InDel primer set for malus micromalus nakai hybrid identification and application thereof
By using InDel-specific primer sets and PCR identification methods, the accuracy and timeliness issues of identifying hybrid offspring of prickly pear were resolved, enabling early, high-throughput, and low-cost hybrid identification, especially accurate differentiation of closely related germplasm.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- GUIZHOU UNIV
- Filing Date
- 2026-05-12
- Publication Date
- 2026-06-30
Smart Images
Figure CN122303476A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of genetic breeding technology, and in particular relates to an InDel primer set for identifying hybrids of prickly pear and its application. Background Technology
[0002] prickly pear ( Rosa roxburghii *Rosa argentea* (also known as prickly pear) is a perennial fruit tree belonging to the genus *Rosa* in the family Rosaceae. It is a distinctive and advantageous fruit in southwestern my country, particularly abundant in Guizhou Province, where it is cultivated over the largest area. The fruit is rich in vitamin C, superoxide dismutase (SOD), flavonoids, triterpenes, and catechins, possessing high nutritional value and health benefits. In recent years, with the rapid development of the prickly pear industry, the demand for superior new varieties has become increasingly urgent.
[0003] Hybrid breeding is an important approach to the selection of new fruit tree varieties and germplasm innovation. By artificially controlling pollination, the superior traits (such as large fruit, thornless, high quality, and disease resistance) of different parents can be recombinated and combined to obtain offspring with excellent overall traits. However, prickly pear is a self-pollinating plant, with anthers distributed around the stigma. During artificial emasculation, its own pollen can easily mix in, leading to self-pollination and fruit setting. Therefore, the authenticity of hybrid offspring is an indispensable part of breeding work.
[0004] Currently, the identification of hybrid offspring of prickly pear mainly relies on morphological observation, such as leaf morphology and fruit characteristics. However, morphological identification has the following limitations: (1) Low accuracy: Different genotypes of prickly pear have high morphological similarity in the seedling stage. Quantitative traits such as leaf length and width are easily affected by environmental conditions and growth stages. There is a large phenotypic overlap between true hybrids and self-pollinated offspring.
[0005] (2) Long cycle: It is necessary to wait until the plant flowers and bears fruit before judging based on the fruit characteristics, which makes early screening impossible.
[0006] (3) Highly subjective: It relies on the researcher's experience and judgment and lacks objective quantitative standards.
[0007] Molecular marker technologies (such as SSR, ISSR, RAPD, etc.) have been applied in hybrid identification of some plants, but the following problems still exist in prickly pear: (1) Poor universality: The existing SSR markers are unstable in polymorphism among different prickly pear hybrid combinations, making them difficult to use directly for hybrid identification.
[0008] (2) Difficulty in distinguishing closely related germplasm: For closely related parents (such as offspring and maternal parent), conventional single molecular markers often fail to produce effective polymorphism.
[0009] Therefore, developing a high-efficiency, accurate, low-cost tool applicable to multiple hybrid combinations of prickly pear for hybrid authenticity identification has significant application value and practical significance. Summary of the Invention
[0010] The purpose of this invention is to provide a set of InDel-specific primers for identifying hybrids of prickly pear, as well as identification methods and applications based on these primers, to solve technical problems such as inaccurate morphological identification and difficulty in distinguishing closely related germplasm using SSR markers, and to achieve early, accurate, and high-throughput identification of hybrid offspring of prickly pear.
[0011] This invention provides an InDel primer set for identifying hybrids of prickly pear, comprising at least one of the following nine primer pairs: Primer pair 41, the nucleotide sequence of which is shown in SEQ ID NO:1-2; Primer pair 122, the nucleotide sequence of which is shown in SEQ ID NO:3-4; Primer pair 132, the nucleotide sequence of which is shown in SEQ ID NO:5-6; Primer pair 133, the nucleotide sequence of which is shown in SEQ ID NO:7-8; Primer pair 137, the nucleotide sequence of which is shown in SEQ ID NO:9-10; Primer pair 36, the nucleotide sequence of which is shown in SEQ ID NO:11-12; Primer pair 39, the nucleotide sequence of which is shown in SEQ ID NO:13-14; Primer pair 144, the nucleotide sequence of which is shown in SEQ ID NO:15-16; Primer pair 149, whose nucleotide sequence is shown in SEQ ID NO:17-18.
