CN117448480A - Molecular marker for detecting pear cold quantity required property and application thereof - Google Patents

Abstract

The invention discloses a molecular marker for detecting pear cold quantity required property and application thereof, belonging to the technical field of plant molecular markers. The molecular marker is shown as SEQ ID NO.1, and single nucleotide polymorphism C & gtA exists at the 216 th position of the CDS region of the DAM1 gene, and the SNP is used as a detection target spot to be applied to the detection of the pear cold demand property. The SNP is obviously associated with the low-cold-demand pear character for the first time, and the cold-demand pear character of different pear varieties can be effectively identified by detecting the single nucleotide polymorphism of the locus, so that the SNP has important significance for low-cold-demand pear variety identification and new variety breeding, shortening the breeding period and improving the breeding efficiency. The invention also provides a method for detecting low-cold-requirement pear varieties/strains by using the KASP technology, which can efficiently detect single nucleotide polymorphism of specific loci of the tested varieties/filial generation genes, and is quick, simple, high in specificity and capable of realizing high-throughput detection.

Description

Molecular marker for detecting pear cold quantity required property and application thereof

Technical Field

The invention relates to the technical field of plant molecular markers, in particular to a genetic molecular marker for detecting pear cold-required traits and application thereof.

Background

Pear is a fallen leaf fruit tree crop which originates from a high-altitude area near a mountain of the southwest of China, is naturally selected and artificially domesticated for a long time, is the temperate fruit tree crop with the widest cultivation range in China at present, and forms a series of local varieties suitable for local cultivation climatic environments in various places in China.

During the long-term evolution process of pears, a biological process of natural dormancy of buds, which is used for coping with cold climate change in winter, is formed. When pear buds enter natural dormancy, the pear buds cannot resume growth even in an environment suitable for growth, dormancy can be naturally released after long-time low-temperature exposure, and the low-temperature time length (cold quantity) required for releasing dormancy has great variation among different varieties. The cold demand of different varieties has close relation with the environmental adaptability, and the varieties originating in the south China generally show the character of low cold demand.

In recent years, with the continuous aggravation of global climate change, the winter low-temperature duration of many areas in China is shortened, and the phenomenon of insufficient dormancy relief of the original suitable varieties occurs. Therefore, local varieties originating in the south of China are valued as important sources of low-demand cold properties. However, how to distinguish low-cold-demand varieties in southern areas by using molecular markers has not been reported yet.

DAM (Dormancy Associated MADS-box) protein is a key transcription factor regulating woody plant dormancy, belonging to the AGL24/SVP branch of the MADS-box family. The pear gene has been cloned into 5 DAM genes, which are located on chromosome 8 and chromosome 15 respectively, and are distributed in tandem and repeated on chromosome 8; in the process of dormancy of the buds of Dangshan pear, the expression level of 5 DAM genes is changed, the expression level is increased continuously along with the increase of dormancy degree, and the expression level is rapidly decreased after dormancy is released (Gao et al, 2021). However, the relationship between the variation of DAM gene and the geographical distribution of the variety has not been reported yet.

With the continued development of the plant science field, plant variety identification has entered the gene level. Compared with the first generation RFLP and the second generation BSA, the Single Nucleotide Polymorphism (SNP) as the third generation molecular marking technology has the advantages of high flux, low cost, easy operation and the like, and is a main matrix of future molecular marking and has great prospect. Competitive allele-specific PCR (Competitive Allele Specific PCR, KASP) is a novel SNP-based genotyping technique that allows for accurate bi-allele determination of SNPs and InDels at specific sites in a broad range of genomic DNA samples, even some complex genomic DNA samples. As pear is a strict selfing variety, most varieties are artificially bred by bud mutation seed selection, genomes among varieties are very similar, and genetic background is single. Molecular markers related to pear character identification are rare at present. Therefore, the development of KASP molecular markers of pears has important significance for variety identification and new variety breeding.

Disclosure of Invention

The invention aims to provide a genetic molecular marker for identifying pear cold-requiring traits, and the low-cold-requiring pear varieties can be distinguished at low cost by using the molecular marker so as to be suitable for planting in areas with shorter low-temperature duration in winter.

