IRAP labeled primer developed based on cherry genome and application of IRAP labeled primer

Abstract

The invention discloses an IRAP marker primer developed based on a cherry genome, develops a specific IRAP marker primer based on an LTR-RT family of the cherry genome, establishes an efficient and stable detection system, not only can solve the technical bottleneck in current cherry germplasm identification and genetic analysis, but also can provide a new tool for cross-species research of rosaceae fruit trees, and has a wide application prospect. The method has important significance on sustainable development of the cherry industry.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of molecular biology technology, and particularly relates to an IRAP marker primer based on a cherry genome and application thereof. BACKGROUND

[0002] Cherry, as an important fruit tree of Rosaceae Prunus subgenus Cerasus, occupies an important position in the global fruit tree industry due to its early maturity, unique flavor, and rich nutrition. With the rapid development of the cherry industry, new variety breeding and germplasm exchange are increasingly frequent. The combination of traditional hybrid breeding and bud mutation selection greatly enriches cherry variety resources, but also brings problems such as variety identification difficulty and germplasm relationship ambiguity.

[0003] At present, the molecular marker technologies used for cherry research mainly include AFLP, RAPD, ISSR, SSR and SRAP. Although the AFLP marker technology has rich polymorphism, it is complex to operate, has high cost, and is strict in DNA quality requirements. The RAPD marker has poor stability, insufficient repeatability, and is easily affected by experimental conditions. The ISSR marker is a dominant marker, which cannot distinguish homozygotes from heterozygotes, and the complex amplification products lead to great difficulty in data analysis. The SSR marker has the advantages of co-dominance and good stability, but the primer design depends on known genome sequences, the development cost is high, and the marker has strong species specificity, and the universality among different cherry germplasms is limited. The SRAP marker has poor amplification effect on centromere and telomere regions, and is sensitive to PCR reaction conditions, so the consistency of the results is difficult to guarantee.

[0004] In recent years, reverse transcription transposon molecular markers have gradually become a research hotspot due to their unique advantages. Reverse transcription transposon is a genetic element widely existing in plant genomes, which can produce a large number of stable genetic variation sites in the genome through RNA-mediated reverse transcription and transposition. Among them, LTR-RT (long terminal repeat retrotransposon) is the largest type of reverse transcription transposon in plant genomes, and has a high copy number in cherry genomes. The conserved region in the long terminal repeat (LTR) provides an ideal target for molecular marker development.

[0005] IRAP (interretrotransposon amplified polymorphism) marker, as a molecular marker technology based on reverse transcription transposon, detects the fragment polymorphism between adjacent members of the same family of reverse transcription transposon in the genome by designing primers in the conserved region of LTR. The IRAP marker has the advantages of simple operation, high polymorphism, good repeatability, and low development cost, and has great potential in the identification of clonal bud mutation and genetic diversity analysis.

[0006] The development of IRAP markers for cherries is still in its early stages. Firstly, there is a lack of specific IRAP primers based on the cherry genome sequence annotation; existing studies mostly use universal primers from other species, resulting in insufficient amplification efficiency and specificity. Secondly, the genetic background varies significantly among different cherry germplasms, especially between wild and cultivated species, and between germplasms of different geographical origins, making it difficult for existing markers to fully cover their genetic diversity. Therefore, there is an urgent need to develop specific IRAP marker primers based on the cherry genome's own LTR-RT family. Summary of the Invention

[0007] The present invention aims to provide an IRAP marker primer developed based on the cherry genome and its application, which solves the limitations of existing markers that rely on known sequences and have strong species specificity, and overcomes the problem of poor consistency of results in traditional techniques.

[0008] To achieve the above objectives, this application provides the following technical solution: An IRAP marker primer developed based on the cherry genome, selected from one or more of the following primer sequences: Primer LTR1-3, sequence 5'-ATGGCTATCTTCACGAGGATGT-3'; Primer LTR1-10, sequence 5'-GGGCTGAAGCAATCTCCAAG-3'; Primer LTR1-47, sequence 5'-ATGGGCTAAAGCAATCACCT-3'; Primer LTR3-11, sequence 5'-GGCCATAATCCACGCACATA-3'; Primer LTR1-1, sequence 5'-GTATGGCCTGAAACAGTCTCCA-3'; Primer LTR1-2, sequence 5'-ATGGGCTGAAGCAATCTCCAAG-3'; Primer LTR1-26, sequence 5'-GTATGGGCTGAAGCAATCTC-3'; Primer LTR1-48, sequence 5'-TATGGGCTAAAGCAATCACC-3'; Primer LTR1-60, sequence 5'-TATGGGCTAAAGCAATCACC-3'.