[0012] Furthermore, when used to identify GN5×DC1 hybrid combinations, primer pairs 36 and 39 are mixed in an equimolar ratio.
[0013] Furthermore, when used to identify WC2×DC1 hybrid combinations, primer pairs 144 and 149 are mixed in an equimolar ratio.
[0014] This invention provides a kit for identifying hybrids of prickly pear, which includes the InDel primer set.
[0015] Furthermore, it also contains 2×Taq Master Mix, dNTPs, and PCR reaction buffer.
[0016] This invention provides a method for identifying the authenticity of prickly pear hybrids, comprising the following steps: (1) Extract genomic DNA from the prickly pear sample to be tested; (2) The extracted DNA was amplified by PCR using the InDel primer set; (3) Electrophoresis detection of PCR amplification products; (4) Determine the result based on the following correspondence: 1) When using primer 41 to identify the WC1×DC1 combination: if the sample shows two bands of 250 bp and 500 bp or only a 500 bp band, the individual is judged as a true hybrid; if only a 250 bp band is shown, the individual is judged as a false hybrid. When using primer 122 to identify the WC1×DC1 combination: individuals with two bands of 500 bp and 250 bp or only a 250 bp band are identified as true hybrids, while individuals with only a 500 bp band are identified as false hybrids. 2) When using primer 41 to identify the DC1×WC1 combination: if the sample shows two bands of 500 bp and 250 bp or only a 250 bp band, the individual is determined to be a true hybrid; if only a 500 bp band is shown, the individual is determined to be a false hybrid. When using primer 122 to identify the DC1×WC1 combination: individuals with two bands of 250 bp and 500 bp or only a 500 bp band are identified as true hybrids, while individuals with only a 250 bp band are identified as false hybrids. 3) When using primer 122 to identify the WC1×GN5 combination: if the sample shows two bands of 300 bp and 100 bp or only shows a 100 bp band, the individual is judged as a true hybrid; if the individual shows only a 300 bp band, the individual is judged as a false hybrid. 4) When using primer 122 to identify the GN5×WC1 combination: if the sample shows two bands of 100 bp and 300 bp or only a 300 bp band, the individual is determined to be a true hybrid; if only a 100 bp band is shown, the individual is determined to be a false hybrid. 5) When using primer 122 to identify the WC2×GN1 combination: if the sample shows two bands of 100 bp and 300 bp or only a 300 bp band, the individual is determined to be a true hybrid; if the individual shows only a 100 bp band, the individual is determined to be a false hybrid. When using primer 132 to identify the WC2×GN1 combination: individuals with two bands of 280 bp and 100 bp or only a 100 bp band are identified as true hybrids, while individuals with only a 280 bp band are identified as false hybrids. 6) When using primer 122 to identify the GN1×WC2 combination: if the sample shows two bands of 300 bp and 100 bp or only shows a 100 bp band, the individual is judged as a true hybrid; if the individual shows only a 300 bp band, the individual is judged as a false hybrid. When using primer 132 to identify the GN1×WC2 combination: individuals with two bands of 100 bp and 280 bp or only a 280 bp band are identified as true hybrids, while individuals with only a 100 bp band are identified as false hybrids. 7) When using primers 144+149 to identify the WC2×DC1 combination: if the sample shows two bands of 250 bp and 500 bp or only a 500 bp band, the individual is determined to be a true hybrid; if only a 250 bp band is shown, the individual is determined to be a false hybrid. 8) When using primers 144+149 to identify the DC1×WC2 combination: if the sample shows two bands of 500 bp and 250 bp or only a 250 bp band, the individual is determined to be a true hybrid; if the individual shows only a 500 bp band, the individual is determined to be a false hybrid. 9) When using primer 41 to identify the WC2×GN5 combination: if the sample shows two bands of 500 bp and 250 bp or only shows a 250 bp band, the individual is determined to be a true hybrid; if only shows a 500 bp band, the individual is determined to be a false hybrid. 10) When using primer 41 to identify the GN5×WC2 combination: if the sample shows two bands of 250 bp and 500 bp or only a 500 bp band, the individual is determined to be a true hybrid; if only a 250 bp band is shown, the individual is determined to be a false hybrid. 11) When using primers 36+39 to identify the GN5×DC1 combination: if the sample shows two bands of 250 bp and 100 bp or only shows a 100 bp band, the individual is judged as a true hybrid; if the individual shows only a 250 bp band, the individual is judged as a false hybrid. 12) When using primers 36+39 to identify the DC1×GN5 combination: if the sample shows two bands of 100 bp and 250 bp or only a 250 bp band, the individual is determined to be a true hybrid; if only a 100 bp band appears, the individual is determined to be a false hybrid.
