CN112080576A - SSR molecular marker for distinguishing and identifying apocarya from pecan, Dabie pecan and Hunan pecan and application thereof - Google Patents
SSR molecular marker for distinguishing and identifying apocarya from pecan, Dabie pecan and Hunan pecan and application thereof Download PDFInfo
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Abstract
The invention relates to the technical field of plant molecular markers, in particular to an SSR molecular marker for distinguishing and identifying carya illinoensis, carya cathayensis, pecan and hickory in Hunan and application thereof. The SSR molecular marker provided by the invention comprises SSR-2 and one or more selected from SSR-1, SSR-3 and SSR-4, wherein the SSR-2 is obtained by amplifying primers SEQ ID NO.3-4, the SSR-1 is obtained by amplifying primers SEQ ID NO.1-2, the SSR-3 is obtained by amplifying primers SEQ ID NO.5-6, and the SSR-4 is obtained by amplifying primers SEQ ID NO. 7-8. The SSR molecular marker can well distinguish apocarya from hickory, Dabie hickory and Hunan hickory, has the advantages of simple operation, good repeatability, high accuracy and the like, and has higher application value.
Description
Technical Field
The invention relates to the technical field of plant molecular markers, in particular to an SSR molecular marker for distinguishing and identifying carya illinoensis, carya cathayensis, pecan and hickory in Hunan and application thereof.
Background
Carya illinoinensis is a plant of the genus Carya of the family Juglandaceae (Jugladaceae), and is one of the dried fruit tree species with high economic value. The pecan has high kernel yield and rich nutrition, is an ideal nutritional and health-care food, and is also an important oil plant.
Hickory nuts (c.cathayensis), pecan davidianus (c.dabieshanensis) and hickory nuts (c.hunanensis) and pecan hickory nuts (c.illinoinensis) belong to the same genus of juglandaceae, but have a large difference from the pecan nuts in terms of nutritional and economic values. Furthermore, the incomplete kernel of apocarya is difficult to distinguish from the kernels of pecans, pecans and hunan pecans in morphology, so that the kernels of pecans, pecans and hunan pecans are easily confused.
With the development of molecular biology, the germplasm resource analysis and variety identification of plants by using molecular means become effective methods, so that the development of molecular markers capable of distinguishing and identifying apocarya from pecans, pecans and Hunan apocarya is of great significance for the production and identification of apocarya products.
Disclosure of Invention
The invention aims to provide an SSR molecular marker for distinguishing and identifying carya illinoensis, carya cathayensis, Dabie carya cathayensis and Hunan carya cathayensis and application thereof.
In order to achieve the purpose, the invention searches for a Simple Sequence Repeat (SSR) of the apocarya which is specific and highly conserved in different apocarya varieties by analyzing the whole genome sequences of the apocarya and the pecans, the pecans and the hickory in Hunan, and develops the SSR molecular markers which can distinguish the apocarya from the pecans, the pecans and the hickory in Hunan. Through a large amount of sequence analysis and verification of samples of apocarya and pecan, Dabie pecan and Hunan pecan, SSR molecular markers capable of effectively distinguishing apocarya from pecan, Dabie pecan and Hunan pecan are finally obtained, and the purpose that the apocarya and pecan, the Dabie pecan and Hunan pecan can be accurately distinguished by utilizing 2-4 SSR molecular markers is achieved.
Based on the above findings, the present invention provides the following technical solutions:
the invention provides SSR molecular markers for distinguishing and identifying carya illinoensis, carya cathayensis, Carya dabieshanensis and Carya hunanensis, which comprise SSR-2 and one or more selected from SSR-1, SSR-3 and SSR-4; wherein, SSR-2 is obtained by amplifying a primer SEQ ID NO.3-4, SSR-1 is obtained by amplifying a primer SEQ ID NO.1-2, SSR-3 is obtained by amplifying a primer SEQ ID NO.5-6, and SSR-4 is obtained by amplifying a primer SEQ ID NO. 7-8.
