta value) of the corresponding SNPs in the two pools were calculated respectively, and finally the average delta value (1MB as the window, 20Kb is the step size) is the ordinate, and the chromosome position is the abscissa. It is found that there is a SNP imbalance between 1522400bp and 7862507bp on
根据已公布的遵辣1号辣椒基因组9号染色体的核苷酸序列设计分子标记,利用两个亲本(辣椒抗根结线虫病品种HDA149和辣椒感根结线虫病8214)和两个混池(抗病池和感病池)对650对SSR分子标记和Indel分子标记的多态性进行筛选。Molecular markers were designed according to the published nucleotide sequence of Zunla No. 1
筛选分子标记Indel537的核苷酸序列所用的上下游引物如下所示:The upstream and downstream primers used to screen the nucleotide sequence of the molecular marker Indel537 are as follows:
Indel537-F:5’-ACAGTCTATCAAAGTGTTCAGGG-3’(SEQ ID No:1)Indel537-F: 5'-ACAGTCTATCAAAGTGTTCAGGG-3' (SEQ ID No: 1)
Indel537-R:5’-CTGGAATGAAAAAAAATTTAAATAA-3’(SEQ ID No:2)Indel537-R: 5'-CTGGAATGAAAAAAAATTTAAAATAA-3' (SEQ ID No: 2)
将分子标记Indel537在根结线虫病辣椒HDA149上扩增出的特异性片段连接到中间载体pEASY-Blunt Clonning Vector(北京全式金生物技术有限公司)上,并用通用引物对M13F和M13R进行测序,得到重组载体的序列如SEQ ID No:3所示。其中,Indel537扩增出来的片段为297bp的DNA序列,所述序列如SEQ ID No:4所示。The specific fragment amplified by the molecular marker Indel537 on the root-knot nematode pepper HDA149 was ligated to the intermediate vector pEASY-Blunt Clonning Vector (Beijing Quanshijin Biotechnology Co., Ltd.), and the universal primers were used to sequence M13F and M13R, The sequence of the obtained recombinant vector is shown in SEQ ID No:3. Wherein, the fragment amplified by Indel537 is a DNA sequence of 297bp, and the sequence is shown in SEQ ID No:4.
3、遗传连锁分析和抗病基因的定位3. Genetic linkage analysis and localization of disease resistance genes
利用在双亲、抗感池中筛选的具有多态性的分子标记Indel537分析F2群体的2148个单株的基因型,并结合F2群体的2148个单株田间的抗病性鉴定结果,利用JoinMap 4.0软件绘制抗病基因的连锁遗传图谱,如图1所示。The genotypes of 2148 individual plants of the F2 population were analyzed using the polymorphic molecular marker Indel537 screened in the parental and susceptible pools, and the results of the field resistance identification of the 2148 individual plants of the F2 population were combined using JoinMap 4.0 The software draws the linked genetic map of disease resistance genes, as shown in Figure 1.
由图1的结果表明:分子标记Indel537与抗线虫基因Me3基因的遗传距离为0.5cM。说明分子标记Indel537与抗线虫基因Me3基因紧密连锁,可用于进一步SCAR分子标记的转化。The results in Figure 1 show that the genetic distance between the molecular marker Indel537 and the nematode resistance gene Me3 gene is 0.5 cM. It shows that the molecular marker Indel537 is closely linked with the nematode resistance gene Me3 gene and can be used for further transformation of SCAR molecular markers.
4、分子标记SCAR537标记的获得4. Obtaining the molecular marker SCAR537
以SEQ ID No:4所示序列为模板,用Primer 5.0软件设计得到优化退火温度的特异性SCAR537分子标记,目的片段大小为153bp,所述序列如SEQ ID No:5。且分子标记SCAR537在双亲、抗感池间具有多态性,该分子标记所扩增出的条带清晰,如图2所示。Using the sequence shown in SEQ ID No: 4 as a template, a specific SCAR537 molecular marker with an optimized annealing temperature was designed with Primer 5.0 software. The size of the target fragment was 153 bp, and the sequence was as shown in SEQ ID No: 5. And the molecular marker SCAR537 has polymorphism between the parental and anti-sense pools, and the band amplified by this molecular marker is clear, as shown in Figure 2.
从图2中可以看出:分子标记SCAR537在具有Me3基因的抗病品种的基因组DNA扩增产物大小为153bp;分子标记SCAR537在不含Me3基因的感病品种的基因组DNA扩增产物不含该特征片段。It can be seen from Figure 2 that the size of the molecular marker SCAR537 in the genomic DNA amplification product of the disease-resistant variety with Me3 gene is 153 bp; the molecular marker SCAR537 in the genomic DNA amplification product of the susceptible variety without the Me3 gene does not contain this Feature Fragments.