In order to achieve the above purpose, the invention adopts the following technical scheme:

the invention provides application of a Single Nucleotide Polymorphism (SNP) locus serving as a detection target spot in preparation of a kit for detecting pear cold quantity required characteristics, wherein the single nucleotide polymorphism locus is a polymorphism with C & gtA at the 216 th position of a nucleotide sequence shown as SEQ ID NO. 1.

According to the invention, the DAM locus sequences of the pear varieties with generally lower cold energy and the DAM locus sequences of the pear varieties with high cold energy are compared through genome resequencing, and a SNP locus with one base substitution exists at the 216 th position of a CDS region (the nucleotide sequence is shown as SEQ ID NO. 1) of a DAM1 gene (NCBI accession No. XM_ 009377956.3) of the pear varieties with low cold energy, the nucleotide mutation type of the locus is C-A, and the mutation locus is positioned at the 18682690 th position of a chromosome 8 of a 'pear' genome.

By analyzing the genotypes of the above sites of a large sample amount of pears, the base at the 216 th position of the CDS region of the DAM1 gene comprises two states of a base C and a base A, and the corresponding genotypes are CC and CA.

Researches show that the base variation of the C > A is obviously related to the low-refrigeration requirement property. When the base is C, the gene is in a normal DAM1 genetic state, when the base is A, a stop codon TAA is formed, so that the translation of the protein is stopped in advance, the formed protein lacks a subsequent active structural domain, the function of the protein is inactivated, and the plant shows the low-refrigeration-requirement property. Compared with the individual spring time of genotype CC, the individual spring time with CA genotype is obviously advanced and corresponds to the low-cold-demand property.

Therefore, the SNP can be used as a molecular marker for identifying the pear varieties with low refrigeration requirement characteristics, or a genetic molecular marker for judging refrigeration requirement of offspring when the pear varieties with low refrigeration requirement characteristics are used as parents for breeding varieties.

As application of the molecular marker, the invention provides a kit for detecting the cold demand property of pears, which comprises the following components: specific primers for detecting the single nucleotide polymorphism at position 216 in the nucleotide sequence shown in SEQ ID NO. 1.

The specific primers are two pairs, and two specific upstream primers and one specific downstream primer are designed aiming at the single nucleotide polymorphism of the specific locus of the DAM gene, each specific upstream primer corresponds to a corresponding C, A base sequence and is subjected to PCR specific amplification with the downstream primer respectively, and the genotype of the corresponding base is judged to be homozygote or heterozygote according to the PCR positive strip amplified by the corresponding paired primers.

Further, the sequences of the upstream primer and the downstream primer of the primer pair I are respectively as follows:

an upstream primer: 5'-TACCAAGGATGTGATTGCAAGGTAC-3' (SEQ ID NO. 2);

a downstream primer: 5'-ATTTGATCCGATTTTTCCCCACCAGTATG-3' (SEQ ID NO. 4);

the upstream and downstream primer sequences of the primer pair II are respectively as follows:

an upstream primer: 5'-TACCAAGGATGTGATTGCAAGGTAA-3' (SEQ ID NO. 3);

a downstream primer: 5'-ATTTGATCCGATTTTTCCCCACCAGTATG-3' (SEQ ID NO. 4).

The invention also provides a KASP kit for detecting the cold quantity required property of pears, which comprises: a primer set for specifically detecting a single nucleotide polymorphism at position 216 in the nucleotide sequence shown in SEQ ID NO. 1.

Specifically, the primer group comprises two upstream typing primers with different universal fluorescent linker sequences and one downstream universal primer. The KASP reaction system contains two fluorescent probes and two quenching probes complementary with the two fluorescent probes, and the fluorescent probes are consistent with the universal fluorescent linker sequence of the upstream typing primer. Along with PCR amplification, a universal fluorescent connector sequence is introduced into a PCR product corresponding to the SNP, a fluorescent probe is added into the PCR product through DNA chain combination complementary to the universal fluorescent connector sequence and is not combined with a quenching probe complementary to the universal fluorescent connector sequence, so that a fluorescent signal is generated, and after the reaction is finished, the genotype of a corresponding base is judged by reading the fluorescent intensities of two different wavelengths in a reaction solution.

