CN109355425B - Molecular marker linked with wheat scab resistance QTL and application thereof - Google Patents
Molecular marker linked with wheat scab resistance QTL and application thereof Download PDFInfo
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Abstract
The invention discloses a molecular marker linked with wheat scab resistance QTL and application thereof, wherein the molecular marker takes the DNA of wheat variety Sumai No.3 as a template, and primers with nucleotide sequences shown as SEQ ID NO.1 and SEQ ID NO.2 respectively carry out PCR amplification and electrophoresis to obtain a DNA fragment with the size of 500 bp; the molecular marker can be applied to screening of wheat scab resistant materials and molecular marker-assisted selective breeding, namely, the resistance of wheat to scab can be judged by detecting whether the molecular marker exists in the wheat, the amplification efficiency is high, the amplification result specificity is good, the breeding time can be shortened, and the method is suitable for popularization and application.
Description
Technical Field
The invention belongs to the field of wheat genetic breeding and molecular biology, and particularly relates to a molecular marker linked with wheat scab resistance QTL and application thereof.
Background
Scab is one of the major diseases of wheat worldwide, and seriously affects the yield and quality of wheat. Wheat scab occurs in large areas in winter wheat areas in the middle and lower reaches of Yangtze river and in northeast spring wheat areas in China, the yield loss of 5-10% can be caused in general epidemic years, and partial field blocks can be harvested absolutely in pandemic years. In recent years, wheat scab has spread to other major wheat producing areas in China with the change of farming modes and climate change. In addition, mycotoxin carried by diseased wheat grains affects the quality of seeds, harms the health of people and animals, and becomes one of the hidden dangers threatening the safety of food. The utilization of gibberellic disease resistance genes for variety improvement is a fundamental way to reduce the harm of wheat scab.
Resistance to wheat scab is a complex quantitative trait controlled by a few major genes plus numerous minor genes. More than 100 QTLs associated with scab resistance to infestation, expansion or DON toxin resistance have been located from common wheat or closely related species. The main effect QTL with better reproducibility in independent experiments comprises the following components: an anti-expansion site Fhb1 located on the short arm of chromosome 3B, an anti-expansion site Fhb2 located on the short arm of chromosome 6BS, and an anti-invasion site Qfhs. ifa-5A located on the short arm of chromosome 5A. Waldron et al used the "Sumai No.3 × Stoa" recombinant inbred line to first locate a major QTL for resistance to expansion at 3 BS. Liu et al developed 27 STS markers for encryption of Qfhs-3BS, which were fine-mapped to the 1.2cM interval by screening and identifying recombinants and named Fhb 1. In addition, Cuthbert et al finely located the resistance QTL on 6BS by using the recombinant inbred line population constructed from the derivative line Sumai No.3, located between Xgwm133 and Xgwm644, and the genetic distance between the markers was about 4 cM. A QTL associated with infection with head blight resistance (qfhs. ifa-5A) was detected in the vicinity of the juxtaglomer of both the derivative line CM82036 from soymbus 3 and the short arm of the 5A chromosome of osbeckia, and accounted for more than 20% of phenotypic variation. Using the recombinant inbred line of Nanda2419 XHope Water white, the infection-resistant sites were finely located between Xwmc415-Xgwm304 at a genetic distance of 0.3 cM. Buerstmayr uses the derivative line "CM-82036" from Sumai No.3 to locate it between Xgwm293-Xgwm156 markers, which are genetically 23.9 cM.
"Sumai No. 3" is a recognized high-quality gibberellic disease resistance source, and Fhb1 and Qfhs. ifa-5A are introduced into different wheat varieties through molecular marker-assisted selection to form new varieties or lines with improved gibberellic disease resistance to different degrees. At present, no report is found about a QTL locus QFLB.2BL linked molecular marker on the long arm of the wheat 2B chromosome.
