A molecular marker, primers, reagent kit, and application for identifying the natural astringency removal trait of Chinese persimmons.

Abstract

The present invention relates to the technical field of molecular biology, and specifically discloses a molecular marker, primer, kit and application for identifying the natural astringency-free trait of Chinese sweet persimmon, wherein the sequence of the molecular marker is shown in SEQ ID NO.3. The molecular marker for identifying Chinese sweet persimmon provided by the present invention has a sequence as shown in SEQ ID NO.3, and the primer for amplifying the molecular marker is shown in SEQ ID NO.1-2. The molecular marker and primer are used to identify the natural astringency-free trait of Diospyros plants, and it is found that the identification accuracy rate reaches 100%.

Description

Technical Field

[0001] The invention relates to the technical field of molecular biology, and in particular to a molecular marker, a primer, a kit and an application thereof for identifying the natural astringency-free trait of Chinese sweet persimmon. Background Art

[0002] Persimmon (Diospyros kaki Thunb., 2n=6x=90; a few varieties 2n=9x=135) is native to my country and is a representative species of Diospyros genus cultivated as a fruit tree. Its traditional production areas are in East Asia, such as China, South Korea and Japan. Persimmon fruit accumulates a large amount of persimmon tannin (persimmon tannin or kakitannin), also known as "proanthocyanidins" (PAs) in the vacuole of its specialized flesh cells, "tannin cells". Its average molecular weight is 14,000Da, which is 3-5 times that of ordinary plant tannins. According to its solubility in alcohol solution, it can be divided into soluble and insoluble tannins. Soluble tannins produce a strong sense of astringency and dryness (commonly known as "astringency") after combining with oral salivary proteins. The critical content of astringency is 0.1%. When fully sweet persimmons are ripe, they can be naturally de-astringent on the tree and can be eaten crisply without de-astringent treatment; the soluble tannin content of non-fully sweet persimmons is still far higher than 0.1% until the fruit is ripe, and they need to be artificially de-astringented before they can be eaten fresh. Artificial de-astringency not only increases production costs, but also makes the fruit soft and inconvenient to transport after de-astringency. Incomplete de-astringency also affects the commercial quality.

[0003] According to whether the fruit can naturally remove astringency during maturity and the genetic characteristics of the trait, persimmon varieties are divided into fully sweet persimmons (pollination-constant and non-astringent, PCNA) whose sweetness and astringency traits are inherited by qualitative traits and non-complete sweet persimmons (non-PCNA) whose sweetness and astringency traits are inherited by quantitative traits. Fully sweet persimmons can be further divided into fully sweet persimmons of Chinese origin (Chinese PCNA, referred to as "Chinese sweet persimmon" or "C-PCNA") whose natural astringency removal trait is controlled by a dominant gene (CPCNA) and fully sweet persimmons of Japanese origin (Japanese PCNA, referred to as "Japanese sweet persimmon" or "J-PCNA") whose natural astringency removal trait is controlled by a recessive gene (ast); according to the role of seeds in removing astringency from the fruit, non-complete sweet persimmons can be further divided into three types: incomplete sweet persimmons (pollination-variant and non-astringent, PVNA) whose seeds have a greater effect on removing astringency, incomplete astringent persimmons (pollination-variant and astringent, PVA) whose seeds have a relatively smaller effect on removing astringency, and fully astringent persimmons (pollination-constant and astringent, PCA) whose seeds have almost no effect on removing astringency. Japanese sweet persimmons have poor soil adaptability, especially some varieties are not compatible or incompatible with the rootstock commonly used in traditional production areas of my country - D.lotus L. in grafting. In addition, its natural astringency is controlled by recessive genes, which leads to frequent cross breeding between sweet persimmon parents. Long-term inbreeding has shown serious decline in tree vigor, yield and single fruit weight. Therefore, the genetic improvement of sweet persimmons urgently needs to break through the "bottleneck" of narrow genetic diversity of breeding parents and long breeding cycle.

