A primer set and kit for identifying kiwifruit waterlogging tolerance resources and a method for identifying the same
By using specific primer sets 78F and 78R for PCR amplification and sequence alignment, the problems of speed and accuracy in identifying the flood tolerance of kiwifruit were solved, enabling rapid screening of flood-tolerant kiwifruit resources and accelerating the breeding process.
Patent Information
- Application Number
- CN202210812595.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2022-07-12
- Publication Date
- 2026-02-24
- Estimated Expiration
- 2042-07-12
AI Technical Summary
Existing technologies make it difficult to quickly and accurately identify the waterlogging tolerance of kiwifruit, leading to inconsistent results during the breeding process and affecting the progress of kiwifruit stress resistance breeding.
PCR amplification was performed using specific primer sets 78F and 78R. Combined with sequencing and sequence alignment, the waterlogging tolerance of kiwifruit was identified by detecting the 7bp sequence CAGATAT at positions 153–159.
It enables rapid and accurate identification of waterlogging tolerance. The results were verified by field pot flooding experiments, ensuring the reliability of the identification results and supporting the screening and breeding of early stress-resistant resources for kiwifruit.
Smart Images
Figure CN115806975B_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of germplasm resource identification technology, and in particular to a primer set and kit for identifying waterlogging-resistant kiwifruit resources, as well as the identification method thereof. Background Technology
[0002] Based on current research on waterlogging stress in plants, it has been clearly established that waterlogging tolerance in plants is a quantitative trait controlled by multiple gene loci. Therefore, it is difficult to develop molecular markers with high accuracy. Preliminary judgment is often made by the expression levels of related differentially expressed genes. However, there are not many differentially expressed gene sequences in resistant and susceptible materials, and it is also difficult to find regularity in the sequences in materials with different resistances. Therefore, it is difficult to develop them into effective molecular markers.
[0003] Kiwifruit plants have a fleshy, shallow root system, with the root system concentrated in the 0-40cm depth below the soil surface. Kiwifruit is extremely sensitive to waterlogging stress and is a fruit tree resource that is highly intolerant of waterlogging. Waterlogging can cause kiwifruit leaves to wilt and fall off, stems to dry out, and even the death of the entire plant. In major kiwifruit producing areas in southern my country, such as Sichuan, Chongqing, and Hunan, the high annual rainfall and heavy clay soils make orchards highly susceptible to waterlogging.
[0004] Currently, the identification of waterlogging-tolerant fruit tree germplasm resources mainly relies on pot flooding tests. It is important to ensure that the growth vigor of the resources used in the initial stage of flooding is basically the same, and the test takes a long time, generally 3 to 10 days. The operation is also relatively cumbersome. Generally, the germplasm resources to be identified need to be planted in containers such as plastic seedling pots of appropriate size. The seedling pots also need to have a relatively uniform cultivation substrate. After the roots are completely submerged for a period of time, the damage symptoms of the leaves and stems above ground are investigated and statistically analyzed in order to obtain relatively accurate identification results. However, in practice, the investigation and statistical analysis of above-ground symptoms is difficult to control. Aside from extreme phenotypes such as complete plant withering or death, other symptoms, such as the degree of yellowing or wilting of leaves, the number and size of withered parts (the proportion of withered area to the entire leaf, sometimes only a portion of a leaf is wilted or withered while the rest remains intact), and the degree of stem withering (upper part of the stem withered, lower part intact), are all subject to the subjective influence of the investigator. This leads to inconsistent identification results, affecting the accurate identification of resistant resources. Therefore, there is an urgent need to develop a rapid and accurate method for identifying flood-resistant resources at the molecular level for molecular marker-assisted breeding to cultivate new kiwifruit varieties with strong flood resistance. Summary of the Invention
[0005] The purpose of this invention is to provide a primer set and reagent kit for identifying waterlogging-tolerant kiwifruit resources, as well as the identification method thereof. The identification method of this invention is simple and easy to operate, and the identification results have been verified by pot waterlogging tolerance tests. The results are accurate and reliable, which can realize the rapid screening of waterlogging-tolerant kiwifruit germplasm resources and accelerate the process of stress-resistant molecular breeding of kiwifruit.
[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution:
[0007] This invention provides a primer set for identifying flood-resistant kiwifruit resources, including a forward primer 78F and a reverse primer 78R; the nucleotide sequence of the forward primer 78F is shown in SEQ ID NO:1, and the nucleotide sequence of the reverse primer 78R is shown in SEQ ID NO:2.
