CN112980999B

CN112980999B - SSR molecular marker of mulberry variety Yuehei 74 and core primer group, kit and application thereof

Info

Publication number: CN112980999B
Application number: CN202110473626.4A
Authority: CN
Inventors: 王振江; 唐翠明; 罗国庆; 戴凡炜; 钟建武; 林森; 陈莲; 李智毅
Original assignee: Sericulture and Agri Food Research Institute GAAS
Current assignee: Sericulture and Agri Food Research Institute GAAS
Priority date: 2021-04-29
Filing date: 2021-04-29
Publication date: 2021-07-23
Anticipated expiration: 2041-04-29
Also published as: CN112980999A

Abstract

The invention discloses an SSR molecular marker of a mulberry variety 'Yueshi 74', a core primer group, a kit and application thereof. The invention utilizes fluorescence SSR technology, 200 pairs of SSR primers developed by mulberry genome sequence design are screened, core primers marked with M792 and M20197 are confirmed to be capable of respectively amplifying two specific bands for a new variety of mulberry, namely Guangdong mulberry 74 ', and the new variety of mulberry, namely Guangdong mulberry 74 ', can be respectively identified from the mulberry strains and can be used for rapid identification and detection of the new variety of mulberry, namely Guangdong mulberry 74 '. The method disclosed by the invention identifies the Guangdong mulberry 74 ' by directly detecting the M792 mark and/or the M20197 mark of the Guangdong mulberry 74 ', overcomes the uncertainty of the identification of external morphological characteristics, is simple to operate, high in detection efficiency, reliable and intuitive in result, provides scientific technical support for the protection of the Guangdong mulberry 74 ' which is a new variety of the mulberry, and is beneficial to popularization, utilization and protection of the variety.

Description

SSR molecular marker of mulberry variety Yuehei 74 and core primer group, kit and application thereof

Technical Field

The invention relates to the technical field of variety resource identification and germplasm innovation, in particular to an SSR molecular marker of a mulberry variety 'Yueshi 74', a core primer group, a kit and application thereof.

Background

The Mori fructus is Morus alba (Moraceae)Morus alba L.) mature ears belonging to the berry family. The mulberry is increasingly popular among people due to its unique taste, rich nutrition and high active substance content, becomes a representative of the third generation fruit, and is listed as a list of 'being food and medicine' by the national ministry of health at present. Except fresh food, the mulberry is processed and developed into various products such as mulberry juice, mulberry wine, mulberry jam, mulberry haematochrome, mulberry vinegar and the like, the mulberry industry is initially large-scale, and the mulberry presents good diversified development momentum.

The 'yueshan 74' new mulberry variety is a new mulberry variety bred by 9 years of oriented cultivation by using Guangdong mulberry as a parent and passing through national plant new variety authorization (CNA 20090138.2) in 2014 and passing through new crop variety approval (Guangdong examined mulberry 20160001) in 2016. The method is characterized in that: the tree shape is upright, branches are more and thick and straight, lateral branches are more, the bark is purple brown, the node shape is straight, phyllotaxis 1/3 is small, and the bark is round and oval; the winter bud is long and triangular, yellow brown, abdominal separation, and small and few side buds. The leaves are long heart-shaped, dark green, long tip tail-shaped, fine sawtooth at the edge of the leaves, deep heart-shaped at the base of the leaves, slightly wrinkled and slightly smooth on the leaf surface, thick and long petioles and obliquely grown leaves. The mature mulberry is purple black, the fruit is cylindrical, the fruit type is good, the long diameter of the mulberry is 2.8-5.2 cm, the transverse diameter is 1.4-2.0 cm, and the average weight of a single fruit is 4.0 g; more seeds, purple black, sour, sweet and delicious taste and good flavor. The practices of introducing the Guangdong mulberry 74' in various places prove that the yield per mu of the variety in the full-bearing period is more than 1600 kg, and the leaf yield per mu is about 1800 kg, so that the method has extremely high popularization and application values.

