SSR molecular markers, methods and applications for identifying interspecific resources of Castanea in East Asia

Abstract

The present invention relates to the field of plant DNA molecular identification, and specifically, to SSR molecular markers, methods and applications for identifying interspecific resources of Castanea plants in East Asia. The SSR molecular markers for identifying interspecific resources of Castanea plants in East Asia include: SSR molecular marker CmSSR01, SSR molecular marker CmSSR02 and SSR molecular marker CmSSR03. The SSR molecular markers can effectively overcome the shortcomings that it is difficult to accurately distinguish chestnut from Castanea mollissima, Castanea henryi and Castanea japonica germplasm only through phenotypic characteristics during field sampling and seedling stages, and have broad application prospects in molecular identification of Castanea germplasm resources and genetic diversity analysis.

Description

Technical Field

[0001] The present invention relates to the field of plant DNA molecular identification, and in particular to SSR molecular markers, methods and applications for identifying interspecific resources of Castanea plants in East Asia. Background Art

[0002] Castanea Castanea ) has high nutritional content, low fat content, rich in riboflavin and multiple vitamins, has good edible value and medicinal value, and is an important dried fruit crop in the world. Today, China's chestnut production ranks first in the world. In 2019, my country's chestnut production exceeded 1.84 million tons, accounting for about 76.83% of the world's annual production. There are four species of Castanea plants distributed in East Asia, namely, Castanea henryi ( Castanea henryi (Skam) Rehd. et Wils.), Chinese chestnut ( Castanea mollissima Bl.)、Castan Castanea seguinii Dode) and Japanese chestnut ( Castanea crenata S. et Z.). Among them, Castanea henryi and Castanea truncatum are mainly distributed in the middle and lower reaches of the Yangtze River in China and the areas to the south. Castanea truncatum is an undeveloped resource, and the main cultivation areas of Castanea henryi are in southern Zhejiang and northern Fujian. The distribution of Japanese chestnut is relatively narrow, mainly distributed in the Korean Peninsula and the Japanese Islands, and it is mainly introduced in Liaoning, Shandong and other places in China.

[0003] The resources of Castanea genus in East Asia show rich genetic diversity in terms of tree vigor, leaf shape, scaly glands, nut size and the number of nuts in the thorny husk. Chinese chestnut is a deciduous tree with an open tree vigor, usually containing 3 nuts, mature leaves are elliptical, the leaf shape index is about 2.58, and the back of the leaves are covered with star-shaped hairs. Japanese chestnut, also known as Dandong chestnut, is a deciduous tree with a tree vigor similar to that of Chinese chestnut. It usually contains 3 nuts and the nuts are large, with a single grain weight of 15-30g. The mature leaves are lanceolate, the leaf shape index is about 3.40, and the back of the leaves are covered with short soft hairs and scaly glands. Castanea chinensis is a deciduous shrub or small tree, usually containing 3 nuts and the nuts are small, weighing about 1g, the mature leaves are elliptical or oblong, the leaf shape index is about 2.41, and the back of the leaves are covered with scaly glands. Castanea henryi is a large deciduous tree with a strong stem and solitary nuts. Mature leaves are oblong or ovate-lanceolate, with a leaf shape index of about 3.22. The back of the leaves is glabrous or occasionally has simple hairs, and a small amount of squamous glands can be seen on young leaves.

[0004] At present, Castanea resources are mainly identified and authenticated through phenotypic characteristics of plants. However, morphological characteristics are often affected by climate, habitat conditions, and natural hybrids in the same area, which leads to errors in subjective species identification. It is difficult to accurately identify them based on the differences in limited phenotypic data. Especially in the seedling stage of chestnuts, there is a phenomenon of atavism between chestnut and Castanea chinensis, Castanea henryi, and Castanea japonica germplasm resources. The leaf shapes are very similar, and the differences in botanical phenotypes are smaller. It is not until the chestnuts bloom and bear fruit that the four East Asian Castanea germplasms are identified through the number of fruits, tree vigor characteristics, and leaf shape index. This greatly limits the use of chestnut germplasms as parent materials to create new germplasms and breed new varieties, and restricts the efficient use of chestnut germplasm resources. Therefore, we urgently need an effective strategy for identifying chestnut germplasm resources to provide accurate materials for the future utilization of chestnut resources.