[0017] This invention provides an application of the InDel primer set for hybrid identification of prickly pear: (1) Application in the identification of genetic diversity in prickly pear; (2) Application in the identification of prickly pear varieties; (3) Application in the identification of hybrid offspring of prickly pear Compared with the prior art, the present invention has the following beneficial effects: (1) High accuracy in identification and reliable results Using the nine InDel primer pairs (including single primer pairs and dual PCR combinations) provided by this invention, the hybrid authenticity of the F1 generation of six hybrid combinations (including reciprocal crosses, totaling 12 combinations) from five backbone parent groups was identified. The true hybridization rate of each combination was consistently above 80%, averaging approximately 89.0%, with a maximum of 91.94% (DC1×GN5 combination). Compared with traditional morphological identification, this invention is not affected by plant development stage or environmental conditions, and the identification results are objective and accurate, effectively avoiding subjective misjudgment.
[0018] (2) Enable early identification and significantly shorten the breeding cycle This invention allows for identification during the seedling stage (two true leaves stage), without waiting for the plant to flower and bear fruit. In contrast, morphological identification requires assessment based on phenotypic characteristics such as fruit traits after the plant has matured, a process that can take several years. This invention enables early, high-throughput screening of hybrid offspring, significantly shortening the breeding cycle and saving land resources and field management costs.
[0019] (3) Easy to operate and low cost This invention employs conventional PCR amplification combined with 2% agarose gel electrophoresis detection, eliminating the need for fluorescent labeling, sequencing, or complex instruments. It offers low cost per sample, making it suitable for screening large-scale breeding populations and easily applicable in general breeding laboratories.
[0020] (4) Wide coverage and strong versatility Nine primer pairs can cover five core parents of prickly pear (Guinong 1 GN1, Guinong 5 GN5, Thornless 1 WC1, Thornless 2 WC2, and Short Thorn 1 DC1) and their six main hybridization combinations (including 12 combinations of reciprocal crosses). Several primers can be used interchangeably between different combinations (such as primers 41 and 122), which has strong practical value.
[0021] (5) Solving the problem of identifying closely related germplasm For closely related germplasm combinations that are difficult to distinguish using conventional single molecular markers—DC1 (a seedling offspring of WC2, i.e., obtained from WC2 seeds and with a very close kinship) and WC2, as well as GN5 and DC1—this invention innovatively employs a dual PCR strategy (primer pairs 144+149 and 36+39) to simultaneously amplify the characteristic bands of both parents in the same reaction tube. This successfully achieves the identification of true and false hybrid combinations with a very close kinship, providing a referable technical solution for the identification of hybrids of similar closely related germplasm.
[0022] (6) Product form protection, high commercial application value This invention is protected in the form of a primer set, a chemical product, which makes infringement determination easy. It can be directly used for early screening of hybrid offspring in prickly pear breeding units or enterprises, and has high commercial application prospects. Attached Figure Description
[0023] Figure 1 The results of identifying the DC1×WC1 hybrid offspring using primer 122 in Example 1 are shown. Lane M: DNA Marker (DM2000), with band sizes of 2000bp, 1000bp, 750bp, 500bp, 250bp, and 100bp respectively; Lane ♀: maternal parent DC1; Lane ♂: paternal parent WC1; the remaining lanes represent the hybrid offspring. Red arrows point to pseudohybrids.
[0024] Figure 2 The results of identifying the WC2×DC1 hybrid offspring using primers 144+149 in Example 2 are shown. Lane M: DNA Marker (DM2000), with band sizes of 2000bp, 1000bp, 750bp, 500bp, 250bp, and 100bp respectively; Lane ♀: maternal parent WC2; Lane ♂: paternal parent DC1; the remaining lanes represent the hybrid offspring. The blue arrows indicate true hybrids that only amplified the paternal characteristic band.
