CN113430300A - SSR molecular marker of mulberry variety Yuehen 123, core primer group and kit thereof, and application of SSR molecular marker - Google Patents
SSR molecular marker of mulberry variety Yuehen 123, core primer group and kit thereof, and application of SSR molecular marker Download PDFInfo
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Abstract
The invention discloses an SSR molecular marker of a mulberry variety 'Yueshan 123', a core primer group, a kit and application thereof. The invention utilizes fluorescence SSR technology, 300 pairs of SSR primers developed by mulberry genome sequence design are screened, and core primers marked with M10360 and M29602 can respectively amplify two specific bands for a new variety of mulberry, namely Guangdong mulberry 123 ', and can respectively identify the new variety of mulberry, namely Guangdong mulberry 123 ', from the mulberry strain, and can be used for rapid identification and detection of the new variety of mulberry, namely Guangdong mulberry 123 '. The method identifies the Guangdong mulberry 123 'by directly detecting the M10360 mark and/or the M29602 mark of the Guangdong mulberry 123', overcomes the uncertainty of identifying the Guangdong mulberry 123 'by external morphological characteristics, has simple operation, high detection efficiency and reliable and intuitive result, provides scientific technical support for protecting the Guangdong mulberry 123' which is a new variety of the Guangdong mulberry, and is favorable for popularization, utilization and protection of the variety.
Description
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 123', a core primer group, a kit and application thereof.
Background
The mulberry is a fruit resource with excellent characteristics, is a representative of fruits of the third generation, is listed as a list of 'food and medicine' by the national ministry of health at present, has increasingly attracted social attention along with the improvement of living standard and the enhancement of health care consciousness of people in recent years, is increasingly popular with people as a new fruit variety due to unique taste, rich nutrition and higher active substances, has gradually gone into families of common people, and is accepted by citizens. 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.
'Guangdong mulberry 123' is a new mulberry variety cultivated by artificial induction and orientation cultivation of seedlings as materials in the silkworm industry and agricultural product processing research of Guangdong province academy of agricultural sciences, belongs to Guangdong mulberry species, and obtains the new plant variety right (CAN 20160060.5) in 2018. The method is characterized in that: the tree shape is slightly developed, branches are thick and long, the number of main branches is large, and the lateral branch germination capacity is weak; the skin color is grey brown, the internodes are straight, the five series of phyllotaxis are provided, the skin holes are thin, the sizes are medium and round; the winter buds are in a regular triangle shape, are brown, are separated from the tips, and have large and few side buds; the anthocyanin of the young leaf is weak in color, the growing posture of the leaf at the top end is inclined upwards, and the growing posture of the petiole is flat and extended; the plant leaves are full-leaf, heart-shaped, dark green, long-tail-shaped tip, thick and round leaf edges, kidney-shaped base leaves, flat and smooth leaf surfaces and strong in glossiness; the mature mulberry is purple black, has long cylindrical shape, good fruit shape, sweet and sour taste and good flavor. The practices of multi-site introduction and production prove that the Yuehen 123 has over 1600 kg of fruit yield per mu in the full-bearing period and about 1800 kg of leaf yield per mu, and has extremely high popularization and application values.
The Guangdong mulberry 123' is a new mulberry variety which is newly bred and obtained in recent years and authorized by a new plant variety of China, and due to the characteristics of high yield and high quality, other varieties are continuously emerged to counterfeit the variety in recent years, but the authenticity of the variety is difficult to distinguish due to the external morphological characteristics in the seedling stage, the effective supervision and arbitration are difficult, and great influence is brought 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 'Yueshu 123', 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 'Yueshu 123', and the SSR molecular marker can be effectively supervised and arbitrated when variety counterfeiting or disputes occur.