The sequences of the primers are as follows:
in the 4 SSR molecular markers provided by the invention, the discrimination of apocarya and pecan, Dabie pecan and Hunan pecan can be realized by adopting SSR-2 and any one selected from SSR-1, SSR-3 and SSR-4.
In order to further improve the identification accuracy, SSR-2 can be used in combination with two or three of SSR-1, SSR-3 and SSR-4 for identification.
As a preferred embodiment of the present invention, the SSR molecular markers include SSR-2, SSR-1, SSR-3 and SSR-4.
Based on the SSR molecular marker, the invention further provides a primer combination for amplifying the SSR molecular marker, which comprises a primer pair shown in SEQ ID NO.3-4 and one or more pairs selected from the primer pairs shown in SEQ ID NO.1-2, SEQ ID NO.5-6 and SEQ ID NO. 7-8.
For the primer combination, when kernel genomic DNA of apocarya and pecan, pecan and Hunan pecan is taken as a template, the length of an amplification product of a primer pair shown by SEQ ID No.1-2 is 163-175 bp, the length of an amplification product of a primer shown by SEQ ID No.3-4 is 264bp, the length of an amplification product of a primer shown by SEQ ID No.5-6 is 254-261 bp, and the length of an amplification product of a primer shown by SEQ ID No.7-8 is 268-280 bp.
For the convenience of detection, the primers in the primer combination labeled by the SSR molecules can be labeled by fluorescence. Alternative fluorescent labels include, but are not limited to, ROX, TRAMA, FAM, and the like.
As an embodiment of the present invention, in the above primer combination, the primer shown in SEQ ID No.1 is labeled with ROX, the primer shown in SEQ ID No.2 is labeled with TRAMA, the primer shown in SEQ ID No.3 is labeled with FAM, the primer shown in SEQ ID No.4 is labeled with TRAMA, the primer shown in SEQ ID No.5 is labeled with TRAMA, the primer shown in SEQ ID No.6 is labeled with ROX, the primer shown in SEQ ID No.7 is labeled with ROX, and the primer shown in SEQ ID No.8 is labeled with FAM.
The invention also provides a kit comprising the primer combination. The kit can be used for distinguishing and identifying carya illinoensis, carya cathayensis, Dabie carya cathayensis and Hunan carya.
Preferably, the kit further comprises other components for PCR amplification including, but not limited to, PCR reaction buffer, dntps, DNA polymerase, negative controls, positive controls, and the like.
As a preferred embodiment of the present invention, the kit comprises primers shown in SEQ ID NO. 1-8.
The invention provides application of the SSR molecular marker or the primer combination or the kit in distinguishing and identifying carya illinoensis, carya cathayensis, Dabie carya cathayensis and Hunan carya.
The invention provides application of the SSR molecular marker or the primer combination or the kit in identifying pecan kernels from four pecan kernels including pecans, pecans and Hunan pecans.
The invention also provides application of the SSR molecular marker or the primer combination or the kit in quality detection of pecan kernel products.
The quality tests described above include, but are not limited to pecan, Dabie pecan, and Hunan pecan adulteration of pecan kernel products of apocarya.
The invention also provides application of the SSR molecular marker or the primer combination or the kit in apocarya genetic breeding or germplasm resource identification.
The invention provides a method for distinguishing and identifying apocarya and pecan, Dabie pecan and Hunan pecan, which comprises the following steps: and (3) taking kernel genome DNA of the walnut to be detected as a template, carrying out PCR amplification by using the primer combination or the kit containing the primer combination, and judging whether the walnut to be detected is the carya illinoensis or not according to the type of a PCR amplification product strip.
The reaction procedure of the PCR amplification described above is: 95 ℃ for 2-5 min; 95 ℃, 1-2 min, 58 ℃, 20-40 s, 72 ℃, 20-40 s, 35 cycles; extending for 3-10 min at 72 ℃.
