CN112695122B - Screening method and application of sclerotinia rot resistant rape strain - Google Patents
Screening method and application of sclerotinia rot resistant rape strain Download PDFInfo
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Abstract
The invention belongs to the technical field of plant molecular breeding, and particularly relates to a screening method and application of an anti-sclerotinia sclerotiorum rape line. The present invention includes a method for identifying the utilization of quantitative trait loci associated with resistance to cabbage type rape sclerotinia sclerotiorum. By polymerizing the resistance QTL, finding the QTL with utilization value, and effectively identifying the locus with the micro-effect by positioning and polymerizing the specific molecular marker and the gene, the resistance of the sclerotinia rot of the rape is improved. These specific markers and polymerized sites can be used for identification screening of resistant materials and improvement of sclerotiniose resistance. The invention can be used for the discovery of new resources of the rape sclerotinia rot resistance and can accelerate the improvement process of the rape sclerotinia rot resistance.
Description
Technical Field
The invention belongs to the technical field of plant molecular breeding, and particularly relates to a screening method and application of an anti-sclerotinia sclerotiorum rape line.
Background
The sclerotinia rot of colza is the most main disease of the main producing area of the rape in China, which leads the yield loss of the colza to be 10 percent to 20 percent all the year round, the yield loss of a large field with serious disease can reach 80 percent (the institute of oil crops of Chinese academy of agricultural science 1979), and the high and stable yield of the colza production in China is seriously influenced. The disease also causes the reduction of the oil content of rape seeds and the change of fatty acid components, and seriously affects the quality of the rape seed oil. Sclerotinia sclerotiorum is a fungal disease caused by infection with Sclerotinia sclerotiorum (Lib.) de pay, which is a typical necrotrophic pathogen. Measures for controlling the hazards of sclerotinia rot of rape in production comprise cultivation management, chemical and biological control, utilization of disease-resistant varieties and the like. Because of numerous hosts of the pathogenic bacteria, the sclerotium of the pathogenic bacteria has strong vitality in uncomfortable environment and has limited preventing and controlling effect of cultivation management. On one hand, chemical and biological control methods increase production cost and easily cause environmental pollution, and on the other hand, the use effect is often influenced by inaccurate control of the disease onset period of sclerotinia sclerotiorum and improper control of the use method. Production practices prove that the cultivation of disease-resistant rape varieties is the most economic and effective way for preventing and treating sclerotinia rot of rape.
At present, germplasm resources for immunizing sclerotinia are not found in rape and related species. However, breeding practice and related research prove that the disease resistance of different rape genotypes to sclerotinia sclerotiorum has obvious difference, and the resistance is expressed as a genetic pattern of quantitative traits. However, there is still no effective method for effectively utilizing this finding.
Liu clarification and the like (1991) find that significant differences exist in sclerotinia resistance of cabbage type rape (Brassica napus, AACC,2n ═ 38) varieties (lines), and a batch of cabbage type rape varieties and lines with strong sclerotinia resistance are obtained by widely screening cabbage type rape quality resources in China, Australia and Sweden. Chenyuqing and the like (1993) carry out resistance screening and identification on more than 300 germplasm materials, obtain 37 germplasm resources with stronger resistance, and find that the resistance of a compound seed is stronger than that of a basic seed in 6 brassica species. Zhouyou et al (1994) performed resistance identification on 2371 materials by a method combining field identification and indoor identification, and found no immune types, but the selected high-resistance germplasm accounts for 6 percent, and found that mustard type (B.juncea, BBCC,2n ═ 34) rape has strong capability of resisting sclerotinia rot. Zhao et al (2004) identified 47 brassica napus from multiple countries and found that winter material resistance was stronger than spring material and double high (high glucosinolate, high erucic acid) material was stronger than double low material. Mei et al (2011) found that C-genome cabbages, especially wild-type cabbages, were highly resistant to sclerotiniose by comparing 68 lines of 3 basic and 3 compound species of brassica. The results show that the rape germplasm resources do not find a variety with high resistance or complete immunity to sclerotinia sclerotiorum at present, but resistance variation of rape variety resources still exists widely, and the C gene group has potential value for improving sclerotinia sclerotiorum resistance breeding of rape.