With the continuous improvement of the state on the new plant variety protection system, the protection consciousness of breeding researchers on the bred new variety is continuously enhanced, and new variety protection is actively applied. In the process of popularizing a new variety of mulberry, namely Guangdong mulberry 74, because the authenticity of the variety is difficult to distinguish and distinguish by external morphological characteristics in the seedling stage, a plurality of other varieties are counterfeited every year, and the variety is difficult to effectively monitor and arbitrate, thereby bringing great influence to the development and utilization of the variety. Therefore, there is a need for a simple, fast and effective identification technique that is real, effective, free from environmental influences and capable of accurately distinguishing the variety.

The traditional new variety identification of the mulberry is mainly based on phenotypic characters, is greatly influenced by environment, has poor stability and long test period, and seriously influences the effectiveness and authority of new variety identification. The molecular marker technology is a future development direction for variety identification and protection due to the characteristics of high polymorphism, short test period, no environmental influence and the like. The Simple Sequence Repeat (SSR) has the advantages of co-dominance, good repeatability, easy detection, Simple operation and the like, so the SSR molecular marker has good application prospect in the variety specificity evaluation and protection of the mulberry.

Disclosure of Invention

The purpose of the invention is: the SSR molecular marker of the new variety of the mulberry, namely Guangdong mulberry 74 ', as well as the core primer group, the kit and the application thereof are provided, the operation is simple, the SSR molecular marker can be used for identifying the authenticity of the new variety of the mulberry, namely Guangdong mulberry 74', and the effective supervision and arbitration can be realized when the variety is counterfeit or disputed.

The first purpose of the invention is to provide an SSR molecular marker for identifying a mulberry variety 'Yuehao 74', which comprises an SSR molecular marker M792 and/or M20197; the repetitive motif of the SSR molecular marker M792 is (AGC)_nWherein n is more than or equal to 6, the right sequence of the protein is shown as SEQ ID NO.6, and the left sequence of the protein is shown as SEQ ID NO. 5; the repetitive motif of the SSR molecular marker M20197 is (TA)_nWherein n is more than or equal to 6, the right sequence is shown as SEQ ID NO.8, and the left sequence is shown as SEQ ID NO. 7.

The second purpose of the invention is to provide a core primer group of SSR molecular markers for identifying a variety of fruit mulberry, namely Guangdong mulberry 74', which comprises a primer aiming at an SSR molecular marker M792 and/or a primer aiming at an SSR molecular marker M20197:

the primer aiming at the SSR molecular marker M792 comprises the following components:

M792-F: 5'-TACAATCCTCCACCTGACAAACT-3' (shown in SEQ ID NO. 1);

M792-R: 5'-GTGGGGACGGAGACTACTACTG-3' (shown in SEQ ID NO. 2);

the 5' end of the primer M792-F is marked with a fluorescent reporter group;

the primer aiming at the SSR molecular marker M20197 comprises the following components:

M20197-F: 5'-TCATTTTGGCTTATCTCTCTTGC-3' (shown in SEQ ID NO. 3);

M20197-R: 5'-TTTGTGTGCTTCTTCTGTAACCA-3' (shown in SEQ ID NO. 4);

the 5' end of the primer M20197-F is marked with a fluorescent reporter group.

Preferably, the fluorescent reporter group is FAM, HEX, TAMRA or ROX.

The third purpose of the invention is to provide a rapid detection kit for the variety of mulberry, namely Guangdong mulberry 74', which comprises the core primer group marked by the SSR molecules.

Preferably, the rapid detection kit further comprises dNTPs, Taq DNA polymerase and ddH₂O。

The fourth purpose of the invention is to provide a method for identifying a mulberry variety 'Yuehao 74' by utilizing SSR molecular markers, which comprises the following steps:

(1) extracting the genome DNA of a mulberry fruit sample to be detected;

(2) performing PCR amplification on the genomic DNA extracted in the step (1) as a template by respectively using the primer pairs M792-F/M792-R and M20197-F/M20197-R;

(3) typing the PCR amplification product in the step (2), and judging the bands of the typing result; if the PCR amplification product obtained by using the primer pair M792-F/M792-R as a primer has only two specific bands of 99 bp and 111 bp, and the PCR amplification product obtained by using the primer pair M20197-F/M20197-R as a primer has only two specific bands of 119 bp and 125 bp, the mulberry sample to be detected is ' Yueshan 74 ', otherwise, the mulberry sample to be detected is non-Yueshan 74 '.