[0004] It has been confirmed that the natural astringency-free trait of Japanese sweet persimmon is controlled by recessive genes. In order to avoid aggravating inbreeding depression, it is necessary to crossbreed with incomplete sweet persimmons. However, when crossbreeding with incomplete sweet persimmons, only a very small number of sweet persimmon individuals may appear in the F1 generation. Even if backcrossing is performed with Japanese sweet persimmons, the sweet persimmon individuals in BC1 only account for about 15% (hexaploid genetic characteristics). Therefore, the genetic improvement cycle of sweet persimmons is longer than that of other fruit trees. When the Chinese sweet persimmon 'Luotian Sweet Persimmon' is used as a parent and crossbred with Japanese sweet persimmons or incomplete sweet persimmons, the sweet persimmon individuals in the F1 generation can account for about 50%, indicating that the natural astringency-free trait of Chinese sweet persimmons is controlled by a dominant single gene. It can be seen that using Chinese sweet persimmons including 'Luotian Sweet Persimmon' as breeding parents can not only obtain more obvious hybrid advantages to avoid inbreeding degeneration, but also shorten the persimmon breeding cycle. The Dabie Mountain area in central my country contains a series of Chinese sweet persimmon resources such as 'Luotian Sweet Persimmon'. These germplasms have the potential for rapid creation of new sweet persimmon varieties with independent intellectual property rights. However, its small fruit, many seeds, and tall and upright tree body limit its application value as a breeding parent. Identifying and cloning the natural astringency-free gene of Chinese sweet persimmon is expected to change the current situation of sweet persimmon breeding, accelerate the breeding process of sweet persimmon, and enhance my country's research status in sweet persimmon genetic breeding. The identification and application of the natural astringency-free gene of Chinese sweet persimmon is the key to the current and future sweet persimmon research, and it is also an important tool and main measure to break through the "bottleneck" of complete sweet persimmon genetic breeding. Persimmon trees have a long juvenile period. It takes a long time and high cost to breed new germplasm obtained through hybridization from seedling breeding to breeding of excellent strains after fruiting. Therefore, it is necessary to develop molecular markers related to the natural astringency-free trait of Chinese sweet persimmon, screen out new germplasm of complete sweet persimmon in the seedling stage, and quickly produce fruit through high grafting, thereby saving time and cost, accelerating the breeding process, and improving the efficiency of germplasm innovation and utilization.

[0005] The existing molecular marker RO2 gene amplification fragment in the prior art is short (about 200bp) and contains primer dimers, whose size is close to the position of the target fragment, making it difficult to distinguish. In addition, the RO2 marker has poor repeatability, and even the same sample or the same DNA template has inconsistent identification in different experimental batches. The above shortcomings of the RO2 marker limit its application in actual breeding practice, and it is urgent to develop new alternative markers. Summary of the invention

[0006] In order to develop a method for identifying Chinese sweet persimmons and Chinese astringent persimmons, the present invention provides a molecular marker, primer, kit and application for identifying the natural astringency-free trait of Chinese sweet persimmons. The present invention uses the primers shown in SEQ ID NO.1-2 and Diospyros plant DNA as a template for amplification. When a product of 2089 bp shown in SEQ ID NO.3 is amplified, it is identified as a Chinese sweet persimmon, otherwise it is a Chinese astringent persimmon, and the identification success rate is 100%.

[0007] The present invention provides a molecular marker for identifying Chinese sweet persimmon, the molecular marker is CPCNA-2k, and its nucleotide sequence is shown in SEQ ID NO. 3. The molecular marker CPCNA-2k is 2089 bp in length, which is significantly longer than the known molecular marker RO2 (about 200 bp), and the molecular marker CPCNA-2k with a length of 2089 bp has higher stability.

[0008] The present invention also provides primers for amplifying the molecular markers, the nucleotide sequences of the primers are shown in SEQ ID NO. 1-2. When the primers for amplifying CPCNA-2k are identified, no primer dimers are generated, the amplified bands are clear, and the identification success rate is 100%.

[0009] The invention also provides a kit containing the primers.

[0010] Furthermore, the kit also includes 2×Taq PCR Master Mix and ddH 2 O.

[0011] The present invention also provides an application of the molecular marker, the primer or the kit in identifying Chinese sweet persimmons and Chinese astringent persimmons.

[0012] Further, the following steps are included:

[0013] Extraction of Diospyros DNA;

[0014] Using the Diospyros plant DNA as a template and the nucleotide fragments shown in SEQ ID NO.1-2 as primers for PCR amplification;

[0015] The products obtained by PCR amplification were detected by electrophoresis. When a product of 2089 bp was amplified, it was considered to be a Chinese sweet persimmon. If a product of 2089 bp was not amplified, it was a Chinese astringent persimmon.

