CN111826455B - Molecular marker VrMLO _ Indel1 for identifying powdery mildew resistance phenotype of mung beans as well as primer and application thereof - Google Patents

Molecular marker VrMLO _ Indel1 for identifying powdery mildew resistance phenotype of mung beans as well as primer and application thereof Download PDF

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CN111826455B
CN111826455B CN201910327322.XA CN201910327322A CN111826455B CN 111826455 B CN111826455 B CN 111826455B CN 201910327322 A CN201910327322 A CN 201910327322A CN 111826455 B CN111826455 B CN 111826455B
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薛晨晨
塞班
陈新
袁星星
陈景斌
普拉给特宋塔
披拉沙斯乃文
闫强
吴然然
张红梅
陈华涛
崔晓艳
刘晓庆
顾和平
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Abstract

The invention discloses a molecular marker VrMLO _ Indel1 for identifying powdery mildew resistance phenotype of mung beans, and primers and application thereof. The molecular marker is used for identifying the powdery mildew resistance phenotype of mung beans, the upstream primer of the molecular marker VrMLO _ Indel1 is shown as SEQ ID No.1, the downstream primer is shown as SEQ ID No.2, a characteristic strip with the size of 235bp can be amplified in a powdery mildew resistance variety through PCR, and a characteristic strip with the size of 226bp can be amplified in a powdery mildew susceptible variety through PCR. According to the invention, through positioning the powdery mildew resistant site of the mung bean, a molecular marker co-separated from the powdery mildew resistant site of the mung bean is discovered, and the marker selection efficiency reaches 100%. The molecular marker primer disclosed by the invention is used for identifying or screening the powdery mildew resistance of mung beans, and the method is quick, simple and convenient, high in selection efficiency and low in cost.