Preferably, the reaction system of the PCR amplification in step (2) is 20 μ L, and includes: 50 ng/. mu.L DNA template 0.5. mu.L, 10 XPCR buffer 2. mu.L, 25 mM MgCl₂mu.L, 0.5. mu.L of 10mM dNTPs, 0.2. mu.L of 5U/. mu.L Taq DNA polymerase, 0.5. mu.L of 10. mu.M upstream primer, 0.5. mu.L of 10. mu.M downstream primer and ddH₂O 13.8 μL。

Preferably, the PCR amplification in step (2) comprises the following reaction procedures: pre-denaturation at 95 ℃ for 5 min; denaturation at 95 ℃ for 30 s, annealing at 60 ℃ for 30 s, cooling to 1 ℃ in each cycle, extension at 72 ℃ for 30 s, and circulating for 10 times; denaturation at 94 ℃ for 30 s, annealing at 50 ℃ for 30 s, and extension at 72 ℃ for 30 s, and circulating for 30 times; finally, extension is carried out for 5min at 72 ℃.

The fifth purpose of the invention is to provide the application of the SSR molecular marker or the core primer group of the SSR molecular marker or the rapid detection kit in identifying the variety of the fruit mulberry, namely the Guangdong mulberry 74.

The invention utilizes fluorescence SSR technology, 200 pairs of SSR primers developed by mulberry transcriptome sequence design are screened, and the core primers M792-F/M792-R of SSR molecular markers M792 can amplify two specific bands of 99 bp and 111 bp to a new variety of mulberry Guangdong mulberry 74 ', the core primers M20197-F/M20197-R of SSR molecular markers M20197 can amplify two specific bands of 119 bp and 125 bp to the new variety of mulberry Guangdong mulberry 74 ', and the new variety of mulberry Guangdong mulberry 74 ' can be respectively identified from the new variety of mulberry (particularly Guangdong mulberry). According to the invention, a specific primer for amplifying a specific strip of 'Guangdong mulberry 74' is obtained through a large amount of experimental data, and the primer is identified by directly detecting the M792 mark and/or the M20197 mark of 'Guangdong mulberry 74', so that the uncertainty of the identification of external morphological characteristics is overcome, the operation is simple, the detection efficiency is high, the result is reliable and intuitive, a scientific technical support is provided for the protection of a new variety of 'Guangdong mulberry 74', and the invention is beneficial to the popularization, utilization and protection of the variety.

Drawings

FIG. 1 is a SSR typing chart of SSR molecular marker M792 core primer amplified ` Yuexi 74 ` and 35 related and phenotypically similar mulberry strain genome DNA; wherein S1 is Guangdong 74, S2 is Pond 10 (female parent), S3 is selected from 851 (male parent), S4 is XN, S5 is NL2, S6 is DX7, S7 is WL4, S8 is WL5, S9 is S1, S10 is S2, S11 is NJ1, S12 is NJ3, S13 is B7, S14 is Z10, S15 is Z12, S16 is NJ6, S17 is ZHAN02, S18 is ZHAN12, S19 is ZHAN26, S20 is CT 20, S20 is WX 20, S20 is WG 20, S20 is ZB 20, S20 is NX 20, S20 is CN, S20 is S20, S20 is S20, S20 is S20, S20 is S20, S20 is S20, S20 is S20, S20 is S20, S36.

FIG. 2 is a SSR typing chart of SSR molecular marker M20197 core primer amplified ` Yuexian 74 ` and 35 related and phenotypically similar mulberry strain genome DNA; wherein S1 is Guangdong 74, S2 is Pond 10 (female parent), S3 is selected from 851 (male parent), S4 is XN, S5 is NL2, S6 is DX7, S7 is WL4, S8 is WL5, S9 is S1, S10 is S2, S11 is NJ1, S12 is NJ3, S13 is B7, S14 is Z10, S15 is Z12, S16 is NJ6, S17 is ZHAN02, S18 is ZHAN12, S19 is ZHAN26, S20 is CT 20, S20 is WX 20, S20 is WG 20, S20 is ZB 20, S20 is NX 20, S20 is CN, S20 is S20, S20 is S20, S20 is S20, S20 is S20, S20 is S20, S20 is S20, S20 is S20, S36.