[0025] Figure 3 The results of primer 36+39 in Example 3 for identifying the GN5×DC1 hybrid offspring. Lane M: DNA Marker (DM2000), with band sizes of 2000bp, 1000bp, 750bp, 500bp, 250bp, and 100bp respectively; Lane ♀: hybrid maternal parent GN5; Lane ♂: hybrid paternal parent DC1; the remaining lanes represent the hybrid offspring. Detailed Implementation
[0026] This invention is based on whole-genome resequencing data of *Prickly pear*, using the T2T nick-free assembled genome of *Prickly pear* as a reference. Sequence alignment was performed using BWA software, and GATK variant detection was conducted. Low-quality sites were removed according to strict filtering criteria (QD<2.0 || FS>200.0 || SOR>10.0 || MQRankSum<-12.5). Specific InDel (insertion / deletion) variant sites with a length ≥30 bp between parents were further screened. Primers were designed and PCR verified based on conserved flanking sequences of these sites, ultimately obtaining 9 pairs of polymorphic, stable, and highly specific InDel functional markers. This primer set can cover the F1 hybrid authenticity identification of 5 core parents (Guinong 1 GN1, Guinong 5 GN5, Thornless 1 WC1, Thornless 2 WC2, and Short-thorned 1 DC1) and their mating pairs, including 12 reciprocal crosses. For the closely related DC1 and WC2 combination (DC1 being the offspring of WC2), dual PCR detection was performed using two pairs of specific primers (144 and 149); for the GN5 and DC1 combination, dual PCR detection was performed using two pairs of specific primers (36 and 39), which can effectively distinguish them.
[0027] Parental Source Description: The five prickly pear parental lines involved in this invention are all known germplasm resources. The codes and corresponding relationships of WC1 (thornless No. 1), WC2 (thornless No. 2), and DC1 (short-thorned No. 1) have been disclosed in Li Jin'e's master's thesis, "Study on the Creation of New Polyploid Germplasm of Prickly Pear by Colchicine Treatment" (Guizhou University, 2024). GN1 (Guinong No. 1) and GN5 (Guinong No. 5) are main cultivated prickly pear varieties approved by Guizhou Province, with approval numbers Qian Shen Guo 2007002 and Qian Shen Guo 2007004 respectively. These can be obtained by the public through commercial channels or from institutions such as Guizhou University. Those skilled in the art can obtain the germplasm resources based on the above-mentioned publicly available information; no preservation is required.
[0028] In this invention: InDel markers: Insertion and Deletion length polymorphism molecular markers, referring to the length polymorphism formed by the insertion or deletion of DNA fragments in specific regions of the genome by different genotypes.
[0029] Core primers: InDel primers that have been screened and verified to have clear bands, stable amplification, and good reproducibility, and can be used for the authenticity identification of prickly pear hybrid offspring.
[0030] Characteristic primers: These are primers whose polymorphic sites, verified by specific hybridization combinations, are unique and stable within that combination, and can effectively distinguish the parents and offspring of that combination.
[0031] Duplex PCR: Two pairs of primers are added simultaneously to the same PCR reaction tube for amplification, used for identification of closely related germplasm.
[0032] True hybrid: After PCR amplification, the test sample amplifies both the maternal and paternal specific bands (heterozygous), or only the paternal specific band is amplified (when the maternal parent is homozygous and the paternal parent is either homozygous or heterozygous).
[0033] 1. Nine pairs of InDel-specific primer sequences: This invention provides nine pairs of InDel-specific primers for identifying hybrids of prickly pear. Their names, sequences, annealing temperatures, number of cycles, and identifiable hybrid combinations are shown in Table 1.
[0034] Table 1. Sequences, amplification parameters, and identifiable parental combinations of 9 InDel-specific primer pairs
[0035] Note: Of the nine primer pairs mentioned above, 41, 122, 132, 133, and 137 are single primer pairs, while 36+39 and 144+149 are duplex PCR primer pairs. For duplex PCR, the two primer pairs are mixed in equimolar proportions and added to the same reaction tube.
