SSR molecular markers, molecular IDs and their applications for identifying cultivars of Carya serrata

Abstract

The present invention relates to the technical field of molecular identification of plant varieties, and specifically to SSR molecular markers, molecular identification cards, and applications thereof for identifying thin-shelled pecan varieties. The SSR molecular markers for identifying thin-shelled pecan varieties provided by the present invention can achieve the identification of 36 major thin-shelled pecan varieties, have high accuracy and stability of discrimination results, are simple to operate, and require a short time for identification, which is convenient for promotion and application in practice. The present invention also establishes a variety-specific molecular fingerprint and its molecular identification card digital encoding, barcode, and QR code, which achieves effective differentiation of 36 major thin-shelled pecan varieties, providing an effective method for thin-shelled pecan variety identification, seedling purity detection, and variety tracing.

Description

Technical Field

[0001] The present invention relates to the technical field of molecular identification of plant varieties, and in particular to SSR molecular markers and molecular identification cards for identifying slender-shell walnut varieties and applications thereof. Background Art

[0002] Carya illinoinensis (Wangenh.) K. Koch, native to the United States and Mexico, belongs to the genus Carya in the Juglandaceae family. Its genome is approximately 690 Mb, with a chromosome number of 2n = 32. Its kernels contain over 70% oil, rich in functional ingredients such as unsaturated fatty acids, polyphenols, flavonoids, vitamins, and sterols. Oleic acid accounts for approximately 90% of the total fatty acid content, making it a high-quality dried fruit and a source of high-quality woody edible oil. Carya illinoinensis combines multiple uses, including fruit, oil, timber, and landscaping, and has promising development prospects. Its cultivation has been steadily increasing in recent years.

[0003] There are over a thousand cultivars of thin-shelled pecans, but only a few dozen are used on a large scale in production, and even fewer have good adaptability and high yields. However, during the introduction and breeding process of thin-shelled pecans, phenomena such as "same name, different species, same species, different names" have occurred. Thin-shelled pecan trees are monoecious, with male and female flowers blooming at different times, necessitating the use of pollinating trees to ensure pollination. Furthermore, different cultivars have varying regional adaptability. If the wrong primary cultivar is used, or the pollinating cultivar is incompatible with the primary cultivar, significant losses can occur to planting companies. The current genetic base of the primary cultivar and its backbone parent is narrow, resulting in a high degree of trait similarity between cultivars bred on this basis. Plant specificity, consistency, and stability (DUS) testing is the primary method for identifying new varieties prescribed by the International Union for the Protection of New Varieties of Plants (UPPO). However, phenotypic characteristics are easily affected by the environment and cultivation practices, resulting in unstable performance. Furthermore, the limited number of phenotypes exhibited by seedlings makes it very difficult to distinguish between similar cultivars. High-quality varieties are the foundation for ensuring high-quality and high-yield economic forests. With the rapid development of the walnut industry, the demand for high-quality seedlings has increased rapidly, and the authenticity of cultivars has become a pressing issue. Therefore, it is urgent to establish a cultivar identification method for walnuts that is highly accurate, simple to operate, and provides stable results.

[0004] Molecular markers can directly detect differences at the DNA level, independent of phenotypic traits. They are highly stable and reproducible, and have been widely used in recent years for molecular fingerprinting, molecular identification, and cultivar identification in plants. Molecular marker technology has been applied to Carya serrata. AFLP and SSR markers have been used to study genetic differences and genetic diversity; RAPD has been used to establish fingerprints for 43 cultivars; RAPD and AFLP have been used to construct genetic linkage maps; and SNP markers have been used to conduct genome-wide association studies related to flowering type. Currently, RAPD, ISSR, SSR, and SCAR techniques have been applied to study genetic diversity and cultivar identification in Carya serrata. However, current cultivar identification techniques primarily rely on dominant markers, with a limited number of available co-dominant markers or require complex techniques and expensive equipment.

[0005] Simple sequence repeats (SSRs), also known as microsatellites, are tandemly repeated sequences of 1 to 6 nucleotides. They are widely distributed throughout the genome and possess advantages such as codominance, high reproducibility, high polymorphism, and ease of detection. Compared to SNP markers, individual SSR markers offer high polymorphism information and require no expensive detection equipment, making them the preferred marker for constructing DNA fingerprints. They are particularly advantageous for distinguishing similar varieties and have been widely used in the identification of various plant varieties and germplasm resources, enabling seedling purity determination and variety traceability. For example, standard systems for variety identification or seedling purity assessment based on SSR molecular fingerprints have been established for food crops such as corn (NY / T 1432-2014), rice (NY / T 1433-2014), and soybeans (NY / T 1788-2009), as well as for economic forests such as apricots (LY / T 2745-2016) and jujubes (LY / T 2426-2015), providing a reference for research on Carya sylvestris. In recent years, high-quality genomic data of Carya illinoinensis has been released, laying the foundation for genome-wide screening of SSR loci and large-scale development of SSR markers. A literature report on 9145 pairs of high-quality SSR primers based on genome-wide screening has provided candidate loci for cultivar identification of Carya illinoinensis (Zhang C.C., Yao XH, Ren HD, Chang J., Wu J., Shao WZ, Fang Q. Characterization and Development of Genomic SSRs in Pecan (Carya illinoinensis). Forests. 2020, 11(1), 61.). Summary of the Invention

[0006] The present invention aims to provide SSR molecular markers, molecular identification cards and applications thereof for identifying thin-shelled walnut varieties.

[0007] The present invention screened and synthesized 80 primer pairs from a previously constructed SSR candidate primer library for pecans. PCR amplification of genomic DNA from eight pecan varieties was performed using these primers. Polyacrylamide gel electrophoresis was used to detect the primers, initially screening for primers with clear target bands and stable detection results. Genotyping was further performed in 36 major pecan varieties that are widely used, including 'Pawnee', 'Mahan', 'Stuart', 'Major', 'Mcmillan', and 'Mohawk'. PCR amplification products were detected using 8% polyacrylamide gel electrophoresis, and band sizes were manually counted. Popgene software was used to calculate parameters such as the number of alleles, observed heterozygosity, genetic distance, genetic similarity, and polymorphism information content (PIC) for each primer pair. A cluster tree of all tested varieties was constructed using the Unified Group Price Measure (UPGMA) method based on Nei's genetic distance. All SSR markers were sorted in descending order according to PIC values. Starting from the primer with the highest PIC value, the number of primers was increased sequentially until each variety could be distinguished. This set of primers was determined as the core primer set, thus obtaining a core marker combination containing 5 SSR markers and its primer combination. After screening, 23 pairs of primers were selected from 80 pairs of primers that amplified clear and stable bands in 8 varieties. These primers were distributed on 13 chromosomes, and a total of 70 alleles were detected in 36 varieties. Among them, 18 pairs of primers showed genetic polymorphism, with 2 to 8 alleles detected and PIC values ​​ranging from 0.03 to 0.72. Cluster analysis found that the genetic distance between different biological replicates of the same variety ranged from 0 to 0.0127, and the genetic distance between different varieties ranged from 0.026 to 0.6359. The samples were mainly clustered according to genetic background. A core primer set containing 5 markers, Ciz91, Ciz85, Ciz81, Ciz140 and Ciz107, was screened, with PIC values ​​ranging from 0.52 to 0.72, which could effectively distinguish all 36 tested varieties. On this basis, 36 variety-specific molecular fingerprint maps were constructed using the core primer set, and the molecular fingerprint maps were digitized and associated with the germplasm information to construct molecular ID barcodes and QR codes.