[0009] Working principle and beneficial effects of the present invention: This application is the first to develop specific IRAP primers based on the LTR-RT family annotation of the cherry genome. It overcomes the limitations of existing markers that rely on known sequences and have strong species specificity. The standardized PCR reaction system and detection method established overcome the problem of poor consistency of results in traditional techniques. The screening of core primers and the expansion of cross-species applications have significantly improved the practicality and coverage of the markers, meeting the diversified needs of cherry and Rosaceae fruit tree germplasm research.

[0010] As a preferred embodiment, the annealing temperature of the primer is 56℃-60℃.

[0011] An application of an IRAP marker primer for cherry germplasm identification, genetic diversity assessment, species phylogenetic analysis, or somatic variation detection.

[0012] As a preferred method, the method includes the following steps: (1) extracting genomic DNA from cherry or target Rosaceae fruit trees; (2) using the extracted genomic DNA as a template, performing PCR amplification with one or more IRAP marker primers as described in claim 1; (3) performing electrophoresis detection on the PCR amplification products, and counting the presence, number and size of amplification bands; (4) establishing a data matrix based on the electrophoresis detection results, calculating genetic parameters and performing cluster analysis, or constructing germplasm fingerprint codes and electronic ID cards.

[0013] As a preferred option, the PCR reaction program in step (2) is as follows: 94℃ pre-denaturation for 3-4 min; 94℃ denaturation for 45 s-1 min, 55℃-59℃ annealing for 45 s-1 min, 72℃ extension for 1 min, for a total of 35-38 cycles; 72℃ final extension for 5-7 min, and 4℃ to terminate the reaction.

[0014] As a preferred option, the electrophoresis detection in step (3) uses 5-8% non-denaturing polyacrylamide gel electrophoresis, voltage 150V, electrophoresis time 1.2h-2.5h, and the bands are observed after fixation, silver staining and color development.

[0015] As a preferred embodiment, the target Rosaceae fruit trees include one or more of the following: pear, peach, plum, apple, loquat, and prickly pear.

[0016] A kit for IRAP-labeled primers, the kit further comprising PCR reaction buffer, dNTPs, Taq DNA polymerase, ddH2O, and molecular weight standard markers. Attached Figure Description

[0017] Figure 1 The amplification of LTR1-3 on several fruit trees in the Rosaceae family; Figure 2 The results show the PCR amplification of loquat using primer LTR1-10. Figure 3 Cluster analysis of several Guizhou loquat species using IRAP; Figure 4 The PCR amplification of Li using primer LTR1-47; Figure 5 Cluster analysis of Lee using IRAP; Figure 6 The PCR amplification of peaches using LRT1-10; Figure 7 Cluster analysis of peaches using IRAP; Figure 8 The PCR amplification of pear using primers LTR1-3; Figure 9 Cluster analysis of pears for IRAP labels; Figure 10 For the 21st generation of agate red cherry; Figure 11 Detection of genetic variations in agate red using IRAP and ISSR (A is IRAP-PCR, B is ISSR). Detailed Implementation

[0018] The following detailed description illustrates the specific implementation method: Source of materials: Cherry germplasm materials: 40 Chinese cherry germplasm accessions were selected, covering major producing areas in Guizhou such as Bijie, Liupanshui, Anshun, and Kaili. These included 35 cultivated varieties (such as Agate Red Cherry and Shandong Daqingye) and 5 wild varieties (such as Leigongshan Microhair Cherry and Yunnan Cherry). All materials were collected from the germplasm resource nursery. Fresh young leaves were collected, treated with liquid nitrogen, and then stored at -80℃.

[0019] Cross-species validation materials: 34 samples from 5 fruit tree species in the Rosaceae family were selected, including 8 varieties of pear (such as Cuiyu and Zhongli No. 1), 6 varieties of peach (such as Yingshuanghong and Chunxue), 6 varieties of plum (such as Qingcui and Cuihong), 8 varieties of apple (such as Sx5 and CXH), and 6 varieties of loquat (such as Panzhou and Ceheng). These samples were collected from the resource nursery of the Fruit Tree Institute of Guizhou Academy of Agricultural Sciences and the farm of the College of Agriculture of Guizhou University.