实施例2标记SCAR537的应用Application of Example 2 Labeling SCAR537
本发明用抗根结线虫病辣椒品种HDA149与感根结线虫病辣椒品种茄门杂交得到F1代,F1植株自交得到的F2群体。对实施例1获得的分子标记SCAR537进行验证,南方根结线虫的病原为南方根结线虫非毒性品种。2018年春季种植该F2群体植株,随机选取其中54株提取DNA。In the invention, the F1 generation is obtained by crossing the root-knot nematode-resistant pepper variety HDA149 with the root-knot nematode-susceptible pepper variety Qimen, and the F2 population is obtained by self-crossing the F1 plants. The molecular marker SCAR537 obtained in Example 1 was verified, and the pathogen of M. incognita was a non-toxic variety of M. incognita. The F2 population plants were planted in the spring of 2018, and 54 of them were randomly selected to extract DNA.
分别以上述供试材料及双亲的基因组DNA为模板,以下述序列为引物对进行PCR扩增,得到PCR扩增产物,Take the above-mentioned test material and the genomic DNA of the parents as templates respectively, and use the following sequences as primer pairs to carry out PCR amplification to obtain PCR amplification products,
上游引物:TTCGGATTAAGCACCCAAAA(SEQ ID NO:6)Upstream primer: TTCGGATTAAGCACCCAAAA (SEQ ID NO: 6)
下游引物:CCGGCTCAAAGCACGAGATA(SEQ ID NO:7)Downstream primer: CCGGCTCAAAGCACGAGATA (SEQ ID NO:7)
将PCR扩增产物用1%琼脂糖凝胶电泳检测。The PCR amplification products were detected by 1% agarose gel electrophoresis.
PCR扩增反应体系(20μl):包括1μl DNA工作液、10μl 2×EasyTaq PCR SuperMix(+dye)、正向引物和反向引物各1μl(10uM/L),用7μl ddH2O将总体积补齐至20μl。PCR amplification reaction system (20 μl): including 1 μl DNA working solution, 10
PCR扩增反应程序:94℃预变性3min,94℃变性30min,53℃退火30s,72℃延伸20s,35个循环;72℃延伸10min。PCR amplification reaction program: 35 cycles of pre-denaturation at 94°C for 3 minutes, denaturation at 94°C for 30 minutes, annealing at 53°C for 30s, extension at 72°C for 20s, and extension at 72°C for 10 minutes.
PCR扩增结果如图3所示,图3的结果表明,此标记鉴定的基因型与植株表型一致,即带型与抗病亲本HDA149基因型一致的辣椒具有抗根结线虫基因Me3基因,带型与感病亲本茄门基因型一致的辣椒为不含抗根结线虫基因Me3基因,对南方根结线虫呈感病性状。The PCR amplification results are shown in Figure 3. The results in Figure 3 show that the genotype identified by this marker is consistent with the plant phenotype, that is, peppers with the same band type as the disease-resistant parent HDA149 genotype have the root-knot nematode resistance gene Me3 gene, The pepper with the same band type as the susceptible parent Solanum genotype does not contain the Me3 gene of the root-knot nematode resistance gene, and is susceptible to the root-knot nematode.
本发明所述SCAR537标记在不同群体中均可准确预测后代是否含有抗根结线虫基因Me3基因,因此可用于辣椒辅助育种或选育抗根结线虫病辣椒品种。The SCAR537 marker of the present invention can accurately predict whether the progeny contains the root-knot nematode resistance gene Me3 gene in different populations, so it can be used for pepper-assisted breeding or selection of root-knot nematode-resistant pepper varieties.
实施例3Example 3
利用实施例1的SCAR537分子标记鉴定8株待测辣椒植株是否抗根结线虫病的方法,该方法包括以下步骤:Utilize the SCAR537 molecular marker of
(1)以SEQ ID No:6、SEQ ID No:7所示序列为上下游引物,以待测早期辣椒植株的DNA为模板进行扩增,得到扩增产物;(1) Take the sequences shown in SEQ ID No: 6 and SEQ ID No: 7 as upstream and downstream primers, and use the DNA of the early pepper plant to be tested as a template to amplify to obtain an amplification product;
将PCR扩增产物用1%琼脂糖凝胶电泳检测。The PCR amplification products were detected by 1% agarose gel electrophoresis.