[0016] Furthermore, the PCR amplification program was as follows: pre-denaturation at 94°C for 3 min, denaturation at 94°C for 30 s, annealing at 54°C for 30 s, extension at 72°C for 1 min 15 s, a total of 35 cycles of denaturation, annealing and extension, and finally extension at 72°C for 5 min.

[0017] Furthermore, the PCR amplification system was: 10 μL, the reaction composition was 5 μL of 2×Taq PCR Master Mix, 0.25 μL of 10 μM / L upstream primer and downstream primer, 3.5 μL of ddHO 2 O, genomic DNA template is 90-180ng, and ddH 2 Add 3% HO to make up to 10 μL.

[0018] Furthermore, Chinese sweet persimmons include: Luotian sweet persimmon, small fruit sweet persimmon, Eshi No. 1, male plant No. 1, male plant No. 2, and male plant No. 8.

[0019] Furthermore, Chinese astringent persimmons include: ox-eye persimmon, Gongcheng water persimmon, black persimmon, millstone persimmon and Fuping pointed persimmon.

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] 1. The present invention provides a molecular marker CPCNA-2k for identifying Chinese sweet persimmon, the sequence of which is shown in SEQ ID NO.3, and the primers for amplifying the molecular marker are shown in SEQ ID NO.1-2. The molecular marker and primers are used to identify the natural astringency-free trait of Diospyros plants, and the identification accuracy rate reaches 100%.

[0022] 2. The molecular marker amplification fragment (2089bp) of the natural astringency-free trait of Chinese sweet persimmon of the present invention is longer than the amplification fragment of the existing marker (about 200bp), which is easier to distinguish and the amplification effect is more stable. The annealing temperature (54°C) of the upstream and downstream primers developed based on the molecular marker is consistent and there are no dimers. The annealing temperature is designed according to the sequence characteristics of the primer itself. 54°C is a temperature suitable for this primer. At this temperature, the primer amplification effect is better, the amplification efficiency of the target band is higher, and the primer dimer is less formed; the dimer will appear similar to a band in the electrophoresis gel. If more dimers are formed, it will affect the judgment of the target fragment.

[0023] 3. The primers and PCR amplification conditions designed by the present invention reduce the formation of dimers to the greatest extent and improve the accuracy of identification of target fragments; the juvenile period of persimmon trees is relatively long, and under the conditions of suitable water and fertilizer conditions, it usually takes 3 years for them to bloom and bear fruit. The molecular markers related to the natural astringency-free trait of Chinese sweet persimmons are of great significance for the identification of complete sweet persimmons in Chinese sweet persimmons and Chinese sweet persimmon hybrid offspring. The complete sweet persimmons can be separated in the seedling stage, and there is no need to wait until the results are found many years later before culling them, which saves time and cost and has broad application prospects.

[0024] Experimental verification found that the molecular marker CPCNA-2k provided by the present invention co-segregates with sweet and astringent traits in molecular genetics, with an identification accuracy rate of 100%, and can be widely used in early molecular marker-assisted breeding with complete sweet persimmon as the breeding target, and can be simply, quickly and efficiently applied to the breeding practice of Diospyros plants. It enriches the types of molecular markers for the identification of the natural astringency-free trait of persimmon trees in my country. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0026] Figure 1 The present invention is a method for early identification of the natural astringency-free trait of Chinese sweet persimmon by molecular markers, and is a PCR amplification electrophoresis diagram of detecting 6 materials with natural astringency-free trait of Chinese sweet persimmon and 5 materials of Chinese astringent persimmon.

[0027] Figure 2-Figure 4 All of them are PCR amplification electrophoresis diagrams of the original RO2 marker method for early identification of the natural astringency-free trait of Chinese sweet persimmon, which was used to detect 6 materials with natural astringency-free trait of Chinese sweet persimmon and 5 materials of Chinese astringent persimmon.

[0028] Figure 5 The present invention is a method for early identification of the natural astringency-free trait of Chinese sweet persimmon by molecular markers, and the electrophoresis diagram of PCR amplification is performed on 7 materials with natural astringency-free trait of fruiting hybrid offspring and 5 materials with astringent persimmons. DETAILED DESCRIPTION

[0029] The specific embodiments of the present invention are described in detail below, but it should be understood that the protection scope of the present invention is not limited by the specific embodiments. Based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in this field without creative work belong to the scope of protection of the present invention. The experimental methods described in the embodiments of the present invention are conventional methods unless otherwise specified, and the materials, reagents, etc. used in the following embodiments can be obtained from commercial channels unless otherwise specified.