[0008] Specifically, the present invention provides the following technical solutions:

[0009] In a first aspect, the present invention provides SSR molecular markers for identifying cultivars of thin-shelled walnuts, wherein the SSR molecular markers include any one or more of the following SSR molecular markers (1) to (5):

[0010] (1) amplified by the primer pair shown in SEQ ID NO. 1-2;

[0011] (2) amplified by a primer pair with a sequence as shown in SEQ ID NO. 3-4;

[0012] (3) amplified by the primer pair whose sequence is shown in SEQ ID NO. 5-6;

[0013] (4) amplified by the primer pair with the sequence shown in SEQ ID NO. 7-8;

[0014] (5) Amplified by the primer pair with the sequence shown in SEQ ID NO.9-10.

[0015] In some embodiments of the present invention, the SSR molecular markers used to identify thin-shelled pecan varieties include: an SSR molecular marker amplified by the primer pair shown in SEQ ID NOs. 3-4 and an SSR molecular marker amplified by the primer pair shown in SEQ ID NOs. 5-6. This molecular marker combination can be used to identify the following 22 thin-shelled pecan varieties: 'Oconee', 'Kiowa', 'Shawnee', 'Waco', 'Hopi', 'Forkert', 'Maramec', 'Mahan', 'Navaho', 'Desirable', 'Osage', 'Chetopa', 'Elliott', 'Major', 'Colby', 'Mcmillan', 'Graking', 'Creek', 'Jayhawk', 'Choctaw', 'Pawnee', and 'Greenriver'.

[0016] In some embodiments of the present invention, the SSR molecular markers used to identify thin-shelled pecan varieties include: SSR molecular markers amplified by primer pairs with sequences such as SEQ ID NO.1-2, SSR molecular markers amplified by primer pairs with sequences such as SEQ ID NO.3-4, and SSR molecular markers amplified by primer pairs with sequences such as SEQ ID NO.5-6. This molecular marker combination can be used to identify the following 33 thin-shelled pecan varieties: 'Oconee', 'Kiowa', 'Shawnee', 'Waco', 'Hopi', 'Forkert', 'Maramec', 'Mahan', 'Navaho', 'Desirable', 'Barton', 'Carter', 'Deerstand', 'Shepherd', 'Osage', 'Chetopa', 'Elliott', 'Major', 'Yates68', 'Posey', 'Colby', 'Mcmillan', 'Mohawk', 'GloriaGrande', 'Silverback', 'Graking', 'Creek', 'Kanza', 'Stuart', 'Jayhawk', 'Choctaw', 'Pawnee', and 'Greenriver'.

[0017] In some embodiments of the present invention, the SSR molecular markers used to identify thin-shelled pecan varieties include: SSR molecular markers amplified by primer pairs with sequences such as SEQ ID NO.1-2, SSR molecular markers amplified by primer pairs with sequences such as SEQ ID NO.3-4, SSR molecular markers amplified by primer pairs with sequences such as SEQ ID NO.5-6, and SSR molecular markers amplified by primer pairs with sequences such as SEQ ID NO.9-10. This molecular marker combination can be used to identify the following 34 thin-shelled pecan varieties: 'Oconee', 'Kiowa', 'Shawnee', 'Waco', 'Hopi', 'Forkert', 'Maramec', 'Mahan', 'Navaho', 'Desirable', 'Barton', 'Carter', 'Deerstand', 'Shepherd', 'Osage', 'Chetopa', 'Elliott', 'Major', 'Yates68', 'Posey', 'Colby', 'Mcmillan', 'Mohawk', 'Gloria Grande', 'Silverback', 'Graking', 'Houma', 'Creek', 'Kanza', 'Stuart', 'Jayhawk', 'Choctaw', 'Pawnee', and 'Greenriver'.

[0018] In some embodiments of the present invention, the SSR molecular markers used to identify thin-shelled walnut varieties include: SSR molecular markers amplified by primer pairs such as SEQ ID NO.1-2, SSR molecular markers amplified by primer pairs such as SEQ ID NO.3-4, SSR molecular markers amplified by primer pairs such as SEQ ID NO.5-6, SSR molecular markers amplified by primer pairs such as SEQ ID NO.7-8, and SSR molecular markers amplified by primer pairs such as SEQ ID NO.9-10. This molecular marker combination can be used to identify the following 36 thin-shelled pecan varieties: 'Oconee', 'Kiowa', 'Shawnee', 'Waco', 'Hopi', 'Forkert', 'Maramec', 'Mahan', 'Navaho', 'Desirable', 'Barton', 'Carter', 'Deerstand', 'Shepherd', 'Osage', 'Chetopa', 'Elliott', 'Major', 'Yates68', 'Posey', 'Colby', 'Mcmillan', 'Mohawk', 'Gloria Grande', 'Silverback', 'Graking', 'Lakota', 'Nacono', 'Houma', 'Creek', 'Kanza', 'Stuart', 'Jayhawk', 'Choctaw', 'Pawnee', and 'Greenriver'.

[0019] In a second aspect, the present invention provides primer pairs for identifying thin-shelled pecan varieties, the primer pairs comprising any one or more of the following primer pairs: a primer pair with a sequence as shown in SEQ ID NO.1-2, a primer pair with a sequence as shown in SEQ ID NO.3-4, a primer pair with a sequence as shown in SEQ ID NO.5-6, a primer pair with a sequence as shown in SEQ ID NO.7-8, and a primer pair with a sequence as shown in SEQ ID NO.9-10.

[0020] The above primer pairs are respectively used to amplify the SSR molecular markers for identifying the thin-shelled walnut varieties described in the first aspect.

[0021] In some embodiments of the present invention, the primer pairs used to identify thin-shelled pecan varieties include primer pairs with sequences as shown in SEQ ID NO.1-2, primer pairs with sequences as shown in SEQ ID NO.3-4, primer pairs with sequences as shown in SEQ ID NO.5-6, primer pairs with sequences as shown in SEQ ID NO.7-8, and primer pairs with sequences as shown in SEQ ID NO.9-10.