[0005] In the past few decades, researchers have developed many molecular markers and widely used them in the analysis of plant genetic diversity, such as restriction fragment length polymorphism (RFLP), random amplified polymorphic DNA (RAPD), amplified fragment length polymorphism (AFLP), simple sequence repeat (SSR) and three-generation marker single nucleotide polymorphism (SNP). At the same time, these molecular markers also provide accurate and reliable identification strategies for plant resources. Among them, SSR (simple sequence repeat, also known as microsatellite) is a tandem repeat sequence with a length of 1 to 6 bases and is widely distributed in the genomes of animals and plants. SSR has good interspecies versatility and specificity, and a small number of SSR marker combinations can provide high detection efficiency. In addition, SSR markers can be developed from the genome and transcriptome sequencing of species, with co-dominance, stability and repeatability, and have become an ideal molecular marker for the identification and identification of animal and plant germplasm resources. Its identification results are highly reliable and repeatable, and are not affected by experimental conditions and equipment, which is of great significance for the collection, preservation, evaluation and utilization of chestnut germplasm resources.

[0006] In view of this, the present invention is proposed. Summary of the invention

[0007] One aspect of the present invention relates to SSR molecular markers for identifying interspecific resources of Castanea plants in East Asia, including: SSR molecular marker CmSSR01, SSR molecular marker CmSSR02 and SSR molecular marker CmSSR03.

[0008] The SSR molecular markers can accurately identify Castanea henryi, Castanea chinensis, Castanea mollissima and Castanea japonica.

[0009] Another aspect of the present invention also relates to a method for identifying the germplasm resources of Castanea genus in East Asia, comprising the following steps:

[0010] (a) using the genomic DNA of the sample to be tested as a template and using the primers of the SSR molecular marker to perform a PCR amplification reaction;

[0011] (b) detecting the fragment size of the PCR amplification reaction product; if the amplification product of the CmSSR01 primer shows at least one of 121bp / 121bp DNA bands, 121bp / 124bp DNA bands or 124bp / 124bp DNA bands, the amplification product of the CmSSR02 primer shows at least two of 128bp / 128bp DNA bands, 128bp / 132bp DNA bands or 132bp / 132bp DNA bands, and the amplification product of the CmSSR03 primer shows at least one of 191bp / 191bp DNA bands, 196bp / 196bp DNA bands or 191bp / 196bp DNA bands, then the sample to be tested is identified as Castanea henryi;

[0012] If the amplification product of the CmSSR01 primers shows 115bp / 115bp DNA bands and / or 109bp / 115bp DNA bands, the amplification product of the CmSSR02 primers shows 138bp / 138bp DNA bands, and the amplification product of the CmSSR03 primers shows 181bp / 181bp DNA bands, the sample to be tested is identified as Castanea truncatula;

[0013] If the amplification product of the CmSSR01 primers shows 118bp / 124bp DNA bands, 118bp / 118bp DNA bands and 124bp / 124bp DNA bands, the amplification product of the CmSSR02 primers shows 140bp / 144bp DNA bands, 140bp / 148bp DNA bands, 148bp / 148bp DNA bands and 144bp / 144bp DNA bands, and the amplification product of the CmSSR03 primers shows at least one of 196bp / 196bp DNA bands, 199bp / 199bp DNA bands, 196bp / 199bp DNA bands, 196bp / 202bp DNA bands, 199bp / 202bp DNA bands or 202bp / 202bp DNA bands, then the sample to be tested is identified as chestnut;

[0014] If the amplification product of the CmSSR01 primers shows at least one of 115bp / 115bp DNA bands, 115bp / 118bp DNA bands or 118bp / 118bp DNA bands, the amplification product of the CmSSR02 primers shows 128bp / 128bp DNA bands, 140bp / 140bp DNA bands and 128bp / 140bp DNA bands, and the amplification product of the CmSSR03 primers shows 205bp / 205bp DNA bands, then the sample to be tested is identified as Japanese chestnut.

[0015] The method for identifying the germplasm resources of the genus Castanea in East Asia is simple to operate, has good reproducibility and high accuracy, and can effectively overcome the disadvantage that it is difficult to accurately distinguish chestnut from Castanea mollissima, Castanea henryi and Castanea japonica germplasm only by phenotypic characteristics during field sampling and seedling stages, and has a very broad application prospect in the molecular identification of Castanea germplasm resources and genetic diversity analysis.