The first purpose of the invention is to provide a SSR molecular marker for identifying a mulberry variety 'Yuehen 123', which comprises an SSR molecular marker M10360 and/or M29602; the repetitive motif of the SSR molecular marker M10360 is (AT) n, wherein n is more than or equal to 6, the right sequence of the SSR molecular marker is shown as SEQ ID NO.6, and the left sequence of the SSR molecular marker is shown as SEQ ID NO. 5; the SSR molecular marker M29602 has a repeating motif (GCA) n, wherein n is more than or equal to 6, the right sequence of the SSR molecular marker is shown as SEQ ID NO.8, and the left sequence of the SSR molecular marker 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 123', which comprises a primer aiming at an SSR molecular marker M10360 and/or a primer aiming at an SSR molecular marker M29602:
the primer aiming at the SSR molecular marker M10360 comprises the following components:
M10360-F: 5'-GAGTTCTTGAGGGGAAAGAGAAA-3' (shown in SEQ ID NO. 1);
M10360-R: 5'-AATTCCTGTGATCATCTGCCTTA-3' (shown in SEQ ID NO. 2);
the primer aiming at the SSR molecular marker M29602 is as follows:
the 5' end of the primer M10360-F is marked with a fluorescent reporter group;
the primer aiming at the SSR molecular marker M29602 is as follows:
M29602-F: 5'-ATGAACCCTCAGATGTTCTCAAT-3' (shown in SEQ ID NO. 3);
M29602-R: 5'-TTAATGATTCGACTCCGGTTTT-3' (shown in SEQ ID NO. 4);
the 5' end of the primer M29602-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 123, 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 'Yueshan 123' 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 by using the genomic DNA extracted in the step (1) as a template and using the primer pairs M10360-F/M10360-R and M29602-F/M29602-R respectively;
(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 M10360-F/M10360-R as a primer has only two specific bands of 158bp and 176 bp, and the PCR amplification product obtained by using the primer pair M29602-F/M29602-R as a primer has only one specific band of 150 bp, the mulberry sample to be detected is 'Yuexi 123', otherwise, the mulberry sample to be detected is non 'Yuexi 123'.
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 123 Yuehong mulberry.
The invention utilizes fluorescence SSR technology, 300 pairs of SSR primers developed by mulberry transcriptome sequence design are screened, core primers M10360-F/M10360-R of an SSR molecular marker M10360 are confirmed to be capable of amplifying two specific bands of 158bp and 176 bpp for a new variety of mulberry, core primers M29602-F/M29602-R of an SSR molecular marker M29602 are capable of amplifying a specific band of 150 bp for the new variety of mulberry, namely Guangdong mulberry 123 ', and the new variety of mulberry, namely Guangdong mulberry 123 ', can be respectively identified from the new variety of mulberry (particularly Guangdong mulberry), and can be used for rapid identification and detection of the new variety of mulberry, namely Guangdong mulberry 123 '. According to the invention, a specific primer of a 'Guangdong mulberry 123' specific band amplified through a large amount of experimental data is obtained, and the 'Guangdong mulberry 123' specific band is identified through directly detecting an M10360 mark and/or an M29602 mark of the 'Guangdong mulberry 123', 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 the 'Guangdong mulberry 123', 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 M10360 core primer amplified DNA of 'Yuehen 123' and 39 related and phenotypically similar mulberry strain genomes; wherein S1 is Yue 123, S2 is Pond 10 (female parent), S3 is air induction 31 (male parent), S4 is Bei7, S5 is Za10, S6 is Za12, S7 is Sh1, S8 is Sh12, S9 is Nja1, S10 is Nja3, S11 is Nja4, S12 is Nja5, S13 is Dj28, S14 is Dj33, S15 is Dj50, S16 is Zi1, S17 is Zi 17, S17 is Xin 17, S17 is Ys798, S17 is Shi Nas 17, S17 is Sha 17-S17, S17 is Shi S17, S17 is Zubs 17-S17, S17 is Shi 17, S17 is Szs 17-S17, S17 is Zubs 17, S17 is Szs 17, S17-S17 is Shi 17, S17 is S17-S17, S17 is Ys 17, S17 is Ys 17-S17, S17 is S17-S17, S17 is S17, S17-S17, S17 is S17, S17 is S17-S17, S17 is S17, S17-S17, S17 is S17, S17-S17, S17 is S17, S17-S17, S17-S17 is S17, S17-S17, S17 is S17, S17 is S17, S17-S17, S17 is S17, S17-S17, S17 is S17.
