A method for identifying interspecific hybrid progeny of walnut and Hebei walnut

Abstract

The present invention provides a method for identifying progeny of interspecific hybrids between walnut and Hebei walnut, relating to the technical field of plant genetic breeding. The present invention provides the use of specific primers in identifying progeny of interspecific hybrids between walnut and Hebei walnut, as well as a method for identifying progeny of interspecific hybrids between walnut and Hebei walnut. The method is simple to operate, has good repeatability, high stability, and is unaffected by the external environment. The method effectively improves the authenticity of hybrid offspring between walnut varieties, increases the efficiency of early selection of hybrid offspring, shortens the breeding cycle, and improves breeding efficiency.

Description

Technical Field

[0001] The invention belongs to the technical field of plant genetic breeding, and particularly relates to a method for identifying interspecific hybrid progenies of walnut and Hebei walnut. Background Art

[0002] Walnut (J. regia L.), a perennial deciduous tree in the Juglans genus of the Juglans family, is one of my country's important ecological and economic forest species. Most walnut varieties currently cultivated in my country are bred through selection or hybridization within the Juglans regia species. Their cold tolerance is low, and walnut production in northern my country is often affected by frost and extreme low temperatures during winter. Hebei walnut (J. hopeiensis Hu), a natural hybrid of Juglans regia and Juglans mandshurica, has low edible value, but Juglans mandshurica is the most cold-resistant species in the Juglans genus. As a hybrid of Juglans regia and Juglans mandshurica, Hebei walnut contains many excellent genes, making it an excellent parent for breeding cold-resistant and disease-resistant walnuts. Cultivating new walnut varieties with strong cold tolerance and high-quality nuts through interspecific hybridization between Juglans regia and Hebei walnut is a key approach to addressing the need for cold protection for young walnut trees in northern my country and further expanding the walnut cultivation area.

[0003] The early species characteristics of the hybrid offspring between walnut and Hebei walnut are not obvious. Through traditional morphological methods, the authenticity of the hybrid offspring is identified from the variation of relatively stable traits such as leaves, flowers, and fruits after the hybrid seedlings are planted. The cycle is long, usually 5-8 years. Planting hybrid offspring requires a large area of ​​land resources and the breeding cost is high. Therefore, the early identification of the authenticity of the hybrid offspring between walnut and Hebei walnut is more important and urgent. Summary of the Invention

[0004] In view of this, the object of the present invention is to provide a method for identifying interspecific hybrid progeny of walnut and Hebei walnut.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0006] The present invention provides an application of specific primers in identifying interspecific hybrid progeny of walnut and Hebei walnut, wherein the specific primers include a combination of one or more of WJR003, WJR076, WJR078, WJR089, WJR150, WJR163, WJR191, WJR258, WJR279, WJR281, ZMZ3, ZMZ5, ZMZ6, ZMZ14, ZMZ27, ZMZ28, ZMZ30, ZMZ33, ZMZ39 and ZMZ46;

[0007] The nucleotide sequences of the specific primer pairs of WJR003, WJR076, WJR078, WJR089, WJR150, WJR163, WJR191, WJR258, WJR279, WJR281, ZMZ3, ZMZ5, ZMZ6, ZMZ14, ZMZ27, ZMZ28, ZMZ30, ZMZ33, ZMZ39 and ZMZ46 are shown in SEQ ID No. 1 to 40.

[0008] Another object of the present invention is to provide a method for identifying interspecific hybrid offspring between walnut and Hebei walnut, comprising the following steps: using the genomic DNA of the sample to be tested as a template, performing PCR amplification using the specific primers in the above application, and using the PCR amplification product to determine the authenticity of the hybrid offspring. When two alleles at the same SSR site inherit one allele from each parent at the same time, they are the hybrid offspring of the two parents.

[0009] Preferably, the genomic DNA is extracted from fresh leaves.

[0010] Preferably, the specific primers include WJR003 and WJR279, the nucleotide sequence of the primer pair of WJR003 is shown as SEQ ID No. 1-2, and the nucleotide sequence of the primer pair of WJR279 is shown as SEQ ID No. 17-18.

[0011] Preferably, the PCR amplification reaction system, based on 20 μL, includes: 10 μL of 2×Taq master mix, 1.0 μL of template DNA, 0.3 μL of F-primer, 0.3 μL of R-primer and the balance of ddH2O.

[0012] Preferably, the reaction procedure of the PCR amplification includes: pre-denaturation at 95°C for 5 min; denaturation at 95°C for 30 s, annealing at 55°C for 30 s, extension at 72°C for 30 s, denaturation at 95°C for 30 s, annealing at 52°C for 30 s, extension at 72°C for 30 s, for a total of 20 cycles; extension at 72°C for 10 min.