Description

Molecular marker VrMLO _ Indel1 for identifying powdery mildew resistance phenotype of mung beans as well as primer and application thereof
Technical Field
The invention belongs to the field of molecular breeding, and relates to a molecular marker VrMLO _ Indel1 for identifying powdery mildew resistance phenotype of mung beans, and a primer and application thereof.
Background
Mung bean (Vigna radiata) is a plant of the genus Vigna of the family leguminosae, the subfamily Papilionaceae, and is an important socio-economic crop of Asia, particularly south and southeast Asia. Widely planted in India, China, Burma, Thailand, Bengal, Pakistan, Cambodia, Indonesia, Philippines, and Australia. Mung beans are usually crops grown separately after rice, wheat and corn, or as part of various planting systems, such as intercropping with sugarcane, because mung beans grow quickly and mature early, only about 75 days. It is estimated that the production area of mung beans exceeds 600 million hectares. India, burma and china are the major planting countries. The average yield of mung beans is not high. Diseases are one of the main causes of low yield. Common diseases infecting mung beans include powdery mildew, leaf spot, virus diseases and the like.
Mung bean powdery mildew is caused by polygonum griseum (Erysiphe polygonii d.c.). It is a major fungal disease of mung beans growing in tropical and subtropical regions, especially in the shade-dry season. The cold season is the main season for producing high quality mung beans in some countries of south and southeast Asia. Powdery mildew can cause the yield loss of susceptible varieties by 20-40%. If the disease occurs in the early growth stage and the weather is proper, the mung bean seedlings can be killed, and the failure of production is caused. The most common method of controlling powdery mildew is spraying a bactericide. Therefore, the breeding of the powdery mildew resistant mung bean variety has great benefits for the mung bean industry in China.
Most of the germplasm resources of the mung beans resisting the powdery mildew come from foreign countries. Among these resistant germplasm, RUM5 and V4718 exhibited broad spectrum resistance. Previous studies showed that resistance to V4718 is controlled by a single dominant gene and RUM5 is controlled by two recessive genes. Quantitative Trait Locus (QTL) studies by predecessors using different resistance sources indicated that resistance was controlled by potentially 1-3 QTLs. However, no report is found on specific molecular markers closely related to resistance.
Disclosure of Invention
The invention aims to overcome the defects in the prior art and provide a molecular marker for identifying the powdery mildew resistance phenotype of mung beans.
Another objective of the invention is to provide molecular marker primers for identifying the powdery mildew resistance phenotype of mung beans.
The invention also aims to provide the molecular marker and the application of the primer thereof.
The purpose of the invention can be realized by the following technical scheme:
the molecular marker is used for identifying the powdery mildew resistance phenotype of mung beans, the upstream primer of the molecular marker VrMLO _ Indel1 is shown as SEQ ID No.1, the downstream primer is shown as SEQ ID No.2, a characteristic strip with the size of 235bp can be amplified in a powdery mildew resistance variety through PCR, and a characteristic strip with the size of 226bp can be amplified in a powdery mildew susceptible variety through PCR. The molecular marker is co-separated from the powdery mildew resistance major gene of mung bean, the powdery mildew resistance marker of mung bean is located on the 9 th chromosome Chro09:9,994,364..9,994,599 of mung bean, and the physical position refers to sequenced mung bean data (http:// plant genetics. snu. ac. kr/media wiki-1.21.3/index. php/Main _ Page).
The primer of the molecular marker for identifying the powdery mildew resistance phenotype of mung beans is shown as an upstream primer in SEQ ID No.1 and a downstream primer in SEQ ID No. 2.
The molecular marker disclosed by the invention is applied to identification of powdery mildew resistant varieties.
The molecular marker primer disclosed by the invention is applied to identification of powdery mildew resistant varieties.
The molecular marker primer disclosed by the invention is applied to identification of powdery mildew resistance genes VrMOL of mung beans.
As the application of the invention is preferable, the characteristic strip with the size of 235bp amplified by the molecular marker primer is a variety containing the anti-powdery mildew gene VrMOL, and the characteristic strip with the size of 226bp amplified by the molecular marker primer is a variety without the anti-powdery mildew gene VrMOL.
The PCR detection kit for screening or identifying the powdery mildew resistance resources of mung beans comprises the molecular marker primer.
A method for screening or identifying a powdery mildew resistant mung bean variety comprises the following steps:
1) extracting the genome DNA of a plant to be detected;
2) taking the genome DNA of a plant to be detected as a template, and carrying out PCR amplification reaction by using the primer of the molecular marker VrMLO _ Indel 1;
3) the PCR amplification product is detected, so that the characteristic strip with the size of 235bp can be amplified to be a mung bean powdery mildew resistant variety, and the characteristic strip with the size of 226bp is amplified to be a mung bean powdery mildew susceptible variety.
Has the advantages that:
according to the invention, through positioning the powdery mildew resistance locus of mung bean, a molecular marker VrMLO _ Indel1 which is co-separated from the powdery mildew resistance major gene of mung bean is discovered, and the selection efficiency of the marker reaches 100%. The molecular marker primer disclosed by the invention is used for identifying or screening the powdery mildew resistance of mung beans, and the method is quick, simple and convenient, high in selection efficiency and low in cost. Only by detecting the characteristics of the amplified bands of the marker, whether the mung beans resist powdery mildew can be judged, so that the powdery mildew resistance of mung bean varieties can be predicted, the powdery mildew-resistant mung bean varieties can be screened rapidly and purposefully, and the method can be applied to mung bean breeding practice and resource and variety identification at high throughput.
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FIG. 1 shows the results of PAGE electrophoresis detection of the molecular marker primers co-separated from the major gene for powdery mildew resistance in powdery mildew resistant varieties RUM5 and V4718, and powdery mildew susceptible varieties CN60 and KPS 1.
FIG. 2 Fine mapping of powdery mildew resistance gene of the present invention
Detailed Description
The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention. Unless otherwise indicated, the examples follow conventional experimental conditions, such as the Molecular cloning handbook, e.g., Sam brook et al (Sam brook J & R ussel DW, Molecular cloning: a laboratory manual,2001), or the conditions suggested by the manufacturer's instructions.
Example 1 CN60X RUM 5F 2 population 190 strains, CN60XRUM5 BC1F1 population 74 strains, population construction and phenotypic characterization
The powdery mildew resistant mung bean wild resource RUM5 is used as a male parent, powdery mildew resistant mung bean resource CN60 is used as a female parent, a hybrid is prepared, an F2 segregation population containing 190 single plants is constructed, each F2 single plant is selfed to obtain a corresponding F2:3 family, and the powdery mildew resistant identification is carried out. In addition, a powdery mildew resistant mung bean wild resource RUM5 is taken as a male parent, a powdery mildew resistant mung bean resource CN60 is taken as a female parent, a hybrid is prepared, a BC1F1 segregation population containing 74 single plants is constructed, and each single plant is selfed to obtain BC1F 1: 2, identifying powdery mildew resistance of the families, and synchronously testing the two groups, wherein the specific method comprises the following steps:
randomly selecting 30 healthy seeds from each material, wherein the healthy seeds comprise 2 parts of powdery mildew-sensitive CN60 and powdery mildew-resistant RUM5, planting the seeds in a field, the row spacing is 50cm, the plant spacing is 12cm, 20d, 25d and 30d after seedling emergence respectively carry out powdery mildew inoculation on leaves, counting is carried out 50d after seedling emergence, and the infection on each leaf is scored according to 1-9 (1 is no disease infection, 3 is 1-25% of leaf area is infected, 5 is 26-50% of leaf area is infected, 7 is 51-75% of leaf area is infected, and 9 is 76-100% of leaf area is infected).
The powdery mildew resistance identification result shows that the RUM5 and F1 seeds obtained by CN60/RUM5 hybridization have full resistance, and the RUM5 powdery mildew resistance is controlled by a dominant gene. The frequency distribution of the resistance level of 190 CN60/RUM 5F 2:3 families to powdery mildew is continuously distributed.
It has been shown that the mung bean powdery mildew resistance locus qPMRUM5-3 is located on linkage group 9, between SSR markers CEDG070 and CEDG259 (Chankaew et al 2013). We performed BLAST searches (http:// plant genetics. snu. ac. kr/sequence server) on these two tagged primer sequences based on the mung bean reference sequence. The sequence is between 9,097,007bp and 19,388,902bp of the 9 th chromosome, the size is 10.292Mb, the sequence between the markers is downloaded, 55 pairs of SSR primers (table 1) are designed, polymorphism is screened between CN60 and RUM5, and PCR reaction is carried out by taking mung bean genome DNA as a template. A10. mu.l PCR reaction system included: 2ng DNA, 1 XTaq enzyme buffer; 2mmol L-1MgCl2, 0.2mmol L-1dNTPs, 5pmol L-1 of upstream and downstream primers and 1U Taq DNA polymerase, ddH2O make up to 10. mu.l. Reaction procedure: pre-denaturation at 94 ℃ for 3 min; denaturation at 94 ℃ for 30s, annealing at 50-60 ℃ for 30s, extension at 72 ℃ for 30s, and 32 cycles; finally, extension is carried out for 10min at 72 ℃. The whole reaction was carried out on an east-wins EDC-810PCR amplimer. The PCR product was separated by 5% polyacrylamide gel electrophoresis and stained by silver staining. The amplified DNA bands were observed in a fluorescent light box.
The acquisition of the anti-powdery mildew marker of the mung bean: 11 pairs of polymorphic SSR markers are screened by using two powdery mildew segregation populations and resistance and infection pools, and a fine positioning diagram of the major QTL of the powdery mildew resistance of the mung beans is shown in figure 2.
TABLE 1
Figure BDA0002036619920000041
Figure BDA0002036619920000051
Figure BDA0002036619920000061
According to the published mung bean genome sequence of NCBI, a 0.046Mb sequence between Vr9gSSR50 and Vr9gSSR55 is searched, primer design is carried out by using software Primer5.0, and through repeated verification of the two groups of separated populations, primers with five molecular markers can be determined and designed to indicate mung bean powdery mildew resistance, VrMLO _ Indel1 is one of the molecular markers, and the upstream primer and the downstream primer are as follows: TAAGGAGGGATGAGTTGCTGA (SEQ ID NO.1) and AAGCATGTGGTGAGTCCAAC (SEQ ID NO.2) (FIG. 1). Characteristic bands with the size of 235bp can be amplified in powdery mildew resistant mung bean varieties RUM5 and V4718 by utilizing SEQ ID NO.1 and SEQ ID NO.2, and characteristic bands with the size of 226bp can be amplified in mung bean powdery mildew resistant mung bean varieties CN60 and KPS 1.
Example 2 validation of molecular tagged primers
Extracting mung bean powdery mildew-susceptible materials KPS1, Sulv No.1, Sulv No. 3 and powdery mildew-resistant materials V4718, Sulv No.2, V2802 and V2709, carrying out PCR amplification on 10 strains of foreign wild mung bean species TC1966 genome DNA, and carrying out powdery mildew resistance identification on each variety by using primers (SEQ ID No.1 and SEQ ID No.2) of the molecular marker, wherein the results are shown in Table 2, and the identification efficiency of the marker is 100%.
TABLE 2
Figure BDA0002036619920000071
The disease resistance of mung bean plants is predicted by identifying the powdery mildew resistance of mung beans through the molecular markers, and the breeding process of powdery mildew resistance varieties of mung beans in China can be improved.
Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.
Sequence listing
<110> agricultural science and academy of Jiangsu province
<120> molecular marker VrMLO _ Indel1 for identifying powdery mildew resistance phenotype of mung beans as well as primer and application thereof
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
taaggaggga tgagttgctg a 21
<210> 2
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
aagcatgtgg tgagtccaac 20