Disclosure of Invention
Aiming at the problems, the invention obtains a new wheat scab resistance QTL locus QFHB.2BL through whole genome correlation analysis and physical position comparison, develops a new molecular marker JAAS52433R1 according to the locus, judges the scab resistance of the wheat plant by detecting whether the wheat plant contains the molecular marker closely linked with the wheat scab resistance QTL locus QFHB.2BL, screens wheat scab resistance materials and accelerates the scab resistance breeding process.
Specifically, the invention is realized by the following steps:
the invention firstly provides a molecular marker linked with wheat scab resistance QTL, the molecular marker takes DNA of Sumai No.3 as a template, sequences with nucleotide sequences respectively shown as SEQ ID NO.1 and SEQ ID NO.2 as primers to carry out PCR amplification, and DNA fragments with the size of 500bp are obtained after electrophoresis of amplification products on agarose gel with the mass percentage of 1%; the molecular marker is closely linked with a new wheat scab resistance QTL site 'QFHB.2BL', and the applicant names the molecular marker as JAAS52433R 1.
Secondly, the invention also provides the application of the molecular marker in predicting the resistance of wheat scab, which comprises the following specific steps: PCR amplification is carried out by taking sample wheat DNA as a template and SEQ ID NO.1 and SEQ ID NO.2 as primers, and after an amplification product is electrophoresed on agarose gel with the mass percent of 1%, if a DNA fragment with the size of 500bp (namely JAAS52433R1) exists, the resistance of the sample wheat to gibberellic disease is predicted to at least reach an anti-resistance level.
In the invention, the resistance detection of the wheat scab is carried out according to the agricultural industry standard NY/T2954-2016 & lt technical specification for identifying scab resistance of wheat regional experimental varieties & gt of the people's republic of China, wherein the ' level of resistance to the scab ' means that the resistance of the wheat variety to the scab reaches the resistance or the water resistance level, and the average severity of the resistance is less than 3.
Thirdly, the invention also provides a pair of primer pairs for predicting the resistance of wheat to scab, and the nucleotide sequences of the primer pairs are respectively shown as NO.1 and SEQ ID NO. 2.
The invention also provides application of the primer pair in predicting the resistance of wheat scab, which comprises the following specific steps: and (2) carrying out PCR amplification by using the DNA of the sample wheat as a template and the SEQ ID NO.1 and SEQ ID NO.2 as primers, and after an amplification product is electrophoresed on agarose gel with the mass percentage of 1%, if a band with the size of 500bp exists, predicting that the resistance of the sample wheat to the gibberellic disease at least reaches an anti-resistance level.
Fifth, the invention also provides a method for predicting wheat scab resistance, which comprises the following specific steps: and (3) carrying out PCR amplification by using the DNA of the sample wheat as a template and using the SEQ ID NO.1 and the SEQ ID NO.2 as primers, and then carrying out electrophoresis, wherein if a DNA fragment with the size of 500bp exists, the resistance of the sample wheat to the gibberellic disease is predicted to at least reach an anti-resistance level.
The PCR reaction system comprises: 10 XBuffer 1. mu.l MgCl at 25mM concentration20.5. mu.l of dNTP at a concentration of 2.5mM, 10. mu.M of primer SEQ ID NO. 10.1. mu.l, 10. mu.M of primer SEQ ID NO. 20.1. mu.l, 5U/. mu.l of Taq polymerase 0.2. mu.l, template DNA 50ng, ddH2O is complemented to 10 mu l;
PCR reaction procedure: 3 minutes at 94 ℃; extension at 94 ℃ for 15 seconds, at 54.5 ℃ for 30 seconds, at 72 ℃ for 30 seconds, for 30 cycles; extension at 72 ℃ for 5 minutes.
The invention has the beneficial effects that: in the wheat scab resistance breeding, the scab resistance of wheat can be selected in early generation through the detection result of the molecular marker, and the breeding process is accelerated. The molecular marker primer pair is obtained through manual comparison and correction, the specificity of an amplification sequence is ensured through the manual comparison, the accuracy of an amplification product is ensured through the manual adjustment of the terminal position of the primer to anchor a mutation site, the amplification efficiency of the obtained primer pair is high, the specificity of an amplification result is good, and the result judgment is accurate and effective.