Further, the sequence of the KASP reaction primer set is:

upstream typing primer F1:5'-GAAGGTGACCAAGTTCATGCTTACCAAGGATGTGATTGCAAGGTAC-3' (SEQ ID NO. 5);

upstream typing primer F2:5'-GAAGGTCGGAGTCAACGGATTTACCAAGGATGTGATTGCAAGGTAA-3' (SEQ ID NO. 6);

downstream universal primer R:5'-ATTTGATCCGATTTTTCCCCACCAGTATG-3' (SEQ ID NO. 4).

Further, the kit also comprises PCR Premix and other reagents required for KSAP reaction, and can be replaced by a commercially available conventional KASP labeling kit, which is a routine knowledge of those skilled in the art.

As the application of the kit, the invention also provides a method for identifying low-refrigeration pear varieties/strains, which comprises the following steps:

(1) Extracting genome DNA of pear of a variety to be detected as a template, and carrying out PCR reaction by using a primer group for specifically detecting single nucleotide polymorphism at the 216 th position in a nucleotide sequence shown in SEQ ID NO. 1;

(2) Judging the cold demand property of the sample to be detected according to the genotype of the product:

if the genotype of the product is CA, judging that the sample to be detected is a low-cooling-capacity variety;

if the genotype of the product is CC, judging that the sample to be detected is a high-cold-demand variety.

When the method is used for analyzing the cold quantity required property of pear individuals, the pear individuals are compared with the normal DAM1 gene base, if the difference exists, namely single nucleotide polymorphism exists: 216C > A, the individual is a low-refrigeration-demand variety, and is suitable for cultivation in warm areas in winter in the south.

Furthermore, the KSAP technology is adopted to identify the cold quantity required property of the pears, and in particular, in the step (1), a primer group with nucleotide sequences shown as SEQ ID NO.5, SEQ ID NO.6 and SEQ ID NO.4 is adopted to carry out KASP reaction detection; in the step (2), fluorescence data reading is carried out on the reaction products, and each sample is genotyped according to the relative intensity of fluorescence with different wavelengths:

if the fluorescence is orange, the genotype is CC;

if the fluorescence is green, the genotype is CA.

Further, the composition of the KASP reaction system includes: based on the total volume of 10 mu L, the kit contains 1 XKASP premix, the final concentration of the upstream typing primer F1 is 0.1 mu M, the final concentration of the upstream typing primer F2 is 0.1 mu M, the final concentration of the downstream universal primer R is 0.2 mu M, and the genome DNA template is 10-50 ng.

Further, the KASP reaction conditions were: pre-denaturation at 95 ℃ for 10min,1 cycle; denaturation at 95 ℃ for 15s, annealing at 61-55 ℃ and extension for 60s,10 cycles, wherein the annealing and extension temperature is reduced by 0.6 ℃ in each cycle; denaturation at 95℃for 15s, annealing at 55℃and extension at 60s, 28-35 cycles.

Compared with the prior art, the invention has the following advantages:

(1) The invention discloses that the single nucleotide polymorphism existing at the 216 th position of the CDS region of the DAM1 gene is obviously associated with the low-cold-requirement traits of pears for the first time, and the single nucleotide polymorphism at the position can be detected to effectively identify the cold-requirement traits of different pear varieties. The method has important significance for low-cold-demand pear variety identification and new variety breeding, shortens the breeding period and improves the breeding efficiency.

(2) The invention provides a kit for detecting low-refrigeration pear varieties/strains, which contains three KASP primers designed based on DAM1 single nucleotide polymorphism. The KASP technology can be used for efficiently detecting single nucleotide polymorphism of specific sites of the test variety/filial generation genes, and the method is quick, simple, high in specificity and capable of realizing high-throughput detection.

Drawings

FIG. 1 is a schematic diagram showing mutation at the mutation site of DAM1 gene.

FIG. 2 is a graph of analysis of the results of KASP typing using 19 pear materials, wherein the regular triangle represents genotype CC samples (homozygous), and the circle represents genotype AC samples (heterozygous).

FIG. 3 shows the results of the identification of the cold requirement genotypes of 4 pears by Sanger sequencing.

Detailed Description

The invention will be further illustrated with reference to specific examples. The following examples are only for illustrating the present invention and are not intended to limit the scope of the present invention. Modifications and substitutions to methods, procedures, or conditions of the present invention without departing from the spirit and nature of the invention are intended to be within the scope of the present invention.