FIG. 3 is an SSR typing chart of genome DNA of 20 bred new varieties of fruit mulberry and new strains for production, popularization and application by SSR molecular marker M792 core primer amplification; wherein S1 is Guangdong 74, S2 is Guangdong 10, S3 is Guangdong 123, S4 is Guangdong mulberry 143, S5 is Guangdong mulberry 145, S6 is Guangdong mulberry 28, S7 is Guangdong 33, S8 is Guangdong mulberry 201, S9 is Red Mulberry No.2, S10 is Yunshen No.1, S11 is Yunshen No.2, S12 is Taiwan Changtuo mulberry, S13 is 46C019, S14 is 72C002, S15 is white pearl, S16 is white jade, S17 is black pearl, S18 is Osmanthus honey, S19 is Xinjiang white mulberry, and S20 is white mulberry No. 2.

FIG. 4 is an SSR typing chart of genome DNA of 20 bred new varieties of fruit mulberry and new strains for production, popularization and application by SSR molecular marker M20197 core primer amplification; wherein S1 is Guangdong 74, S2 is Guangdong 10, S3 is Guangdong 123, S4 is Guangdong mulberry 143, S5 is Guangdong mulberry 145, S6 is Guangdong mulberry 28, S7 is Guangdong 33, S8 is Guangdong mulberry 201, S9 is Red Mulberry No.2, S10 is Yunshen No.1, S11 is Yunshen No.2, S12 is Taiwan Changtuo mulberry, S13 is 46C019, S14 is 72C002, S15 is white pearl, S16 is white jade, S17 is black pearl, S18 is Osmanthus honey, S19 is Xinjiang white mulberry, and S20 is white mulberry No. 2.

Detailed Description

To further illustrate the technical means and effects of the present invention adopted to achieve the predetermined objects, the present invention will be further described with reference to specific embodiments.

Example 1 screening of novel Mulberry variety 'Yueshi 74' specific primer sequences

1. SSR primer selection

Searching a genome sequence in a mulberry genome database by using MISA software, searching SSR loci in the genome sequence, setting the length of a searched SSR motif repeating unit to be 2-6 nucleotides, setting the minimum search repetition times of 2, 3, 4, 5 and 6 nucleotides to be 6, 5, 4 and 4, setting the length of a flanking sequence of the SSR loci to be more than or equal to 150 bp, and designing SSR primers by using Primer3v2.3.4 (http:// Primer3. sourcego-large.net) software according to the searched SSR loci, wherein the design standard is as follows: the length of the primer is 18-25bp, the annealing temperature is 57-63 ℃, the GC content is 40-70%, the length of the PCR product is 100-300bp, and 200 pairs of primers are synthesized by randomly selecting SSR sites which can be compared with an NCBI non-redundant protein database, wherein each pair of primers consists of an upstream primer A and a downstream primer B. The primer sequence is synthesized by Shenzhen Hua Dagen science and technology Limited.

2. Extraction of DNA of mulberry variety

The new variety 'yue shen 74', the female parent 'pool 10', the male parent '851' and 33 other excellent Guangdong mulberry strains with similar relatives and phenotypes are selected as 36 (the serial numbers 1 to 36 comprise 15 mulberry strains with the parent blood system of 'yue shen 74', the 15 mulberry strains are relatively close to the relatives of 'yue shen 74', and the rest 20 mulberry strains have similar phenotypes to the 'yue shen 74') for preliminary screening. The lines of mulberry used for the preliminary screening are shown in table 1 (from national mulberry germplasm resource garden-south China sub garden).