[0016] Another aspect of the present invention also relates to a kit, comprising the SSR molecular markers for identifying interspecific resources of Castanea plants in East Asia.

[0017] The kit can accurately and quickly distinguish and identify Castanea henryi, Castanea chinensis, Castanea mollissima and Castanea japanensis.

[0018] Another aspect of the present invention also relates to the application of the kit in identifying the germplasm resources of Castanea genus in East Asia.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] (1) The SSR molecular markers for identifying interspecific resources of Castanea plants in East Asia provided by the present invention can accurately identify Castanea henryi, Castanea chinensis, Castanea mollissima and Castanea japonica.

[0021] (2) The method for identifying East Asian Castanea germplasm resources provided by the present invention is simple to operate, has good reproducibility and high accuracy, and can effectively overcome the shortcoming that it is difficult to accurately distinguish chestnut from Castanea mollissima, Castanea henryi and Castanea japonica germplasm only by phenotypic characteristics during field sampling and seedling stages. It has a broad application prospect in the molecular identification of Castanea germplasm resources and genetic diversity analysis.

[0022] (3) The kit provided by the present invention can accurately and quickly distinguish and identify Castanea henryi, Castanea mollissima, Castanea mollissima and Castanea japonica. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0024] Figure 1 Schematic diagram of the target fragment obtained by primer CmSSR03 in interspecies samples using capillary electrophoresis;

[0025] Figure 2 The clustering dendrogram of 123 Castanea species was constructed using three SSR primers. DETAILED DESCRIPTION

[0026] The technical scheme of the present invention will be clearly and completely described below in conjunction with the accompanying drawings and specific embodiments, but it will be understood by those skilled in the art that the following described embodiments are part of embodiments of the present invention, rather than all embodiments, and are only used to illustrate the present invention, and should not be considered as limiting the scope of the present invention. Based on the embodiments in the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative work are within the scope of protection of the present invention. If specific conditions are not specified in the embodiments, they are carried out according to normal conditions or conditions recommended by the manufacturer. If the manufacturer is not specified in the reagents or instruments used, they are all conventional products that can be purchased commercially.

[0027] One aspect of the present invention relates to SSR molecular markers for identifying interspecific resources of Castanea plants in East Asia, including: SSR molecular marker CmSSR01, SSR molecular marker CmSSR02 and SSR molecular marker CmSSR03.

[0028] The SSR molecular marker can accurately identify Castanea henryi, Castanea chinensis, Castanea mollissima and Castanea japonica.

[0029] Preferably, the primer nucleotide sequence of the SSR molecular marker CmSSR01 is as follows:

[0030] Forward primer: 5′-CAAGTCCGATCCTTCCTCTG-3′;

[0031] Reverse primer: 5′-AGCTGGGTTTTGAGTAGCGA-3′.

[0032] Preferably, the primer nucleotide sequence of the SSR molecular marker CmSSR02 is as follows:

[0033] Forward primer: 5′-ACGCCTTCAGTTGCTGTTTC-3′;

[0034] Reverse primer: 5′-CAACGGTCTTACCCTTTGGA-3′.

[0035] Preferably, the primer nucleotide sequence of the SSR molecular marker CmSSR03 is as follows:

[0036] Forward primer: 5′-CAGCATCAGCACTCGTTCA-3′;

[0037] Reverse primer: 5′-GGGATTGAGAGGATGAAGCA-3′.

[0038] Another aspect of the present invention also relates to a method for identifying the germplasm resources of Castanea genus in East Asia, comprising the following steps:

[0039] (a) using the genomic DNA of the sample to be tested as a template and using the primers of the SSR molecular marker to perform a PCR amplification reaction;

[0040] (b) detecting the fragment size of the PCR amplification reaction product; if the amplification product of the CmSSR01 primer shows at least one of 121bp / 121bp DNA bands, 121bp / 124bp DNA bands or 124bp / 124bp DNA bands, the amplification product of the CmSSR02 primer shows at least two of 128bp / 128bp DNA bands, 128bp / 132bp DNA bands or 132bp / 132bp DNA bands, and the amplification product of the CmSSR03 primer shows at least one of 191bp / 191bp DNA bands, 196bp / 196bp DNA bands or 191bp / 196bp DNA bands, then the sample to be tested is identified as Castanea henryi;