[0008] 78F (SEQ ID NO: 1): 5′-ATGTGCCGGCGGTTCAATCC-3′;
[0009] 78R (SEQ ID NO:2): 5'-TCAGGCCACCGTAGGGGTAA-3'.
[0010] The present invention also provides a kit for identifying flood-resistant kiwifruit resources, comprising the above-mentioned primer set and detection reagent.
[0011] Furthermore, the detection reagent includes PCR mix and ddH2O.
[0012] This invention also provides a method for identifying flood-tolerant kiwifruit resources using the above-mentioned primer set or kit, comprising the following steps:
[0013] (1) Extract genomic DNA from the kiwifruit to be tested;
[0014] (2) Using the genomic DNA extracted in step (1) as a template, perform PCR amplification using the primer set described in claim 1;
[0015] (3) Sequencing the amplification products from step (2) yields sequencing results;
[0016] (4) Compare the sequencing results of step (3) with the waterlogging-resistant resource sequence. If a 7bp CAGATAT sequence is present at position 153-159, the kiwifruit to be tested is a waterlogging-resistant resource; otherwise, it is a non-waterlogging-resistant resource.
[0017] Further, the method for extracting kiwifruit genomic DNA in step (1) is as follows: take 2-3 tender kiwifruit leaves, add liquid nitrogen to completely submerge the leaves, grind the leaves for 2-3 minutes, take 100-200 mg of the ground leaves, extract kiwifruit leaf genomic DNA using a plant genomic DNA extraction kit, and adjust the concentration of the DNA stock solution to 20-40 ng / μL.
[0018] Further, the PCR reaction system in step (2), in 20 μl, includes the following components: 2 μL of 100 nmol / L forward primer 78F, 2 μL of 100 nmol / L reverse primer 78R, 12 μL of PCR mix, 2 μL of DNA template, and 2 μL of ddH2O.
[0019] Furthermore, the amplification program for the PCR reaction in step (2) is as follows: 94℃ pre-denaturation for 90s; 94℃ denaturation for 20s, 58℃ annealing for 20s, 72℃ extension for 60s, denaturation, annealing and extension are performed for 35 cycles; final extension at 72℃ for 5min.
[0020] Furthermore, the specific method for comparing the sequencing results with the flood-resistant resource sequence in step (4) is as follows: the sequencing results are compared with the flood-resistant resource sequence using the "multiple sequence alignment" function of the biological software DNAMAN.
[0021] Compared with the prior art, the beneficial effects of the present invention are as follows:
[0022] This invention uses specific primer sets for amplification and sequence alignment of the amplification results with flood-resistant resource sequences to identify flood-resistant resources based on the differences in loci. The identification results have been verified by field pot flooding experiments, ensuring the accuracy and reliability of the marker identification results. This marker can be used for flood-resistant molecular breeding of kiwifruit, accelerating the early screening and breeding process of stress-resistant resources in kiwifruit. Attached Figure Description
[0023] Figure 1 The figures show the amplification results of specific primer pairs 78F and 78R in 18 kiwifruit resources; in the figure, M: DNA Marker, standard molecular weight, DL-2000; 1 to 18 represent kiwifruit resource numbers, namely KR5, KR6, KR7, ZMH, DZH, DAZ, ZM2, HJG, HYA, HWD, BOSX, M52, BLN, XUX, CHY, CHG, HR1 and HR2 respectively;
[0024] Figure 2 The sequence alignment results of amplification products of specific primer pairs 78F and 78R in 18 kiwifruit resources;
[0025] Figure 3Symptoms of the above-ground parts of 12 kiwifruit resources after 10 days of waterlogging in pots. Detailed Implementation
[0026] This invention provides a primer set for identifying flood-tolerant kiwifruit resources, including a forward primer 78F and a reverse primer 78R; the nucleotide sequence of the forward primer 78F is shown in SEQ ID NO:1, and the nucleotide sequence of the reverse primer 78R is shown in SEQ ID NO:2; specifically as follows:
[0027] 78F (SEQ ID NO: 1): 5′-ATGTGCCGGCGGTTCAATCC-3′;
[0028] 78R (SEQ ID NO:2): 5'-TCAGGCCACCGTAGGGGTAA-3'.
[0029] The present invention also provides a kit for identifying flood-resistant kiwifruit resources, comprising the above-mentioned primer set and detection reagent.