Table 1 preliminary screening 36 mulberry lines used

The specific screening steps are as follows:

the DNA of a new variety of mulberry, namely Guangdong mulberry 74 and other 35 excellent strains of mulberry (shown in Table 1) which are also Guangdong mulberry seeds is extracted by using an improved CTAB method, and the specific steps of the DNA extraction are as follows:

(1) the formula for preparing CTAB extracting solution (Hexadecyl trimethyl ammonium bromide ) is as follows: 2% CTAB, 0.1M Tris-HCl, 20mM EDTA, 1.4M NaCl, TE buffer (10 mM Tris-HCl, 1mM EDTA, pH 8.0);

(2) putting 1g of material (tender material is selected as much as possible) into a mortar, and grinding the material into powder by liquid nitrogen;

(3) adding the ground material into a 10mL centrifuge tube, adding 4 mL CTAB extracting solution and 80 μ L beta-mercaptoethanol (preheated at 65 ℃) and uniformly mixing, then carrying out water bath at 65 ℃ for 45 min, and uniformly mixing for 3-4 times;

(4) adding 1mL of 5M KAc, and carrying out ice bath for 20 min;

(5) 4 mL of chloroform was added: isoamyl alcohol (24:1), and emulsifying for 10 min;

(6) centrifuging at 12000 rpm for 10 min at room temperature, and transferring the supernatant into a new 10mL centrifuge tube;

(7) taking the supernatant, adding 3M NaAc (pH 5.2) with the volume of 1/10-1/5, turning and uniformly mixing, adding isopropanol with the same volume, and uniformly mixing until DNA precipitation occurs;

(8) adding 1mL of 75% ethanol into the precipitate, rinsing for 3-4 times, and rinsing for 1 time by using absolute ethanol; precipitating DNA and drying;

(9) adding 100 mu L of TE buffer solution containing 10 mu g/ml RNase A to dissolve the precipitate, and carrying out water bath at 37 ℃ for 1h to degrade RNA;

(10) detecting quality by electrophoresis, detecting DNA concentration by spectrophotometer, adjusting DNA concentration to 50 ng/. mu.L, and storing at-20 deg.C.

3. PCR amplification

Taking the mulberry genome DNA extracted in the step 2 as a template, and performing PCR amplification by using an SSR fluorescence labeling detection technology, wherein the primers comprise an upstream primer and a downstream primer, the 5' end of the upstream primer is labeled with a fluorescence reporter group (FAM, HEX, TAMRA or ROX, and the HEX fluorescence labeling is used in the invention), the upstream primer is designed and synthesized in the step 1, and the downstream primer is designed and synthesized in the step 1. Upstream primer-directed PCR amplification with a fluorescent reporter group labeled at the 5' end produces a PCR product with fluorescence.

The total PCR reaction is 20. mu.L, wherein 50 ng/. mu.L DNA template is 0.5. mu.L, 10 XPCR buffer is 2.0. mu.L, and 25 mM MgCl₂2.0. mu.L, 10mM dNTPs 0.5. mu.L, 5U/. mu.L Taq DNA polymerase 0.2. mu.L, 10. mu.M upstream primer 0.5. mu.L, 10. mu.M downstream primer 0.5. mu.L, ddH₂O 13.8 μL。

The PCR reaction condition is pre-denaturation at 95 ℃ for 5 min; denaturation at 95 ℃ for 30 s, annealing at 60 ℃ (1 ℃ per cycle) for 30 s, extension at 72 ℃ for 30 s, and 10 cycles; denaturation at 94 ℃ for 30 s, annealing at 50 ℃ for 30 s, and extension at 72 ℃ for 30 s, and circulating for 30 times; finally, extension is carried out for 5min at 72 ℃ to obtain an amplification product.

4. Screening for polymorphic primers

And 3, taking 2.5 mu L of the amplification product in the step 3, adding 1.5 mu L of Loading Buffer, uniformly mixing and Loading, carrying out band detection after 2% agarose gel electrophoresis for 25min, and screening the product with the amplification band for sequencing and typing. The products with amplified product bands screened in the above steps were diluted to within 0.5ng, 0.5. mu.L of the product was put into Hidi containing liz500, and typing was carried out using a 3730XL DNA sequencer (ABI, USA), and the typing results were band-discriminated using GeneMarker (soft Genetics LLC, USA).