[0030] Example 1: A molecular marker for early identification of the natural astringency-free trait of Chinese sweet persimmon.

[0031] The present invention designs marker primers based on the segment associated with CPCNA (chr13B:35748961-35751050) on chromosome 13 where the CPCNA genome is located in the Chinese sweet persimmon genome (NCBI SRA database BioProject ID No. PRJNA810977), and develops a molecular marker CPCNA-2k that can be used to identify the natural astringency-free trait of Chinese sweet persimmon.

[0032] Wherein: the upstream primer sequence is: 5'-TCACGAAGAAAGTCTACGATG-3', 21 bp, as shown in SEQ ID No. 1 in the sequence list.

[0033] The downstream primer sequence is: 5'-TCAGCATCTGTGCCACTAACC', 21 bp as shown in SEQ ID No. 2 in the sequence table.

[0034] According to the characteristics of this molecular marker primer, a suitable annealing temperature and other common PCR amplification conditions were designed.

[0035] Example 2: The molecular markers provided in Example 1 were used to conduct early identification of the natural astringency-free traits of 6 materials with known traits that can naturally remove astringency and 5 materials with known traits of astringent persimmon.

[0036] (1) The modified CTAB method was used to extract DNA from individual Diospyros plants;

[0037] ① Weigh 50 mg of fresh plant leaves, grind them in liquid nitrogen, and transfer them to a 2 mL centrifuge tube.

[0038] ② Prepare the extract immediately before use, and prepare Solution A and Solution B. 100mL Solution A includes 10mL 1mol·L -1 Tris-HCl (PH=8.0), 1mL 0.5mol·L -1 EDTA-Na 2 , 5mL 5mol·L -1 NaCl, 2g of pvp-4.0 powder, and finally 84mL of water. 100mL of liquid B includes 10mL of 1mol·L -1 Tris-HCl (PH=8.0), 10mL 0.5mol·L -1 EDTA-Na 2 , 30mL 5mol·L - 1 NaCl, 2g CTAB solid powder, 1g pvp-4.0 powder, and finally add 50mL water.

[0039] ③ After grinding, open the lid, add 1 mL of liquid A, and place on ice for 15 min, inverting 2-3 times during the process. Centrifuge at 12000g for 10 min, discard the supernatant; add 1 mL of liquid A again, and repeat this step.

[0040] ④ After the B solution is heated in a water bath at 65°C, it is boiled in a microwave oven and mercaptoethanol is added to the B solution at a ratio of 1:50; 700 μL of B solution is added to the sample, shaken well, heated in a water bath at 65°C for 1 hour, shaken every 15 minutes, and centrifuged at 12000g for 10 minutes.

[0041] ⑤ Take a new 2mL centrifuge tube, add 600μL of chloroform:isoamyl alcohol (24:1) mixture in a fume hood, invert and mix for 10 minutes, and centrifuge at 12000g for 10 minutes; take a new 2mL centrifuge tube, aspirate the upper supernatant of the layers (upper, middle and lower layers), add chloroform:isoamyl alcohol (24:1) mixture, and repeat this step.

[0042] ⑥ Take a new 1.5mL centrifuge tube, aspirate the upper supernatant, add 500μL isopropanol (pre-cooled at -20℃ 30min in advance), shake well, and precipitate at -20℃ for 1-2h; centrifuge at 11000g for 5min, discard the supernatant, and a transparent white precipitate can be seen at the bottom of the tube. Add 500mL 75% ethanol, shake gently to mix, centrifuge at 11000g for 5min, discard the supernatant, centrifuge for 5-10s, and air-dry in a fume hood.

[0043] ⑦ The liquid in the centrifuge tube is the obtained persimmon genomic DNA. The quality and concentration of the sample are detected using 1.8% agarose gel and ultraviolet spectrophotometer. After the sample passes the test, it is stored in a -20°C refrigerator for later use.