The preferred reaction procedure for PCR amplification is: pre-denaturation at 95 ℃ for 3 min; denaturation at 95 ℃ for 2min, annealing at 58 ℃ for 30s, extension at 72 ℃ for 30s, and 35 cycles; extension at 72 ℃ for 5 min.
The 15 μ L reaction system for PCR amplification described above is: 7.5. mu.L of 2 XPCR reaction buffer, 1. mu.L of forward primer (concentration: 10 pmol/. mu.L), 1. mu.L of reverse primer (concentration: 10 pmol/. mu.L), 1. mu.L of DNA template (concentration: 50 ng/. mu.L), ddH2O 4.5μL。
The detection of the PCR amplification product band type can be carried out by capillary electrophoresis.
The method for judging whether the walnut to be detected is the apocarya comprises the following steps: if the primer pair shown in SEQ ID NO.3-4 can amplify a 264bp strip, the primer pair shown in SEQ ID NO.1-2, the primer pair shown in SEQ ID NO.5-6 and the primer pair shown in SEQ ID NO.7-8 can respectively amplify 163-175 bp, 254-261 bp and 268-280 bp strips, the walnut to be detected is the carya illinoensis; if the primer pair shown in SEQ ID NO.3-4 can amplify a 264bp strip, and the primer pair shown in SEQ ID NO.1-2, the primer pair shown in SEQ ID NO.5-6 and the primer pair shown in SEQ ID NO.7-8 do not obtain a target amplification strip, the walnut to be detected is a hickory nut, a Dabie hickory nut or a Hunan hickory nut.
Specifically, the method for distinguishing and identifying the carya illinoensis, the pecans, the Dabie pecans and the Hunan pecans provided by the invention comprises the following steps:
(1) extracting genome DNA of a kernel sample to be detected;
(2) respectively carrying out PCR amplification on the genomic DNA extracted in the step (1) by using a primer pair shown in SEQ ID NO.3-4, a primer pair shown in SEQ ID NO.1-2, a primer pair shown in SEQ ID NO.5-6 and a primer pair shown in SEQ ID NO. 7-8;
(3) detecting the PCR amplification product of the step (2) by using agarose gel electrophoresis;
(4) detecting the size of the PCR amplification product band in the step (2) by using a capillary electrophoresis method;
(5) and judging whether the sample to be detected is the pecan kernel or not according to the size of the band of the amplified product.
The invention has the beneficial effects that:
the invention develops 4 SSR molecular markers capable of distinguishing and identifying apocarya and pecan, Dabie pecan and Hunan pecan for the first time, wherein 1 SSR molecular marker can be amplified in the apocarya and pecan, Dabie pecan and Hunan pecan, and the other 3 SSR molecular markers are apocarya specific molecular markers, so that the apocarya can be well distinguished from the pecan, the Dabie pecan and Hunan pecan. The apocarya and pecan, the Dabie pecan and the Hunan pecan materials for developing and verifying the validity of the SSR molecular marker cover most of the existing apocarya and pecan, Dabie pecan and Hunan pecan varieties, so that the SSR molecular marker has reliability and repeatability.
The SSR molecular marker provided by the invention has the advantages of simple operation, good repeatability and high accuracy in distinguishing and identifying the apocarya from the pecan, the Dabie pecan and the Hunan pecan, and effectively solves the problem that the seeds of the apocarya and the pecan, the Dabie pecan and the Hunan pecan are difficult to distinguish by utilizing the identification of forms and the like.
Drawings
FIG. 1 is an electrophoresis chart of kernel genomic DNA extracted in example 2 of the present invention, wherein lanes 1-6 are Carya illinoensis kernel samples, lanes 7-11 are Carya cathayensis kernel samples, lanes 12-16 are Carya dabieshanensis kernel samples, and lanes 17-21 are Carya hunanensis kernel samples.