[0020] Material for mutation detection: Agate Red Cherry tissue culture seedlings that have been subcultured 21 times in the laboratory, used for somatic cell mutation detection.

[0021] Example: An IRAP marker primer developed based on the cherry genome, selected from one or more of the following primer sequences: Primer LTR1-3, sequence 5'-ATGGCTATCTTCACGAGGATGT-3'; Primer LTR1-10, sequence 5'-GGGCTGAAGCAATCTCCAAG-3'; Primer LTR1-47, sequence 5'-ATGGGCTAAAGCAATCACCT-3'; Primer LTR3-11, sequence 5'-GGCCATAATCCACGCACATA-3'; Primer LTR1-1, sequence 5'-GTATGGCCTGAAACAGTCTCCA-3'; Primer LTR1-2, sequence 5'-ATGGGCTGAAGCAATCTCCAAG-3'; Primer LTR1-26, sequence 5'-GTATGGGCTGAAGCAATCTC-3'; Primer LTR1-48, sequence 5'-TATGGGCTAAAGCAATCACC-3'; Primer LTR1-60, sequence 5'-TATGGGCTAAAGCAATCACC-3'.

[0022] See Table 1: Table 1 IRAP Primer Information

[0023] Optimal PCR reaction volume (25 μL): 2.0 mmol L -1 MgCl2, 1.0 U Taq enzyme, 0.2 mmol L -1 Primers, 20 ng template DNA, 0.3 mmol L - ¹ dNTPs, 2.5 μL 10×PCR Buffer, with the remainder being ddH2O. The optimal PCR program was determined as follows: 94℃ pre-denaturation for 3 min; 94℃ denaturation for 1 min, 57℃ annealing for 1 min, 72℃ extension for 1 min, for a total of 38 cycles; 72℃ final extension for 7 min, and storage at 4℃.

[0024] Detection of amplified products: PCR products were detected by 8% non-denaturing polyacrylamide gel electrophoresis at 150V for 1.5-2 hours. After fixation with 5% glacial acetic acid + 10% ethanol for 15 minutes, silver staining with 0.1% silver nitrate for 15 minutes, and color development with 1.5% NaOH + 1.1% formaldehyde, the band information was photographed and recorded.

[0025] As shown in Tables 2 and 3, these 9 primers amplified 222 bands, averaging 24.67 bands per primer. A total of 200 polymorphic sites were identified, averaging 22.22 per primer, with an average polymorphism rate of 90.09%. The band range was between 150 and 2500 bp. The observed average effective allele count was 1.81, the average effective allele count was 1.35, the Nei' genetic diversity was 0.21, and the Shannon index was 0.33. Primers LTR1-3, LTR1-10, LTR3-11, and LTR1-47 were able to distinguish all 40 samples and can be used as core primers for cherry IRAP molecular markers.

[0026] Table 2. Statistical results of cherry IRAP primer bands

[0027] Table 3 Comparison of primer genetic diversity

[0028] (2) Using the optimized PCR system and procedure, 9 core primers were used to amplify 5 Rosaceae fruit trees, and the number of effective primers, total number of loci, number of polymorphic loci and polymorphism ratio of each species were counted.

[0029] Reference Figure 1 Tables 4 and 5 show the effective primers for pear (10 primers, 88.67% polymorphism), peach (9 primers, 71.50% polymorphism), apple (8 primers, 86.27% polymorphism), plum (8 primers, 88.81% polymorphism), and loquat (6 primers, 82.61% polymorphism). Among these, primers LTR1-3, LTR1-10, LTR3-11, and LTR1-8 showed high polymorphism in all species and can be used as universal core primers for Rosaceae fruit trees.

[0030] Table 4. IRAP primer information and their universality in several Rosaceae fruit trees.

[0031] Table 5. IRAP primer information and their universality in several Rosaceae fruit trees.

[0032] (3) Cluster analysis Reference Figure 2 and Figure 3 PCR amplification of loquat using primers LTR1-10.

[0033] Cluster family analysis was performed on loquat resources collected from six counties in Guizhou Province. The results were as follows: Figure 2 and Figure 3 The results show that the similarity coefficient is between 0.59 and 0.77. With a threshold of 0.64, the loquats can be divided into two groups: Panzhou and Luodian are grouped into one group, and the other four loquats are grouped into another group.