[0022] In a third aspect, the present invention provides SSR molecular markers for identifying thin-shelled walnut varieties, wherein the SSR molecular markers include any one or more of the following SSR molecular markers (1) to (23):

[0023] (1) amplified by the primer pair shown in SEQ ID NO. 1-2;

[0024] (2) amplified by a primer pair with a sequence as shown in SEQ ID NO. 3-4;

[0025] (3) amplified by the primer pair whose sequence is shown in SEQ ID NO. 5-6;

[0026] (4) amplified by the primer pair with the sequence shown in SEQ ID NO. 7-8;

[0027] (5) amplified by the primer pair whose sequence is shown in SEQ ID NO. 9-10;

[0028] (6) amplified by the primer pair with the sequence shown in SEQ ID NO. 11-12;

[0029] (7) amplified by the primer pair whose sequence is shown in SEQ ID NO. 13-14;

[0030] (8) amplified by the primer pair whose sequence is shown in SEQ ID NO. 15-16;

[0031] (9) amplified by the primer pair whose sequence is shown in SEQ ID NO. 17-18;

[0032] (10) amplified by the primer pair whose sequence is shown in SEQ ID NO. 19-20;

[0033] (11) amplified by the primer pair with the sequence shown in SEQ ID NO. 21-22;

[0034] (12) amplified by the primer pair whose sequence is shown in SEQ ID NO. 23-24;

[0035] (13) amplified by the primer pair whose sequence is shown in SEQ ID NO. 25-26;

[0036] (14) amplified by the primer pair whose sequence is shown in SEQ ID NO. 27-28;

[0037] (15) was amplified by the primer pair with the sequence shown in SEQ ID NO. 29-30;

[0038] (16) was amplified by the primer pair whose sequence is shown in SEQ ID NO. 31-32;

[0039] (17) was amplified by the primer pair whose sequence is shown in SEQ ID NO. 33-34;

[0040] (18) was amplified by the primer pair whose sequence is shown in SEQ ID NO. 35-36;

[0041] (19) was amplified by the primer pair whose sequence is shown in SEQ ID NO. 37-38;

[0042] (20) was amplified by the primer pair whose sequence is shown in SEQ ID NO. 39-40;

[0043] (21) was amplified by the primer pair whose sequence is shown in SEQ ID NO. 41-42;

[0044] (22) was amplified by the primer pair whose sequence is shown in SEQ ID NO. 43-44;

[0045] (23) was amplified using the primer pair shown in SEQ ID NO. 45-46.

[0046] In some embodiments of the present invention, an SSR molecular marker combination consisting of the SSR molecular markers in (1) to (23) above is provided. The molecular marker combination can be used for genetic diversity analysis or phylogenetic relationship analysis of thin-shelled pecan varieties.

[0047] In a fourth aspect, the present invention provides primer pairs for identifying thin-shelled pecan varieties, the primer pairs comprising any one or more of the following primer pairs: primer pairs with sequences as shown in SEQ ID NOs. 1-2, primer pairs with sequences as shown in SEQ ID NOs. 3-4, primer pairs with sequences as shown in SEQ ID NOs. 5-6, primer pairs with sequences as shown in SEQ ID NOs. 7-8, primer pairs with sequences as shown in SEQ ID NOs. 9-10, primer pairs with sequences as shown in SEQ ID NOs. 11-12, primer pairs with sequences as shown in SEQ ID NOs. 13-14, primer pairs with sequences as shown in SEQ ID NOs. 15-16, primer pairs with sequences as shown in SEQ ID NOs. 17-18, primer pairs with sequences as shown in SEQ ID NOs. 19-20, primer pairs with sequences as shown in SEQ ID NOs. 21-22, primer pairs with sequences as shown in SEQ ID NOs. 23-24, primer pairs with sequences as shown in SEQ ID NOs. 25-26, primer pairs with sequences as shown in SEQ ID NOs. The primer pair shown in NO.27-28, the primer pair with the sequence shown in SEQ ID NO.29-30, the primer pair with the sequence shown in SEQ ID NO.31-32, the primer pair with the sequence shown in SEQ ID NO.33-34, the primer pair with the sequence shown in SEQ ID NO.35-36, the primer pair with the sequence shown in SEQ ID NO.37-38, the primer pair with the sequence shown in SEQ ID NO.39-40, the primer pair with the sequence shown in SEQ ID NO.41-42, the primer pair with the sequence shown in SEQ ID NO.43-44, and the primer pair with the sequence shown in SEQ ID NO.45-46.

[0048] In some embodiments of the present invention, a primer combination consisting of primers shown in SEQ ID NOs. 1 to 46 is provided. The primer combination can be used for genetic diversity analysis or phylogenetic relationship analysis of slender-shelled pecan varieties.

[0049] In a fifth aspect, the present invention provides a kit comprising the above-mentioned primer pair for identifying thin-shelled pecan varieties.

[0050] The above kit can be used to identify thin-shelled pecan varieties.

[0051] In a sixth aspect, the present invention provides a DNA chip comprising the primer pair described above for identifying thin-shelled pecan varieties.

[0052] The DNA chip can be used to identify thin-shelled walnut varieties through hybridization technology.

[0053] In a seventh aspect, the present invention provides any of the following applications of the above-mentioned SSR molecular marker for identifying thin-shelled pecan varieties, the primer pair for identifying thin-shelled pecan varieties, the kit, or the DNA chip:

[0054] (1) Application in identifying thin-shelled pecan varieties;

[0055] (2) Application in constructing DNA fingerprint database of thin-shelled pecan varieties;

[0056] (3) Application in genetic diversity analysis of walnut germplasm resources and purity detection of seeds or seedlings;

[0057] (4) Application in the tracing of varieties or analysis of kinship of thin-shelled pecans;

[0058] (5) Application in molecular marker-assisted breeding of pecan.

[0059] The thin-shell pecan varieties of the present invention are preferably one or more of 'Oconee', 'Kiowa', 'Shawnee', 'Waco', 'Hopi', 'Forkert', 'Maramec', 'Mahan', 'Navaho', 'Desirable', 'Barton', 'Carter', 'Deerstand', 'Shepherd', 'Osage', 'Chetopa', 'Elliott', 'Major', 'Yates 68', 'Posey', 'Colby', 'Mcmillan', 'Mohawk', 'Gloria Grande', 'Silverback', 'Graking', 'Lakota', 'Nacono', 'Houma', 'Creek', 'Kanza', 'Stuart', 'Jayhawk', 'Choctaw', 'Pawnee', and 'Greenriver'. More preferably, the thin-shell pecan varieties are the 36 varieties mentioned above.

[0060] In an eighth aspect, the present invention provides a method for constructing a molecular ID card for a thin-shelled pecan variety, the method comprising: using genomic DNA of the thin-shelled pecan as a template, performing PCR amplification using a primer pair with sequences shown in SEQ ID NOs. 1-2, a primer pair with sequences shown in SEQ ID NOs. 3-4, a primer pair with sequences shown in SEQ ID NOs. 5-6, a primer pair with sequences shown in SEQ ID NOs. 7-8, and a primer pair with sequences shown in SEQ ID NOs. 9-10 to obtain a PCR amplification product, performing electrophoresis detection on the PCR amplification product, distinguishing different band patterns of the electrophoretic bands of the PCR amplification product by different assignments, and constructing a molecular ID card for the thin-shelled pecan variety;

[0061] The thin-shelled pecan variety is one or more of 'Oconee', 'Kiowa', 'Shawnee', 'Waco', 'Hopi', 'Forkert', 'Maramec', 'Mahan', 'Navaho', 'Desirable', 'Barton', 'Carter', 'Deerstand', 'Shepherd', 'Osage', 'Chetopa', 'Elliott', 'Major', 'Yates68', 'Posey', 'Colby', 'Mcmillan', 'Mohawk', 'Gloria Grande', 'Silverback', 'Graking', 'Lakota', 'Nacono', 'Houma', 'Creek', 'Kanza', 'Stuart', 'Jayhawk', 'Choctaw', 'Pawnee', and 'Greenriver'.

[0062] Preferably, the band patterns of the electrophoresis bands of the PCR amplification products corresponding to 36 varieties of thin-shelled pecans and their corresponding assignments (codes) are shown in Table 1.

