技术领域technical field
本发明属于葡萄育种技术领域，涉及对毁灭性害虫葡萄根瘤蚜抗性的快速检测。The invention belongs to the technical field of grape breeding and relates to the rapid detection of the resistance to the destructive pest phylloxera.
背景技术Background technique
葡萄根瘤蚜是葡萄的一种毁灭性虫害，主要通过取食根系危害葡萄，常规的植保技术难以防治。目前最有效的预防方法是采用葡萄根瘤蚜抗性砧木，进行嫁接栽培。然而在葡萄砧木育种过程中，选育的砧木是否具有根瘤蚜抗性，需要进行砧木对葡萄根瘤蚜抗性的鉴定，通常采用葡萄砧木根系人工接种葡萄根瘤蚜后统计感染形成的根瘤数量进行鉴定，无根瘤形成或根瘤形成数量少的为具有抗性，根瘤数量多的为感性。但该抗性鉴定需要在特殊的隔离条件下进行以避免虫害扩散，此外鉴定需要2年以上的重复试验鉴定，耗费时间较长，操作过程中的环境条件可能会影响鉴定结果的准确性。Grape phylloxera is a destructive insect pest of grapes. It mainly harms grapes by feeding on the roots. It is difficult to prevent and control with conventional plant protection techniques. Currently the most effective preventive method is to use phylloxera-resistant rootstocks for grafting. However, in the process of grape rootstock breeding, whether the selected rootstock has phylloxera resistance needs to be identified for the rootstock’s resistance to phylloxera. Usually, the roots of grape rootstocks are artificially inoculated with phylloxera and the number of nodules formed by infection is counted for identification. , No nodules or a small number of nodules are resistant, and those with a large number of nodules are susceptible. However, the identification of resistance needs to be carried out under special isolation conditions to avoid the spread of pests. In addition, the identification requires more than 2 years of repeated test identification, which takes a long time, and the environmental conditions during the operation may affect the accuracy of the identification results.
发明内容Contents of the invention
本发明的目的在于提供一种用于葡萄根瘤蚜抗性的快速鉴定方法。The purpose of the present invention is to provide a rapid identification method for grape phylloxera resistance.
为达到上述目的，本发明采用了以下技术方案。In order to achieve the above object, the present invention adopts the following technical solutions.
以待检测的葡萄砧木的DNA为模板，对与葡萄根瘤蚜抗性相连锁的DNA分子标记进行聚合酶链式反应扩增，对扩增获得的产物进行电泳检测，若电泳结果中存在与所述DNA分子标记对应的DNA条带，则说明对应的葡萄砧木具有葡萄根瘤蚜抗性，若电泳结果中不存在与所述DNA分子标记对应的DNA条带，则说明对应的葡萄砧木不具有葡萄根瘤蚜抗性。Using the DNA of the grape rootstock to be detected as a template, perform polymerase chain reaction amplification on the DNA molecular markers linked to the resistance of grape phylloxera, and perform electrophoresis detection on the amplified products. If there is no DNA band corresponding to the DNA molecular marker in the electrophoresis result, it means that the corresponding grape rootstock has no phylloxera resistance. Phylloxera resistance.
所述DNA分子标记为微卫星分子标记。The DNA molecular marker is a microsatellite molecular marker.
所述聚合酶链式反应扩增采用寡聚脱氧核苷酸序列Seq01和Seq02作为引物对，Seq01具有如SEQ.ID.NO.1所示的核苷酸序列，Seq02具有如SEQ.ID.NO.2所示的核苷酸序列。The polymerase chain reaction amplification uses oligodeoxynucleotide sequences Seq01 and Seq02 as a primer pair, Seq01 has a nucleotide sequence as shown in SEQ.ID.NO.1, and Seq02 has a nucleotide sequence as shown in SEQ.ID.NO. .2 the nucleotide sequence shown.
所述DNA条带的大小约为169bp。The size of the DNA band is about 169bp.
本发明提供了一种在DNA水平上快速鉴定葡萄砧木对葡萄根瘤蚜有无抗性的方法，可以快速区分葡萄砧木有无葡萄根瘤蚜抗性，解决了现有葡萄根瘤蚜抗性鉴定需要特定隔离条件、易受环境条件影响且需要年限较长的问题。The invention provides a method for quickly identifying whether grape rootstocks are resistant to phylloxera at the DNA level, can quickly distinguish whether grape rootstocks are resistant to phylloxera, and solves the need for specific identification of phylloxera resistance in the existing grapes. Problems that are isolated, susceptible to environmental conditions, and require long years.