[0034] Reference Figure 4 and Figure 5 PCR amplification of Li was performed using primer LTR1-47.

[0035] IRAP markers were used to amplify PCR and perform cluster analysis on six plum varieties. At a genetic similarity coefficient of 0.55, they clustered into two groups: the Qingcui plum from Sichuan clustered with the Cuihong plum, while the other four varieties—'April Plum', 'Fengtang Plum', 'Jiuqian Plum', and 'Hongxin Plum'—all originated from Guizhou. 'Jiuqian Plum' is an early-maturing plum from Jiuqian Town, Sandu County, Guizhou, introduced to Sandu from elsewhere in the 19th century. 'April Plum' comes from Zhenfeng County, 'Fengtang Plum' is a local variety bred by the Anshun Agricultural Research Institute from Zhenning County, and 'Hongxin Plum' is a local variety from Shiqian County. The clustering results largely corresponded to their geographical origins. The two varieties from Sichuan were genetically close, but far from the Guizhou plums. Among the Guizhou plums, 'April Plum' and 'Fengtang Plum' were genetically close.

[0036] Reference Figure 6 and Figure 7 PCR amplification of peaches using primers LRT1-10.

[0037] Cluster analysis of several peach varieties by IRAP showed that the similarity coefficients ranged from 0.62 to 0.83. With a similarity coefficient threshold of 0.77, the tested materials could be divided into two groups: 'Yingshuanghong' and 'Chunmei Peach' were in one group, while the other four varieties were clustered together. Among them, 'Xiwei Yellow Peach' and 'Jinxiu Yellow Peach' had the highest similarity coefficients (0.83). 'Yingshuanghong' (Qingzhou Winter Snow Peach × Zhonghua Shoutao) is a variety bred by the Qingzhou Fruit Tree Selection Institute. 'Chunxue Peach' is an early-maturing variety introduced from the United States (Xin Xianmu et al., 2006). The male parent of Chunmei Peach is a semi-dwarf nectarine single plant SD9238, and the female parent is a peach hybrid single plant 89-3-16 (Niu Liang et al., 2011). 'Jinxiu Huangtao' ('Baihua × Yunshu No. 1') and 'Zhongyoutao No. 13' are nectarines. Nectarines are common peaches, and due to the insertion of retrotransposons, the peel has undergone hairless mutation, thus they can cluster with common peaches such as Jinxiu Huangtao. 'Chunxue' has nectarine lineage and clusters with 'Zhongyoutao No. 13'; 'Xiwei Huangtao' and 'Jinxiu Huangtao' both belong to the yellow-fleshed peach family, therefore they cluster together. The clustering results demonstrate the accuracy of the labeling.

[0038] Reference Figure 8 and Figure 9PCR amplification of pear using primers LTR1-3.

[0039] 'Cuiyu' is an early-maturing sand pear variety bred by crossing Xizi Green and Cuiguan. It is grouped with 'Zhongli No. 1' and 'Yunhong'. 'Zhongli No. 1' is a hybrid of 'Xinshiji' and 'Zaosu Li'. 'Yunhong' is a superior local variety selected by the Yunnan Academy of Agricultural Sciences from local varieties. 'Ruoguang', 'Zhongshu 32', 'Zaobaimi', and 'Huangjin Li' are grouped together. 'Ruoguang' is an early-maturing variety bred by the Chiba Prefectural Agricultural Experiment Station in Japan using a cross between Shinsui and Fengshui (Liu Xueping et al., 2008). 'Huangjin Li' is a new sand pear variety bred by the Naju Branch of the Korean Horticultural Experiment Station using a cross between 'Xin Gao' and '20th Century'. 'Zaobaimi' is a new early- to mid-maturing high-quality pear variety bred by crossing 'Xingshui' as the female parent and 'Huobali' (Pyrus pyrifolia Nakai cv. Huobali) as the male parent. 'Xingshui' pear was bred by the Shizuoka Horticultural Experiment Station in Japan, with 'Kikusui' and 'Wase Xingzang' as its parents. 'Zhongli No. 4' has a relatively large genetic distance from other varieties. This variety is a new early-maturing, large-fruited, high-quality pear variety selected from the offspring of the 'Zaomeisu × Qiyuesu' combination.