[0044] (2) PCR amplification of the extracted DNA template was performed using the upstream primer and downstream primer obtained in Example 1; the PCR amplification procedure was: 94°C pre-denaturation for 3 min, 94°C denaturation for 30 s, 54°C annealing for 30 s, 72°C extension for 1 min 15 s, denaturation, annealing and extension for 35 cycles, and finally 72°C extension for 5 min. (The annealing temperature is designed based on the Tm value of the primer sequence itself, and is generally the middle value of the Tm values ​​of the upstream and downstream primer sequences. The Tm values ​​of the upstream and downstream sequences of this pair of primers are both 54°C, and the PCR amplification effect is better according to this temperature.) The PCR amplification system was 10 μL, and the reaction composition was 5 μL of 2×Taq PCR MasterMix, 0.25 μL of 10 μM / L upstream primer and downstream primer, and 3.5 μL of ddH 2 O, 150ng of genomic DNA template, and finally ddH 2 Add 3% HO to make up to 10 μL.

[0045] (3) The PCR amplification products were separated by 1.8% agarose gel electrophoresis and photographed using a gel imager; the electrophoresis separation conditions were: 220V 15min, GoldView nucleic acid dye staining, and the marker was a DNA molecular weight standard of 100-5000bp; the photographs were taken using a BIO-RAD Gel Doc XR gel imager.

[0046] (4) The natural astringency characteristics of Chinese sweet persimmons were identified based on the bands in the electrophoresis diagram. The individuals with a band at 2089 bp (sequence as shown in SEQ ID NO.3) on the electrophoresis gel were Chinese sweet persimmons; the individuals without a band at 2089 bp were Chinese astringent persimmons. Table 1 shows the identification results of 6 Chinese sweet persimmons and 5 Chinese astringent persimmons that have produced fruits. The bands in the electrophoresis diagram are as follows: Figure 1 shown.

[0047] SEQ ID NO.3:

[0048]

[0049] Table 1 Identification results of 11 samples of Chinese sweet persimmon cultivars

[0050] serial number Germplasm name Whether it has natural astringency 1 Luotian sweet persimmon yes 2 Small sweet persimmon yes 3 Eshi No.1 yes 4 Male plant No. 1 yes 5 Male plant No. 2 yes 6 Male plant No. 8 yes 7 Ox-eye persimmon no 8 Gongcheng water persimmon no 9 Black Persimmon no 10 Mopan Persimmon no 11 Fuping persimmon no

[0051] The natural astringency-free characteristics of Chinese persimmon were identified based on the bands in the electrophoresis diagram. The results showed that the accuracy of the identification results of this method can be considered to be 100%. It can be inferred that the present invention can accurately identify the natural astringency-free characteristics of Chinese persimmon.

[0052] Example 3: Identification of the natural astringency-free properties of hybrid offspring

[0053] Twelve hybrid offspring with known natural astringency-free characteristics that have produced fruit were collected from the persimmon germplasm demonstration garden of Huazhong Agricultural University, and their DNA was extracted from each of them.

[0054] (1) The modified CTAB method was used to extract DNA from individual Diospyros plants;

[0055] ① Weigh 50 mg of fresh plant leaves, grind them in liquid nitrogen, and transfer them to a 2 mL centrifuge tube.

[0056] ② Prepare the extract solution immediately before use, and prepare solution A and solution B. 100mL solution A includes 10mL 1mol·L-1Tris-HCl (PH=8.0), 1mL 0.5mol·L-1EDTA-Na2, 5mL 5mol·L-1NaCl, 2g pvp-4.0 powder, and finally add 84mL water. 100mL solution B includes 10mL 1mol·L-1Tris-HCl (PH=8.0), 10mL 0.5mol·L-1EDTA-Na2, 30mL5mol·L-1NaCl, 2g CTAB solid powder, 1g pvp-4.0 powder, and finally add 50mL water.

[0057] ③ After grinding, open the lid, add 1 mL of liquid A, and place on ice for 15 min, inverting 2-3 times during the process. Centrifuge at 12000g for 10 min, discard the supernatant; add 1 mL of liquid A again, and repeat this step.

[0058] ④ After the B solution is heated in a water bath at 65°C, it is boiled in a microwave oven and mercaptoethanol is added to the B solution at a ratio of 1:50; 700 μL of B solution is added to the sample, shaken well, heated in a water bath at 65°C for 1 hour, shaken every 15 minutes, and centrifuged at 12000g for 10 minutes.

[0059] ⑤ Take a new 2mL centrifuge tube, add 600μL of chloroform:isoamyl alcohol (24:1) mixture in a fume hood, invert and mix for 10 minutes, and centrifuge at 12000g for 10 minutes; take a new 2mL centrifuge tube, aspirate the upper supernatant of the layers (upper, middle and lower layers), add chloroform:isoamyl alcohol (24:1) mixture, and repeat this step.