附图说明Description of drawings
图1为具有根瘤蚜抗性葡萄砧木和不具有根瘤蚜抗性葡萄砧木采用本方法检测的结果图；图1中1、2、3泳道为已知不具有根瘤蚜抗性的葡萄砧木经检测的结果，4、5、6泳道为已知有根瘤蚜抗性的葡萄砧木经检测的结果，M泳道为分子量标准，数字250、200以及150表示DNA条带大小，单位为碱基对(basepair,bp)。Fig. 1 is a graph of the detection results of grape rootstocks with and without phylloxera resistance by this method; lanes 1, 2, and 3 in Fig. 1 are known grape rootstocks without phylloxera resistance after detection 4, 5, and 6 swimming lanes are the results of the detection of grape rootstocks known to have phylloxera resistance, M swimming lanes are molecular weight standards, and the numbers 250, 200 and 150 indicate the size of the DNA band, and the unit is base pair (base pair ,bp).
具体实施方式Detailed ways
下面结合附图对本发明作详细说明。The present invention will be described in detail below in conjunction with the accompanying drawings.
本发明提供一种通过检测与葡萄根瘤蚜抗性性状基因紧密连锁的DNA分子标记来判断葡萄根瘤蚜抗性是否存在的方法。本发明基于微卫星(Simplesequencerepeat,SSR)分子标记技术，以本发明提供的寡聚脱氧核苷酸序列Seq01和Seq02作为SSR引物对，以待检测的葡萄砧木的DNA为模板，对与葡萄根瘤蚜抗性相连锁的特定DNA片段即DNA分子标记进行聚合酶链式反应(Polymerasechainreaction,PCR)扩增，对扩增获得产物进行电泳检测后，筛选出了大小约为169bp的特定DNA条带（品种间长度无差异），与葡萄根瘤蚜抗性存在紧密连锁关系，即具有该特定DNA条带的待检测葡萄砧木具有葡萄根瘤蚜抗性，无该特定条带的待检测葡萄砧木无葡萄根瘤蚜抗性。The invention provides a method for judging whether the grapevine phylloxera resistance exists by detecting the DNA molecular marker closely linked with the grapevine phylloxera resistance trait gene. The present invention is based on the microsatellite (Simple sequence repeat, SSR) molecular marker technology, with the oligodeoxynucleotide sequences Seq01 and Seq02 provided by the present invention as the SSR primer pair, with the DNA of the grape rootstock to be detected as a template, for the phylloxera The specific DNA fragment linked to the resistance, that is, the DNA molecular marker, was amplified by Polymerase chain reaction (PCR), and after the amplified product was detected by electrophoresis, a specific DNA band (variety) with a size of about 169 bp was screened out. There is no difference in length between them), and there is a close linkage relationship with phylloxera resistance, that is, the grape rootstocks to be tested with this specific DNA band have phylloxera resistance, and the grape rootstocks to be tested without this specific band have no phylloxera resistance.
本发明的具体步骤如下：Concrete steps of the present invention are as follows:
(1)采用常规的CTAB法或SDS法分别提取待检测葡萄砧木的叶片、茎段或根系部位的基因组DNA（即样品DNA），CTAB法提取样品中DNA的步骤如下：①0.4g左右叶片经液氮冷冻快速研磨成粉末状，转入2mL离心管中；②立即加入65℃预热的CTAB提取缓冲液700μL以及β-巯基乙醇20μL，剧烈震荡至充分混匀，置65℃水浴中保温30min，取出后冷却至室温；③加等体积的氯仿/异戊醇=24:1，轻轻地颠倒混匀，于室温下12000rpm离心10min，转移上清液至新的离心管中；④经过③后，向上清液中加等体积的氯仿/异戊醇=24:1，轻轻地颠倒混匀，于室温下12000rpm离心10min，转移上清液至新离心管中；⑤经过④后，向上清液中加入等体积经-20℃预冷的异丙醇，轻轻地颠倒混匀至有白色絮状沉淀出现后，静置于-20℃30min使沉淀生成,于4℃12000rpm离心10min，倒掉上清液，保留沉淀物（DNA）；⑥加入500μL预冷的70%乙醇洗涤沉淀物（13000rpm离心1min），倒掉上清液，保留沉淀，并重复本步骤一次；⑦加入500μL预冷的无水乙醇洗涤沉淀（13000rpm离心1min），倒掉上清液，沉淀静置风干后溶于40μLddH2O中4℃过夜；⑧取溶解后的样品DNA0.5μL用琼脂糖凝胶电泳法检测DNA浓度、纯度和降解程度。经检测样品DNA的OD260/280介于1.8-2.0之间、经0.7%的琼脂糖核酸电泳检测DNA样品无杂质、无明显降解的条件下，将各待检测葡萄砧木样品DNA加水稀释至终浓度为100ng/uL待用；(1) Use the conventional CTAB method or SDS method to extract the genomic DNA (i.e. sample DNA) of the leaves, stems or roots of the grape rootstock to be tested. The steps for extracting the DNA in the sample by the CTAB method are as follows: Freeze with liquid nitrogen and quickly grind into powder, transfer to a 2mL centrifuge tube; ② Immediately add 700 μL of CTAB extraction buffer preheated at 65 °C and 20 μL of β-mercaptoethanol, shake vigorously until fully mixed, and place in a 65 °C water bath for 30 minutes , take it out and cool to room temperature; ③add an equal volume of chloroform/isoamyl