Currently, genetic analysis identifies some cabbage type rape sclerotinia rot resistance Quantitative Trait (QTL) sites. For example, Zhao et al (2006) stably detected the presence of a QTL (Sll16) at N16 in three trials, which accounts for 5.9-14.9% of phenotypic variation; yin et al (2010) detected a QTL on N12 in both 2004 and 2005 using toothpick inoculation identification, accounting for 10.2-17.3% phenotypic variation; the detection by Wei et al (2014) of two QTLs located at C2 was detectable in a two year field natural onset survey, accounting for 10.84-17.44% and 9.86-16.25% phenotypic variation, respectively. However, since different researchers use different markers (RAPD and RFLP markers are used in the early days, SSR markers are mainly used in 2010) and the marker density is still low, it is difficult to determine whether QTLs located on the same linkage group and located by different researchers are the same, and further research is needed.
Disclosure of Invention
Aiming at the problems in the prior art, the invention provides a screening method of an sclerotinia sclerotiorum strain and application thereof, aiming at solving part of the problems in the prior art or at least relieving part of the problems in the prior art.
The present invention includes a method for identifying the utilization of quantitative trait loci associated with resistance to cabbage type rape sclerotinia sclerotiorum. By polymerizing the resistance QTL, the QTL with utilization value is discovered, the locus with the micro-effect is polymerized by gene positioning and is effectively identified, thereby improving the sclerotinia rot resistance of the rape. These polymerized sites can be used for improvement of sclerotinia sclerotiorum resistance and identification screening of resistant materials. The invention can provide new resources for rape sclerotinia rot resistance breeding and accelerate the improvement process of rape disease resistance.
The invention is realized in such a way that the screening method of the sclerotinia sclerotiorum strain comprises the following steps: carrying out quantitative statistic analysis on QTL (quantitative trait locus) resistance loci on single plants in a rape group, wherein the more the QTL resistance loci are in the same single plant, the better the resistance to sclerotinia sclerotiorum is; the QTL resistance loci are respectively:
further, primers for amplifying the QTL resistance locus are respectively as follows:
further, PCR conditions for amplifying the QTL resistance locus are as follows:
and (3) PCR reaction system: the volume of the reaction was 10. mu.l. In a 96-well PCR plate, 1. mu.l of 10 Xbuffer (750mmol/L Tris-HCl, pH 8.8; 200mmol/L (NH4)2SO 4; 0.1% Tween20), 0.16. mu.l of 10mmol/L dNTPs, 0.8. mu.l of 25mmol/L MgCl2, 0.5U Taq DNA polymerase, 10ng DNA template, 0.4. mu.l of 10. mu. mol/L (R + F) primer, plus ddH, were added to each reaction2O to 10. mu.l, and then covered with 10. mu.l of mineral oil.
PCR procedure: pre-denaturation at 94 ℃ for 4 min; denaturation at 94 ℃ for 1min, annealing at 60 ℃ for 30s, extension at 72 ℃ for 45s, 10 cycles, each cycle decreasing by 0.5 ℃; denaturation at 94 ℃ for 1min, annealing at 55 ℃ for 30s, extension at 72 ℃ for 45s, and 30 cycles; extending for 10min at 72 ℃; 10min at 25 ℃.
The invention also provides application of the screening method of the sclerotinia sclerotiorum strain in breeding sclerotinia sclerotiorum resistant materials for rape.
The invention also provides application of the QTL resistance locus in the breeding of the sclerotinia rot resistant material of rape.
The specific process of the invention comprises:
1. identifying the resistance QTL based on linkage analysis of parents;
a) group construction and marker development: the DH (doubled haploid) population was generated by microspore culture of F1 produced by crossing Huashuang No. 5 (female parent, semi-winter variety, sclerotinia resistance, etc.) and A7005 (male parent, semi-winter pure line, more resistant to sclerotinia). Both parents were purified by microspore culture prior to crossing. 190 lines were randomly selected for subsequent experiments. The SSR marker sequences are designed and developed according to a cabbage genome (BoGMS: Brassica oleracea genome micro-satellite) and an EST sequence of Brassica napus (BnEMS: Brassica napus EST micro-satellite). After screening SSR primer polymorphisms by using parents and F1, carrying out genotype analysis on DH populations on primers with polymorphisms on parents.