5. Data analysis

The screening result shows that 142 polymorphic primers are screened out from 200 primers synthesized in the step 1; further selecting a core primer with good repeatability and stability and capable of amplifying specific allelic sites of a new variety of mulberry 'Yueshu 74', and finally obtaining a primer pair M792-F/M792-R (M792-F: 5'-TACAATCCTCCACCTGACAAACT-3', shown in SEQ ID NO.1, M792-R: 5'-GTGGGGACGGAGACTACTACTG-3', shown in SEQ ID NO. 2) and a primer pair M20197-F/M20197-R (M20197-F: 5'-TCATTTTGGCTTATCTCTCTTGC-3', shown in SEQ ID NO.3, M20197-R: 5'-TTTGTGTGCTTCTTCTGTAACCA-3', shown in SEQ ID NO. 4) which can be used as specific core primers for identifying 'Yueshu 74'. The SSR molecular marker corresponding to the core primer M792-F/M792-R is M792 marker, and the repetitive motif is (AGC)_nN is not less than 6, the M792 markThe right sequence is shown in SEQ ID NO.6, and the left sequence is shown in SEQ ID NO. 5. The SSR molecular marker corresponding to the core primer M20197-F/M20197-R is an M20197 marker, and the repetitive motif is (TA)_nN is more than or equal to 6, the right sequence of the M20197 marker is shown as SEQ ID NO.8, and the left sequence is shown as SEQ ID NO. 7.

The SSR fluorescence labeling primers M792-F/M792-R are used as primers, amplification products of a new mulberry variety Yue Shen 74 ' labeled at M792 are two specific bands of 99 bp (shown as SEQ ID NO. 9) and 111 bp (shown as SEQ ID NO. 10), the SSR fluorescence labeling primers M20197-F/M20197-R are used as primers, amplification products of the new mulberry variety Yue Shen 74 ' labeled at M20197 are two specific bands of 119 bp (shown as SEQ ID NO. 11) and 125 bp (shown as SEQ ID NO. 12), and the new mulberry variety Yue Shen ' Yue Shen 74 ' can be identified from the new mulberry variety E Shen 74 ' and 35 related mulberry strains with similar phenotypes only by using the SSR fluorescence labeling primers M20197-F/M20197-R or M792-F/M792-R (see the finger print data in Table 2, wherein the sequences of the primer are shown in the figure 1 and the figure 2.

TABLE 2 statistics of capillary electrophoresis bands of 36 mulberry lines with primer pairs

6. Further validation of the primers

Further verifying 2 pairs of specific core primers M792-F/M792-R, M20197-F/M20197-R for identifying 'Yuehen 74' obtained by screening, selecting 19 new varieties of mulberry cultivated nationwide at present, carrying out amplification detection on the new varieties of mulberry and 'Yuehen 74' with certain popularization and application in production, carrying out DNA extraction, amplification and detection methods, and finding that the 2 pairs of specific core primers can distinguish 'Yuehen 74' from other varieties (figures 3 and 4) and amplification specific allelic sites of 'Yuehen 74' and other varieties are shown in Table 3.

Table 3 shows the statistics of capillary electrophoresis bands of new varieties of cultivated mulberries and new varieties for production, popularization and application

As can be seen from the results in tables 2 and 3, the SSR molecular markers M792 and M20197 have high polymorphism, and can rapidly identify 'yuehen 74' from 36 related and phenotypically similar mulberry strains, 20 bred new mulberry varieties, and new production, popularization and application strains. However, considering that more new varieties may appear in the future, in order to identify the scientificity, the invention suggests that the core primers of the 2 SSR molecular markers are adopted for simultaneous detection, and if the result obtained by amplifying the SSR fluorescent marker primers by 2 is consistent with the invention, the variety can be identified as a new mulberry variety 'Yuexian 74'.

In the process of obtaining and identifying the specific molecular marker of 'Guangdong mulberry 74', the invention firstly attaches importance to the acquisition of primers, and 200 pairs of SSR primers are randomly designed and acquired from a mulberry genome database so as to objectively evaluate the characteristics of varieties. The primer screening is carried out by properly selecting the mulberry strains, firstly, 36 mulberry strains with similar relativity and phenotype are preliminarily screened, the strains are similar to 'Yueshi 74' in phenotypic characters, and certain difficulty exists in phenotypic identification, so that the screening of the strains to obtain the specific core primer capable of amplifying the specific band of the 'Yueshi 74' of the new variety of the mulberry has very important significance. On the basis of obtaining the specific core primer by preliminary screening, 20 bred new mulberry varieties and new production, popularization and application varieties are selected for verification, the banding result is consistent with the previous performance, and the Guangdong mulberry 74' can be quickly and accurately identified from the new varieties, so that the pair of primers is good in specificity and stability.