[0063] Table 1 PCR amplification product lengths and molecular ID codes of 36 cultivars of Carya sylvestris

[0064]

[0065]

[0066] Preferably, the method for constructing the above-mentioned molecular identity card also includes: a step of assigning the germplasm resource information of the thin-shelled walnut, wherein the germplasm resource information includes species information, variety origin information and germplasm resource category.

[0067] In a ninth aspect, the present invention provides a method for identifying thin-shelled pecan varieties, the method comprising: using the genomic DNA of thin-shelled pecan as a template, using the primer pair for identifying thin-shelled pecan varieties described above to perform PCR amplification, performing length and / or sequence analysis on the obtained PCR amplification products, and determining the type of thin-shelled pecan variety based on the length and / or sequence characteristics.

[0068] In the above method, the thin-shell pecan variety is one or more of 'Oconee', 'Kiowa', 'Shawnee', 'Waco', 'Hopi', 'Forkert', 'Maramec', 'Mahan', 'Navaho', 'Desirable', 'Barton', 'Carter', 'Deerstand', 'Shepherd', 'Osage', 'Chetopa', 'Elliott', 'Major', 'Yates 68', 'Posey', 'Colby', 'Mcmillan', 'Mohawk', 'Gloria Grande', 'Silverback', 'Graking', 'Lakota', 'Nacono', 'Houma', 'Creek', 'Kanza', 'Stuart', 'Jayhawk', 'Choctaw', 'Pawnee', and 'Greenriver'. Preferably, the 36 thin-shell pecan varieties mentioned above are used.

[0069] Preferably, the method for identifying the thin-shelled pecan variety includes: using the genomic DNA of the thin-shelled pecan as a template, using the primer pair with sequences such as SEQ ID NO.1-2, the primer pair with sequences such as SEQ ID NO.3-4, the primer pair with sequences such as SEQ ID NO.5-6, the primer pair with sequences such as SEQ ID NO.7-8, and the primer pair with sequences such as SEQ ID NO.9-10 to perform PCR amplification, performing length and / or sequence analysis on the obtained PCR amplification products, and judging the thin-shelled pecan variety type based on the length and / or sequence characteristics.

[0070] The basis for judging the type of thin-shelled pecan variety based on the length characteristics of the PCR amplification products is shown in Table 1.

[0071] The beneficial effects of the present invention are as follows: the present invention first developed 23 SSR markers that are polymorphic in 36 major thin-shelled pecan varieties, and these 23 SSR markers can be used to detect the genetic diversity of the 36 major thin-shelled pecan varieties. A core primer set containing 5 highly polymorphic sites was screened and obtained from these 23 SSR markers. The core primer set can be used to identify the 36 major thin-shelled pecan varieties, with high accuracy and stability of the discrimination results, simple operation, and a short time required for identification, which is convenient for promotion and application in practice. Based on the core primer set, a variety-specific molecular fingerprint map and its molecular ID digital encoding, barcode and QR code were established, which achieved effective differentiation of the 36 major thin-shelled pecan varieties, and provided an effective method for thin-shelled pecan variety identification, seedling purity detection, and variety tracing. BRIEF DESCRIPTION OF THE DRAWINGS

[0072] Figure 1 The electrophoretic bands of primers Ciz91 and Ciz150 in Example 1 of the present invention in the test samples, wherein lanes 1 and 47 are DNA markers; lanes 2 to 46 are: 'Kanza', 'Mohawk', 'Shawnee', 'Mahan', 'Mahan', 'Osage', 'Pawnee', 'Mcmillan', 'Lakota', 'Silver back', 'Carter', 'Colby', 'Stuart', 'Greenriver', 'Waco', 'Major', 'Oconee', 'Oconee', 'Navaho', 'GloriaGrande', 'Forkert', 'Choctaw', 'Creek', 'Mohawk', 'Elliott', 'Barton', 'Creek', 'Des irable', 'Graking', 'Greenriver', 'Hopi', 'Jayhawk', 'Kiowa', 'Lakota', 'Maramec', 'Mohawk', 'Nacono', 'Navaho', 'Oconee', 'Posey', 'Houma', 'Yates68', 'Shepherd', 'Deerstand', 'Chetopa'.

[0073] Figure 2 The figure shows the distribution of 23 SSR markers on chromosomes in Example 1 of the present invention.

[0074] Figure 3This is the UPGMA genetic cluster diagram of 45 samples from 36 varieties in Example 1 of the present invention, where the numbers in brackets indicate biological replicates; the different colored squares corresponding to the variety name represent that the variety has a Schley, Success, or Major genetic background, respectively.

[0075] Figure 4 This is the molecular fingerprint of 36 varieties in Example 2 of the present invention.

[0076] Figure 5 This is the molecular ID of the thin-shelled pecan variety 'Pawnee' in Example 2 of the present invention.

[0077] Figure 6 This is the molecular identification card of the 36 thin-shelled pecan varieties in Example 2 of the present invention. DETAILED DESCRIPTION

[0078] The following examples are used to illustrate the present invention but are not intended to limit the scope of the present invention.

[0079] The 36 pecan varieties involved in the following examples can be obtained through commercial purchase channels or from the germplasm resource bank of the Institute of Subtropical Forestry, Chinese Academy of Forestry. Among them, varieties such as 'Mahan', 'Pawnee', 'Greenriver', 'Mohawk', 'Osage', 'Lakota', 'Mcmillan', 'Carter', 'Colby', and 'Stuart' have been disclosed in the literature (Zhang CC, Yao XH, Ren HD, Chang J., Wu J., Shao WZ, Fang Q. Characterization and Development of Genomic SSRs in Pecan (Caryaillinoinensis). Forests. 2020, 11(1), 61.).

[0080] Example 1 Development and validation of SSR molecular markers for identification of walnut varieties

[0081] 1 Materials and Methods

[0082] 1.1 Experimental materials and DNA extraction

[0083] Thirty-six cultivars of Carya serrata were used as experimental materials (Table 2). Among them, 'Mahan' (2), 'Oconee' (3), 'Mohawk' (3), 'Creek' (2), 'Greenriver' (2), 'Lakota' (2), and 'Navaho' (2) were introduced in different years (1999, 2007, and 2008) with 2 to 3 biological replicates, totaling 45 samples. All samples were collected from the Carya serrata germplasm resource conservation base of the Subtropical Forestry Research Institute of the Chinese Academy of Forestry, located in Jiande City, Zhejiang Province (29°N, 119°W). Young leaves were collected in spring and quickly frozen in liquid nitrogen and stored in a -80°C ultra-low temperature freezer. Genomic DNA was extracted using the Tiangen Plant Genomic DNA Extraction Kit, and the DNA integrity and concentration were detected using 1% agarose gel and a Quawell Q5000 spectrophotometer (Quawell Technology, Inc., USA). The DNA was diluted to 50 ng / μL and stored at -20°C until use.

[0084] Table 2 Information on 36 thin-shelled pecan varieties①

[0085]

[0086]

[0087] ①All tested varieties were introduced from the United States.