FIG. 2 is an electrophoresis chart of PCR amplification products of primer sets P3-1 and P3-2 on a part of Carya illinoensis, Carya cathayensis, Carya dabieshanensis and Carya hunanensis samples in example 2 of the present invention, wherein M is DNAmarker, Lane 1 is Carya illinoensis kernel sample, Lane 2-3 is Carya illinoensis kernel sample, Lane 4-5 is Carya dabieshanensis kernel sample, and Lane 6-7 is Carya hunanensis kernel sample.
Fig. 3A and 3B are capillary electrophoresis detection diagrams of PCR amplification products of a part of apocarya main cultivars (boni and mahhan), improved species of apocarya (yayou XK89), and apocarya and hunan apocarya samples by using primer sets P1-1 and P1-2 in example 2 of the present invention, wherein the apocarya cultivars boni and mahhan can detect target products (indicated by arrows), and the apocarya, hunan apocarya, and alopecan cannot detect the target products.
Detailed Description
The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention. Example 1 development of SSR molecular markers for discriminating and identifying Carya illinoensis from Carya cathayensis, Carya dabieshanensis and Carya hunanensis
The basic process for the development of the SSR molecular markers for distinguishing and identifying the apocarya and the pecans, the pecans and the hickories in Hunan province of the invention is as follows:
(1) analyzing the whole genome sequence of apocarya, pecan, Dabie pecan and Hunan pecan, searching the SSR molecular marker specific to the apocarya, designing and synthesizing the primer for amplifying the SSR molecular marker according to the whole genome sequence of the apocarya.
(2) The complete mature kernels of different varieties of apocarya (10 improved varieties), carya (2 improved varieties and 8 excellent clones), Dabie hickory (5 seedling individuals in different regions) and Hunan hickory (5 seedling individuals in different regions) are collected in the main production area of China.
(3) Extracting kernel genome DNA of different materials of the four pecan plants by using a kit method, detecting the quality of the extracted DNA by using 0.8-1.5% agarose gel electrophoresis, and detecting the concentration of the DNA by using a nucleic acid determinator.
(4) And (3) carrying out PCR amplification on genome DNA of different apocarya, pecan, Dabie pecan and Hunan pecan materials in the step (3) by using primers of SSR molecular markers designed in the step (1), and detecting PCR products by using capillary electrophoresis. Screening a large number of SSR molecular markers and combinations thereof to finally obtain 4 SSR molecular markers which are respectively SSR-1, SSR-2, SSR-3 and SSR-4, wherein the SSR molecular markers are obtained by amplifying primers P1-1, P1-2(SEQ ID NO.1-2), P2-1, P2-2(SEQ ID NO.3-4), P3-1, P3-2(SEQ ID NO.5-6) and P4-1 and P4-2(SEQ ID NO.7-8) in sequence.
For different apocarya, pecan, Dabie pecan and Hunan pecan materials, the primers shown in SEQ ID NO.3-4 can obtain amplification products in the kernels of the apocarya, pecan, Dabie pecan and Hunan pecan, the length of the amplification products is 264bp, the primers shown in SEQ ID NO.1-2, SEQ ID NO.5-6 and SEQ ID NO.7-8 are specific SSR molecular markers of the apocarya, and the amplification products can only be obtained in the apocarya, wherein the length of the amplification products of the primers shown in SEQ ID NO.1-2 is 163-175 bp, the length of the amplification products of the primers shown in SEQ ID NO.5-6 is 254-261 bp, and the length of the amplification products of the primers shown in SEQ ID NO.7-8 is 268-280 bp.
Synthesizing the above primer marked by fluorescence, specifically, the primer shown in SEQ ID NO.1 is marked by ROX, the primer shown in SEQ ID NO.2 is marked by TRAMA, the primer shown in SEQ ID NO.3 is marked by FAM, the primer shown in SEQ ID NO.4 is marked by TRAMA, the primer shown in SEQ ID NO.5 is marked by TRAMA, the primer shown in SEQ ID NO.6 is marked by ROX, the primer shown in SEQ ID NO.7 is marked by ROX, and the primer shown in SEQ ID NO.8 is marked by FAM.