[0041] If the amplification product of the CmSSR01 primers shows 115bp / 115bp DNA bands and / or 109bp / 115bp DNA bands, the amplification product of the CmSSR02 primers shows 138bp / 138bp DNA bands, and the amplification product of the CmSSR03 primers shows 181bp / 181bp DNA bands, the sample to be tested is identified as Castanea truncatula;

[0042] If the amplification product of the CmSSR01 primers shows 118bp / 124bp DNA bands, 118bp / 118bp DNA bands and 124bp / 124bp DNA bands, the amplification product of the CmSSR02 primers shows 140bp / 144bp DNA bands, 140bp / 148bp DNA bands, 148bp / 148bp DNA bands and 144bp / 144bp DNA bands, and the amplification product of the CmSSR03 primers shows at least one of 196bp / 196bp DNA bands, 199bp / 199bp DNA bands, 196bp / 199bp DNA bands, 196bp / 202bp DNA bands, 199bp / 202bp DNA bands or 202bp / 202bp DNA bands, then the sample to be tested is identified as chestnut;

[0043] If the amplification product of the CmSSR01 primers shows at least one of 115bp / 115bp DNA bands, 115bp / 118bp DNA bands or 118bp / 118bp DNA bands, the amplification product of the CmSSR02 primers shows 128bp / 128bp DNA bands, 140bp / 140bp DNA bands and 128bp / 140bp DNA bands, and the amplification product of the CmSSR03 primers shows 205bp / 205bp DNA bands, then the sample to be tested is identified as Japanese chestnut.

[0044] The method for identifying East Asian Castanea germplasm resources is simple to operate, has good reproducibility and high accuracy, and can effectively overcome the shortcoming that it is difficult to accurately distinguish chestnut from Castanea mollissima, Castanea henryi and Castanea japonica germplasm only by phenotypic characteristics during field sampling and seedling stages, and has broad application prospects in molecular identification of Castanea germplasm resources and genetic diversity analysis.

[0045] The present invention adopts capillary electrophoresis detection technology to detect the site information of chestnut resources. The specific reading band method is described as follows: 1. Chestnut is a diploid plant, and the number of target fragments is 1 or 2, that is, the capillary electrophoresis shows a single peak (homozygote) or a double peak (heterozygote); 2. The reading band method adopts A / B (heterozygote) or A / A (homozygote), and the letters represent the target fragment size of each chromosome. The homozygote reading band - for example, the CmSSR01 primer amplification result has only one peak at the 121bp position, indicating that the SSR fragments from the two chromosomes of the parents in the test material are consistent, and then it is read as 121bp / 121bp, and the heterozygote reading band - for example, the CmSSR01 primer amplification result has a peak at each of the 121bp and 124bp positions, indicating that the SSR fragments from the two chromosomes of the parents in the test material are inconsistent, and then it is read as 121bp / 124bp.

[0046] Preferably, the genomic DNA of the sample to be tested comes from at least one of bark, leaves or roots.

[0047] Preferably, the primers of the SSR molecular markers are fluorescently labeled. Different primers of different SSR molecular markers are respectively fluorescently labeled.

[0048] Preferably, the type of fluorescent label includes: one of FAM, HEX, ROX and TAMRA.

[0049] There are two methods for preparing primers for fluorescently labeled SSR molecular markers: one is to directly modify the primer sequence with different types of fluorescent substances; the other is to add a universal primer M13 (-CACGACGTTGTAAAACGAC-) to the 5' end of each primer and only modify M13 with different types of fluorescent substances.

[0050] Preferably, the genus Castanea includes Castanea henryi, Castanea chinensis, Castanea mollissima and Castanea japonica.