alcohol=24:1, gently invert and mix well, centrifuge at 12000rpm at room temperature for 10min, transfer the supernatant to a new centrifuge tube; ④After ③ Finally, add an equal volume of chloroform/isoamyl alcohol = 24:1 to the supernatant, gently invert and mix well, centrifuge at 12000rpm at room temperature for 10min, and transfer the supernatant to a new centrifuge tube; ⑤After ④, upward Add an equal volume of isopropanol pre-cooled at -20°C to the supernatant, gently invert and mix until a white flocculent precipitate appears, then stand at -20°C for 30 minutes to form a precipitate, and centrifuge at 12,000 rpm at 4°C for 10 minutes. Pour off the supernatant and keep the precipitate (DNA); ⑥ Add 500 μL pre-cooled 70% ethanol to wash the precipitate (centrifuge at 13000 rpm for 1 min), discard the supernatant, keep the precipitate, and repeat this step once; ⑦ Add 500 μL pre-cooled Wash the precipitate with cold absolute ethanol (centrifuge at 13000rpm for 1min), discard the supernatant, let the precipitate stand and air-dry, then dissolve it in 40 μL ddH2 O overnight at 4°C; ⑧ Take 0.5 μL of the dissolved sample DNA and use agarose gel electrophoresis Detect DNA concentration, purity, and degree of degradation. After the OD260/280 of the sample DNA is between 1.8-2.0, and the DNA sample is detected by 0.7% agarose nucleic acid electrophoresis without impurities and no obvious degradation, the DNA of each grape rootstock sample to be tested is diluted with water to the final concentration 100ng/uL for use;
(2)将按照本发明提供的寡聚脱氧核苷酸序列seq01和seq02合成的单链寡聚DNA，分别加水稀释成10umol/L水溶液，序列分别为：(2) Dilute the single-stranded oligomeric DNA synthesized according to the oligodeoxynucleotide sequences seq01 and seq02 provided by the present invention into a 10umol/L aqueous solution with water respectively, and the sequences are respectively:
Seq01:5’-TGTTGGTGTGTGTTTGTACGTG-3’Seq01: 5'-TGTTGGTGTGTGTGTTTGTACGTG-3'
Seq02:5’-TGTTATTGAGTTAGAATGAAGTGTTGG-3’Seq02: 5'-TGTTATTGAGTTAGAATGAAGTGTTGG-3'
(3)在每个0.2mL薄壁PCR管中加入：2uL2mmol/L的dNTP,1.7uL25mmol/L的MgCl2,2ul10Xtaq缓冲液，步骤(2)配制的seq01、seq02溶液各2.0uL,步骤(1)配制的待测样品DNA溶液2uL,加入Taq聚合酶1个单位，加水至总体积20ul，轻微震荡混匀，瞬时离心；(3) Add in each 0.2mL thin-walled PCR tube: dNTP of 2uL2mmol/L,MgCl2 of 1.7uL25mmol/L, 2ul10Xtaq buffer solution, each 2.0uL of seq01 and seq02 solutions prepared in step (2), step (1 ) prepared 2uL of the DNA solution of the sample to be tested, added 1 unit of Taq polymerase, added water to a total volume of 20ul, vortexed slightly, and centrifuged instantaneously;
(4)把上述PCR管置于PCR仪上，执行下述程序：94度，5分钟；94度，60秒，55度，90秒，72度，90秒，循环35次；72度，5分钟；程序结束；(4) Place the above-mentioned PCR tube on the PCR instrument and perform the following procedures: 94 degrees, 5 minutes; 94 degrees, 60 seconds, 55 degrees, 90 seconds, 72 degrees, 90 seconds, 35 cycles; 72 degrees, 5 minutes minutes; end of program;
(5)把PCR管中的产物进行6%的聚丙烯凝胶电泳或2.5%的琼脂糖凝胶电泳，检查不同待测样品DNAPCR反应中产生的DNA条带；(5) The product in the PCR tube is subjected to 6% polypropylene gel electrophoresis or 2.5% agarose gel electrophoresis to check the DNA bands produced in the DNA PCR reactions of different samples to be tested;
本发明选择了已知具有根瘤蚜抗性的葡萄砧木品种（包括Rici、Boerner、V.Riparia181g等）和已知无根瘤蚜抗性的葡萄砧木品种（包括华佳8号、山葡萄、刺葡萄等）共计30余种进行了试验，其中一次试验中（参见图1），泳道1～3依次为华佳8号、山葡萄、刺葡萄；泳道4～6依次为Rici、Boerner、V.Riparia181g，以上材料均采集于西北农林科技大学葡萄种质资源圃；The present invention has selected grape rootstock varieties known to have phylloxera resistance (including Rici, Boerner, V.Riparia181g, etc.) and known grape rootstock varieties without phylloxera resistance (including Huajia No. ), a total of more than 30 species were tested, and in one test (see Figure 1), lanes 1 to 3 were Huajia No. 8, mountain grape, and thorn grape; lanes 4 to 6 were Rici, Boerner, V.Riparia 181g, and above The materials were all collected from the grape germplasm resource nursery of Northwest A&F University;