Example 2 application of SSR molecular markers for distinguishing and identifying apocarya from pecans, pecans and hickories in Hunan
Mature kernels of full-thin-shell hickories, large-mount hickories and Hunan hickories are collected in main production areas of Chinese thin-shell hickories, large-mount hickories and Hunan hickories, wherein 10 samples of each thin-shell hickories and 10 samples of each hickories and 5 samples of each large-mount hickories and Hunan hickories are taken as samples to be detected.
(I) amplification of SSR molecular marker target fragment
1. Extraction and detection of kernel genomic DNA:
extracting kernel genome DNA by using a TSINGKE plant DNA extraction kit (universal type), and specifically comprising the following steps:
(1) placing the adsorption column in a collection tube, adding 250 μ l Buffer BL, and centrifuging at 12000rpm for 1min to activate the silica gel membrane;
(2) taking dry kernel tissue (not more than 20mg) of a sample to be detected, adding liquid nitrogen, and fully grinding. Grinding, placing in 1.5ml centrifuge tube, adding 400 μ l Buffer gP1, vortex oscillating for 1min, and water bath at 65 deg.C for 10-30min, taking out, reversing, and mixing to thoroughly crack;
(3) adding 150 μ l Buffer gP2, vortex and shake for 1min, ice bath for 5 min;
(4) centrifuging at 12000rpm for 5min, and transferring the supernatant into a new centrifuge tube;
(5) adding absolute ethanol with the same volume as the supernatant, immediately and fully oscillating and uniformly mixing, transferring all the liquid into an adsorption column, centrifuging at 12000rpm for 30s, and discarding the waste liquid;
(6) adding 500 μ l Buffer Pw (anhydrous ethanol is added before use) into the adsorption column, centrifuging at 12000rpm for 30s, and discarding the waste liquid;
(7) adding 500 μ l of Wash Buffer (absolute ethyl alcohol is added before use) into the adsorption column, centrifuging at 12000rpm for 30s, and discarding the waste liquid;
(8) repeating the operation step 7;
(9) putting the adsorption column back into the collecting tube, centrifuging at 12,000rpm for 2min, opening the cover, and air drying for 1 min;
(10) taking out the adsorption column, putting into a clean centrifuge tube, adding 50-100 μ l TE Buffer (preheating TE Buffer at 65 ℃) at the center of the adsorption membrane, standing for 2min at 20-25 ℃, and centrifuging for 2min at 12,000 rpm.
(11) The quality of the genomic DNA was checked by electrophoresis on a 1% agarose gel and the concentration of the genomic DNA was checked by UV spectrophotometer (FIG. 1).
2. PCR amplification of SSR fragments
Taking the extracted genome DNA of different sample kernels as templates, respectively using a primer group 1(P1-1 and P1-2), a primer group 2(P2-1 and P2-2), a primer group 3(P3-1 and P3-2) and a primer group 4(P4-1 and P4-2) to perform PCR amplification on different samples, wherein the PCR reaction system is as follows:
the PCR reaction conditions were as follows:
(II) detection of target fragment of SSR molecular marker
1. Agarose gel electrophoresis detection of PCR products
(1) A1% agarose gel was prepared, and 2. mu.L of the amplification product obtained in the above (I) was pipetted and mixed with 0.5. mu.L bromophenol blue, and applied thereto at a voltage of 300V for 12 minutes.
(2) The electrophoresis result was observed under an ultraviolet lamp and photographed (fig. 2).
2. Capillary electrophoresis detection strip size
(1) The internal standards of ABI HiDi Formamide and ABI GeneScan 500LIZ Size Standard were scaled to 130: 1, mixing to prepare a mixed solution.
(2) Mix was dispensed from a domestic 96-well plate and 10. mu.l of the mixture was added to each well.