The test methods used in the following examples are conventional methods unless otherwise specified; the materials, reagents and the like used, unless otherwise specified, are those commercially available.

Example 1

1. Analysis of nucleotide polymorphism (SNP) sites of DAM1 Gene

The study obtained genome re-sequencing (about 10X) data of 9 local pear varieties such as 'night deep pear', 'nanning big sand pear', 'taiwan blue-and-white' and the like in the south China areas such as Fujian, guangdong, guangxi and the like and 12 varieties such as 'Dangshan crisp', 'green crown', 'pear', 'red early crisp' and the like in other areas of China, and by comparing single nucleotide polymorphisms in DAM locus sequences of the varieties with InDel variation, referring to FIG. 1, a single base substitution SNP locus is found at a 216 th base in a CDS region of DAM1 originating from the pear varieties in the south China areas, and the nucleotide mutation type of the locus is C.fwdarw.A. The normal genetic state of the DAM1 gene when the base is C, and the formation of the stop codon TAA when the base is A, results in premature termination of protein translation, resulting in the formation of a protein with only a portion of the MADS-box domain and the lack of subsequent K-boxes and other active domains, resulting in functional inactivation of the protein.

The nucleotide sequence of CDS region of DAM1 gene is shown in SEQ ID NO. 1.

2. Correlation analysis of nucleotide polymorphism and refrigeration requirement property of DAM1 gene

SNP genotypes of the sites in 264 pear samples from different regions of the country are analyzed by utilizing a genome resequencing method, the existence of both CC and CA types is found, and the pear varieties with AA type are not found.

As is well known, there is a significant correlation between the spring time and the cold demand characteristics, and the relative relationship between the spring time and cold demand is generally represented in research (castde et al, 2014). And counting the difference value between the flowering time of the rest varieties and the zero point by using the flowering time of the first variety in the population as the zero point. The spring time of the 264 samples was further analyzed, and a significant correlation was found between the SNP locus and the spring time of the variety, and the results are shown in Table 1.

TABLE 1 DAM1 base variation is related to the time of initial flowers in spring

As can be seen from Table 1, the individual showing the CA genotype had a significant advance of about 6 days in spring time for low-calorie traits, where varieties identified as CA include 'dark pear', 'Nanning big-sand pear', 'Taiwan blue-white', 'Taiwan red-colored', 'Zhenxian astringent pear', 'Zhenxian big-heart pear', 'Huiyang pear', 'goose pear' and 'red Pi Li'.

Therefore, the SNP can be used as a molecular marker for the low-refrigeration requirement property of the variety, or as a genetic molecular marker for judging the refrigeration requirement of offspring when the variety is used as a parent for breeding the variety.

Example 2

1. Based on the results of example 1, we found that the above base variation of C > a was significantly correlated with low refrigeration requirement traits. We have therefore devised KASP-labeled primers for detecting single nucleotide polymorphisms at specific sites of the DAM1 gene, consisting of two specific upstream primers and one specific downstream primer, as follows:

forward primer F1:5'-GAAGGTGACCAAGTTCATGCTTACCAAGGATGTGATTGCAAGGTAC-3';

forward primer F2:5'-GAAGGTCGGAGTCAACGGATTTACCAAGGATGTGATTGCAAGGTAA-3';

reverse primer R:5'-ATTTGATCCGATTTTTCCCCACCAGTATG-3';

each specific upstream primer corresponds to a corresponding C, A base sequence, and PCR specific amplification is carried out with the downstream primer, and the length of an amplified product is 60bp.

The front end of the forward primer 1 is marked by a universal fluorescent sequence FAM, and the front end of the forward primer 2 is marked by a universal fluorescent sequence VIC. And after the reaction is finished, the fluorescence intensities of two different wavelengths in the reaction liquid are read, and the genotype of the corresponding base is judged.

2.PCR amplification by using KASP (KASP-labeled primer) to identify genotypes of specific sites of DAM1 genes of different pear varieties

2.1 there are 19 varieties of pear to be tested, specifically: "red Pi Li", "Nanning Dazheng pear", "Huiyang pear", "night deep pear", "Qing county Daxin pear", "Taiwan blue and white", "Nanjing Chi Pi Li", "Zhangpu red Pi Li", "Zhuanxian astringent pear", "Taiwan red flower", "sweet osmanthus flower", "Fushan mountain", "Nanjing red heart pear", "Qinghua pear", "red flower pear", "Huiyang sour pear", "goose pear", "Dangshan crisp and" red early crisp ".

