Molecular marker subjected to co-segregation with pea powdery mildew resistance allele er1-6 and application thereof

Abstract

The invention provides a molecular marker subjected to co-segregation with a pea powdery mildew resistance allele er1-6. The molecular marker is located on a VI linkage group of a pea genetic map, and the genetic distance between the molecular marker and the allele er1-6 is 0cM. According to the single base difference (at the position of 1121, T-C) between a disease-resistant variety G0001778 containing the resistance allele er1-6 and an er1 candidate gene PsMLO1cDNA sequence of infected variety pea number 6, primers are designed on the two sides of an SNP mutation site, a high resolution melting curve analysis technology is used for developing the molecular marker subjected to co-segregation with the pea powdery mildew resistance allele er1-6, the marker is subjected to group detection of F2 and F3 which are derived by the disease-resistant variety G0001778 and the infected variety pea number 6 in a hybridization mode, and it is verified that the marker is a co-dominance functional marker subjected to co-segregation with the gene er1-6. Afterwards, the effectiveness is verified in pea powdery mildew resistance resource identification through the marker, the functional marker can be used for molecular marker-assisted selection breeding of the pea powdery mildew resistance, and therefore the breeding process is accelerated.

Description

technical field

[0001] The invention relates to the technical fields of plant pathology, genetic breeding and molecular biology detection, in particular to a molecular marker co-separated with pea powdery mildew resistance allele er1-6 and its application. Background technique

[0002] Pea powdery mildew caused by Erysiphepisi D.C. is a worldwide disease (Nisar et al., 2011; Fondevilla et al., 2012). Pea powdery mildew has occurred in the southern and northern pea producing areas of my country. Under favorable climatic conditions, the infection rate of susceptible varieties can be as high as 100%, greatly affecting the yield and quality of pea, and causing serious economic losses (Peng Huaxian et al., 1991). The most economical, effective and environmentally safe way to control pea powdery mildew is to plant disease-resistant varieties. At present, a large number of powdery mildew resistant pea resources have been identified abroad, and three independently inherited powder...

Examples

Embodiment 1

[0037] Example 1 Identification of the er1 candidate gene PsMLO1cDNA sequence of the resistance resource and identification of the new allele er1-6

[0038] The RNAprep plant total RNA extraction kit (spin column type, purchased from Tiangen Company) was used to extract the total RNA of 15 pea disease-resistant resources and the susceptible control variety Baying No. 6. The specific operation method is as follows:

[0039] 1) Take about 100 mg of young pea leaves and quickly grind them into powder in liquid nitrogen, add 450 μL of RL (before use, add β-mercaptoethanol to a final concentration of 1%), and vortex vigorously to mix; 2) Transfer all solutions to On the filter column CS (the filter column CS is placed in the collection tube), centrifuge at 12000rpm for 5min, carefully draw the supernatant in the collection tube into the RNase-free centrifuge tube; 3) Slowly add 0.5 times the volume of anhydrous Ethanol, mix well, transfer the obtained solution and the precipitate ...

Embodiment 2

[0046] Example 2 Development of molecular markers co-segregated with pea powdery mildew resistance allele er1-6

[0047] According to the results of the gene PsMLO1 cDNA sequence comparison between the pea powdery mildew resistant variety G0001778 and the susceptible variety Bawan 6 and the wild-type PsMLO1 sequence, it was found that G0001778 contained a new allele er1-6, which was opened in the PsMLO1 sequence. A single base substitution (T→C) occurred at the 1121st position of the reading frame, and PrimerPremier5.0 software was used to design upstream and downstream primers on both sides of the SNP mutation site to develop functional molecular markers.

[0048] Upstream primer F: 5'-CTGGAGATCACCTTTTCTGGTT-3'

[0049] Downstream primer R: 5'-CATGTACAAACACACATACACACG-3'

[0050] The upstream and downstream primers of the functional marker are respectively located on the 11th exon and the 11th intron of the PsMLO1 gene. Primers were synthesized by Sangon Bioengineering (Sha...