[0060] ⑥ Take a new 1.5mL centrifuge tube, aspirate the upper supernatant, add 500μL isopropanol (pre-cooled at -20℃ 30min in advance), shake well, and precipitate at -20℃ for 1-2h; centrifuge at 11000g for 5min, discard the supernatant, and a transparent white precipitate can be seen at the bottom of the tube. Add 500mL 75% ethanol, shake gently to mix, centrifuge at 11000g for 5min, discard the supernatant, centrifuge for 5-10s, and air-dry in a fume hood.

[0061] ⑦ The liquid in the centrifuge tube is the obtained persimmon genomic DNA. The quality and concentration of the sample are detected using 1.8% agarose gel and ultraviolet spectrophotometer. After the sample passes the test, it is stored in a -20°C refrigerator for later use.

[0062] (2) PCR amplification of the extracted DNA template was performed using the upstream primer and downstream primer obtained in Example 1; the PCR amplification procedure was: 94°C pre-denaturation for 3 min, 94°C denaturation for 30 s, 54°C annealing for 30 s, 72°C extension for 1 min 15 s, denaturation annealing extension for 35 cycles, and finally extension at 72°C for 5 min. (The annealing temperature is designed based on the Tm value of the primer sequence itself, and is generally the middle value of the Tm value of the upstream and downstream primer sequences. The Tm value of the upstream and downstream sequences of this pair of primers is 54°C, and the PCR amplification effect is better according to this temperature.) The PCR amplification system is 10 μL, and the reaction composition is 5 μL of 2×Taq PCR MasterMix, 0.25 μL of 10 μM / L upstream primer and downstream primer, 3.5 μL of ddH2O, and the genomic DNA template is 90-180 ng. Finally, ddH2O is added. 2 Add 3% HO to make up to 10 μL.

[0063] (3) The PCR amplification products were separated by 1.8% agarose gel electrophoresis and photographed using a gel imager; the electrophoresis separation conditions were: 220V 15min, GoldView nucleic acid dye staining, and the marker was a DNA molecular weight standard of 100-5000bp; the photographs were taken using a BIO-RAD Gel Doc XR gel imager.

[0064] Table 2 shows the identification results and parental information of 7 fruit-bearing hybrid offspring Chinese sweet persimmons and 5 Chinese astringent persimmons.

[0065] Table 2 Hybrid progeny sample information

[0066]

[0067]

[0068] The results are as follows Figure 5 As shown, the sweet persimmon with natural astringency-free trait in the hybrid offspring of Chinese sweet persimmon has a clear band at 2089 bp (sequence as shown in SEQ ID NO.3) on the electrophoresis gel, while the astringent persimmon of the hybrid offspring has no band at 2089 bp.

[0069] Comparative Example 1: The original RO2 marker was used to identify the natural astringency-free properties of 6 naturally astringent materials with known properties and 5 astringent persimmon materials.

[0070] Comparative Example 1 The natural astringency-free properties of 6 materials with known properties that can be naturally de-astringent and 5 materials with astringent persimmon provided in Table 1 of Example 2 were identified using the original RO2 marker. The identification process is as follows:

[0071] The extracted cultivated persimmon DNA template was used for PCR amplification; the PCR amplification program was as follows: 95°C pre-denaturation for 60s, 94°C denaturation for 30s, 58.5°C annealing for 20s, 72°C extension for 30s, denaturation, annealing and extension for 35 cycles, and finally 72°C extension for 5min. The PCR amplification system was 10μL, and the reaction composition was 5μL of 2×Taq PCR Master Mix, 0.25μL of 10μM / L upstream primer and downstream primer, and 3.5μL of ddH 2 O, 150ng of genomic DNA template, and finally ddH 2 Add 3% HO to make up to 10 μL.

[0072] The sequence of the upstream primer RO2F is shown in SEQ ID NO.4, and the sequence of the downstream primer RO2R is shown in SEQ ID NO.5.

[0073] SEQ ID NO.4: 5'-TCTTTACAACTGAGAAGGGGGGG-3';

[0074] SEQ ID NO. 5: 5'-TGTCTTACAGACGTCGTCGGT-3'.