[0036] Table 2. Locations of the InDel sites corresponding to the 9 primer pairs on the prickly pear genome.
[0037] 2. Identification methods (1) DNA extraction Genomic DNA was extracted from the prickly pear samples using a plant genomic DNA extraction kit (BioTeke, Beijing, DP3211). DNA concentration and purity (OD260 / OD280 between 1.8 and 2.0) were measured using an ultra-micro spectrophotometer and stored at -20℃ for later use.
[0038] (2) PCR amplification PCR amplification system (total volume 20 μL): 8 μL ddH2O, 0.5 μL upstream primer (10 μM), 0.5 μL downstream primer (10 μM), 1 μL DNA template (50 ng / μL), 10 μL 2×Taq Master Mix (Tiangen, KT201). PCR amplification program: 94℃ pre-denaturation for 3 min; 30-35 cycles: 94℃ denaturation for 1 min, annealing (temperatures see Table 1) for 1 min, 72℃ extension for 1 min; 72℃ final extension for 5 min; store at 4℃.
[0039] Double PCR procedure: Add 0.5 μL of each of the two primer pairs (e.g., 36 and 39, or 144 and 149) (final concentration 0.25 μM each) to the same reaction tube, 7 μL of ddH2O, and the other components as above. Set the annealing temperature to 53 ℃ (144+149) or 57 ℃ (36+39). 144+149 is for 35 cycles, and 36+39 is for 30 cycles.
[0040] (3) Electrophoresis detection Take 5 μL of PCR amplification product and separate it by electrophoresis using a 2% agarose gel (containing nucleic acid dye) at 120V for 30-40 min. After electrophoresis, observe and photograph the gel using a gel imaging system.
[0041] (4) Result determination Based on the electrophoresis results of the PCR amplification products, the following three scenarios will be used for judgment: Scenario 1 (True Hybrid): The test sample simultaneously amplifies specific bands from both the maternal and paternal parents (heterozygous).
[0042] Scenario 2 (True hybrid, special case): The sample to be tested only amplifies the paternal-specific band, and the maternal parent is homozygous at this locus (i.e., the maternal parent only produces one characteristic band, and the paternal parent is homozygous or heterozygous).
[0043] Scenario 3 (false hybrid, i.e., self-crossed offspring): The test sample only amplifies the maternal-specific band, and no paternal-specific band appears.
[0044] Example 1 Primer 122 is used to identify the offspring of DC1×WC1 hybridization. Leaves from DC1 (maternal parent), WC1 (paternal parent), and 220 F1 seedlings were collected. Genomic DNA was extracted using a commercial plant DNA extraction kit. The DNA concentration and purity (OD260 / OD280 between 1.8 and 2.0) were measured using a micro spectrophotometer and stored at -20℃ for later use. PCR amplification was performed using primer 122 from Table 1. The PCR reaction system was as described in step (2) of the amplification method, and the electrophoresis detection was as described in step (3) of the amplification method. The electrophoresis results are shown below. Figure 1The maternal parent DC1 amplified a band of approximately 100 bp, and the paternal parent WC1 amplified a band of approximately 300 bp. In the F1 generation, individuals exhibiting both 100 bp and 300 bp bands, or only the 300 bp band, were classified as true hybrids. Individuals amplifying only the maternal 100 bp band and not the paternal 300 bp band were classified as false hybrids (i.e., offspring of maternal self-pollination or individuals contaminated by pollen). The results showed 201 true hybrids, of which 196 exhibited double bands, and 5 exhibited only the paternal band, resulting in a true hybridity rate of 91.36% (Table 2).
[0045] Example 2 Double PCR (144+149) for identification of WC2×DC1 hybrid progeny Leaves from WC2 (maternal parent), DC1 (paternal parent), and 170 F1 seedlings were collected. Genomic DNA was extracted using a commercial plant DNA extraction kit. The DNA concentration and purity (OD260 / OD280 between 1.8 and 2.0) were measured using a micro spectrophotometer and stored at -20℃ for later use. Double PCR amplification was performed using primers 144 and 149 as shown in Table 1. The PCR reaction system was as described in step (2) of the amplification method, and the electrophoresis detection was as described in step (3) of the amplification method. The results are as follows: Figure 2 As shown, the maternal parent WC2 amplified a band of approximately 250 bp, and the paternal parent DC1 amplified a band of approximately 500 bp. In the F1 generation, individuals exhibiting the paternal characteristic band (500 bp) were classified as true hybrids. The identification results showed 142 true hybrids, of which 140 exhibited two bands, and 2 exhibited only the paternal band, resulting in a true hybridity rate of 83.53% (Table 3). Three of these individuals exhibited only the paternal band (no maternal band) and were still classified as true hybrids.