[0030] In this invention, the concentration of the forward primer 78F is 80–120 nmol / L, preferably 90–110 nmol / L, and more preferably 95–105 nmol / L; the concentration of the reverse primer 78R is 80–120 nmol / L, preferably 90–110 nmol / L, and more preferably 95–105 nmol / L.
[0031] In this invention, the detection reagent includes PCR mix and ddH2O.
[0032] In this invention, the PCR mix was purchased from Zhengzhou Pulehai Technology Co., Ltd.
[0033] This invention also provides a method for identifying flood-tolerant kiwifruit resources using the above-mentioned primer set or kit, comprising the following steps:
[0034] (1) Extract genomic DNA from the kiwifruit to be tested;
[0035] (2) Using the genomic DNA extracted in step (1) as a template, perform PCR amplification using the primer set described in claim 1;
[0036] (3) Sequencing the amplification products from step (2) yields sequencing results;
[0037] (4) Compare the sequencing results of step (3) with the waterlogging-resistant resource sequence. If a 7bp CAGATAT sequence is present at position 153-159, the kiwifruit to be tested is a waterlogging-resistant resource; otherwise, it is a non-waterlogging-resistant resource.
[0038] In this invention, the genomic DNA of the kiwifruit to be tested is first extracted. The method for extracting the kiwifruit genomic DNA is as follows: take 2-3 young kiwifruit leaves, add liquid nitrogen to completely submerge the leaves, grind the leaves for 2-3 minutes, and then grind the leaves for 3-5 minutes, preferably 3.5-4.5 minutes, and more preferably 4 minutes; take the ground leaves and extract the kiwifruit leaf genomic DNA using a plant genomic DNA extraction kit (purchased from Zhengzhou Pulehai Technology Co., Ltd.), and the extraction method refers to the instructions of the DNA extraction kit; the amount of ground leaves used is 100-200 mg, preferably 120-180 mg, and more preferably 140-160 mg.
[0039] In this invention, ddH2O is used to adjust the concentration of the extracted DNA mother liquor to 20-40 ng / μL, preferably 25-35 ng / μL, and more preferably 28-32 ng / μL.
[0040] After obtaining the genomic DNA of the kiwifruit to be tested, the present invention uses the extracted genomic DNA as a template and performs PCR amplification using forward primer 78F and reverse primer 78R to obtain the amplification product.
[0041] In this invention, the PCR reaction system, in 20 μl units, comprises the following components: 2 μL of 100 nmol / L forward primer 78F, 2 μL of 100 nmol / L reverse primer 78R, 12 μL of PCR mix, 2 μL of DNA template, and 2 μL of ddH2O.
[0042] In this invention, the preferred amplification program for the PCR reaction is: 94℃ pre-denaturation for 90s; 94℃ denaturation for 20s, 58℃ annealing for 20s, 72℃ extension for 60s, with 35 cycles of denaturation, annealing, and extension; and a final extension at 72℃ for 5min.
[0043] After obtaining the amplification product, the present invention performs sequencing on the amplification product to obtain sequencing results.
[0044] After obtaining the sequencing results, this invention compares the sequencing results with the sequences of flood-resistant resources. If a 7bp CAGATAT sequence is present at positions 153-159, the kiwifruit to be tested is a flood-resistant resource; if a 7bp CAGATAT sequence is not present at positions 153-159, the kiwifruit to be tested is a non-flood-resistant resource.
[0045] In this invention, the method for comparing the sequencing results with the flood-resistant resource sequence is as follows: the sequencing results are compared with the flood-resistant resource sequence using the "multiple sequence alignment" function of the biological software DNAMAN.
[0046] The technical solutions provided by the present invention will be 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.
[0047] Example 1
[0048] This embodiment provides a method for identifying flood-resistant kiwifruit resources, including the following steps:
[0049] (1) Select 3 tender kiwifruit leaves and place them in a ceramic mortar. Add an appropriate amount of liquid nitrogen to ensure that the leaves are completely submerged. After 3 minutes, the leaves are completely frozen. Use a ceramic grinding pestle to quickly grind the leaves for 5 minutes to grind them into powder. Use a stainless steel spoon to scrape 200mg of the powder and put it into 2ml of EP. Use a plant genomic DNA extraction kit to extract kiwifruit leaf genomic DNA. Dilute the DNA stock solution with ddH2O to 30ng / μL and store it in a -20℃ freezer for later use.