[0013] Preferably, after the PCR amplification, the PCR amplification product is subjected to capillary electrophoresis detection, and the obtained raw data is subjected to genotype analysis using the analysis software Genemarker 2.2.0. When AA×BB, AB×CD, AB×CC, or AA×BC type markers are present, it can be determined whether the progeny is a hybrid of the two parents.

[0014] Preferably, when WJR279 is used for PCR amplification, if the PCR amplification product simultaneously contains a group of bands including 218bp and 226bp, 218bp and 234bp, 222bp and 226bp, or 222bp and 234bp, it indicates that the sample to be tested is an interspecific hybrid offspring of walnut and Hebei walnut.

[0015] Compared with the prior art, the present invention provides an application of specific primers in identifying interspecific hybrid progenies of walnut and Hebei walnut, and a method for identifying interspecific hybrid progenies of walnut and Hebei walnut, which have the following beneficial effects:

[0016] The invention has the advantages of simple operation, good repeatability, high stability, and is not affected by the external environment. The invention effectively improves the discrimination of the authenticity of hybrid offspring between walnut varieties, improves the early selection efficiency of hybrid offspring, shortens the breeding cycle, and improves the breeding efficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 This is the amplification result of WJR279 on Liaoning No. 7;

[0018] Figure 2 This is the amplification result of WJR279 on 'Yihe No. 1';

[0019] Figure 3 The amplification results of WJR279 in some hybrid progenies are shown. DETAILED DESCRIPTION

[0020] In order to achieve the above technical objectives, the present invention provides the following technical solutions:

[0021] The present invention provides an application of specific primers in identifying interspecific hybrid progeny of walnut and Hebei walnut, wherein the specific primers include a combination of one or more of WJR003, WJR076, WJR078, WJR089, WJR150, WJR163, WJR191, WJR258, WJR279, WJR281, ZMZ3, ZMZ5, ZMZ6, ZMZ14, ZMZ27, ZMZ28, ZMZ30, ZMZ33, ZMZ39 and ZMZ46;

[0022] The nucleotide sequences of the specific primer pairs of WJR003, WJR076, WJR078, WJR089, WJR150, WJR163, WJR191, WJR258, WJR279, WJR281, ZMZ3, ZMZ5, ZMZ6, ZMZ14, ZMZ27, ZMZ28, ZMZ30, ZMZ33, ZMZ39 and ZMZ46 are shown in SEQ ID No. 1 to 40.

[0023] The SSR primers described in the present invention are all SSR primers, and their primer pair information is shown in Table 1. The SSR primers described in the present invention can be used to specifically identify whether the sample to be tested is an interspecific hybrid progeny of walnut and Hebei walnut.

[0024] Table 1 SSR primer information

[0025]

[0026]

[0027]

[0028]

[0029] The present invention also provides a method for identifying interspecific hybrid offspring between walnut and Hebei walnut, comprising the following steps: using the genomic DNA of the sample to be tested as a template, performing PCR amplification using the specific primers in the above application, and using the PCR amplification product to judge the authenticity of the hybrid offspring. When two alleles at the same SSR site inherit one allele from each parent at the same time, they are the hybrid offspring of the two parents.

[0030] The genomic DNA described herein is preferably extracted from fresh leaves. The present invention does not specifically limit the method for extracting the genomic DNA. A kit method is preferred. While the PlantZol kit method is used in the examples, this method is not intended to be the sole protection scope of the present invention. Other methods, such as the CTAB method, may also be used.

[0031] The present invention utilizes the primers shown in Table 1 for PCR amplification. The PCR amplification reaction system, in a volume of 20 μL, preferably comprises: 10 μL of 2× Taq master mix, 1.0 μL of template DNA, 0.3 μL of F-primer, 0.3 μL of R-primer, and the balance of ddH₂O. The PCR amplification reaction procedure of the present invention preferably comprises: pre-denaturation at 95°C for 5 min; denaturation at 95°C for 30 s, annealing at 55°C for 30 s, extension at 72°C for 30 s, 20 cycles of denaturation at 95°C for 30 s, annealing at 52°C for 30 s, and extension at 72°C for 30 s; and extension at 72°C for 10 min.

[0032] The present invention uses PCR amplification products to judge the authenticity of hybrid progeny, preferably including capillary electrophoresis. For example, when WJR279 is used for PCR amplification, when the PCR amplification product simultaneously contains a group of bands of 218bp and 226bp, 218bp and 234bp, 222bp and 226bp, or 222bp and 234bp, it indicates that the sample to be tested is an interspecific hybrid progeny of walnut and Hebei walnut. When only the maternal characteristic band or other paternal characteristic bands are present, it indicates that the sample to be tested is a self-pollinated progeny or a pseudo-hybrid progeny, and does not belong to the interspecific hybrid progeny of walnut and Hebei walnut.