(6)根据试验结果可以确定，具有根瘤蚜抗性的葡萄砧木能够产生图1中箭头所示位置的特定大小（169bp）的DNA片段所形成的条带，不具有根瘤蚜抗性的葡萄砧木不能产生相应的所示位置的特定大小DNA片段所形成的条带；若加入了某一待检测DNA的泳道有169bp这一特定大小的DNA条带产生，则该待测样品DNA为来源于具有根瘤蚜抗性的葡萄砧木植株，若无特定大小(169bp)的DNA条带产生，则该待测样品DNA为来源于无根瘤蚜抗性的葡萄砧木植株。(6) According to the test results, it can be determined that grape rootstocks with phylloxera resistance can produce bands formed by DNA fragments of a specific size (169 bp) at the position indicated by the arrow in Figure 1, and grape rootstocks without phylloxera resistance The band formed by the DNA fragment of a specific size corresponding to the indicated position cannot be produced; if a DNA band of a specific size of 169 bp is added to the swimming lane of a certain DNA to be detected, the DNA of the sample to be tested is derived from a If the phylloxera-resistant grape rootstock plants do not produce a DNA band of a specific size (169bp), the DNA of the sample to be tested is derived from the phylloxera-resistant grape rootstock plants.
采用无性繁殖的葡萄砧木可以用叶片、根系、茎段在任何季节提取DNA进行检测，取样部位不影响检测结果的一致性，取样部位和取样时间不影响检测结果。Grape rootstocks that are propagated asexually can be tested by extracting DNA from leaves, roots, and stems in any season. The sampling site does not affect the consistency of the test results, and the sampling site and sampling time do not affect the test results.
本发明可以用于葡萄根瘤蚜抗性性状的DNA分子标记辅助选择育种，用于葡萄根瘤蚜抗性育种中的抗性材料的早期选择和非抗性材料的淘汰；同时，以该特定的DNA条带为路标，可以进行葡萄根瘤蚜抗性基因的精细定位或通过染色体步移法接近葡萄根瘤蚜抗性基因，从而为葡萄根瘤蚜抗性基因的克隆打下基础。The present invention can be used for DNA molecular marker assisted selection breeding of grape phylloxera resistance traits, for early selection of resistant materials and elimination of non-resistant materials in grape phylloxera resistance breeding; at the same time, with the specific DNA The strips are signposts, which can be used for fine mapping of grapevine phylloxera resistance genes or close to grapevine phylloxera resistance genes by chromosome walking, thus laying the foundation for the cloning of grapevine phylloxera resistance genes.
| Application Number | Priority Date | Filing Date | Title |
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| CN201410131800.7ACN103937887B (en) | 2014-04-02 | 2014-04-02 | A Rapid Identification Method for Grape Phylloxera Resistance |
| Application Number | Priority Date | Filing Date | Title |
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| CN201410131800.7ACN103937887B (en) | 2014-04-02 | 2014-04-02 | A Rapid Identification Method for Grape Phylloxera Resistance |
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| CN201410131800.7AExpired - Fee RelatedCN103937887B (en) | 2014-04-02 | 2014-04-02 | A Rapid Identification Method for Grape Phylloxera Resistance |
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| CN101646769A (en)* | 2007-02-20 | 2010-02-10 | 孟山都技术公司 | Invertebrate micrornas |
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| CN101646769A (en)* | 2007-02-20 | 2010-02-10 | 孟山都技术公司 | Invertebrate micrornas |
| Title |
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| Junke Zhang et al.A framework map from grapevine V3125 (Vitis vinifera ‘Schiava grossa’ × ‘Riesling’) × rootstock cultivar ‘Bö* |
| rner’ (Vitis riparia × Vitis cinerea) to localize genetic determinants of phylloxera root resistance.《Theor Appl Genet》.2009,第119卷第1039－1051页.* |
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