[0088] 1.2 PCR amplification and electrophoresis detection

[0089] From the candidate SSR primer library established earlier (Zhang CC, Yao XH, Ren HD, Chang J., Wu J., Shao WZ, Fang Q. Characterization and Development of Genomic SSRs in Pecan (Carya illinoinensis). Forests. 2020, 11 (1), 61.), a total of 80 primer pairs with SSR repeat motifs of 2 to 4 bases were screened. First, genomic DNA of 8 varieties ('Mahan', 'Oconee', 'Pawnee', 'Kanza', 'Stuart', 'Creek', 'Kiowa', 'Shawnee') was used as the material to screen the 80 primer pairs; the primers with better amplification effect were screened and genotyped for 45 samples of 36 varieties ( Figure 1 ).

[0090] PCR reaction system: 2× Taq PCR MasterMix 10 μL (Nanozyme, Nanjing), upstream and downstream primers 1 μL each, DNA template 1 μL, ddH2O 7 μL.

[0091] The PCR reaction program was as follows: pre-denaturation at 95°C for 2 min, 29 cycles including denaturation at 94°C for 40 s, annealing at the specific annealing temperature for 45 s, extension at 72°C for 1 min, and finally extension at 72°C for 7 min, and storage at 4°C.

[0092] Polyacrylamide gel electrophoresis and silver staining were used to separate and detect target bands. The following 8% non-denaturing polyacrylamide gel recipe: 14 mL of 30% gel preparation solution (29:1) (Beijing Solebo), 15 mL of 5× TBE, 20 mL of H₂O, 400 μL of 10% ammonium persulfate (AP), and 40 μL of TEMD (tetramethyldiethylamine). The electrophoresis buffer was 0.5× TBE, and an AL50 DNA Marker (Nanjing Zhongding Biotechnology) was used. Electrophoresis was performed using a JY300HC universal electrophoresis power supply (Beijing Junyi) at 180 V and 400 mA for 90–180 min. After electrophoresis, the gel was removed and stained with 1 g / L AgNO₃ solution for 10–15 min. The gel was then developed with a colorimetric solution (20 g / L NaOH solution with 10 mL of formaldehyde) for 5–8 min. The gel was rinsed twice with purified water and then placed on a light box for photography.

[0093] 1.3 Data Processing

[0094] Electrophoresis bands were manually interpreted and assigned the values ​​A, B, C, D, and so on, from largest to smallest. Missing bands were indicated by a dot (.). Genotypes for each marker were counted for each sample. The observed heterozygosity (HO), expected heterozygosity (HE), number of alleles (NA), and allele frequency (F) of each marker were calculated using Popgene software (http: / / cc.oulu.fi / ~jaspi / popgen / popgen.htm). Polymorphism information content (PIC) was calculated using the formula PIC = 1 - ∑Pi², where Pi is the frequency of the i-th allele for the current primer. Genetic distance (GD) and genetic similarity (GS) were calculated (Nei et al., 1979). Cluster analysis was performed on 45 samples from 36 cultivars using the UPGMA method. A cluster tree was drawn using NTSYS-pc software. The sequences of each marker were aligned to the pecan genome (Lovell JT, Bentley N.B., Bhattarai G., et al. 2021. Four chromosome scale genomes and a pan-genome annotation to accelerate pecan tree breeding. Nature Communication, 12: 4125.) using online software (https: / / phytozome-next.jgi.doe.gov / blast-search). The distribution of each marker on the chromosome was plotted using TBtools software (Chen C., Chen H., Zhang Y., et al. 2020. TBtools: an integrative toolkit developed for interactive analyses of big biological data. Molecular Plant, 13: 1194–1202.).

[0095] 1.4 Screening of core primers

[0096] Cluster analysis of 36 varieties was performed by increasing the number of markers in descending order of SSR marker PIC values, and the minimum SSR primer combination used to effectively distinguish all participating varieties was screened. This set of primers was used as the core primer set for the subsequent construction of molecular fingerprints and molecular identity cards for each variety.

[0097] 2. Results and Analysis

[0098] 2.1 Development of SSR Markers

[0099] 47 pairs (58.75%) of the 80 primer pairs were able to amplify the target bands in the 8 varieties, of which 23 pairs (28.75%) had clear target bands with few miscellaneous bands and were easy to read ( Figure 1 ). These 23 primer pairs were further used to perform PCR amplification in 45 samples of 36 varieties (Table 3, Table 4, Figure 1 ). These 23 markers were mapped to the genome of Hickory chinensis and were found to be distributed on 13 chromosomes except Chr07, Chr10 and Chr13. Chr15 and Chr05 had 3 and 4 markers, respectively, and the remaining chromosomes had 1 to 2 markers ( Figure 2 ).

[0100] Table 3 Primer information of 23 SSR markers

[0101]

[0102] Table 4 Genetic diversity parameters of 23 SSR markers

[0103]

[0104]

[0105] 2.2 Genetic diversity and cluster analysis

[0106] The amplified bands for 23 primer pairs in 36 varieties were counted, and data were analyzed using Popgene software (Table 4). Eighteen markers were found to be genetically polymorphic across the 36 varieties, while five markers were not polymorphic in the tested varieties. The number of alleles detected (NA) for the 23 markers ranged from 1 to 8, for a total of 70 alleles, with an average of 3.04 loci detected per marker. Ciz91 detected the most loci, with 8, followed by Ciz85 and Ciz52, both with 5 alleles detected. Among the 18 polymorphic markers, the observed heterozygosity (Ho) ranged from 0.03 to 0.86, the expected heterozygosity (HE) ranged from 0.03 to 0.73, and the polymorphism information content (PIC) ranged from 0.03 to 0.72. Genetic distances between biological replicates of the same cultivar ranged from 0 to 0.0127. 'Oconee' (Ciz139) and 'Navaho' (Ciz75) differed from their respective biological replicates at one locus, while the remaining cultivars had consistent banding patterns with their own biological replicates. Genetic distances between cultivars ranged from 0.026 to 0.636, with 'Mohawk' and 'Graking' being the closest, while 'Jayhawk' and 'Stuart' were the farthest apart. Genetic similarity coefficients ranged from 0.53 to 0.97 among cultivars.

[0107] Cluster analysis found that when the genetic similarity coefficient was 0.721, the 36 varieties could be divided into three categories: Category I contained 32 varieties, Category II contained 3 varieties, and Category III contained only 1 variety. In Category I, 16 varieties had the genetic background of 'Schley', 15 varieties had the genetic background of 'Success', 5 varieties had the genetic background of 'Major', and the parentage of the other 9 varieties was unknown (Table 2, Figure 3 ). In Group II, the parentage of 'Elliott' is unknown, while 'Gloria Grande' is a seedling offspring of 'Stuart'. Group II contains only one variety, 'Jayhawk', which was bred from a seedling offspring of 'Giles'.

[0108] 2.3 Core primer screening

[0109] Among the 18 polymorphic SSR loci, 6 had PIC values ​​greater than 0.5 (Table 4), indicating high polymorphism. Ciz91 had the highest PIC value (0.72), followed by Ciz85 (0.70). Ciz81 and Ciz140 were greater than 0.6, while Ciz107 and Ciz142 were 0.57 and 0.52, respectively. By increasing the number of markers in descending order of PIC value, we identified 36 varieties. We found that at least five markers, Ciz91, Ciz85, Ciz81, Ciz140, and Ciz107, were sufficient to effectively distinguish all tested varieties (Table 5). These markers are located on chromosomes 15, 8, 1, 9, and 4, respectively. Figure 2 These five SSR primer pairs were determined as the core primer set for the subsequent construction of molecular fingerprints and molecular ID cards for each variety.