b) Field test and resistance identification: data for resistance identification are from the field trials described below. The DH colony and the parent thereof, F1, were planted in the rape test field of agriculture university in Wuhan Huazhong Hubei (2009-. The field test adopts a completely random block test, and each material is planted in a single-row area for 3 times of repetition. The identification adopts a method for identifying the living stalks in the adult stage: inoculating and identifying when the field material is in the final flowering stage, punching new edge hypha by using a 7mm puncher, placing the new edge hypha on a preservative film with the length of 10cm and the width of 5cm, inoculating the new edge hypha on a stem internode with the height of 50cm, fixing hypha blocks by using the preservative film without exposing the hypha blocks in the air, and investigating the length of disease spots 7 days after inoculation.
c) Genetic linkage map construction and QTL positioning: the map was constructed using MAPMAKER 3.0 software. The genetic distance between the loci was calculated according to the Kosambi function. QTL detection is carried out by a Composite Interval Mapping (CIM) of Windows QTL Cartographer V2.5 software. Interval tests of QTL scanning have a window size of 10cM without considering the cofactor, and the parameters Pin-0.05 and Pout-0.05 (model 6). At a significance level of 0.05, 1000 assignment tests were used to determine LOD thresholds for traits in each environment. The confidence interval of the QTL is determined according to the interval corresponding to the LOD-1 at the peak position of the QTL.
2. Screening resistance materials by comparing the quantity of different resistance QTLs;
QTL positioning is carried out by constructing a linkage map, and QTL sites related to the sclerotinia rot are found. These sites are located on a1, a2, A3, a6, A8, a9, C7 and C88 linkage groups, respectively, designated qSRA1, qSRA2, qSRA3, qSRA6, qSRA8, qSRA9, qSRC7, qSRC8, respectively. 2 of these QTLs were resistant alleles from the parent Hua 5, and the other loci were resistant alleles from the parent A7005. The taxonomic statistical analysis revealed that the materials containing different numbers of resistance sites were very different in resistance. When the synergistic effect of 9 resistance loci is contained, the lesion spots of the stalks after 7 days of inoculation are 1.29cm, when the synergistic effect of only one resistance locus is contained, the lesion spots of the stalks after 7 days of inoculation are 4.9cm, and when the synergistic effect of 2-8 resistance loci is contained, the rule that the lesion spots are gradually reduced is presented. At the same time, we screened several individuals containing multiple resistance sites.
In summary, the advantages and positive effects of the invention are as follows:
(1) the QTL sites for controlling the sclerotinia rot resistance of the cabbage type rape are identified, and the micro-effect sites are introduced into the existing common rape variety, so that the sclerotinia rot resistance can be improved, and the problem of the lack of sclerotinia rot resistance resources at present can be solved.
(2) The invention analyzes and identifies some linkage markers of the micro-effect QTL, and the linkage markers can be effectively used for resisting the genotype of disease identification in the rape and can screen disease-resistant materials in the early period.
(3) Materials with different resistance degrees can be obtained by screening materials by using the resistance QTL locus, and the materials can be used for breeding research of the antibacterial nuclear disease.
Drawings
FIG. 1 shows lesion lengths containing different numbers of resistant sites.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following examples, and the apparatuses and reagents used in the examples and test examples are commercially available without specific reference. The specific embodiments described herein are merely illustrative of the invention and are not intended to be limiting.
Various modifications to the precise description of the invention will be readily apparent to those skilled in the art from the information contained herein without departing from the spirit and scope of the appended claims. It is to be understood that the scope of the invention is not limited to the procedures, properties, or components so defined, as these embodiments, as well as others described, are intended to be illustrative of specific aspects of the invention only. Indeed, various modifications of the embodiments of the invention which are obvious to those skilled in the art or related fields are intended to be included within the scope of the following claims.
For a better understanding of the invention, and not as a limitation on the scope thereof, all numbers expressing quantities, percentages, and other numerical values used in this application are to be understood as being modified in all instances by the term "about". Accordingly, unless expressly indicated otherwise, the numerical parameters set forth in the specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained. At the very least, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. As used herein, "about" means within 10%, preferably within 5%, of a given value or range.
The normal temperature in the following embodiments of the present invention refers to a natural room temperature condition in four seasons, and is not subjected to additional cooling or heating treatment, and is generally controlled at 10 to 30 ℃, preferably 15 to 25 ℃.
The invention discloses a screening method and application of an sclerotinia sclerotiorum strain, which are shown in the following embodiments.