Drawings
FIG. 1 shows the result of amplification of primer pairs of the software designed molecular markers P1 and P2 in resistant varieties;
wherein, M: the lanes DL2000 and 1-8 are respectively Ningmai No. 9, Ningmai No. 13, Wangshuibai, Sumai No.3, Jimai No. 22, Annong 8455, Huai mai 18 and Xiaoyan 54.
FIG. 2 shows the amplification result of primer pair of molecular marker JAAS52433R1 in resistant variety;
wherein, M: the lanes DL2000 and 1-8 are respectively Ningmai No. 9, Ningmai No. 13, Wangshuibai, Sumai No.3, Jimai No. 22, Annong 8455, Huai mai 18 and Xiaoyan 54.
Detailed Description
The experimental procedures used in the following examples are all conventional procedures unless otherwise specified. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
The following examples relate to nucleotide sequences:
SEQ ID NO.1：GTCTTCCCTAAGCGACGGTATCAT；
SEQ ID NO.2：GAAACAGGTCAAGATGATGAAAAGAT；
SEQ ID NO.3:GAAACAGGTCAAGATGATGAAAAGTT；
SEQ ID NO.4:ATCCTGGAGATGTGATGTGTGTTGA；
SEQ ID NO.5:CTTCTTGAAGATTTGGATGATCGT；
SEQ ID NO.6:GCCCTCTAATGACAAGACTGCTAAA。
examples relate to material sources:
the following examples relate to wheat varieties or lines such as sumai No.3 (table 1): the wheat is preserved by the wheat research institute of agricultural biotechnology of agricultural academy of sciences of Jiangsu province;
gibberella: is Asian fusarium Fa0609, which is preserved by the wheat crop research institute of food crop research institute of agricultural academy of sciences of Jiangsu province.
Example 1 verification of molecular marker JAAS52433R1 in resistant varieties
The DNA templates for PCR amplification in this example were: ningmai No. 9, Ningmai No. 13, Wangshuibai and Sumai No.3, the average severity of the wheat varieties is respectively 2.25, 2.53, 0.96 and 1.33, and the wheat varieties achieve or are more than or equal to resistance to gibberellic disease; the average disease spikelet rates of the Jimai 22, the Annong 8455, the Huai mai 18 and the Xiaoyan 54 are 8.41, 8.14, 7.44 and 7.94 respectively, and the disease variety is an infectious disease variety. Extracting the genomic DNA of the wheat leaves by using a CTAB method, and quantifying the genomic DNA by using a micro-spectrophotometer Nanodrop.
When the software Primer premier5.0 is used for designing the molecular marker Primer of the variation site, the Primer design software cannot carry out sequence comparison and correction, cannot anchor the variation site, and cannot adjust the base type at the tail end of the Primer. The primer pair P1 (the nucleotide sequences of which are respectively shown as SEQ ID NO.3 and SEQ ID NO. 4) and P2 ((the nucleotide sequences of which are respectively shown as SEQ ID NO.5 and SEQ ID NO. 6)) designed by software are used for PCR amplification by taking the anti-susceptible variety as a template:
the PCR reaction system was 10. mu.l: 10 XBuffer 1. mu.l, MgCl2(25mM)0.5. mu.l, 0.5. mu.l dNTP (2.5mM), 0.1. mu.l primer SEQ ID NO.1 (10. mu.M), 0.1. mu.l primer SEQ ID NO.3 (10. mu.M), 0.2. mu.l Taq polymerase (5U/. mu.l), 50ng template DNA, ddH2O is complemented to 10 mu l;
PCR reaction procedure: 3 minutes at 94 ℃; extension at 94 ℃ for 15 seconds, at 54.5 ℃ for 30 seconds, at 72 ℃ for 30 seconds, for 30 cycles; extension at 72 ℃ for 5 min;
the amplification products were detected by 1.0% agarose gel electrophoresis, the electrophoresis results are shown in FIG. 1, the amplification products of primer pair P1 and P2 contain non-specific amplification bands, and the amplification results cannot effectively identify the resistant varieties.