[0075] The PCR amplification products were separated by 1.8% agarose gel electrophoresis and photographed under a gel imager; the electrophoresis separation conditions were: 220V 15min, GoldView nucleic acid dye staining, and the marker was a DNA molecular weight standard of 100-2000bp; the photos were taken using a BIO-RAD Gel Doc XR gel imager.

[0076] Repeat the above process three times to obtain Figure 2-Figure 4 , Figure 2 In the middle, no obvious bands were found in material 3 Eshi No. 1 and material 6 Xiongzhu No. 8; Figure 3 There were no obvious bands between the Luotian sweet persimmon of material 1 and the small-fruit sweet persimmon of material 2; and Figure 3 Chinese material 8 Gongcheng persimmon, Figure 4 Both material 10, Mopan persimmon, and material 11, Fupingjian persimmon, showed bands at about 200 bp, which were inconsistent with the natural astringency removal of the actual materials. Figure 2-Figure 4 In all experiments, primer dimers were present, and their sizes were close to the target fragments, making it difficult to distinguish. In the three repeated experiments, the RO2 labeling had poor repeatability, and even the same sample or the same DNA template had inconsistent identification in different experimental batches.

[0077] In summary, the molecular marker CPCNA-2k provided by the present invention, the molecular marker and primers identify the natural astringency-free traits of Diospyros plants, and the identification accuracy rate reaches 100%. It is longer than the amplified fragment (about 200bp) of the existing marker, easier to distinguish and the amplification effect is more stable. The annealing temperature (54°C) of the upstream and downstream primers developed based on the molecular marker is consistent and there is no dimer. The annealing temperature is designed according to the sequence characteristics of the primer itself. 54°C is a temperature suitable for this primer. At this temperature, the primer amplification effect is better, the amplification efficiency of the target band is higher, and the primer dimer is less formed. It solves the problem that the RO2 gene amplified fragment in the prior art is short, there are primer dimers, and its size is close to the position of the target fragment, which makes it difficult to distinguish and has poor repeatability. Even the same sample or the same DNA template has inconsistent identification in different experimental batches.

[0078] Although the preferred embodiments of the present invention have been described, those skilled in the art may make other changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

[0079] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. A molecular marker for identifying the natural astringency-free trait of Chinese persimmon, characterized in that: The molecular marker is CPCNA-2k, and its nucleotide sequence is shown in SEQ ID NO.

3.

2. A primer for amplifying the molecular marker according to claim 1, characterized in that: The nucleotide sequences of the primers are shown in SEQ ID NO.1-2.

3. A kit comprising the primer according to claim 2.

4. The kit according to claim 3, characterized in that The kit also includes 2×Taq PCR MasterMix and ddH2O.

5. Use of the molecular marker according to claim 1, the primer according to claim 2 or the kit according to claim 3 in distinguishing Chinese sweet persimmon and Chinese astringent persimmon.

6. The use according to claim 5, comprising the following steps: Extraction of Diospyros DNA; Using the Diospyros plant DNA as a template and the nucleotide fragments shown in SEQ ID NO.1-2 as primers, PCR amplification is performed; The products obtained by PCR amplification were detected by electrophoresis. When a product of 2089 bp was amplified, it was considered to be a Chinese sweet persimmon. If a product of 2089 bp was not amplified, it was a Chinese astringent persimmon.

7. The use according to claim 6, characterized in that: The PCR amplification program was as follows: pre-denaturation at 94°C for 3 min, denaturation at 94°C for 30 s, annealing at 54°C for 30 s, extension at 72°C for 1 min 15 s, a total of 35 cycles of denaturation, annealing and extension, and finally extension at 72°C for 5 min.

8. The use according to claim 6, characterized in that: The PCR amplification system was: 10 μL, the reaction composition was 5 μL of 2×Taq PCR Master Mix, 0.25 μL of 10 μM upstream primer and downstream primer, 3.5 μL of ddH2O, 90-180 ng of genomic DNA template, and finally ddH2O was added to make up to 10 μL.

9. The use according to claim 5, characterized in that: Chinese sweet persimmons include: Luotian sweet persimmon, small fruit sweet persimmon, E Persimmon No. 1, Male Plant No. 1, Male Plant No. 2, and Male Plant No.

8.

10. The use according to claim 9, characterized in that: Chinese astringent persimmons include: ox-eye persimmon, Gongcheng water persimmon, black persimmon, grinding disc persimmon, and Fuping pointed persimmon.

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