Wherein 9 varieties of 'dark pear', 'nanning Chinese pear', 'taiwan blue-and-white', 'taiwan red-and-white', 'county astringent pear', 'county big heart pear', 'Huiyang sour pear', 'goose pear' and 'red Pi Li' are varieties which have been determined to be CA type by genome re-sequencing;

'Nanjing Chi Pi Li', 'Zhangpu Chi Pi Li', 'Huiyang Chinese pear', 'sweet osmanthus', 'mountain cross', 'Nanjing Chili', 'Chihua pear' and 'Qinghua pear' are pear varieties collected from Fujian, guangdong, guangxi and other provinces respectively, adapt to the climate environment in south China, can normally bloom and fruit in the environment with low-temperature accumulation of 0-7.2 ℃ in winter of the original place for less than 300 hours, and are varieties with low cold quantity;

the Dangshan crisp and the Dangshan crisp are typical northern pear varieties, and researches show that the cold required at 0-7.2 ℃ of the Dangshan crisp is more than 1230 hours (lightning sealing and the like, 2013), and the Dangshan crisp is a high-cold required variety.

2.2 genomic DNA extraction: extracting genome DNA from young pear tree tissue in 0.2g by means of CATB process or available plant genome extracting kit, dissolving extracted genome DNA in deionized water, regulating concentration to 100 ng/. Mu.L and low temperature storing.

2.3PCR reaction System composition: based on a total volume of 10. Mu.L, the primer contains FLu-arms 2 XPCR Mix 5. Mu.L, 10. Mu.M forward primer F1.1. Mu.L, 10. Mu.M forward primer F2.1. Mu.L, 10. Mu.M reverse primer 0.2. Mu.L, and 20ng of genomic DNA template.

In the genotyping test, KASP genotyping mixture (except for DNA template) was prepared according to the above formulation, 50 parts of mixture was prepared, and after pipetting the KASP genotyping mixture with a pipetting gun and centrifuging, it was packed in 96-well plates with 10. Mu.L/well and 20ng DNA template was added per well, and each variety was repeated 2 times. The PCR plates were sealed with a sealing plate membrane and then centrifuged.

2.4PCR amplification: PCR amplification was performed using a conventional PCR instrument with a touchdown PCR program, the amplification program being shown in Table 2.

TABLE 2 PCR amplification procedure

After the PCR amplification was completed, the 96-well plate was taken out, and fluorescence was read using a fluorescent quantitative PCR instrument (Bio-Rad), and the reading procedure was set as follows: 30 ℃ for 5s+plate read. Fluorescence types FAM, HEX (i.e. 550nm channel, applicable VIC labeling) were selected.

2.5 carrying out clustering typing on all the tested varieties according to the nucleotide types of the SNP loci.

As shown in fig. 2, 19 varieties of the tested pears are clustered into two different types, wherein 'red Pi Li', 'nanning large pears', 'Huiyang pears', 'night deep pears', 'county large pears', 'taiwan blue flowers', 'south red Pi Li', 'Zhongpu red Pi Li', 'county astringent pears', 'taiwan red flowers', 'sweet osmanthus', 'mountain', 'south red pears', 'blue flowers', 'red pears', 'Huiyang pears', 'white pears', 'Huiyang pears', 'gei pears' are expressed as CA heterozygotes, wherein all 9 varieties determined by genome re-sequencing in the early stage and 8 newly collected local pears from south China are expressed as low-required cold quantity on production, and the lower-temperature accumulation is only required to break dormancy flowering results; and the northern Dangshan crisp and red early crisp pears are CC-shaped, are consistent with the early resequencing result, are produced to be in sleep, and are free from the accumulation of cold required quantity, so that the pear is not suitable for climatic conditions with higher winter temperature.

3. Sequencing of PCR products by Sanger method to verify the accuracy of KASP typing

The PCR products of 3 CA samples ('night deep pears', 'red pears' and 'taiwan red flowers') and 1 CC sample ('red early shortcake') were selected for Sanger sequencing, as shown in fig. 3, in the sequencing peak diagram of the 3 CA heterozygote samples, two peaks corresponding to a/C amplification products appear at the corresponding SNP sites, while in the 1 CC sample, the corresponding SNP sites are single peaks corresponding to C amplification products. These results demonstrate that the KASP typing results are consistent with Sanger sequencing results, verifying the accuracy of the KASP labeling typing.