FIG. 2 is a SSR typing chart of SSR molecular marker M29602 core primer amplified DNA of Guangdong mulberry 123' and 39 related and phenotypically similar mulberry strain genomes; wherein S1 is Yue 123, S2 is Pond 10 (female parent), S3 is air induction 31 (male parent), S4 is Bei7, S5 is Za10, S6 is Za12, S7 is Sh1, S8 is Sh12, S9 is Nja1, S10 is Nja3, S11 is Nja4, S12 is Nja5, S13 is Dj28, S14 is Dj33, S15 is Dj50, S16 is Zi1, S17 is Zi 17, S17 is Xin 17, S17 is Ys798, S17 is Shi Nas 17, S17 is Sha 17-S17, S17 is Shi S17, S17 is Zubs 17-S17, S17 is Shi 17, S17 is Szs 17-S17, S17 is Zubs 17, S17 is Szs 17, S17-S17 is Shi 17, S17 is S17-S17, S17 is Ys 17, S17 is Ys 17-S17, S17 is S17-S17, S17 is S17, S17-S17, S17 is S17, S17 is S17-S17, S17 is S17, S17-S17, S17 is S17, S17-S17, S17 is S17, S17-S17, S17-S17 is S17, S17-S17, S17 is S17, S17 is S17, S17-S17, S17 is S17, S17-S17, S17 is S17.
FIG. 3 is SSR typing chart of genome DNA of 20 bred new varieties of fruit mulberry and new strains for production, popularization and application, amplified by SSR molecular marker M10360 core primer; wherein S1 is Guangdong 123, 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 M29602 core primer amplification; wherein S1 is Guangdong 123, 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 'Yueshan 123' 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, 300 pairs of primers are synthesized by randomly selecting SSR sites which can be compared with an NCBI non-redundant protein database, and 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 primary screening is carried out by using a new variety of mulberry, 'Guangdong mulberry 123', a female parent, 'Pond 10', a male parent, 'Aerosol 31', and 37 other excellent Guangdong mulberry strains with similar relativity and phenotype, wherein the number of the excellent strains is from 1 to 40 (the number of the excellent strains comprises 16 mulberry strains with the ancestral of the 'Guangdong mulberry 123', the 16 mulberry strains are closer to the relativity of the 'Guangdong mulberry 123', and the rest 21 mulberry strains are similar to the phenotype of the 'Guangdong mulberry 123'). 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 of 40 lines of Morus alba
The specific screening steps are as follows:
the DNA of a new variety of mulberry, namely Guangdong mulberry 123 and other 39 excellent strains of mulberry (shown in Table 1) which are also Guangdong mulberry seeds are 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 a10 mL 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 results showed that 124 pairs of polymorphic primers were screened out of the 300 pairs of primers synthesized in step 1; further selecting a core primer with good repeatability and stability and capable of amplifying specific allelic sites for a new variety of mulberry ' Yuehu 123 ', and finally obtaining a primer pair M10360-F/M10360-R (M10360-F: 5'-GAGTTCTTGAGGGGAAAGAGAAA-3', shown in SEQ ID NO.1, M10360-R: 5'-AATTCCTGTGATCATCTGCCTTA-3', shown in SEQ ID NO. 2) and a primer pair M29602-F/M29602-R (M29602-F: 5'-ATGAACCCTCAGATGTTCTCAAT-3', shown in SEQ ID NO.3, M29602-R: 5'-TTAATGATTCGACTCCGGTTTT-3' -3 ', shown in SEQ ID NO. 4) which can be used as a specific core primer for identifying ' Yuehu 123 '. The SSR molecule corresponding to the core primer M10360-F/M10360-R is marked as M10360, the repetitive motif is (AT) n, n is more than or equal to 6, the right sequence of the M10360 mark is shown as SEQ ID NO.6, and the left sequence is shown as SEQ ID NO. 5. The SSR molecular marker corresponding to the core primer M29602-F/M29602-R is M29602 marker, and the repetitive motif is (GCA)nN is more than or equal to 6, the right sequence of the M29602 marker is shown as SEQ ID NO.8, and the left sequence is shown as SEQ ID NO. 7.