[0110] Table 5: Differentiation of varieties by adding SSR primers one by one

[0111]

[0112] Example 2 Construction of molecular fingerprints and molecular ID cards of thin-shelled pecan varieties

[0113] 1 Materials and Methods

[0114] Based on the genotypes of the core primer set determined in Example 1 (a total of five markers: Ciz91, Ciz85, Ciz81, Ciz140, and Ciz107) in the test varieties, the markers A, B, C, D, etc. were sequentially converted to 1, 2, 3, 4, etc., and missing sites were replaced with "0" to construct a molecular fingerprint specific to each variety. By combining the germplasm resource information with the molecular fingerprint, molecular ID cards for 36 thin-shelled pecan varieties were constructed using a barcode generator and a QR code generator (http: / / qr-batch.com / ).

[0115] 2 Results and Analysis

[0116] According to the band patterns detected by 5 pairs of core primers in 36 varieties, A, B, C, D... were converted into 1, 2, 3, 4... in sequence to construct a molecular fingerprint map specific to each variety ( Figure 4, Table 1). Among them, the single marker Ciz91 can identify 'Colby', 'Greenriver', 'Mahan', and 'Pawnee'; Ciz85 can identify 'Mcmillan'; Ciz81 can identify 'Elliott' and 'Posey'; Ciz140 can identify 'Chetopa' and 'Gloria Grande'; and Ciz107 can identify 'Houma'. The banding patterns of the five core primer pairs in each variety were further converted into a 10-digit fingerprint code. This was then combined with the germplasm information code to construct a variety-specific 15-digit molecular ID code. The germplasm information code has 5 digits and is divided into 3 parts. The first digit represents the species information. There are 5 species of Carya in China, including Carya cathayensis Sarg., Carya dabieshanensis WCheng & R.H.Chang, Carya hunanensis WCLiu, Carya kweichowensis Kuang & A.M.Liu and Carya tonkinensis Lecomte), the thin-shelled pecan is an introduced species, so the first species information code of the thin-shelled pecan is 6; the second to fourth digits are the origin information of the variety, such as 156 for China (different provinces in China can be replaced by provincial codes), 840 for the United States; the fifth digit is the germplasm resource category, referring to the "Technical Procedures for Survey and Cataloging of Thin-shelled Pecan Genetic Resources" (LY / T2804-2017), 1 represents seed resources (population), 2 represents seed resources (family), 3 represents seed resources (individual), 4 represents introduced varieties, and 5 represents selected varieties. Take 'Pawnee' as an example ( Figure 5 ), whose molecular ID number is 684042225771222, indicating that the species is Carya serrata, an introduced variety from the United States. The electrophoretic band patterns of the five core markers (Ciz81, Ciz85, Ciz91, Ciz140, and Ciz107) are 22, 25, 77, 12, and 22, respectively. Finally, using the 15-digit molecular ID number of each variety, variety-specific barcodes and QR codes were created ( Figure 6 ).

[0117] Pecan (Hyacinthus serrata) is a world-renowned woody oil-bearing and dried fruit tree with a beautiful shape and excellent wood properties. It has been introduced to China for over 100 years and has demonstrated good adaptability and high productivity in various regions. Pecan (Hyacinthus serrata) has been introduced by numerous institutions, using various forms such as cuttings and seeds. This has led to irregular introductions, resulting in the phenomenon of "same name, different species, same species, different names." During seedling propagation and distribution, insufficient attention or an excessive pursuit of profitability has raised questions about the authenticity of some seedling varieties. Variety is a core element of agricultural production, and inauthentic or non-expected varieties often result in significant losses for growers, production companies, and research institutions. Therefore, it is urgent to establish a comprehensive and efficient identification technology system for Pecan (Hyacinthus serrata) varieties.

[0118] Currently, identification of thin-shelled pecan varieties is primarily based on morphological characteristics of organs such as flowers, fruits, and leaves. However, these morphological indicators are susceptible to environmental influences and lack stability. The genetic base of the main cultivated varieties is narrow, and phenotypic differences between varieties are minimal. Furthermore, fewer morphological indicators are available for evaluation during the seedling stage, making authenticating seedling varieties based on phenotypic traits extremely challenging. Molecular markers can detect differences in DNA sequences and offer strong stability and reproducibility. They have been widely used in variety and germplasm identification research for agricultural and forestry crops, and have significant advantages in identifying phenotypically similar germplasm. SSR markers and SNPs are the two most widely used molecular markers for variety identification research. They are widely distributed across the genome, co-dominant, and offer advantages such as high reproducibility and ease of detection. Compared to SNP markers, individual SSR markers have higher polymorphism information content, making them the preferred marker for constructing DNA fingerprints. However, the number of available SSR markers for thin-shelled pecans is limited, and research on genetic diversity and variety identification based on SSRs is just beginning. Previously, Zhang et al. (Zhang CC, Yao XH, Ren HD, Chang J., Wu J., Shao WZ, Fang Q. Characterization and Development of Genomic SSRs in Pecan (Carya illinoinensis). Forests. 2020, 11(1), 61.) screened more than 9,000 pairs of high-quality SSR primers across the entire genome of Carya illinoinensis, providing a large number of candidate molecular markers for genetic breeding research on this species. This study developed 23 new SSR markers and studied the genetic diversity of 36 introduced varieties, of which 6 sites had a polymorphic information content greater than 0.5, belonging to highly polymorphic sites.

[0119] Cluster analysis based on UPGMA found that different varieties and their biological replicates were clustered together, indicating that the SSR markers provided by the present invention have good stability and repeatability. Among them, two 'Navaho' samples had an allele difference at the Ciz75 locus, and one sample of 'Oconee' differed from the other two samples in the Ciz139 marker. The phenomenon of differences in bands of the same variety has also been reported in other crops. In peppers, two pairs of primers detected differential bands in different individuals of the same variety. The reason may be that the main focus of the variety breeding process is on phenotypic traits, and SSR sites are not selected, resulting in residual variation between different individuals of the variety. Different varieties are clustered according to kinship. Class I includes 32 varieties. Except for 9 unknown parents, the other varieties have one or two varieties of 'Schley', 'Success' and 'Major' (Table 2, Figure 3 For example, 'Mahan' was selected from the seedlings of 'Schley', and 'Mohawk' was bred by hybridizing 'Success' (♀) and 'Mahan' (♂), and 'Pawnee' and 'Creek' were further bred with 'Mohawk' as parents (Bentley N., Grauke LJ, Klein P. 2019. Genotyping bysequencing (GBS) and SNP marker analysis of diverse accessions of pecan (Caryaillinoinensis). Tree Genetics and Genomes, 15(1): 1-17.). Therefore, 'Schley', 'Success' and 'Major' as backbone parents provide excellent genetic resources for the breeding of a large number of varieties, but it also shows that the genetic background of existing varieties is relatively narrow and the genetic diversity is relatively low. The present invention utilizes 23 SSR markers to better invert the genetic relationships of 36 varieties, further demonstrating that these 23 SSR markers have great application potential in the study of genetic diversity of walnuts.