Embodiment 3

[0055] Example 3 Application of molecular markers co-segregated with pea powdery mildew resistance allele er1-6

[0056]The developed er1-6 functional molecular markers were applied and functionally verified on pea variety resources. The results showed that the molecular marker amplified a 158bp target band in all detected disease-resistant and susceptible pea resources. Through HRM analysis technology, the pea resources containing the disease-resistant allele er1-6 (G0001752, G0001763, G0001764, G0001768, G0001778, identified by the PsMLO1cDNA sequence, all have a single base mutation T→C at 1121bp of the PsMLO1cDNA sequence) formed The same melting curve ( image 3 ). Disease-resistant resources containing other er1 alleles er1-1 (Tara), er1-2 (X9002 and G0005576), er1-4 (YI), and susceptible resources (Bawan 6, Longwan 1, G0001747, G0003839, G0003840) all form a melting curve which is obviously different from that of er1-6 ( image 3 ). The results showed that the mark...

technical field

[0001] The invention relates to the technical fields of plant pathology, genetic breeding and molecular biology detection, in particular to a molecular marker co-separated with pea powdery mildew resistance allele er1-6 and its application. Background technique

[0002] Pea powdery mildew caused by Erysiphepisi D.C. is a worldwide disease (Nisar et al., 2011; Fondevilla et al., 2012). Pea powdery mildew has occurred in the southern and northern pea producing areas of my country. Under favorable climatic conditions, the infection rate of susceptible varieties can be as high as 100%, greatly affecting the yield and quality of pea, and causing serious economic losses (Peng Huaxian et al., 1991). The most economical, effective and environmentally safe way to control pea powdery mildew is to plant disease-resistant varieties. At present, a large number of powdery mildew resistant pea resources have been identified abroad, and three independently inherited powder...

Examples

Embodiment 1

[0037] Example 1 Identification of the er1 candidate gene PsMLO1cDNA sequence of the resistance resource and identification of the new allele er1-6

[0038] The RNAprep plant total RNA extraction kit (spin column type, purchased from Tiangen Company) was used to extract the total RNA of 15 pea disease-resistant resources and the susceptible control variety Baying No. 6. The specific operation method is as follows:

[0039] 1) Take about 100 mg of young pea leaves and quickly grind them into powder in liquid nitrogen, add 450 μL of RL (before use, add β-mercaptoethanol to a final concentration of 1%), and vortex vigorously to mix; 2) Transfer all solutions to On the filter column CS (the filter column CS is placed in the collection tube), centrifuge at 12000rpm for 5min, carefully draw the supernatant in the collection tube into the RNase-free centrifuge tube; 3) Slowly add 0.5 times the volume of anhydrous Ethanol, mix well, transfer the obtained solution and the precipitate ...

Embodiment 2

[0046] Example 2 Development of molecular markers co-segregated with pea powdery mildew resistance allele er1-6

[0047] According to the results of the gene PsMLO1 cDNA sequence comparison between the pea powdery mildew resistant variety G0001778 and the susceptible variety Bawan 6 and the wild-type PsMLO1 sequence, it was found that G0001778 contained a new allele er1-6, which was opened in the PsMLO1 sequence. A single base substitution (T→C) occurred at the 1121st position of the reading frame, and PrimerPremier5.0 software was used to design upstream and downstream primers on both sides of the SNP mutation site to develop functional molecular markers.

[0048] Upstream primer F: 5'-CTGGAGATCACCTTTTCTGGTT-3'

[0049] Downstream primer R: 5'-CATGTACAAACACACATACACACG-3'

[0050] The upstream and downstream primers of the functional marker are respectively located on the 11th exon and the 11th intron of the PsMLO1 gene. Primers were synthesized by Sangon Bioengineering (Sha...

Embodiment 3

[0055] Example 3 Application of molecular markers co-segregated with pea powdery mildew resistance allele er1-6

[0056]The developed er1-6 functional molecular markers were applied and functionally verified on pea variety resources. The results showed that the molecular marker amplified a 158bp target band in all detected disease-resistant and susceptible pea resources. Through HRM analysis technology, the pea resources containing the disease-resistant allele er1-6 (G0001752, G0001763, G0001764, G0001768, G0001778, identified by the PsMLO1cDNA sequence, all have a single base mutation T→C at 1121bp of the PsMLO1cDNA sequence) formed The same melting curve ( image 3 ). Disease-resistant resources containing other er1 alleles er1-1 (Tara), er1-2 (X9002 and G0005576), er1-4 (YI), and susceptible resources (Bawan 6, Longwan 1, G0001747, G0003839, G0003840) all form a melting curve which is obviously different from that of er1-6 ( image 3 ). The results showed that the mark...

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