In summary, the SNP markers developed according to the invention in this example developed KASP marker detection kit for DAM1 gene single nucleotide polymorphism site, successfully verified the genotypes of varieties with CA heterozygous genotypes found by using the resequencing technique, further identified 8 varieties with the CA heterozygous genotypes, and distinguished varieties with CA heterozygous and CC homozygous genotypes, further compared the typing result of KASP technique with the result of Sanger sequencing, and confirmed the accuracy of KASP marking typing developed by the invention.

Claims (9)

1. The application of the single nucleotide polymorphism site as a detection target in preparing a kit for detecting the cold quantity required property of pears is characterized in that the single nucleotide polymorphism site is the polymorphism with C & gtA at the 216 th position of a nucleotide sequence shown as SEQ ID NO. 1.

2. The use of claim 1, wherein the kit comprises: specific primers for detecting the single nucleotide polymorphism at position 216 in the nucleotide sequence shown in SEQ ID NO. 1.

3. The use according to claim 2, wherein the specific primers are two pairs, wherein the nucleotide sequence of the upstream primer of primer pair i is shown in SEQ ID No.2 and the nucleotide sequence of the downstream primer is shown in SEQ ID No. 4; the nucleotide sequence of the upstream primer of the primer pair II is shown as SEQ ID NO.3, and the nucleotide sequence of the downstream primer is shown as SEQ ID NO. 4.

4. A KASP kit for detecting a cold requirement trait of a pear, the KASP kit comprising: a primer set for specifically detecting a single nucleotide polymorphism at position 216 in the nucleotide sequence shown in SEQ ID NO. 1.

5. The KASP kit for detecting the cold requirement property of pears according to claim 4, wherein the primer group comprises an upstream parting primer F1, an upstream parting primer F2 and a downstream universal primer R, and the nucleotide sequences of the upstream parting primer F2 and the downstream universal primer R are respectively shown as SEQ ID NO.5, SEQ ID NO.6 and SEQ ID NO. 4.

6. A method for identifying low-refrigeration pear varieties/strains, comprising the steps of:

(1) Extracting genome DNA of pear of a variety to be detected as a template, and carrying out PCR reaction by using a primer group for specifically detecting single nucleotide polymorphism at the 216 th position in a nucleotide sequence shown in SEQ ID NO. 1;

(2) Judging the cold demand property of the sample to be detected according to the genotype of the product:

if the genotype of the product is CA, judging that the sample to be detected is a low-cooling-capacity variety/strain;

if the genotype of the product is CC, judging that the sample to be detected is a high-cold-demand variety/strain.

7. The method for identifying low-cold pear varieties/strains according to claim 6, wherein in the step (1), KASP reaction detection is performed by using a primer group with nucleotide sequences shown as SEQ ID NO.5, SEQ ID NO.6 and SEQ ID NO. 4; in the step (2), fluorescence data reading is carried out on the reaction products, and each sample is genotyped according to the relative intensity of fluorescence with different wavelengths:

if the fluorescence is orange, the genotype is CC;

if the fluorescence is green, the genotype is CA.

8. The method for identifying low refrigeration pear varieties/lines according to claim 7, wherein the composition of the KASP reaction system comprises: based on the total volume of 10 mu L, the kit contains 1 XKASP premix, the final concentration of the upstream typing primer F1 is 0.1 mu M, the final concentration of the upstream typing primer F2 is 0.1 mu M, the final concentration of the downstream universal primer R is 0.2 mu M, and the genome DNA template is 10-50 ng.

9. The method for identifying low refrigeration pear varieties/lines according to claim 7, wherein the KASP reaction conditions are: pre-denaturation at 95 ℃ for 10min,1 cycle; denaturation at 95 ℃ for 15s, annealing at 61-55 ℃ and extension for 60s,10 cycles, wherein the annealing and extension temperature is reduced by 0.6 ℃ in each cycle; denaturation at 95℃for 15s, annealing at 55℃and extension at 60s, 28-35 cycles.