[0120] Molecular IDs abstract the molecular fingerprint of a target variety, creating a variety-specific digital ID. These are crucial for identifying varieties and germplasm, and for guiding their application. They are widely used in commercial tree species such as peaches, pears, apples, tea trees, and walnuts. However, research on pecans is just beginning. The present invention developed 23 SSR markers, and used 2 to 3 biological replicates of different varieties to verify the repeatability and stability of these 23 SSR markers. It was found that the genetic similarity coefficient of 36 varieties was 0.53 to 0.97, which was not much different from previous studies (He Xudong, Zheng Jiwei, Tian Xueyao, et al. 2021. Analysis of kinship and construction of fingerprints of thin-shelled pecan varieties. Forestry Science Research, 34(4):95-102.; Shi Juanjuan, Ye Shengyue, Yu Shiqun, et al. 2013. SSR analysis of genetic diversity of 37 newly introduced thin-shelled pecan varieties. Journal of Anhui Agricultural University, 40(1):42-46.); a core primer set containing 5 markers was screened, which can realize the effective identification of all tested varieties. The number of alleles detected by the five markers is 8, 5, 4, 4, and 4, respectively. The maximum number of materials that can be distinguished by the primer combination is theoretically 2560 (Chen Liang, Zheng Yuhong, Fan Xuhong, et al. 2016. Construction of SSR fingerprint identity cards of newly bred soybean varieties in Jilin Province. Soybean Science, 35(6):896–901.). The present invention specifically adds a germplasm information code to the constructed molecular identity card, which contains information on species, origin, and germplasm type. This provides a reference for the subsequent construction of molecular identity cards for various germplasm resources such as different Carya plants, bred varieties, introduced varieties, families, and superior plants.

[0121] In summary, the present invention developed 23 new SSR markers for thin-shelled pecans, analyzed the genetic diversity and genetic relationships of 36 varieties, and determined five primer pairs, namely Ciz81, Ciz85, Ciz91, Ciz140 and Ciz107, as the core primer set based on the polymorphic information content and variety identification ability of the markers, which can effectively distinguish the 36 varieties. Based on this, molecular fingerprints and molecular identity cards of the 36 tested varieties were constructed, providing an effective method for thin-shelled pecan variety identification, traceability, seed authenticity and purity detection, etc.

[0122] Although the present invention has been described in detail above using general descriptions and specific embodiments, it will be apparent to those skilled in the art that modifications and improvements may be made based on the present invention. Therefore, such modifications and improvements, which do not depart from the spirit of the present invention, are intended to be within the scope of protection claimed herein. Sequence Listing <110> Research Institute of Subtropical Forestry, Chinese Academy of Forestry <120> SSR molecular markers, molecular IDs and their applications for identifying cultivars of Carya serrata <130> KHP221114568.0 <160> 46 <170> SIPOSequenceListing 1.0 <210> 1 <211> twenty one <212> DNA <213> Artificial Sequence <400> 1 tgaaagtgag atggtggtgt g 21 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <400> 2 aaggactaga tggatgccca 20 <210> 3 <211> twenty two <212> DNA <213> Artificial Sequence <400> 3 cgtcccatta tgaatgaatg aa 22 <210> 4 <211> twenty three <212> DNA <213> Artificial Sequence <400> 4 tgtaatgaca gacctatcca cga 23 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <400> 5 gaccacctta cgtgggagaa 20 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <400> 6 gcatcgagac acatcctttg 20 <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <400> 7 gctgagatgc ctagctgctt 20 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <400> 8 agtcgattgg cctctgatga 20 <210> 9 <211> twenty two <212> DNA <213> Artificial Sequence <400> 9 ggctagccta tcattattta tg 22 <210> 10 <211> 20 <212> DNA <213> Artificial Sequence <400> 10 gatcatcggg tttctgcatt 20 <210> 11 <211> twenty one <212> DNA <213> Artificial Sequence <400> 11 ttggactacg ttgaaacacg a 21 <210> 12 <211> twenty two <212> DNA <213> Artificial Sequence <400> 12 aaagaatatt cggccaactt gt 22 <210> 13 <211> twenty one <212> DNA <213> Artificial Sequence <400> 13 aggtcatgtc ccacaatcat c 21 <210> 14 <211> 20 <212> DNA <213> Artificial Sequence <400> 14 cctagaatat gcccagcacg 20 <210> 15 <211> twenty one <212> DNA <213> Artificial Sequence <400> 15 cagtttctgt gccttcgaat c 21 <210> 16 <211> 20 <212> DNA <213> Artificial Sequence <400> 16 atgctcactt caccggagtt 20 <210> 17 <211> 20 <212> DNA <213> Artificial Sequence <400> 17 cgttgaacgt ccattaccaa 20 <210> 18 <211> 20 <212> DNA <213> Artificial Sequence <400> 18 ccaaccacgt tgtgtctcac 20 <210> 19 <211> 20 <212> DNA <213> Artificial Sequence <400> 19 cgagggcaat taggacacat 20 <210> 20 <211> 20 <212> DNA <213> Artificial Sequence <400> 20 catgttcacc aacctcatcg 20 <210> twenty one <211> 20 <212> DNA <213> Artificial Sequence <400> twenty one ttagctgctt gttaggcggt 20 <210> twenty two <211> 20 <212> DNA <213> Artificial Sequence <400> twenty two gcagctgctt tgttgttgtt 20 <210> twenty three <211> 20 <212> DNA <213> Artificial Sequence <400> twenty three ttcttcgccg agtgctctat 20 <210> twenty four <211> 20 <212> DNA <213> Artificial Sequence <400> twenty four tcttggcaac acaggttctg 20 <210> 25 <211> 20 <212> DNA <213> Artificial Sequence <400> 25 atagcctcct caatcccacc 20 <210> 26 <211> 20 <212> DNA <213> Artificial Sequence <400> 26 catctgagca gattgcgtgt 20 <210> 27 <211> 20 <212> DNA <213> Artificial Sequence <400> 27 gtttctagca ggtgcggaag 20 <210> 28 <211> 20 <212> DNA <213> Artificial Sequence <400> 28 tggtcgaatt ggagtcctct 20 <210> 29 <211> 19 <212> DNA <213> Artificial Sequence <400> 29 ccaaacaaat ggaccgttg 19 <210> 30 <211> 20 <212> DNA <213> Artificial Sequence <400> 30 ttccaaacta ggcaaagcgt 20 <210> 31 <211> 20 <212> DNA <213> Artificial Sequence <400> 31 tatggtggtt gccagtgtgt 20 <210> 32 <211> 20 <212> DNA <213> Artificial Sequence <400> 32 tttcggcatc ctcctatgtc 20 <210> 33 <211> 20 <212> DNA <213> Artificial Sequence <400> 33 cgcatcgatt ggaaggttat 20 <210> 34 <211> 20 <212> DNA <213> Artificial Sequence <400> 34 ggacattggt cttgcatgtg 20 <210> 35 <211> 20 <212> DNA <213> Artificial Sequence <400> 35 tgtttcattc cctttcctgg 20 <210> 36 <211> 20 <212> DNA <213> Artificial Sequence <400> 36 cctgtatcac gcatttgcag 20 <210> 37 <211> 20 <212> DNA <213> Artificial Sequence <400> 37 ggtctctcaa gatgccttgg 20 <210> 38 <211> 20 <212> DNA <213> Artificial Sequence <400> 38 aactgatctc tggttgcgct 20 <210> 39 <211> 20 <212> DNA <213> Artificial Sequence <400> 39 ttatggaagc gcaggatacc 20 <210> 40 <211> 20 <212> DNA <213> Artificial Sequence <400> 40 gcggatagct ggaatggtta 20 <210> 41 <211> 20 <212> DNA <213> Artificial Sequence <400> 41 aaaccgaatt tgcattctgc 20 <210> 42 <211> 20 <212> DNA <213> Artificial Sequence <400> 42 agaagcacat ttccaaccca 20 <210> 43 <211> 20 <212> DNA <213> Artificial Sequence <400> 43 cttaagtgga acggcatcgt 20 <210> 44 <211> 20 <212> DNA <213> Artificial Sequence <400> 44 atgtctgtta aaccgcgacc 20 <210> 45 <211> 20 <212> DNA <213> Artificial Sequence <400> 45 aattatggga agctgcatgg 20 <210> 46 <211> 20 <212> DNA <213> Artificial Sequence <400> 46 actttcctgc acaaacccac 20