(3) 0.5. mu.l of PCR sample was added to a 96-well plate and centrifuged at 4000 rpm.
(4) The mixed plate was pre-denatured by heating at 95 ℃ for 5 minutes using a metal bath heater, and immediately taken out and placed at-20 ℃.
(5) Taking out after cooling, centrifuging at 4000rpm, thawing, and mixing uniformly.
(6) Loading to 3730 sequencer for capillary electrophoresis.
(7) The SSR molecular marker amplification product site information was analyzed using the software Gene mapper 4.1 (FIGS. 3A and 3B).
The detection results of the band sizes of the amplification products obtained from four different pecan kernel samples are shown in table 1.
TABLE 1 band size obtained by four pairs of primers amplifying four different pecan kernels
"in table 1 represents that no fragments were amplified.
(III) judging whether the sample to be detected is the pecan kernel or not according to the SSR molecular marker typing result
1. Interpretation of SSR target bands
(1) Primer sets P1-1 and P1-2
The size of a target band obtained by the primer groups P1-1 and P1-2 in the genomic DNA of the carya illinoensis kernel is 163 bp-175 bp, and the target band cannot be amplified in the genomic DNA of the carya thunbergii, the carya hunanensis and the carya macrolepis (Table 1, and figures 3A and 3B).
(2) Primer sets P2-1 and P2-2
The primer groups P21-1 and P21-2 can amplify four kinds of hickory kernel genome DNA to obtain 264bp target bands (Table 1).
(3) Primer sets P3-1 and P3-2
The size of the target band obtained by the primer groups P3-1 and P3-2 in the genomic DNA of the apocarya illinoensis kernel is 254 bp-261 bp, and the target band cannot be amplified in the genomic DNA of the apocarya thunbergii, the apocarya hunanensis and the apocarya macrolepigone (Table 1).
(4) Primer sets P4-1 and P4-2
The size of the target band obtained by the primer groups P4-1 and P4-2 in the genomic DNA of the apocarya illinoensis kernel is 270bp to 280bp, and the target band cannot be amplified in the genomic DNA of the apocarya thunbergii, the apocarya hunanensis and the apocarya macrolepigone (Table 1).
2. Determination of whether sample is pecan kernel
(1) Judging to be the pecan kernel
If the primer groups P1-1 and P1-2, the primer groups P2-1 and P2-2, the primer groups P3-1 and P3-2 and the primer groups P4-1 and P4-2 can be amplified in the genomic DNA of the sample to be detected to obtain a target strip, the sample is judged to be the pecan kernel (Table 2).
(2) Judging as non-thin shell hickory kernel
If the primer groups P2-1 and P2-2 can amplify to obtain a target band in the genomic DNA of the sample to be detected, and the primer groups P1-1 and P1-2, the primer groups P2-1 and P2-2 and the primer groups P3-1 and P3-2 cannot amplify to obtain the target band, judging that the sample is not pecan kernels (Table 2).
TABLE 2 determination criteria for whether the kernel sample to be tested is a pecan kernel
Comparative example 1
The method comprises the following steps of distinguishing and identifying the apocarya sample from hickory, Hunan hickory and Dabie hickory by using the combination of four other SSR molecular markers (amplification primers are shown as SEQ ID NO.9-16) different from SSR-1, SSR-2, SSR-3 and SSR-4, and the specific method and results are as follows:
PCR amplification was performed on Carya illinoensis samples as well as Carya cathayensis, Hunan Carya cathayensis and Dabie Carya cathayensis samples using primer sets P5-1(GTTGCAAGCATAACTTGTAACCA) and P5-2(TCAAACCAAACCAACAAGCA), primer sets P6-1(TGTGTCGTGCAACGTTTGT) and P6-2(TCTTCGTAGAATGACGCTTCC), primer sets P7-1(TGCCGTCATAGGAAGAAAGG) and P7-2(AAAGCCATTTGGCACGTTAG), and primer sets P8-1(GCGCCGTTATGGATTTAAGA) and P8-2 (GCATCTTCTTACCGAGCGAG).