PCR扩增反应体系(20μl):包括1μl DNA工作液、10μl 2×EasyTaq PCR SuperMix(+dye)、正向引物和反向引物各1μl(10uM/L),用7μl ddH2O将总体积补齐至20μl。PCR amplification reaction system (20 μl): including 1 μl DNA working solution, 10
PCR扩增反应程序:94℃预变性3min,94℃变性30min,53℃退火30s,72℃延伸20s,35个循环;72℃延伸10min。PCR amplification reaction program: 35 cycles of pre-denaturation at 94°C for 3 minutes, denaturation at 94°C for 30 minutes, annealing at 53°C for 30s, extension at 72°C for 20s, and extension at 72°C for 10 minutes.
(2)如果扩增产物大小为153bp,则鉴定待测辣椒植株为抗根结线虫病品种;如果扩增产物不含有该特征片段,则鉴定待测辣椒植株为感根结线虫病品种。(2) If the size of the amplified product is 153bp, then identify the pepper plant to be tested as a root-knot nematode-resistant variety; if the amplified product does not contain the characteristic fragment, identify the pepper plant to be tested as a root-knot nematode-susceptible variety.
PCR扩增结果如图3所示,图3的结果表明,植株1、3、5、8的扩增产物大小为153bp,初步鉴定待测辣椒植株1、3、5、8为抗根结线虫病品种;植株2、4、6、7的扩增产物不含有该特征性片段,初步鉴定待测辣椒植株2、4、6、7为感根结线虫病品种。The PCR amplification results are shown in Figure 3. The results in Figure 3 show that the size of the amplified products of
对所述辣椒植物做进一步的抗性鉴定,具体步骤如下:待辣椒苗长出4片真叶时,在临近根系的土壤上打两个1.5cm深的孔,将南方根结线虫二龄幼虫分别接种到两个孔内,每株接种南方根结线虫600条,接种时注意摇匀线虫悬浮液。接种后的辣椒在25-28℃的温室培养,期间光照16h,黑暗8h,正常管理。接种线虫60d后,取出辣椒根系,轻轻抖下根表的土壤,用清水小心冲洗干净,统计并记录每个单株的根结数及卵块数。Further resistance identification of the pepper plant is carried out, and the specific steps are as follows: when the pepper seedling grows 4 true leaves, two 1.5cm deep holes are made on the soil near the root system, and the second instar larvae of Nematode incognita Inoculate into two wells respectively, and each strain is inoculated with 600 M. incognita. When inoculating, pay attention to shaking the nematode suspension. The inoculated peppers were cultured in a greenhouse at 25-28°C with 16 hours of light and 8 hours of darkness during normal management. 60 days after inoculation with nematodes, take out the pepper root system, shake off the soil on the root surface gently, rinse it carefully with clean water, and count and record the number of root knots and egg masses per plant.
本实验所采用方法是Djian-Caporalino等人使用的抗性鉴定方法,若植株卵块数≤5,则记为抗病单株;若植株卵块数>5。则记为感病单株;若植株卵块数<5,但根结数≥6,记为抗性不确定单株。The method used in this experiment is the resistance identification method used by Djian-Caporalino et al. If the number of egg masses in a plant is less than or equal to 5, it is recorded as a disease-resistant single plant; if the number of egg masses in a plant is >5. If the number of egg masses is less than 5, but the number of root knots is greater than or equal to 6, it is recorded as a single plant with uncertain resistance.
上述辣椒的抗性鉴定结果表明:辣椒植株1、3、5、8所有单株未发病,辣椒植株2、4、6、7均表现为高度感病,与本发明鉴定方法的结果一致。The above resistance identification results of pepper show that all individual plants of
以上所述仅为本发明的实施例,并非因此限制本发明的专利范围,凡是利用本发明说明书内容所作的等效结构或等效流程变换,或直接或间接运用在其他相关的技术领域,均同理包括在本发明的专利保护范围内。The above descriptions are only the embodiments of the present invention, and are not intended to limit the scope of the patent of the present invention. Any equivalent structure or equivalent process transformation made by using the contents of the description of the present invention, or directly or indirectly applied in other related technical fields, are all applicable. Similarly, it is included in the scope of patent protection of the present invention.