Example 1 identification of resistance QTL by linkage analysis of population construction by parent construction with differences in resistance
1. Construction and field test of cabbage type rape DH positioning group
DH (doubled haploid) population was generated by microspore culture of F1 generated by crossing Huashuang No. 5 and Jia 7005. Before crossing, two parents were purified by microspore culture, and 190 lines were randomly selected for subsequent experiments. The DH group and the parent thereof, F1, are planted in the rape test field of agriculture university in Wuhan Huazhong Hubei (2009-. The field test adopts a completely random block test, each material is planted in a single-row area, and the process is repeated for 3 times. The identification adopts a method for identifying the living stalks in the adult stage: inoculating and identifying when the field material is in the final flowering stage, punching new edge hypha by using a 7mm puncher, placing the new edge hypha on a preservative film with the length of 10cm and the width of 5cm, inoculating the new edge hypha on a stem internode with the height of 50cm, fixing hypha blocks by using the preservative film without exposing the hypha blocks in the air, and investigating the length of disease spots 7 days after inoculation. The stalk sclerotinia resistance of the parents and DH colony is identified by the identification method of the adult stalk. The Stalk Resistance (SR) of parents in three environments has obvious difference, the length of disease spots on J7005 stalks after 7 days of inoculation is 2-3cm smaller than that of Hua 5, then the disease spots on the parents are more and more different, the disease spots already extend to most areas of main stalks after 30 days of inoculation, and therefore plants die, and the disease spots on the J7005 stalks are only 10-15cm at the moment.
2. Construction of DH colony genetic linkage map and QTL analysis
Genetic linkage map construction and QTL positioning: maps were constructed using MAPMAKER 3.0 software and genetic distances were calculated according to Kosambi function. QTL detection adopts a composite interval mapping method of Windows QTL Cartographer V2.5 software. Interval tests of QTL scanning have a window size of 10cM without considering the cofactor, and the parameters Pin-0.05 and Pout-0.05 (model 6). At a significance level of 0.05, 1000 assignment tests were used to determine LOD thresholds for traits in each environment. The DH genetic linkage map contains 272 pairs of SSR markers, the total length of the map is 1579cM, and the average interval between the markers is 5.2 cM. QTL positioning is carried out on the resistance of the stalks in the adult plant period by utilizing a composite interval mapping method (CIM). Totally identifying 9 stalk micro-effect QTLs (Table 1, wherein 3 of the QTLs can be detected at the same time in different years, but are identical in position, so that only 9 positions are identified, and the names of the positions are identical, namely identical positions), wherein the single QTL explains that the phenotypic variation is 3.43-32.61%, and the phenotypic variation is respectively positioned on 8 linkage groups of A1, A2, A3, A6, A8, A9, C7 and C8 and respectively named as qSRA1, qSRA2, qSRA3, qSRA6, qSRA8, qSRA9, qSRC7 and qSRC 8. 2 of these QTLs were resistant alleles from parent Hua 5, and the other loci were resistant alleles from parent A7005.
Table 1 Stem Resistance (SR) QTL sites located in the DH population
Example 2 selection of resistant Material by comparing the amount of different resistance QTLs
QTL quantity statistical analysis is carried out on single plants in DH groups, and the material resistance difference containing different numbers of resistance sites is found to be large. When 9 resistance sites are contained, the effect is enhancedWhen the synergistic effect is achieved, the lesion spots are 1.29cm after the stalk is inoculated for 7 days, the lesion spots are 4.9cm after the stalk is inoculated for 7 days, and when the synergistic effect is achieved, the lesion spots are gradually reduced (figure 1) after the stalk is inoculated for 2-8 resistance sites, and the length of the lesion spots is reduced by 0.45cm when the effect of one resistance site is averagely increased, and the resistance is gradually and correspondingly enhanced. Meanwhile, 4 individuals (shown in Table 2) containing more than 8 resistance sites are screened, namely C4-133B, C4-139B, C18-83A, C4-283A, scab is 1.29cm, 1.61 cm, 1.84 cm and 1.86cm respectively, and the resistance materials can be used as new resources for breeding the anti-sclerotinia sclerotiorum. These germplasm resources containing different numbers of resistance sites can provide materials for resistance breeding. The markers of the sites can be used as linkage markers to be applied to the screening of early resistant materials in molecular marker-assisted selective breeding, and the marker names and sequences are shown in Table 3. And (3) PCR reaction system: the volume of the reaction was 10. mu.l. In a 96-well PCR plate, 1. mu.l of 10 Xbuffer (750mmol/L Tris-HCl, pH 8.8; 200mmol/L (NH4)2SO 4; 0.1% Tween20), 0.16. mu.l of 10mmol/L dNTPs, 0.8. mu.l of 25mmol/L MgCl2, 0.5U Taq DNA polymerase, 10ng DNA template, 0.4. mu.l of 10. mu. mol/L (R + F) primer, plus ddH, were added to each reaction2O to 10. mu.l, and then covered with 10. mu.l of mineral oil.