[0051] GMATA software was used to identify and verify SSRs in the published whole genome sequence of chestnut (http: / / castaneadb.net / ). The repetition number of the repeated sequence and the location information of the chromosome were filtered, and 330 SSRs with high potential were preliminarily identified. Primer 3 software was used to design primers for related SSR molecular markers. Subsequently, 12 chestnut resources distributed in 11 provinces were used as the first screening materials for polymorphism screening, and the polymorphism of primers was detected on 8% polyacrylamide gel. Finally, 18 pairs of primers with good polymorphism and clear main bands were screened as candidates. Subsequently, the inventors collected 100 chestnut resources from four species, including Chinese chestnut, Castanea henryi, Castanea chinensis and Japanese chestnut, across the country as the next screening and verification materials. Among them, there were 25 Chinese chestnuts, 20 Castanea henryi, 25 Castanea chinensis and 25 Japanese chestnuts. Using higher-resolution capillary electrophoresis detection technology, three pairs of SSR primers with strong interspecies specificity and conservation were finally screened out. These three pairs of primers can accurately identify chestnut resources such as Castanea mollissima, Castanea henryi, Castanea truncatula and Castanea japonica.

[0052] Another aspect of the present invention also relates to a kit, comprising the SSR molecular markers for identifying interspecific resources of Castanea plants in East Asia.

[0053] Another aspect of the present invention also relates to the use of the kit in identifying Castanea germplasm resources in East Asia.

[0054] Example

[0055] 1. Test plant materials

[0056] A total of 123 test materials were collected, of which 74 wild resources were surveyed and collected during 2019-2021 (25 wild Castanea resources, 25 wild Castanea mollissima resources and 24 wild Castanea henryi resources), 24 cultivated chestnut varieties (lines) were collected from the National Chestnut Germplasm Resource Garden in Tai'an City, Shandong Province, and 25 cultivated Japanese chestnut resources were collected from the Germplasm Resource Garden of Liaoning Provincial Economic Forestry Research Institute (see Table 1 for details). Ten young leaves were collected from each material, treated with liquid nitrogen and stored in a -80℃ refrigerator.

[0057] 2. DNA Extraction

[0058] The modified CTAB method was used to extract chestnut gDNA:

[0059] (1) Grind about 500 mg of young leaves with liquid nitrogen, pour into a 2 mL centrifuge tube, add 1 mL of 2% CTAB extract containing 2% β-mercaptoethanol preheated to 65°C, and incubate in a 65°C water bath for 30 min, shaking every 10 min.

[0060] (2) Centrifuge at room temperature, 12,000 rpm, for 10 min. Transfer the supernatant to a new 2 ml centrifuge tube, add an equal volume of chloroform and isoamyl alcohol (24:1), shake gently for 10 min, and centrifuge at 12,000 rpm for 5 min.

[0061] (3) Pipette the supernatant into a new 2 mL centrifuge tube and add 6 μL RNase. Shake well and let stand for 10 min.

[0062] (4) Pipette the supernatant into a new 2 mL centrifuge tube, add an equal volume of chloroform and isoamyl alcohol (24:1), shake gently for 10 min, and centrifuge at 12,000 rpm for 5 min;

[0063] (5) Transfer the supernatant into a new 1.5 mL centrifuge tube, add two volumes of pre-cooled -20 °C anhydrous ethanol to precipitate the DNA, and centrifuge at 12000 rpm for 1 min.

[0064] (6) Add 75% and 100% ethanol precooled at -20°C, rinse, and centrifuge at 12,000 rpm for 1 min.

[0065] (7) Add 100 μL TE buffer to dissolve;

[0066] (8) DNA quality was detected by 1.5% agarose gel electrophoresis and concentration was determined by spectrophotometer.

[0067] 3. Capillary electrophoresis SSR-PCR amplification and detection

[0068] PCR amplification uses a 20 μL reaction system: it contains 1 μL template DNA (20-50 ng / μL), 10 μL 2× TaqPCR Master Mixes, 0.1 μL forward primer (10 uM), 0.3 μL reverse primer (10 uM), 0.2 μL fluorescently labeled (FAM, HEX, ROX or TAMRA) M13 primer (10 uM) and 8.4 μL ddH2O. The reaction program is: 94℃ pre-denaturation for 3 min, 94℃ denaturation for 30 s, 56℃ annealing for 30 s, 72℃ extension for 30 s, 35 cycles, and finally 72℃ extension for 10 min, and storage at 10℃.

[0069] The PCR amplification products of fluorescent SSR primers were combined. Then, the fragment position information in the mixture was detected using a capillary electrophoresis instrument or sequencer, and the specific fragment size information of the allele site was read using Gene Marker v 2.2.0 software (Soft Genetics LLC, StateCollege, PA, USA).