On the basis, the applicant artificially adjusts the anchoring position of the tail end of a primer pair and adjusts the base type of the tail end on the basis of artificially comparing and correcting the sequence of the variation site, artificially changes the 25 th base of the sequence of SEQ ID NO.3 from 'T' to 'A', further obtains a primer pair (SEQ ID NO.1 and SEQ ID NO. 2) with molecular markers JAAS52433R1, and performs PCR amplification on the primer pair SEQ ID NO.1 and SEQ ID NO.2 by using the genomic DNA of the variety as a template and the primer pair of the molecular markers JAAS52433R 1:
the PCR reaction system is a 10 mu l system and is suitable for screening high-throughput samples: 10 XBuffer 1. mu.l, MgCl2(25mM) 0.5. mu.l, dNTP (2.5mM) 0.5. mu.l, primer SEQ ID NO.1 (10. mu.M) 0.1. mu.l, primer SEQ ID NO.2 (10. mu.M) 0.1. mu.l, Taq polymerase (5U/. mu.l) 0.2. mu.l, template DNA 50ng, ddH2Make up to 10. mu.l of O.
PCR reaction procedure: 3 minutes at 94 ℃; extension at 94 ℃ for 15 seconds, at 54.5 ℃ for 30 seconds, at 72 ℃ for 30 seconds, for 30 cycles; extension at 72 ℃ for 5 min;
the amplification products were detected by electrophoresis on a 1.0% agarose gel and the results recorded by a gel imaging system. The detection results are shown in FIG. 2, including Ningmai No. 9, Ningmai No. 13, Wangshuibai and Sumai No.3 containing the specific amplification product band (500bp) of the molecular marker JAAS52433R1, and Jimai 22, Annong 8455, Huai mai 18 and Xiaoyan 54 containing no specific amplification product band (500 bp). The above results can be estimated as: the resistance of nimi No. 9, nimi No. 13, wangshuibai and sumai No.3 to scab is: at least to an anti-moderate level; the Jimai 22, the Annong 8455, the Huai mai 18 and the Xiaoyan 54 are the varieties of the scab.
The above results show that: the resistance result presumed by the molecular marker is consistent with the actual resistance level, the resistance of the wheat variety to the gibberellic disease can be judged by detecting whether the wheat variety contains the specific band of the molecular marker JAAS52433R1, and the detection result is correct and effective.
Example 2 screening of wheat Material resistant to Gibberella scab Using molecular marker JAAS52433R1
The test varieties (lines) are wheat varieties 'Ningmai' series: ningmai No.3, Ningmai No.6, Ningmai No. 7, Ningmai No. 8, Ningmai No. 9, Ningmai No.10, Ningmai No. 11, Ningmai No. 12, Ningmai No. 13, Ningmai No. 14, Ningmai No. 15, Ningmai No. 16, Ningmai No. 18 and Ningmai No. 20, which are bred by grain crop research institute of agriculture academy of sciences of Jiangsu province and are commercial wheat varieties; the wheat lines have: ning 0076, ning 0149, ning 0311, ning 0320, ning 0331, ning 05450, ning 05-51, ning 0588, ning 06-174, ning 0668, ning 0670, ning 07169, ning 0798, ning 07-F307, ning 61799, ning 7840, ning 8940, ning 894013, ning 894037, ning 9-11, ning 9181 and ning 9 Da 78.
The above wheat cultivar (line) was examined by the method for detecting the molecular marker JAAS52433R1 established in example 1, and the result of the detection of the marker was described as "presence" or "absence".