Claims (2)

1. The application of the molecular marker primer in identifying the mung bean powdery mildew resistant variety is characterized in that an upstream primer of the molecular marker is shown as SEQ ID No.1, a downstream primer of the molecular marker is shown as SEQ ID No.2, a characteristic strip with the size of 235bp can be amplified by PCR in the powdery mildew resistant variety, and a characteristic strip with the size of 226bp can be amplified by PCR in the powdery mildew resistant variety.
2. A method for screening or identifying a powdery mildew resistant mung bean variety is characterized by comprising the following steps:
1) Extracting the genome DNA of a plant to be detected;
2) Carrying out PCR amplification reaction by using the genomic DNA of a plant to be detected as a template and using the molecular marker primer in claim 1;
3) The PCR amplification product is detected, so that the characteristic strip with the size of 235bp can be amplified to be a mung bean powdery mildew resistant variety, and the characteristic strip with the size of 226bp is amplified to be a mung bean powdery mildew susceptible variety.
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CN105154442A (en) * 2015-09-18 2015-12-16 中国农业科学院作物科学研究所 Indel mark of powdery mildew resistance allele er1-7 of peas and application thereof

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CN105950736B (en) * 2016-05-26 2020-06-09 中国农业科学院作物科学研究所 Molecular marker co-separated from bruchid-resistant gene VrPGIP of mung bean and application thereof

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