[0046] Example 3 Double PCR (36+39) to identify GN5×DC1 hybrid progeny Leaves from GN5 (maternal parent), DC1 (paternal parent), and 174 F1 seedlings were collected. Genomic DNA was extracted using a commercial plant DNA extraction kit. The DNA concentration and purity (OD260 / OD280 between 1.8 and 2.0) were measured using a micro spectrophotometer and stored at -20℃ for later use. Double PCR amplification was performed using primers 36 and 39 from Table 1. The PCR reaction system was as described in step (2) of the amplification method, and the electrophoresis detection was as described in step (3) of the amplification method. The results are as follows: Figure 3As shown, the maternal parent GN5 amplified a band of approximately 250 bp, and the paternal parent DC1 amplified a band of approximately 100 bp. In the F1 generation, individuals exhibiting both parental characteristic bands or only the paternal characteristic band were identified as true hybrids. The results showed 156 true hybrids, all of which exhibited two bands, resulting in a true hybridization rate of 89.66% (Table 3).
[0047] The F1 generation of six hybridization combinations (including 12 combinations from reciprocal crosses) were identified using the above nine primer pairs. The true hybridization rate of each combination is shown in Table 3. The average true hybridization rate was approximately 89.0%.
[0048] Table 3. Results of authenticity identification of F1 hybrids from different prickly pear hybrid combinations
[0049] Note: In the primer column, the " / " in "41 / 122, 122 / 132" indicates that only one of the two primer pairs needs to be used for independent PCR detection; the "+" in "144+149, 36+39" indicates that the two primer pairs are mixed in equal molar amounts and then used for duplex PCR detection in the same reaction tube.
[0050] Comparative Example 1: Morphological identification vs. Molecular identification of this invention One hundred F1 seedlings of the same GN5×WC2 hybrid combination were taken and morphologically identified and molecularly identified using primer 41 of this invention at the two true leaf stages.
[0051] Morphological identification group: During the seedling stage, the leaf morphology (length-to-width ratio, leaf shape index, leaf margin serrations, etc.) of hybrid offspring and self-pollinated offspring are highly similar, and the variation between individuals is large and the overlap is high, making it impossible to accurately distinguish between true hybrids and false hybrids, and identification work cannot be carried out effectively.
[0052] The molecular identification group of this invention uses primer 41 for PCR amplification and electrophoresis detection, which can clearly distinguish the genotypes of offspring at the same seedling stage. The results showed that among 100 seedlings, 91 individuals amplified both parental characteristic bands or only the paternal band (true hybridity rate of 91.80%), and 9 individuals amplified only the maternal band (false hybrids).
[0053] Conclusion: Morphological identification cannot be completed in the seedling stage, while this invention can achieve accurate and high-throughput screening in the seedling stage, significantly shortening the breeding cycle.
[0054] Comparative Example 2: Single InDel marker vs. Duplex PCR of the present invention Fifty F1 seedlings of the WC2×DC1 hybrid combination were taken and identified using a single InDel marker (primer 144 only or primer 149 only) and the dual PCR of this invention (144+149).
[0055] Single marker identification group: Single marker 144 amplified a 500 bp band in the paternal DC1, but no amplification product was found in the maternal WC2 due to the homozygous deletion at this site; single marker 149 amplified a 250 bp band in the maternal WC2, but no amplification product was found in the paternal DC1 due to the deletion of this insert. Therefore, single markers cannot simultaneously detect genetic information from both parents.
[0056] The present invention employs a dual PCR identification method: using primers 144 and 149 for dual PCR amplification, simultaneously amplifying the 250 bp characteristic band of the maternal parent WC2 and the 500 bp characteristic band of the paternal parent DC1 in the same reaction tube. Testing was performed on 50 F1 generation seedlings, successfully identifying 44 true hybrids (88.0% true hybridity rate) and 6 false hybrids.