Claims

1. An SSR molecular marker for identifying walnut varieties, characterized in that: The SSR molecular markers include the following combinations of SSR molecular markers (1) to (5): (1) amplified by the primer pair shown in SEQ ID NO. 1-2; (2) amplified by a primer pair with a sequence as shown in SEQ ID NO. 3-4; (3) amplified by the primer pair whose sequence is shown in SEQ ID NO. 5-6; (4) amplified by the primer pair with the sequence shown in SEQ ID NO. 7-8; (5) amplified by the primer pair whose sequence is shown in SEQ ID NO. 9-10; The thin-shell pecan varieties are the following 36 thin-shell pecan varieties: 'Oconee', 'Kiowa', 'Shawnee', 'Waco', 'Hopi', 'Forkert', 'Maramec', 'Mahan', 'Navaho', 'Desirable', 'Barton', 'Carter', 'Deerstand', 'Shepherd', 'Osage', 'Chetopa', 'Elliott', 'Major', 'Yates68', 'Posey', 'Colby', 'Mcmillan', 'Mohawk', 'Gloria Grande', 'Silverback', 'Graking', 'Lakota', 'Nacono', 'Houma', 'Creek', 'Kanza', 'Stuart', 'Jayhawk', 'Choctaw', 'Pawnee', and 'Greenriver'; The band sizes of 36 walnut varieties amplified using the primer pair are as follows:

2. A primer pair for identifying a variety of walnuts, characterized in that: The primer pairs include the following primer pairs: primer pairs with sequences as shown in SEQ ID NO.1-2, primer pairs with sequences as shown in SEQ ID NO.3-4, primer pairs with sequences as shown in SEQ ID NO.5-6, primer pairs with sequences as shown in SEQ ID NO.7-8, and primer pairs with sequences as shown in SEQ ID NO.9-10.

3. A kit, characterized in that The invention comprises the primer pair for identifying the thin-shelled pecan variety according to claim 2.

4. A DNA chip, characterized in that The invention comprises the primer pair for identifying the thin-shelled pecan variety according to claim 2.

5. Any of the following uses of the SSR molecular marker for identifying thin-shelled pecan varieties according to claim 1, the primer pair for identifying thin-shelled pecan varieties according to claim 2, the kit according to claim 3, or the DNA chip according to claim 4: (1) Application in identifying thin-shelled pecan varieties; (2) Application in constructing DNA fingerprint database of thin-shelled pecan varieties; (3) Application in genetic diversity analysis of walnut germplasm resources and purity detection of seeds or seedlings; (4) Application in the tracing of varieties or analysis of kinship of thin-shelled pecans; (5) Application in molecular marker-assisted breeding of pecan; The thin-shell pecan varieties are the following 36 thin-shell pecan varieties: 'Oconee', 'Kiowa', 'Shawnee', 'Waco', 'Hopi', 'Forkert', 'Maramec', 'Mahan', 'Navaho', 'Desirable', 'Barton', 'Carter', 'Deerstand', 'Shepherd', 'Osage', 'Chetopa', 'Elliott', 'Major', 'Yates68', 'Posey', 'Colby', 'Mcmillan', 'Mohawk', 'Gloria Grande', 'Silverback', 'Graking', 'Lakota', 'Nacono', 'Houma', 'Creek', 'Kanza', 'Stuart', 'Jayhawk', 'Choctaw', 'Pawnee', and 'Greenriver'; The band sizes of 36 walnut varieties amplified using the primer pair are as follows:

6. A method for constructing a molecular ID card for a thin-shelled pecan variety, characterized in that: The method comprises: using genomic DNA of Carya serrata as a template, performing PCR amplification using a primer pair with sequences shown in SEQ ID NOs. 1-2, a primer pair with sequences shown in SEQ ID NOs. 3-4, a primer pair with sequences shown in SEQ ID NOs. 5-6, a primer pair with sequences shown in SEQ ID NOs. 7-8, and a primer pair with sequences shown in SEQ ID NOs. 9-10 to obtain PCR amplification products, performing electrophoresis detection on the PCR amplification products, distinguishing different band types of the electrophoresis bands of the PCR amplification products with different assignments, and constructing a molecular identification card of the Carya serrata variety; The thin-shell pecan varieties are the following 36 thin-shell pecan varieties: 'Oconee', 'Kiowa', 'Shawnee', 'Waco', 'Hopi', 'Forkert', 'Maramec', 'Mahan', 'Navaho', 'Desirable', 'Barton', 'Carter', 'Deerstand', 'Shepherd', 'Osage', 'Chetopa', 'Elliott', 'Major', 'Yates68', 'Posey', 'Colby', 'Mcmillan', 'Mohawk', 'Gloria Grande', 'Silverback', 'Graking', 'Lakota', 'Nacono', 'Houma', 'Creek', 'Kanza', 'Stuart', 'Jayhawk', 'Choctaw', 'Pawnee', and 'Greenriver'; The band sizes of 36 walnut varieties amplified using the primer pair are as follows:

7. A method for identifying thin-shelled pecan varieties, characterized in that: The method comprises: using genomic DNA of thin-shelled pecan as a template, performing PCR amplification using the primer pair for identifying thin-shelled pecan varieties according to claim 2, performing length and / or sequence analysis on the obtained PCR amplification product, and determining the thin-shelled pecan variety type based on the length and / or sequence characteristics; The thin-shell pecan varieties are the following 36 thin-shell pecan varieties: 'Oconee', 'Kiowa', 'Shawnee', 'Waco', 'Hopi', 'Forkert', 'Maramec', 'Mahan', 'Navaho', 'Desirable', 'Barton', 'Carter', 'Deerstand', 'Shepherd', 'Osage', 'Chetopa', 'Elliott', 'Major', 'Yates68', 'Posey', 'Colby', 'Mcmillan', 'Mohawk', 'Gloria Grande', 'Silverback', 'Graking', 'Lakota', 'Nacono', 'Houma', 'Creek', 'Kanza', 'Stuart', 'Jayhawk', 'Choctaw', 'Pawnee', and 'Greenriver'; The band sizes of 36 walnut varieties amplified using the primer pair are as follows:

Images