The field resistance identification adopts a single flower drip method: culturing strong pathogenic wheat gibberellic disease F0609 conidium liquid (5 × 10)5Conidia/ml), dripping conidia solution into 1 floret at the middle part of ear at the early stage of flowering, spraying water mist 3-4 times a day after each variety of 10 ears is bagged by a plastic bag for 3 days, and spraying water mist for about 5 minutes each time to achieve the moisturizing effect. Investigating the severity of the inoculated ear 21 days after inoculation, and calculating the average severity, wherein the wheat variety with the average severity less than 2 is judged as "resistant", and the wheat variety with the average severity more than or equal to 2 and less than 3 is judged as "resistant"; a wheat variety with an average severity of 3 or more was judged as "susceptible", i.e., a wheat variety with a mild or high susceptibility to gibberellic disease (agricultural industry Standard NY/T2954-2016).
The results of the marker detection and determination and the results of the field identification and determination are shown in table 1:
TABLE 1 screening of wheat varieties (lines) resistant to gibberellic disease using the molecular marker JAAS52433R1
As can be seen from Table 1, the results judged by using the molecular marker JAAS52433R1 are consistent with the field identification results, which proves that the molecular marker can be used for screening the wheat scab resistance.
Sequence listing
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<120> molecular marker linked with wheat scab resistance QTL and application thereof
<141> 2018-12-12
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Claims (3)
1. A molecular marker linked with wheat scab resistance QTL takes DNA of Sumai No.3 as a template and sequences with nucleotide sequences respectively shown as SEQ ID NO.1 and SEQ ID NO.2 as primers to carry out PCR amplification, and DNA fragments with the size of 500bp are obtained after electrophoresis of amplification products on agarose gel with the mass percentage of 1%.
2. A pair of primer pairs for predicting the resistance of wheat to scab, wherein the nucleotide sequences of the primer pairs are respectively shown as SEQ ID NO.1 and SEQ ID NO. 2.
3. A prediction method of wheat scab resistance is characterized by comprising the following specific steps: and (3) carrying out PCR amplification by using the DNA of the sample wheat as a template and using the SEQ ID NO.1 and the SEQ ID NO.2 as primers, and then carrying out electrophoresis, wherein if a DNA fragment with the size of 500bp exists, the resistance of the sample wheat to the gibberellic disease is predicted to at least reach an anti-resistance level.
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| US20100269216A1 (en) * | 2009-04-16 | 2010-10-21 | Syngenta Participations Ag | Network population mapping |
| WO2014011859A2 (en) * | 2012-07-13 | 2014-01-16 | Pioneer Hi-Bred International, Inc. | Molecular markers for various traits in wheat and methods of use |
| CN103290002B (en) * | 2013-05-13 | 2014-09-10 | 四川农业大学 | Molecular marker of wheat tiller QTL (quantitative trait locus) QMtn. sicau-2B.1 and application thereof |
| CN104560976B (en) * | 2015-01-04 | 2017-11-24 | 山东农业大学 | A kind of molecular labeling of quick detection E. elongata anti gibberellic disease gene and application |
| WO2017053247A1 (en) * | 2015-09-21 | 2017-03-30 | Dow Agrosciences Llc | Wheat with elevated fructan, arabinoxylan |
| CN108796117A (en) * | 2018-06-20 | 2018-11-13 | 山东省农业科学院作物研究所 | One main effect QTL and its application for influencing wheat quality and yield traits |
| CN108486278B (en) * | 2018-06-26 | 2020-12-01 | 江苏省农业科学院 | Primer pair for identifying gibberellic disease resistance of Ningmai No. 9 and derivative variety thereof and application |
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| CN101892307A (en) * | 2010-05-12 | 2010-11-24 | 江苏省农业科学院 | SSCP marker closely linked with major wheat scab resistance QTL and application thereof |
| WO2017034971A1 (en) * | 2015-08-21 | 2017-03-02 | Monsanto Technology Llc | Enhanced recombination of genomic loci |
| CN106834527A (en) * | 2017-04-01 | 2017-06-13 | 江苏省农业科学院 | Molecular labeling and its application with wheat seedling sharp eyespot resistance QTL close linkages |
| CN108467901A (en) * | 2018-03-23 | 2018-08-31 | 河南科技大学 | The common wheat alien translocation line and its selection of a kind of gene containing anti gibberellic disease and application |
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