[0040] (4) IRAP detection of genetic variation in agate red cherry Reference Figure 10 and Figure 11 The black arrows point to the polymorphic bands. M: marker D2000; LTR-3, LTR-8, LTR47 are IRAP primers; M05, M03, 812, 811, 866 are ISSR primers.

[0041] Agate Red cherry tissue culture seedlings, 21st subculture using 9 IRAP primers and 14 ISSR primers ( Figure 10 Genetic variation detection was performed, and the results showed that the nine IRAP primers amplified a total of 212 loci, of which 7 were polymorphic, with a polymorphism rate of 3.30%. The ISSR primers amplified a total of 98 loci, with 0 polymorphic loci, a polymorphism rate of 0.00%. The IRAP polymorphic loci occurred on primers LTR1-8 and LTR1-47. Figure 11 This indicates that the PpRT8 and PpRT47 sequences may be active and involved in the somatic cell variation process during long-term tissue culture subculture, and also shows that the IRAP molecular marker is more sensitive than ISSR in detecting variation.

[0042] It should be noted that those skilled in the art can make various modifications and improvements without departing from the technical solution of this invention. These modifications and improvements should also be considered within the scope of protection of this invention, and will not affect the effectiveness of the invention or the practicality of the patent. The scope of protection claimed in this application shall be determined by the content of its claims, and the specific embodiments described in the specification can be used to interpret the content of the claims.

Claims

1. IRAP marker primers developed based on the genome of Prunus avium, characterized in that, selected from one or more of the following primer sequences: primer LTR1-3, sequence 5'-ATGGCTATCTTCACGAGGATGT-3'; primer LTR1-10, sequence 5'-GGGCTGAAGCAATCTCCAAG-3'; primer LTR1-47, sequence 5'-ATGGGCTAAAGCAATCACCT-3'; primer LTR3-11, sequence 5'-GGCCATAATCCACGCACATA-3'; primer LTR1-1, sequence 5'-GTATGGCCTGAAACAGTCTCCA-3'; primer LTR1-2, sequence 5'-ATGGGCTGAAGCAATCTCCAAG-3'; primer LTR1-26, sequence 5'-GTATGGGCTGAAGCAATCTC-3'; primer LTR1-48, sequence 5'-TATGGGCTAAAGCAATCACC-3'; primer LTR1-60, sequence 5'-TATGGGCTAAAGCAATCACC-3'.

2. The IRAP marker primer of claim 1, wherein, The annealing temperature of the primer is 56-60℃.

3. The use of the IRAP marker primer according to claim 1 or 2, wherein, For cherry germplasm identification, genetic diversity evaluation, species relationship analysis or somatic mutation detection.

4. Use according to claim 3, characterized in that, The method comprises the following steps: (1) extracting genomic DNA of cherry or target Rosaceae fruit tree; (2) using the extracted genomic DNA as template, performing PCR amplification with one or more IRAP marker primers according to claim 1; (3) performing electrophoretic detection on the PCR amplification product, and counting the presence or absence, number and size of the amplified bands; (4) establishing a data matrix based on the electrophoretic detection result, calculating genetic parameters and performing cluster analysis, or constructing a germplasm fingerprint code and electronic identity card.

5. Use according to claim 4, characterized in that, The PCR reaction program in step (2) is as follows: 94℃ pre-denaturation for 3-4 min; 94℃ denaturation for 45 s-1 min, 55-59℃ annealing for 45 s-1 min, 72℃ extension for 1 min, a total of 35-38 cycles; 72℃ final extension for 5-7 min, and 4℃ termination reaction.

6. Use according to claim 5, characterized in that, The electrophoretic detection in step (3) uses 5-8% non-denaturing polyacrylamide gel electrophoresis, voltage 150V, electrophoresis time 1.2-2.5h, and the bands are observed after fixation, silver staining and color development.

7. Use according to claim 6, characterized in that, The target Rosaceae fruit tree includes one or more of pear, peach, plum, apple, loquat and prickly pear.

8. A kit comprising the IRAP marker primer according to claim 1.

9. The kit of claim 8, wherein The kit further comprises PCR reaction buffer, dNTP, Taq DNA polymerase, ddH2O and molecular weight marker Marker. The primer LTR1-3, sequence 5'-ATGGCTATCTTCACGAGGATGT-3';

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