PCR procedure: pre-denaturation at 94 ℃ for 4 min; denaturation at 94 ℃ for 1min, annealing at 60 ℃ for 30s, extension at 72 ℃ for 45s, 10 cycles, each cycle decreasing by 0.5 ℃; denaturation at 94 ℃ for 1min, annealing at 55 ℃ for 30s, extension at 72 ℃ for 45s, and 30 cycles; extending for 10min at 72 ℃; 10min at 25 ℃.
TABLE 2 plaque Length 7 days after inoculation of the selected antibacterial Nuclear disease Material
TABLE 3 molecular markers linked to the resistance locus QTL
The above description is intended to be illustrative of the preferred embodiment of the present invention and should not be taken as limiting the invention, but rather, the intention is to cover all modifications, equivalents, and alternatives falling within the spirit and scope of the invention.
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Claims (3)
1. A screening method of an antibacterial sclerotinia rot rape strain is characterized by comprising the following steps: carrying out quantitative statistic analysis on QTL (quantitative trait locus) resistance loci on single plants in a rape group, wherein the more the QTL resistance loci are in the same single plant, the better the resistance to sclerotinia sclerotiorum is; the QTL resistance loci are respectively:
primers for amplifying the QTL resistance locus are respectively as follows:
。
2. the method of claim 1, wherein the method comprises the steps of: PCR conditions for amplifying the QTL resistance locus are as follows:
and (3) PCR reaction system: the volume of the reaction is 10 mul, 1 mul of 10 Xbuffer solution, 0.16 mul of 10mmol/L dNTPs, 0.8 mul of 25mmol/L MgCl20.5U Taq DNA polymerase, 10ng DNA template, 0.4. mu.l 10. mu. mol/L forward primer and reverse primer, and ddH2Carrying out covering by adding 10 mul of mineral oil after O is changed to 10 mul; the 10 × buffer comprises: 750mmol/L Tris-HCl, pH8.8; 200mmol/L (NH4)2SO4；0.1% Tween20；
PCR procedure: pre-denaturation at 94 ℃ for 4 min; denaturation at 94 ℃ for 1min, annealing at 60 ℃ for 30s, extension at 72 ℃ for 45s, 10 cycles, each cycle decreasing by 0.5 ℃; denaturation at 94 ℃ for 1min, annealing at 55 ℃ for 30s, extension at 72 ℃ for 45s, and 30 cycles; extending for 10min at 72 ℃; 10min at 25 ℃.
3. Use of a method of screening a sclerotinia sclerotiorum line according to claim 1 or 2 in the breeding of sclerotinia sclerotiorum resistant material for oilseed rape.
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|---|---|---|---|---|
| CN108271685A (en) * | 2018-01-21 | 2018-07-13 | 孙晓敏 | A kind of method of the anti-sclerotiniose cabbage type rape breeding material of selection and breeding |
| CN109680097A (en) * | 2019-02-28 | 2019-04-26 | 西南大学 | The molecular labeling and application that a kind of and sclerotinia sclerotiorum stem resistance is closely related |
| CN110564762A (en) * | 2019-09-25 | 2019-12-13 | 湖北大学 | Elongation factor BnELP4 gene for regulating cabbage type rape sclerotinia sclerotiorum resistance and application thereof |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108271685A (en) * | 2018-01-21 | 2018-07-13 | 孙晓敏 | A kind of method of the anti-sclerotiniose cabbage type rape breeding material of selection and breeding |
| CN109680097A (en) * | 2019-02-28 | 2019-04-26 | 西南大学 | The molecular labeling and application that a kind of and sclerotinia sclerotiorum stem resistance is closely related |
| CN110564762A (en) * | 2019-09-25 | 2019-12-13 | 湖北大学 | Elongation factor BnELP4 gene for regulating cabbage type rape sclerotinia sclerotiorum resistance and application thereof |
Non-Patent Citations (1)
| Title |
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| 甘蓝型油菜抗菌核病QTL定位;周李鹏;《万方数据》;20141013;第2章 * |
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