The SSR fluorescence labeling primers M10360-F/M10360-R are used as primers, amplification products of the new mulberry variety Guangdong mulberry 123 'marked at M10360 are two specific bands of 158bp (shown as SEQ ID NO. 9) and 176 bp (shown as SEQ ID NO. 10), the SSR fluorescence labeling primers M29602-F/M29602-R are used as primers, the amplification products of the new mulberry variety Guangdong mulberry 123' marked at M29602 are a specific band of 150 bp (shown as SEQ ID NO. 11), the new mulberry variety Guangdong mulberry 123 'can be identified from the new mulberry variety Guangdong mulberry 123' and the 39 related phenotypically similar mulberry strains thereof only by using the SSR fluorescence labeling primers M29602-F/M29602-R or M10360-F/M10360-R (see FIGS. 1 and 2), and fingerprint data are shown in Table 2.
TABLE 2 statistics of capillary electrophoresis bands of 40 mulberry lines with primer pairs
6. Further validation of the primers
Further verifying 2 pairs of specific core primers M10360-F/M10360-R, M29602-F/M29602-R for identifying 'Yueshen 123' obtained by screening, selecting 19 new mulberry varieties bred nationwide at present, carrying out amplification detection on the new mulberry varieties and 'Yueshen 123' with certain popularization and application in production at the same time, and finding that the 2 pairs of specific core primers can distinguish 'Yueshen 123' from other varieties (figures 3 and 4) and amplification specific allelic sites of 'Yueshen 123' 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 M10360 and M29602 both have high polymorphism, and can rapidly identify 'yuehu 123' from 40 related and phenotypically similar mulberry strains, 20 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 123'.
In the process of obtaining and identifying the 'Guangdong mulberry 123' specific molecular marker, the invention firstly attaches importance to the acquisition of primers, and 300 pairs of SSR primers are randomly designed and acquired from a mulberry genome database so as to objectively evaluate the variety characteristics. The primer screening is carried out by properly selecting the mulberry strains, firstly, 40 mulberry strains with similar relativity and phenotype are preliminarily screened, the strains are similar to 'Yueshi 123' in phenotype character, and certain difficulty exists in phenotype identification, so that the screening of the strains to obtain the specific core primer capable of amplifying the specific band of the new variety 'Yueshi 123' of the mulberry has very important significance. On the basis of obtaining the specific core primer through 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 'Yueshi 123' can be quickly and accurately identified from the band result, so that the pair of primers is good in specificity and stability.
The method adopts specific core primers M10360-F/M10360-R and M29602-F/M29602-R to provide better guarantee for the rapid and accurate detection and identification of the < 123 > Yuesheng.
The specific core primers M10360-F/M10360-R and M29602-F/M29602-R can also be directly used as a part of a rapid detection kit for rapidly identifying a new mulberry variety 'Yueshi 123'.
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.
Sequence listing
<110> Bombycis of Guangdong province academy of agricultural sciences and institute of agricultural product processing
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<210> 6
<211> 96
<212> DNA
<213> Mulberry (Morus alba L.)
<400> 6
gttgttgaga gacctttcta agagtgagta gtgctaaata ttgatattaa tggaatctaa 60
taatattagc ctataaggca gatgatcaca ggaatt 96
<210> 7
<211> 23
<212> DNA
<213> Mulberry (Morus alba L.)
<400> 7
atgaaccctc agatgttctc aat 23
<210> 8
<211> 109
<212> DNA
<213> Mulberry (Morus alba L.)