[0057] Conclusion: For hybrid combinations such as WC2 and DC1, which are closely related (DC1 is a seedling offspring of WC2), a single InDel marker cannot effectively identify them. The dual PCR technology used in this invention is a necessary and effective means to identify the authenticity of the hybrids, rather than a simple superposition of a single marker.
[0058] Comparative Example 3: Reported SSR primers vs. the characteristic primers of this invention Fifty F1 seedlings of the WC2×GN1 hybrid combination were taken and identified using the previously reported prickly pear SSR primers and the characteristic primer 132 of this invention.
[0059] The previously reported SSR primer identification group: PCR amplification was performed using four randomly selected, publicly published pairs of prickly pear SSR primers 5, 6, 8, and 24 (Yan Xiuqin. Transcriptome Analysis of Prickly Pear Fruit and Development and Application of EST-SSR [D]. Guizhou University, 2015). The results showed that the SSR primers amplified single, uniformly sized bands in the maternal parent WC2, paternal parent GN1, and F1 individuals, with no polymorphism between the parents, making it impossible to distinguish between true and false hybrids.
[0060] The characteristic primer identification group of this invention: PCR amplification was performed using primer 132 of this invention. The maternal parent WC2 amplified a band of approximately 300 bp, and the paternal parent GN1 amplified a band of approximately 100 bp. Fifty F1 seedlings were tested. Individuals that amplified both parental bands simultaneously or only the paternal band were identified as true hybrids, while individuals that amplified only the maternal band were identified as false hybrids. This method allows for clear identification.
[0061] Conclusion: The reported prickly pear SSR primers lack polymorphism in this hybrid combination and cannot be used for hybrid identification; while the characteristic primer 132 of this invention has stable length polymorphism between the parents, which can achieve accurate identification of offspring.
[0062] The technical concept provided by this invention, which is based on whole-genome resequencing to screen for large fragment InDel (≥30 bp) sites and then designing combined primers (including dual PCR strategies), is not only applicable to the five currently disclosed parents and their pairings, but also provides a directly referable technical path for developing hybrid identification primers for other prickly pear backbone parents and their derived lines, and even other fruit crops.
[0063] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.
Claims
1. An InDel primer set for identifying Malus x domestica hybrids, characterized in that, Includes at least one of the following 9 primer pairs: Primer pair 41, the nucleotide sequence of which is shown in SEQ ID NO:1-2; Primer pair 122, the nucleotide sequence of which is shown in SEQ ID NO:3-4; Primer pair 132, the nucleotide sequence of which is shown in SEQ ID NO:5-6; Primer pair 133, the nucleotide sequence of which is shown in SEQ ID NO:7-8; Primer pair 137, the nucleotide sequence of which is shown in SEQ ID NO:9-10; Primer pair 36, the nucleotide sequence of which is shown in SEQ ID NO:11-12; Primer pair 39, the nucleotide sequence of which is shown in SEQ ID NO:13-14; Primer pair 144, the nucleotide sequence of which is shown in SEQ ID NO:15-16; Primer pair 149, whose nucleotide sequence is shown in SEQ ID NO:17-18.
2. The InDel primer set according to claim 1, characterized in that, When used to identify GN5×DC1 hybrid combinations, primer pairs 36 and 39 are mixed in an equimolar ratio.
3. The InDel primer set according to claim 1, characterized in that, When used to identify WC2×DC1 hybrid combinations, primer pairs 144 and 149 are mixed in an equimolar ratio.
4. A kit for identifying hybrid varieties of prickly pear, characterized in that, It includes the InDel primer set as described in any one of claims 1 to 4.
5. The reagent kit according to claim 4, characterized in that, It also contains 2×Taq Master Mix, dNTPs, and PCR reaction buffer.