[0070] 4. Analysis of electrophoresis results

[0071] The above primers were used to statistically analyze the PCR amplification products of 123 chestnut germplasm resources in the form of a matrix table. Figure 1 The figure shows a schematic diagram of the target fragment obtained by using the capillary electrophoresis detection technology with primer CmSSR03. The gray peak is the internal standard peak, and the black peak is the target fragment peak. For the specific site reading method, please refer to the information detection method in the specific implementation method section. Figure 1 -A1 is the electrophoresis result of the Castanea henryi sample, Figure 1 -A2, Figure 1 -A3 and Figure 1 -A4 is the electrophoresis result of the chestnut sample. Figure 1 -A5 and Figure 1 -A6 is the electrophoresis result of the Japanese chestnut sample tested, Figure 1 -A7 is the electrophoresis result legend of the tested Castanea truncatula sample. Table 1 is the result statistics of each resource, and the attribution of the samples is determined based on the site groups and differences of the target bands of different samples in different primers. Figure 1 The X-axis is the fragment value, the Y-axis is the peak height value, the gray is the internal standard peak graph, and the internal standard is the marker value in capillary electrophoresis (the GS500 marker gradient range is 0~500bp); the black is the target fragment peak graph.

[0072] Table 1 Sample information and SSR loci of 123 Castanea species in East Asia

[0073]

[0074] It can be seen from Table 1 that in the Castanea henryi resources, the molecular marker CmSSR01 showed three DNA bands of 121bp / 121bp (homozygous) or 121bp / 124bp or 124bp / 124bp (homozygous); the molecular marker CmSSR02 showed two DNA bands of 128bp / 128bp (homozygous) or 128bp / 132bp or 132bp / 132bp (homozygous); the molecular marker CmSSR03 showed three DNA bands of 191bp / 191bp, 196bp / 196bp or 191bp / 196bp.

[0075] In the Castanea resources, the molecular marker CmSSR01 showed two DNA bands of 115bp / 115bp (homozygous) or 109bp / 115bp; the molecular marker CmSSR02 showed one DNA band of 138bp / 138bp (homozygous), and the molecular marker CmSSR03 showed one DNA band of 181bp / 181bp.

[0076] In the chestnut resources, the molecular marker CmSSR01 showed three DNA bands of 118bp / 118bp or 118bp / 121bp or 118bp / 124bp; the molecular marker CmSSR02 showed three DNA bands of 140bp / 140bp, 140bp / 148bp or 144bp / 144bp (homozygous) or 148bp / 148bp (homozygous) and 140bp / 144bp; CmSSR03 showed six DNA bands of 196bp / 196bp or 199bp / 199bp or 196bp / 199bp or 196bp / 202bp or 199bp / 202bp or 202bp / 202bp.

[0077] In Japanese chestnut resources, the molecular marker CmSSR01 showed three DNA bands of 115bp / 115bp, 115bp / 118bp or 118bp / 118bp; the molecular marker CmSSR02 showed three DNA bands of 128bp / 128bp, 140bp / 140bp and 128bp / 140bp; and the molecular marker CmSSR03 showed 205bp / 205bp.

[0078] The experimental results show that the three molecular markers have specific bands in various inter-species resources, and inter-species resources rarely share bands, and there are obvious differences between species. The cluster tree results show that the chestnut, castanea henryi, Japanese chestnut and castanea resources are each independently clustered ( Figure 2), in the figure, Cluster1 represents chestnut, Cluster2 represents Japanese chestnut, Cluster3 represents Castanea chinensis, and Cluster4 represents Castanea henryi, indicating that the three pairs of SSR primers can effectively distinguish the four Castanea plant resources. Any one of the three fluorescent SSR molecular markers can distinguish the germplasm resources of Chinese chestnut, Castanea chinensis, Castanea henryi and Japanese chestnut, but considering that there may be more and larger chestnut resources in practical applications that need to be detected, especially the phenomenon of natural introgression between wild Castanea materials, therefore, in order to ensure the accuracy and scientificity of identification, the present invention recommends using three pairs of SSR primers to perform species detection on the samples to be tested. If the specific peak graphs described above appear at the same time, it can be determined as various internal resources; if the peak graphs with mixed sites appear in the CmSSR01 and CmSSR03 primers, it can be determined as an interspecific hybrid resource.