The results are shown in table 3, and the results show that effective discrimination of the apocarya sample from carya, carya hunanensis and pecan cannot be realized by adopting the SSR molecular marker combination.
TABLE 3 amplification results of 4 additional primer pairs on different hickory nut samples
"in table 3 represents that no fragments were amplified.
Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.
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Claims (10)
1. The SSR molecular marker for distinguishing and identifying the carya illinoensis from the carya cathayensis, the pecan walnuts and the hickory nuts in Hunan province is characterized by comprising SSR-2 and one or more selected from SSR-1, SSR-3 and SSR-4, wherein the SSR-2 is obtained by amplifying a primer SEQ ID NO.3-4, the SSR-1 is obtained by amplifying a primer SEQ ID NO.1-2, the SSR-3 is obtained by amplifying a primer SEQ ID NO.5-6, and the SSR-4 is obtained by amplifying a primer SEQ ID NO. 7-8.
2. SSR molecular marker according to claim 1, characterized in that it comprises SSR-2, SSR-1, SSR-3 and SSR-4.
3. A primer combination for amplifying SSR molecular markers of claim 1 or 2 comprising a primer pair shown in SEQ ID nos. 3-4 and one or more pairs selected from the group consisting of the primer pairs shown in SEQ ID nos. 1-2, 5-6 and 7-8.
4. A kit comprising the primer combination of claim 3.
5. Use of an SSR molecular marker according to claim 1 or 2 or a primer combination according to claim 3 or a kit according to claim 4 for distinguishing apocarya from pecan, pecan and hickory in Hunan.
6. Use of an SSR molecular marker according to claim 1 or 2 or a primer combination according to claim 3 or a kit according to claim 4 in the quality detection of pecan kernel products.
7. Use of an SSR molecular marker according to claim 1 or 2 or a primer combination according to claim 3 or a kit according to claim 4 in apocarya genetic breeding or in germplasm resource identification.
8. A method for distinguishing and identifying apocarya from pecans, Dabie pecans and Hunan pecans is characterized in that the kernel genomic DNA of a walnut to be detected is used as a template, the primer combination of claim 3 or the kit of claim 4 is used for PCR amplification, and whether the walnut to be detected is the apocarya is judged according to the type of a PCR amplification product strip.
9. The method of claim 8, wherein the reaction procedure of the PCR amplification is: 95 ℃ for 2-5 min; 95 ℃, 1-2 min, 58 ℃, 20-40 s, 72 ℃, 20-40 s, 35 cycles; extending for 3-10 min at 72 ℃.
10. The method as claimed in claim 8 or 9, wherein the method for judging whether the walnut to be detected is carya illinoensis comprises the following steps: if the primer pair shown in SEQ ID NO.3-4 can amplify a 264bp strip, the primer pair shown in SEQ ID NO.1-2, the primer pair shown in SEQ ID NO.5-6 and the primer pair shown in SEQ ID NO.7-8 can respectively amplify 163-175 bp, 254-261 bp and 268-280 bp strips, the walnut to be detected is the carya illinoensis; if the primer pair shown in SEQ ID NO.3-4 can amplify a 264bp strip, the primer pair shown in SEQ ID NO.1-2, the primer pair shown in SEQ ID NO.5-6 and the primer pair shown in SEQ ID NO.7-8 are not amplified to obtain a target strip, the walnut to be detected is a hickory nut, a Dabie hickory nut or a Hunan hickory nut.
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| CN114182033A (en) * | 2021-11-04 | 2022-03-15 | 中国林业科学研究院亚热带林业研究所 | SSR molecular markers of carya illinoensis Mahan, Pawnee and Greenriver and application thereof |
| CN114182034A (en) * | 2021-11-04 | 2022-03-15 | 中国林业科学研究院亚热带林业研究所 | SSR molecular marker of apocarya variety McMillian and application thereof |
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