序列表sequence listing
<110> 中国农业科学院蔬菜花卉研究所<110> Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences
<120> 与抗根结线虫基因Me3连锁的SCAR分子标记及其应用<120> SCAR Molecular Marker Linked to Root-knot Nematode Gene Me3 and Its Application
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acagtctatc aaagtgttca ggg 23acagtctatc aaagtgttca ggg 23
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<213> 人工序列(未知)<213> Artificial Sequence (Unknown)
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ctggaatgaa aaaaaattta aataa 25ctggaatgaa aaaaaattta aataa 25
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<212> DNA<212> DNA
<213> 人工序列(未知)<213> Artificial Sequence (Unknown)
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ggatatctgc agaattgccc tttcgcttga aacttctgga taagatatgg tataataggg 120ggatatctgc agaattgccc tttcgcttga aacttctgga taagatatgg tataataggg 120
atcaattttt tgttaaaaaa aatgaagtta tgtggatcaa tttttaacac tcaagcaact 180atcaattttt tgttaaaaaa aatgaagtta tgtggatcaa ttttttaacac tcaagcaact 180
tataagttcg gattaagcac ccaaaacagt gctctgttac aacttctaac ttaaattagg 240tataagttcg gattaagcac ccaaaacagt gctctgttac aacttctaac ttaaattagg 240
gccattgtct actcacagaa acaaaaaaaa tggacagaaa aatttgataa ataaatatga 300gccattgtct actcacagaa acaaaaaaaa tggacagaaa aatttgataa ataaatatga 300
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tctacttctt tagaggtaga agggcaattc cagcacactg gcggccgtta ctagtggatc 420tctacttctt tagaggtaga agggcaattc cagcacactg gcggccgtta ctagtggatc 420
cgagctcggt accaagcttg gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt 480cgagctcggt accaagcttg gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt 480
atccgctcac aattccacac aacatacgag ccggaagcat aaagtgtaaa gcctggggtg 540atccgctcac aattccacac aacatacgag ccggaagcat aaagtgtaaa gcctggggtg 540
cctaatgagt gag 553cctaatgagt gag 553
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<212> DNA<212> DNA
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tcgcttgaaa cttctggata agatatggta taatagggat caattttttg ttaaaaaaaa 60tcgcttgaaa cttctggata agatatggta taatagggat caattttttg ttaaaaaaaa 60
tgaagttatg tggatcaatt tttaacactc aagcaactta taagttcgga ttaagcaccc 120tgaagttatg tggatcaatt tttaacactc aagcaactta taagttcgga ttaagcaccc 120
aaaacagtgc tctgttacaa cttctaactt aaattagggc cattgtctac tcacagaaac 180aaaacagtgc tctgttacaa cttctaactt aaattagggc cattgtctac tcacagaaac 180
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ctcgtgcttt gagccggggg tctatcggaa acagcctttc tacttcttta gaggtag 297ctcgtgcttt gagccgggggg tctatcggaa acagcctttc tacttcttta gaggtag 297
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<212> DNA<212> DNA
<213> 人工序列(未知)<213> Artificial Sequence (Unknown)
<400> 5<400> 5
ttcggattaa gcacccaaaa cagtgctctg ttacaacttc taacttaaat tagggccatt 60ttcggattaa gcacccaaaa cagtgctctg ttacaacttc taacttaaat tagggccatt 60
gtctactcac agaaacaaaa aaaatggaca gaaaaatttg ataaataaat atgagaggtt 120gtctactcac agaaacaaaa aaaatggaca gaaaaatttg ataaataaat atgagaggtt 120
agtgtttgtg tggtatctcg tgctttgagc cgg 153agtgtttgtg tggtatctcg tgctttgagc cgg 153
<210> 6<210> 6
<211> 20<211> 20
<212> DNA<212> DNA
<213> 人工序列(未知)<213> Artificial Sequence (Unknown)
<400> 6<400> 6
ttcggattaa gcacccaaaa 20ttcggattaa gcacccaaaa 20
<210> 7<210> 7
<211> 20<211> 20
<212> DNA<212> DNA
<213> 人工序列(未知)<213> Artificial Sequence (Unknown)
<400> 7<400> 7
ccggctcaaa gcacgagata 20ccggctcaaa gcacgagata 20
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CN101153318A (en) * | 2006-09-29 | 2008-04-02 | 中国农业科学院蔬菜花卉研究所 | Molecular markers for assisting selection of root-knot nematode resistant N genes |
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Non-Patent Citations (6)
Title |
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Root-knot nematode (Meloidogyne spp.) Me resistance genes in pepper (Capsicum annuum L.) are clustered on the P9 chromosome;C Djian-Caporalino等;《Theor Appl Genet》;20061129;第114卷(第3期);第473-486页 * |
对辣椒抗性基因Me3表现毒性的南方根结线虫群体的SCAR分子标记;王刚等;《中国农业科学》;20130301;第46卷(第5期);第943-949页 * |
快速鉴定辣椒抗根结线虫基因Me3的SCAR标记开发;曹鸿一;《中国优秀博硕士学位论文全文数据库(硕士)农业科技辑(月刊)》;20190930(第09期);D046-102 * |
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辣椒抗根结线虫基因Me3的精细定位研究;许小艳等;《园艺学报》;20110228;第38卷(第02期);第288-294页 * |
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