The method adopts specific core primers M792-F/M792-R and M20197-F/M20197-R to provide a better guarantee for the rapid and accurate detection and identification of the Guangdong mulberry 74'.

The specific core primers M792-F/M792-R and M20197-F/M20197-R can also be directly used as a part of a rapid detection kit for rapidly identifying a new mulberry variety 'Yueshi 74'.

The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.

It should be noted that the above-mentioned examples only represent some embodiments of the present invention, but should not be construed as limiting the scope of the invention. All modifications which can be derived or suggested by a person skilled in the art from the disclosure of the present invention are to be considered within the scope of the invention.

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Claims

1. An SSR molecular marker for identifying a variety of fruit mulberry, namely Guangdong mulberry 74', is characterized by comprising an SSR molecular marker M792 and/or M20197; the repetitive motif of the SSR molecular marker M792 is (AGC)_nWherein n is more than or equal to 6, the right sequence of the protein is shown as SEQ ID NO.6, and the left sequence of the protein is shown as SEQ ID NO. 5; the repetitive motif of the SSR molecular marker M20197 is (TA)_nWherein n is more than or equal to 6, the right sequence is shown as SEQ ID NO.8, and the left sequence is shown as SEQ ID NO. 7.

2. A core primer group of SSR molecular markers for identifying a fruit mulberry variety, namely Guangdong mulberry 74', is characterized by comprising a primer aiming at an SSR molecular marker M792 and/or a primer aiming at an SSR molecular marker M20197:

M792-F：5’-TACAATCCTCCACCTGACAAACT-3’；

M792-R：5’-GTGGGGACGGAGACTACTACTG-3’；

the 5' end of the primer M792-F is marked with a fluorescent reporter group;

M20197-F：5’- TCATTTTGGCTTATCTCTCTTGC-3’；

M20197-R：5’- TTTGTGTGCTTCTTCTGTAACCA -3’；

the 5' end of the primer M20197-F is marked with a fluorescent reporter group.

3. A core primer set of SSR molecular markers for the identification of the Mulberry species, Guangdong Mulberry 74, according to claim 2, wherein said fluorescence reporter group is FAM, HEX, TAMRA or ROX.

4. A rapid detection kit for identifying the variety of Morus yuehensis 74', comprising the SSR molecular marker core primer set of claim 2 or 3.

5. A method for identifying a mulberry variety 'Yuehen 74' by utilizing SSR molecular markers is characterized by comprising the following steps:

(1) extracting the genome DNA of a mulberry fruit sample to be detected;

(2) performing PCR amplification on the genomic DNA extracted in the step (1) as a template by using the primer pairs M792-F/M792-R and M20197-F/M20197-R of claim 2 or 3 respectively;

6. The method for identifying the variety of fruit mulberry, namely yuehen 74', by using SSR molecular markers according to claim 5, wherein said PCR amplification in step (2) is performed in a reaction system of 20 μ L, and comprises: 50 ng/. mu.L DNA template 0.5. mu.L, 10 XPCR buffer 2. mu.L, 25 mM MgCl₂mu.L, 0.5. mu.L of 10mM dNTPs, 0.2. mu.L of 5U/. mu.L Taq DNA polymerase, 0.5. mu.L of 10. mu.M upstream primer, 0.5. mu.L of 10. mu.M downstream primer and ddH₂O13.8 mu L; the PCR amplification of the step (2) comprises the following reaction procedures: pre-denaturation at 95 ℃ for 5 min; denaturation at 95 ℃ for 30 s, annealing at 60 ℃ for 30 s, cooling to 1 ℃ in each cycle, extension at 72 ℃ for 30 s, and circulating for 10 times; denaturation at 94 ℃ for 30 s, annealing at 50 ℃ for 30 s, and extension at 72 ℃ for 30 s, and circulating for 30 times; finally, extension is carried out for 5min at 72 ℃.

7. Use of a SSR molecular marker detection reagent according to claim 1 for the identification of the mulberry variety Guangdong 74'.

8. Use of the core primer set of claim 2 or 3 or the rapid test kit of claim 4 for identifying the variety of mulberry, yuehen 74.