[0079] Although the present invention has been illustrated and described with specific embodiments, it should be appreciated that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit it. Those skilled in the art should understand that the technical solutions described in the above embodiments may be modified, or some or all of the technical features thereof may be replaced by equivalents without departing from the spirit and scope of the present invention. However, these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present invention. Therefore, this means that all such replacements and modifications within the scope of the present invention are included in the appended claims.

Claims

1. A method for identifying Castanea germplasm resources in East Asia, characterized in that: The following steps are involved: (a) Using the genomic DNA of the sample to be tested as a template, the PCR amplification reaction is performed using primers of SSR molecular markers; (b) detecting the fragment size of the PCR amplification reaction product; if the amplification product of the CmSSR01 primer shows at least one of 121bp / 121bp DNA bands, 121bp / 124bp DNA bands or 124bp / 124bp DNA bands, the amplification product of the CmSSR02 primer shows at least two of 128bp / 128bp DNA bands, 128bp / 132bp DNA bands or 132bp / 132bp DNA bands, and the amplification product of the CmSSR03 primer shows at least one of 191bp / 191bp DNA bands, 196bp / 196bp DNA bands or 191bp / 196bp DNA bands, then the sample to be tested is identified as Castanea henryi; If the amplification product of the CmSSR01 primers shows 115bp / 115bp DNA bands and / or 109bp / 115bp DNA bands, the amplification product of the CmSSR02 primers shows 138bp / 138bp DNA bands, and the amplification product of the CmSSR03 primers shows 181bp / 181bp DNA bands, the sample to be tested is identified as Castanea truncatula; If the amplification product of the CmSSR01 primers shows 118bp / 124bp DNA bands, 118bp / 118bp DNA bands and 124bp / 124bp DNA bands, the amplification product of the CmSSR02 primers shows 140bp / 144bp DNA bands, 140bp / 148bp DNA bands, 148bp / 148bp DNA bands and 144bp / 144bp DNA bands, and the amplification product of the CmSSR03 primers shows at least one of 196bp / 196bp DNA bands, 199bp / 199bp DNA bands, 196bp / 199bp DNA bands, 196bp / 202bp DNA bands, 199bp / 202bp DNA bands or 202bp / 202bp DNA bands, then the sample to be tested is identified as chestnut; If at least one of 115bp / 115bp DNA bands, 115bp / 118bp DNA bands or 118bp / 118bp DNA bands appears in the amplification product of the CmSSR01 primers, 128bp / 128bp DNA bands, 140bp / 140bp DNA bands and 128bp / 140bp DNA bands appear in the amplification product of the CmSSR03 primers, and 205bp / 205bp DNA bands appear in the amplification product of the CmSSR03 primers, the sample to be tested is identified as Japanese chestnut; The SSR molecular markers include: SSR molecular marker CmSSR01, SSR molecular marker CmSSR02 and SSR molecular marker CmSSR03; The primer nucleotide sequence of the SSR molecular marker CmSSR01 is as follows: Forward primer: 5′-CAAGTCCGATCCTTCCTCTG-3′; Reverse primer: 5′-AGCTGGGTTTTGAGTAGCGA-3′; The primer nucleotide sequence of the SSR molecular marker CmSSR02 is as follows: Forward primer: 5′-ACGCCTTCAGTTGCTGTTTC-3′; Reverse primer: 5′-CAACGGTCTTACCCTTTGGA-3′; The primer nucleotide sequence of the SSR molecular marker CmSSR03 is as follows: Forward primer: 5′-CAGCATCAGCACTCGTTCA-3′; Reverse primer: 5′-GGGATTGAGAGGATGAAGCA-3′; The East Asian Castanea genus includes Castanea henryi, Castanea chinensis, Castanea mollissima and Castanea japonica.

2. The method for identifying Castanea germplasm resources in East Asia according to claim 1, characterized in that: The genomic DNA of the sample to be tested comes from at least one of bark, leaves or roots.

3. The method for identifying the germplasm resources of Castanea genus in East Asia according to claim 1, characterized in that: The primers of the SSR molecule marker are fluorescently labeled.

4. The method for identifying Castanea germplasm resources in East Asia according to claim 3, characterized in that: The type of fluorescent label includes: one of FAM, HEX, ROX and TAMRA.

5. Application of the method for identifying East Asian Castanea germplasm resources as described in any one of claims 1 to 4 in identifying East Asian Castanea germplasm resources.

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