[0033] The present invention can also use genotype to determine the authenticity of hybrid offspring. Specifically, after the PCR amplification, the PCR amplification product is subjected to capillary electrophoresis detection, and the raw data is subjected to genotyping analysis using the analysis software Genemarker2.2.0. When AA×BB, AB×CD, AB×CC, or AA×BC type markers are present, it can be determined whether the hybrid offspring is a hybrid of the two parents. However, AA×AB, AB×BB, AB×BC, and AC×BC type markers cannot fully identify whether the hybrid offspring is a hybrid because one identical allele locus exists between the parents, and further verification is required. The present invention does not specifically limit the capillary electrophoresis detection method.

[0034] The technical solutions provided by the present invention are described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0035] Example 1

[0036] 1. Experimental Materials

[0037] The experimental samples were collected from the Liaoning Provincial Economic Forestry Research Institute's Research and Demonstration Base in Songmudao Village, Paotai Subdistrict, Jinpu New District, Dalian City, Liaoning Province. Newly emerged leaves from the hybrid parents, 'Liaoning No. 7' (walnut) and 'Yihe No. 1' (Hebei walnut), were collected and brought back to the laboratory and stored at -20°C until further use. Five replicates were performed for each variety.

[0038] 2. Extraction of genomic DNA

[0039] Extract using the PlantZol kit. Take 100 mg of fresh leaves, grind thoroughly in liquid nitrogen, and place in a clean centrifuge tube. Add 400 μL of PlantZol and vortex to completely resuspend the sample. Add 7.5 μL of RNase A to the lysate and mix thoroughly. Incubate at 55°C for 15 min. Add an equal volume of phenol-chloroform, vortex to mix, and centrifuge at 12,000 × g for 5 min. Carefully transfer the upper aqueous phase to a clean centrifuge tube, add an equal volume of isopropanol, and invert to mix thoroughly. Centrifuge at 12,000 × g for 5 min and discard the supernatant. Add 500 μL of 70% ethanol, vortex for 5 seconds, and centrifuge at 12,000 × g for 5 min. Discard the supernatant. Centrifuge again for 1-2 min and aspirate any remaining liquid. Air dry the DNA pellet, add 50-200 μL of TE, and incubate at 65°C for 10 min-1 h to dissolve the DNA, gently flicking the tube several times to dissolve it.

[0040] 3. PCR amplification

[0041] The PCR reaction system (20 μL) consisted of 10 μL of 2× Taq master mix, 1.0 μL of template DNA, 0.3 μL of F-primer, 0.3 μL of R-primer, and 8.4 μL of ddH₂O. The forward primer was fluorescently labeled. The PCR protocol was as follows: 95°C initial denaturation for 5 min; 20 cycles of 95°C denaturation for 30 s, 55°C annealing for 30 s, and 72°C extension for 30 s; 95°C denaturation for 30 s, 52°C annealing for 30 s, and 72°C extension for 30 s; and 72°C extension for 10 min. After amplification, the reaction mixture was stored at 4°C until use. Primer information is shown in Table 1.

[0042] 4. Capillary electrophoresis detection

[0043] A mixture of 0.3 μL of PCR product, 0.5 μL of molecular weight internal standard, and 9.5 μL of deionized formamide was added to a PCR plate. The mixture was denatured at 95°C for 5 minutes, cooled at 4°C, and centrifuged. The DNA was then analyzed using 1× Buffer. The raw data was analyzed using Genemarker 2.2.0 software, and peak plots and Excel locus information tables were exported by locus.

[0044] 5. Effects of different primers on gene DNA amplification

[0045] All 20 primer pairs amplified samples from both Liaoning 7 and Yihe 1. The genotypes of the parents for all 20 primer pairs are shown in Table 2, where NN indicates the absence of a band. Markers with the AA×BB, AB×CD, AB×CC, and AA×BC patterns theoretically allow for direct identification of hybrid progeny. However, markers with the AA×AB, AB×BB, AB×BC, and AC×BC patterns cannot fully identify hybrid progeny due to the presence of an identical allele between the parents, requiring further verification.

[0046] Table 2 Genotypes of 20 primer pairs in parents

[0047]

[0048] Example 2

[0049] 1. Experimental Materials

[0050] Samples were collected from the Liaoning Provincial Economic Forestry Research Institute's Research Demonstration Base in Songmudao Village, Paotai Street, Jinpu New District, Dalian City, Liaoning Province. Sixty hybrid progenies of 'Liaoning 7' and 'Yihe 1' were collected. Two primer pairs, WJR003 and WJR279, were used to repeatedly verify the authenticity of the hybrid progenies. Newly sprouted leaves from the hybrid progenies were collected, brought back to the laboratory, and stored at -20°C for future use.