6. A method for identifying the authenticity of a prickly pear hybrid, characterized in that, Includes the following steps: (1) Extract genomic DNA from the prickly pear sample to be tested; (2) The extracted DNA was amplified by PCR using the InDel primer set described in any one of claims 1 to 3; (3) Electrophoresis detection of PCR amplification products; (4) Determine the result based on the following correspondence: 1) When using primer 41 to identify the WC1×DC1 combination: if the sample shows two bands of 250 bp and 500 bp or only a 500 bp band, the individual is judged as a true hybrid; if only a 250 bp band is shown, the individual is judged as a false hybrid. When using primer 122 to identify the WC1×DC1 combination: individuals with two bands of 500 bp and 250 bp or only a 250 bp band are identified as true hybrids, while individuals with only a 500 bp band are identified as false hybrids. 2) When using primer 41 to identify the DC1×WC1 combination: if the sample shows two bands of 500 bp and 250 bp or only a 250 bp band, the individual is determined to be a true hybrid; if only a 500 bp band is shown, the individual is determined to be a false hybrid. When using primer 122 to identify the DC1×WC1 combination: individuals with two bands of 250 bp and 500 bp or only a 500 bp band are identified as true hybrids, while individuals with only a 250 bp band are identified as false hybrids. 3) When using primer 122 to identify the WC1×GN5 combination: if the sample shows two bands of 300 bp and 100 bp or only shows a 100 bp band, the individual is judged as a true hybrid; if the individual shows only a 300 bp band, the individual is judged as a false hybrid. 4) When using primer 122 to identify the GN5×WC1 combination: if the sample shows two bands of 100 bp and 300 bp or only a 300 bp band, the individual is determined to be a true hybrid; if only a 100 bp band is shown, the individual is determined to be a false hybrid. 5) When using primer 122 to identify the WC2×GN1 combination: if the sample shows two bands of 100 bp and 300 bp or only a 300 bp band, the individual is determined to be a true hybrid; if the individual shows only a 100 bp band, the individual is determined to be a false hybrid. When using primer 132 to identify the WC2×GN1 combination: individuals with two bands of 280 bp and 100 bp or only a 100 bp band are identified as true hybrids, while individuals with only a 280 bp band are identified as false hybrids. 6) When using primer 122 to identify the GN1×WC2 combination: if the sample shows two bands of 300 bp and 100 bp or only shows a 100 bp band, the individual is judged as a true hybrid; if the individual shows only a 300 bp band, the individual is judged as a false hybrid. When using primer 132 to identify the GN1×WC2 combination: individuals with two bands of 100 bp and 280 bp or only a 280 bp band are identified as true hybrids, while individuals with only a 100 bp band are identified as false hybrids. 7) When using primers 144+149 to identify the WC2×DC1 combination: if the sample shows two bands of 250 bp and 500 bp or only a 500 bp band, the individual is determined to be a true hybrid; if only a 250 bp band is shown, the individual is determined to be a false hybrid. 8) When using primers 144+149 to identify the DC1×WC2 combination: if the sample shows two bands of 500 bp and 250 bp or only a 250 bp band, the individual is determined to be a true hybrid; if the individual shows only a 500 bp band, the individual is determined to be a false hybrid. 9) When using primer 41 to identify the WC2×GN5 combination: if the sample shows two bands of 500 bp and 250 bp or only shows a 250 bp band, the individual is determined to be a true hybrid; if only shows a 500 bp band, the individual is determined to be a false hybrid. 10) When using primer 41 to identify the GN5×WC2 combination: if the sample shows two bands of 250 bp and 500 bp or only a 500 bp band, the individual is determined to be a true hybrid; if only a 250 bp band is shown, the individual is determined to be a false hybrid. 11) When using primers 36+39 to identify the GN5×DC1 combination: if the sample shows two bands of 250 bp and 100 bp or only shows a 100 bp band, the individual is judged as a true hybrid; if the individual shows only a 250 bp band, the individual is judged as a false hybrid. 12) When using primers 36+39 to identify the DC1×GN5 combination: if the sample shows two bands of 100 bp and 250 bp or only a 250 bp band, the individual is determined to be a true hybrid; if only a 100 bp band appears, the individual is determined to be a false hybrid.
7. The application of the InDel primer set according to any one of claims 1 to 3, the kit according to any one of claims 4 to 5, or the method according to claim 6 in any of the following aspects: (1) Application in the identification of genetic diversity in prickly pear; (2) Application in the identification of prickly pear varieties; (3) Application in the identification of hybrid offspring of prickly pear.