[0050] (2) Using the genomic DNA extracted in step (1) as a template, PCR amplification was performed using the following primer set:
[0051] 78F (SEQ ID NO: 1): 5′-ATGTGCCGGCGGTTCAATCC-3′;
[0052] 78R (SEQ ID NO: 2): 5′-TCAGGCCACCGTAGGGGTAA-3′;
[0053] The PCR reaction system, in 20 μl units, includes the following components: 2 μL of 100 nmol / L forward primer 78F, 2 μL of 100 nmol / L reverse primer 78R, 12 μL of PCR mix, 2 μL of DNA template, and 2 μL of ddH2O.
[0054] The PCR amplification program was as follows: 94℃ pre-denaturation for 90s; 94℃ denaturation for 20s, 58℃ annealing for 20s, 72℃ extension for 60s, with 35 cycles of denaturation, annealing, and extension; and a final extension at 72℃ for 5min.
[0055] (3) Perform agarose gel electrophoresis on the amplification products of step (2), and use an agarose gel DNA recovery kit to recover the amplification band at 1000 bp (DNA marker), and send it to a sequencing company to perform bidirectional sequencing using the Sanger method to obtain sequencing results.
[0056] (4) The sequencing results of step (3) are compared with the sequence of the flood-resistant resource using the "multiple sequence alignment" function of the biological software DNAMAN. If a 7bp sequence of CAGATAT is present at position 153-159, the kiwifruit to be tested is a flood-resistant resource; otherwise, it is a non-flood-resistant resource.
[0057] Example 2
[0058] This embodiment provides a method for identifying flood-resistant kiwifruit resources, including the following steps:
[0059] (1) Select two tender kiwifruit leaves and place them in a ceramic mortar. Add an appropriate amount of liquid nitrogen to ensure that the leaves are completely submerged. After 2 minutes, the leaves are completely frozen. Use a ceramic grinding pestle to quickly grind the leaves for 3 minutes to grind them into powder. Use a stainless steel spoon to scrape 100mg of the powder and put it into 2ml of EP. Use a plant genomic DNA extraction kit to extract kiwifruit leaf genomic DNA. Dilute the DNA stock solution with ddH2O to 35ng / μL and store it in a -20℃ freezer for later use.
[0060] (2) Using the genomic DNA extracted in step (1) as a template, PCR amplification was performed using the following primer set:
[0061] 78F (SEQ ID NO: 1): 5′-ATGTGCCGGCGGTTCAATCC-3′;
[0062] 78R (SEQ ID NO: 2): 5′-TCAGGCCACCGTAGGGGTAA-3′;
[0063] The PCR reaction system, in 20 μl units, includes the following components: 2 μL of 100 nmol / L forward primer 78F, 2 μL of 100 nmol / L reverse primer 78R, 12 μL of PCR mix, 2 μL of DNA template, and 2 μL of ddH2O.
[0064] The PCR amplification program was as follows: 94℃ pre-denaturation for 90s; 94℃ denaturation for 20s, 58℃ annealing for 20s, 72℃ extension for 60s, with 35 cycles of denaturation, annealing, and extension; and a final extension at 72℃ for 5min.
[0065] (3) Perform agarose gel electrophoresis on the amplification products of step (2), and use an agarose gel DNA recovery kit to recover the amplification band at 1000 bp (DNA marker), and send it to a sequencing company to perform bidirectional sequencing using the Sanger method to obtain sequencing results.
[0066] (4) The sequencing results of step (3) are compared with the sequence of the flood-resistant resource using the "multiple sequence alignment" function of the biological software DNAMAN. If a 7bp sequence of CAGATAT is present at position 153-159, the kiwifruit to be tested is a flood-resistant resource; otherwise, it is a non-flood-resistant resource.
[0067] Experimental Example
[0068] The identification method described in Example 1 was used to identify 18 kiwifruit germplasm resources preserved by the research group. The resource codes are: KR5, KR6, KR7, ZMH, DZH, DAZ, ZM2, HJG, HYA, HWD, BOSX, M52, BLN, XUX, CHY, CHG, HR1, and HR2. The PCR amplification results of the genomic DNA of the 18 kiwifruit germplasm resources are shown in the figure. Figure 1 The results after sequence alignment using biological software are shown below. Figure 2 ,Depend on Figure 2 It can be seen that KR5, KR6, KR7, ZMH, DZH and DAZ are flood-resistant resources, and each contains a 7bp sequence (CAGATAT) at positions 153-159. The other 12 resources are flood-intolerant resources and do not contain this sequence.