[0051] 2. Extraction of genomic DNA

[0052] Extract using the PlantZol kit. Take 100 mg of fresh leaves, grind thoroughly in liquid nitrogen, and place in a clean centrifuge tube. Add 400 μL of PlantZol and vortex to completely resuspend the sample. Add 7.5 μL of RNase A to the lysate and mix thoroughly. Incubate at 55°C for 15 min. Add an equal volume of phenol-chloroform, vortex to mix, and centrifuge at 12,000 × g for 5 min. Carefully transfer the upper aqueous phase to a clean centrifuge tube, add an equal volume of isopropanol, and invert to mix thoroughly. Centrifuge at 12,000 × g for 5 min and discard the supernatant. Add 500 μL of 70% ethanol, vortex for 5 seconds, and centrifuge at 12,000 × g for 5 min. Discard the supernatant. Centrifuge again for 1-2 min and aspirate any remaining liquid. Air dry the DNA pellet, add 50-200 μL of TE, and incubate at 65°C for 10 min-1 h to dissolve the DNA, gently flicking the tube several times to dissolve it.

[0053] 3. PCR amplification

[0054] PCR reaction system (20 μL): 10 μL 2× Taq master mix, 1.0 μL template DNA, 0.3 μL F-primer, 0.3 μL R-primer, 8.4 μL ddH2O. The forward primer was fluorescently labeled. The PCR reaction program was as follows: 95°C initial denaturation for 5 min; 95°C denaturation for 30 s, 55°C annealing for 30 s, 72°C extension for 30 s; 95°C denaturation for 30 s, 52°C annealing for 30 s, 72°C extension for 30 s, for a total of 20 cycles; 72°C extension for 10 min. After amplification, store in a refrigerator at 4°C until use.

[0055] 4. Capillary electrophoresis detection

[0056] A mixture of 0.3 μL of PCR product, 0.5 μL of molecular weight internal standard, and 9.5 μL of deionized formamide was added to a PCR plate. The mixture was denatured at 95°C for 5 minutes, cooled at 4°C, and centrifuged. The DNA was then analyzed using 1× Buffer. The raw data was analyzed using Genemarker 2.2.0 software, and peak plots and Excel locus information tables were exported by locus.

[0057] 5. Authenticity Identification of Hybrid Progeny

[0058] Primers WJR003 and WJR279 were used to identify 60 hybrid progenies of 'Liaoning 7' and 'Yihe 1'. The amplification results of WJR279 on 'Liaoning 7' are shown in the following figure. Figure 1 The amplification results of WJR279 on 'Yihe No. 1' are shown in Figure 2 As shown; the amplification results of WJR279 on some hybrid progenies are shown Figure 3 As shown, the results showed that 18 individual plants inherited one allele from each parent and were identified as true hybrid offspring, and 42 individual plants inherited one allele from the mother or two alleles, one inherited from the mother and the other not from the father, and were identified as false hybrid offspring.

[0059] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. The application of specific primers in identifying progeny of interspecific hybridization between walnut and Hebei walnut, characterized in that: The specific primers are WJR003 and WJR279; The nucleotide sequence of WJR003 is shown in SEQ ID No. 1-2; The nucleotide sequence of WJR279 is shown in SEQ ID No. 17-18; The walnut is 'Liaoning No. 7', and the Hebei walnut is 'Yihe No. 1'.

2. A method for identifying progeny of interspecific hybridization between walnut and Hebei walnut, characterized in that: The following steps are involved: The genomic DNA of the sample to be tested is used as a template, and PCR amplification is performed using the specific primers in the application described in claim 1. The authenticity of the hybrid offspring is determined by using the PCR amplification product. When two alleles of the same SSR locus inherit one allele from each parent, they are hybrid offspring of the two parents; the walnut is 'Liaoning No. 7', and the Hebei walnut is 'Yihe No. 1'.

3. The method according to claim 2, characterized in that The genomic DNA was extracted from fresh leaves.

4. The method according to claim 2, characterized in that The PCR amplification reaction system, calculated as 20 μL, includes: 10 μL of 2×Taq master mix, 1.0 μL of template DNA, 0.3 μL each of upstream and downstream primers, and the balance of ddH 2 O.

5. The method according to claim 2 or 4, characterized in that: The PCR amplification reaction procedure includes: pre-denaturation at 95°C for 5 minutes; denaturation at 95°C for 30 seconds, annealing at 55°C for 30 seconds, extension at 72°C for 30 seconds, denaturation at 95°C for 30 seconds, annealing at 52°C for 30 seconds, extension at 72°C for 30 seconds, for a total of 20 cycles; and extension at 72°C for 10 minutes.

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