<400> 8
ggctcctacc caattcaaca tgcctcacca gcaactgcag cagcagcaac agcaagcttt 60
gccactatcg cagaatggtg gcacttcaaa accggagtcg aatcattaa 109
<210> 9
<211> 158
<212> DNA
<213> Mulberry (Morus alba L.)
<400> 9
gagttcttga ggggaaagag aaaagcccaa ccagttcatg gaagccatat atatatatat 60
atgttgttga gagacctttc taagagtgag tagtgctaaa tattgatatt aatggaatct 120
aataatatta gcctataagg cagatgatca caggaatt 158
<210> 10
<211> 176
<212> DNA
<213> Mulberry (Morus alba L.)
<400> 10
gagttcttga ggggaaagag aaaagcccaa ccagttcatg gaagccatat atatatatat 60
atatatatat atatatatat gttgttgaga gacctttcta agagtgagta gtgctaaata 120
ttgatattaa tggaatctaa taatattagc ctataaggca gatgatcaca ggaatt 176
<210> 11
<211> 150
<212> DNA
<213> Mulberry (Morus alba L.)
<400> 11
atgaaccctc agatgttctc aatgcagcag cagcagcagc aggctcctac ccaattcaac 60
atgcctcacc agcaactgca gcagcagcaa cagcaagctt tgccactatc gcagaatggt 120
ggcacttcaa aaccggagtc gaatcattaa 150
Claims (8)
1. A SSR molecular marker for identifying the variety of fruit mulberry, yuehu 123, comprising SSR molecular markers M10360 and/or M29602; the repetitive motif of the SSR molecular marker M10360 is (AT)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 M29602 is (GCA)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 Yuehen 123' is characterized by comprising primers aiming at an SSR molecular marker M10360 and/or primers aiming at an SSR molecular marker M29602:
the primer aiming at the SSR molecular marker M10360 comprises the following components:
M10360-F：5’- GAGTTCTTGAGGGGAAAGAGAAA -3’；
M10360-R：5’- AATTCCTGTGATCATCTGCCTTA -3’；
the 5' end of the primer M10360-F is marked with a fluorescent reporter group;
the primer aiming at the SSR molecular marker M29602 is as follows:
M29602-F：5’-ATGAACCCTCAGATGTTCTCAAT-3’；
M29602-R：5’-TTAATGATTCGACTCCGGTTTT-3’；
the 5' end of the primer M29602-F is marked with a fluorescent reporter group.
3. A core primer set of SSR molecular markers for the identification of the Mulberry variety, Guangdong Mulberry 123', 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 123', comprising the SSR molecular marker core primer set of claim 2 or 3.
5. A method for identifying a mulberry variety 'Yuehen 123' 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 by using the genomic DNA extracted in the step (1) as a template and using the primer pairs M10360-F/M10360-R and M29602-F/M29602-R of claims 2 or 3, respectively;
(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 M10360-F/M10360-R as a primer has only two specific bands of 158bp and 176 bp, and the PCR amplification product obtained by using the primer pair M29602-F/M29602-R as a primer has only one specific band of 150 bp, the mulberry sample to be detected is 'Yuexi 123', otherwise, the mulberry sample to be detected is non 'Yuexi 123'.
6. The method for identifying the variety of fruit mulberry, namely Yuehong 123', by using SSR molecular markers according to claim 5, wherein the PCR amplification in step (2) is performed in 20 μ L reaction system, 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 an SSR molecular marker according to claim 1 for identifying the mulberry variety yueher 123'.
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 123.
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Application publication date: 20210924 Assignee: Panzhihua Lvyuan Agricultural Technology Co., Ltd. Assignor: SERICULTURE & AGRI FOOD Research Institute GAAS Contract record no.: X2022440000067 Denomination of invention: SSR molecular marker of a fruit mulberry cultivar 'Yueling 123' and its core primer set, kit and application Granted publication date: 20211109 License type: Common License Record date: 20220801 |