[0069] Twelve germplasm resources were selected, designated as DZH, DAZ, ZM2, HJG, HYA, HWD, BOSX, M52, BLN, XUX, CHY, and CHG. Since KR5, KR6, KR7, ZMH, and DZH belong to different strains of the same kiwifruit species, only DZH was selected for the flooding experiment. Similarly, HR1, HR2, and XUX belong to different strains of the same kiwifruit species, so only XUX was selected for the flooding experiment. The results of the flood-tolerant resources in Example 1 were verified using a potted flooding experiment. The results are shown in [Figure 1]. Figure 3 ,Depend on Figure 3 It was found that after 10 days of flooding, the above-ground parts of samples DZH and DAZ still showed new shoots and normal growth, without obvious symptoms of leaf wilting, yellowing, or stem drying, indicating they were flood-tolerant resources. The other eight samples showed obvious damage and were not flood-tolerant. The identification results from the pot experiment were consistent with the results obtained by the identification method of this invention, namely, DZH and DAZ are flood-tolerant resources, as the 78F and 78R primer amplification sequences both contain a 7bp sequence (CAGATAT) at positions 153-159; the other 10 resources are not flood-tolerant resources, as the 78F and 78R primer amplification sequences do not contain this 7bp sequence. This demonstrates the accuracy and reliability of the identification method of this invention in identifying flood-tolerant kiwifruit resources.
[0070] As can be seen from the above embodiments and experimental examples, the present invention provides a primer set and kit for identifying waterlogging-resistant kiwifruit resources and its identification method. The identification method of the present invention is simple and easy to operate, and the identification results have been verified by potted waterlogging resistance tests. The results are accurate and reliable, which can realize the rapid screening of waterlogging-resistant kiwifruit germplasm resources and accelerate the process of stress-resistant molecular breeding of kiwifruit.
[0071] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.
Claims
1. A method for identifying flood-resistant resources of kiwifruit, characterized in that, Includes the following steps: (1) Extract genomic DNA from the kiwifruit to be tested; (2) Using the genomic DNA extracted in step (1) as a template, PCR amplification was performed using the primer set for identifying the waterlogging-resistant resources of kiwifruit; The primer set includes a forward primer 78F and a reverse primer 78R; the nucleotide sequence of the forward primer 78F is shown in SEQ ID NO:1, and the nucleotide sequence of the reverse primer 78R is shown in SEQ ID NO:
2. (3) Sequencing the amplification products from step (2) yields sequencing results; (4) Compare the sequencing results of step (3) with the waterlogging-resistant resource sequence. If a 7bp CAGATAT sequence is present at position 153-159, the kiwifruit to be tested is a waterlogging-resistant resource; otherwise, it is a non-waterlogging-resistant resource.
2. The method for identifying flood-resistant kiwifruit resources according to claim 1, characterized in that, The method for extracting kiwifruit genomic DNA in step (1) is as follows: Take 2-3 young kiwifruit leaves, add liquid nitrogen to completely submerge the leaves, grind the leaves for 2-3 minutes, take 100-200 mg of the ground leaves, and extract kiwifruit leaf genomic DNA using a plant genomic DNA extraction kit, and adjust the concentration of the DNA stock solution to 20-40 ng / μL.
3. The method for identifying flood-resistant kiwifruit resources according to claim 1, characterized in that, The PCR reaction system in step (2), in 20 μl, includes the following components: 2 μL of 100 nmol / L forward primer 78F, 2 μL of 100 nmol / L reverse primer 78R, 12 μL of PCR mix, 2 μL of DNA template, and 2 μL of ddH2O.
4. The method for identifying flood-resistant kiwifruit resources according to claim 1, characterized in that, The amplification program for the PCR reaction in step (2) is as follows: 94℃ pre-denaturation for 90s; 94℃ denaturation for 20s, 58℃ annealing for 20s, 72℃ extension for 60s, denaturation, annealing and extension are performed for 35 cycles; final extension at 72℃ for 5min.
5. The method for identifying flood-resistant kiwifruit resources according to claim 1, characterized in that, The specific method for comparing the sequencing results with the flood-resistant resource sequence in step (4) is as follows: the sequencing results are compared with the flood-resistant resource sequence using the "multiple sequence alignment" function of the biological software DNAMAN.