CN116640871A - Morchella SSR molecular marker, primer and application thereof - Google Patents

Morchella SSR molecular marker, primer and application thereof Download PDF

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CN116640871A
CN116640871A CN202310450501.9A CN202310450501A CN116640871A CN 116640871 A CN116640871 A CN 116640871A CN 202310450501 A CN202310450501 A CN 202310450501A CN 116640871 A CN116640871 A CN 116640871A
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刘伟
杨振艳
时晓菲
于富强
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Kunming Institute of Botany of CAS
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Abstract

The invention belongs to the technical field of molecular biological processing, and particularly relates to an SSR molecular marker of morchella conica, a primer and application thereof. The invention provides an SSR molecular marker of morchella conica, which specifically comprises 40 SSR polymorphic sites; the nucleotide sequences of 35 SSR polymorphism molecular markers are successfully developed and are respectively shown as SEQ ID NO. 1-35. The invention integrates the genome data of 4 new morchella strains 20D11A, 20D13A, 20D21A and 20D24A based on the genome data of morchella terranei M04M24 and morchella terranei M04M26, develops the general SSR molecular marker of the morchella terranei through the comparative analysis of the whole genome level, finally obtains 35 SSR sites with high polymorphism and good stability through the population amplification verification, can be used for the identification of the morchella terranei species, the analysis of the population genetic diversity and the like, and has good application value.

Description

Morchella SSR molecular marker, primer and application thereof
Technical Field
The invention belongs to the technical field of molecular biology, and particularly relates to an SSR molecular marker of morchella conica, a primer and application thereof.
Background
Morchella is a precious edible fungus, has delicious taste and crisp and tender mouthfeel, is rich in various bioactive components, has the effects of resisting cancer, resisting tumor, resisting fatigue, improving immunity, protecting liver and the like, and is deeply favored by human beings. Morchella conica is one of cultivars which are pushed in a large area at present, has the advantages of strong temperature difference fluctuation resistance, wide adaptability and stable yield, and is widely popularized in China in recent years. However, due to the weak foundation of the genetic breeding research at the front end, the breeding work of the variety is difficult to advance, and the phenomenon of vitality degradation caused by the random passage of the variety on the market also often occurs, such as the extreme cases that the same strain has obvious difference in each expression, even some areas have high yield, and some areas are dead.
The simple repeated sequence (SSRs, simpleSequenceRepeats), also called microsatellite locus (microsatellite), is a sequence formed by tandem repeat of 1-6 bp repeated units, has the advantages of high sequence variation degree, high stability, co-dominance and the like, is widely used in animals, plants and microorganisms, and simultaneously, SSR molecular markers are widely used in researches such as genetic map construction, target gene calibration, fingerprint map drawing, variety identification, pedigree analysis, inter-population genetic distance analysis, evolution, genetic diversity and the like.
Traditional SSR marker development is carried out by means of conventional repeated sequence enrichment PCR amplification, ISSR (inter-simple sequence repeat) transformation, EST-SSR (expressed sequence tag-simple sequence repeat) sequence development and simple development detection by means of searching SSR sites by means of a single genome sequence. SSR distribution and sequence characterization of Morchella crassipes transcriptome in prior art 1 (Liu Wei, et al. Light industry report 2017,32 (02): 33-39) analyzed the SSR distribution and sequence characterization of Morchella crassipes based on their transcriptome data. SSR information analysis and molecular marker development of the M12-10 transcriptome of Morchella hainanensis in the prior art 2 (Meng Qing, et al, university of Qinghai, 2019,37 (06): 1-10) are also based on Morchella hainanensis transcriptome data for SSR information analysis and molecular marker development, wherein the randomly screened 12 pairs of SSR primers show stable and repeatable polymorphism of 4 pairs of primers, and the collected 19 Morchella hainanensis are subjected to genetic diversity analysis. In the prior art 3 (Ma Jie, et al; morchella EST-SSR marker development and genetic diversity analysis based on transcriptome sequences; jiangsu agricultural report, 2020,36 (05): 1282-1290), morchella was developed by using transcriptome data as well, 15 pairs of SSR primers with better stability were obtained by screening, and 33 pairs of morchella specimens were subjected to genetic diversity analysis. Prior art 4 (Du XH et al hybridization, characterization and transferability of SSRs in the genus Morchella. Functional Biology,2019,123 (7): 528-538) designed a set of SSR molecular markers suitable for M.importuna, M.sextelata, M.eximia, M.exuberans, mel-13 and Mel-21 based on SSR characterization of one Morchella genome data. The SSR marker designed by the prior art has low polymorphism and poor stability, and can not effectively distinguish Morchella conica.
Disclosure of Invention
The invention aims to make up the defects of the prior art, provides an SSR molecular marker of morchella conica with high polymorphism and good stability, and a primer and application thereof, and can be used for effectively identifying the morchella conica species and analyzing population genetic diversity.
The invention provides a Morchella esculenta SSR molecular marker, which comprises SSR01, SSR02, SSR03, SSR04, SSR05, SSR06, SSR07, SSR08, SSR09, SSR10, SSR11, SSR12, SSR13, SSR14, SSR15, SSR16, SSR17, SSR18, SSR19, SSR20, SSR21, SSR22, SSR23, SSR24, SSR25, SSR26, SSR27, SSR28, SSR29, SSR30, SSR31, SSR32, SSR33, SSR34 and SSR35, wherein the nucleotide sequences of the SSR01, SSR02, SSR03, SSR04, SSR05, SSR06, SSR07, SSR08, SSR09, SSR10, SSR11, SSR12, SSR13, SSR14, SSR16, SSR17, SSR18, SSR19, SSR20, SSR21, SSR24, SSR25, SSR26 and SSR35 are shown in SEQ ID NO.
The invention also provides a primer combination for amplifying the Morchella terraced SSR molecular marker, which comprises a forward primer combination and a reverse primer combination;
the nucleotide sequence of the forward primer combination is shown as SEQ ID NO. 36-70;
the nucleotide sequence of the reverse primer combination is shown as SEQ ID NO. 71-105.
The invention also provides application of the SSR molecular marker or primer combination in one or more of the following (a) - (d):
(a) Constructing a molecular fingerprint of Morchella conica;
(b) Screening Morchella conica;
(c) Identifying strain quality resources of the morchella;
(d) Analysis of genetic diversity of Morchella conica.
The invention also provides a molecular fingerprint of Morchella conica, which is constructed by the primer combination in the technical scheme.
The invention also provides a morchella conica genetic diversity analysis kit, which comprises the primer combination in the technical scheme.
The invention also provides an analysis method of morchella conica genetics, which comprises the following steps:
taking Morchella conica genome DNA as a template, and carrying out PCR amplification by using the primer combination in the technical scheme to obtain an amplification product;
detecting the amplified product by electrophoresis to obtain an SSR locus polymorphism amplified strip;
genetic diversity analysis was performed based on the amplified bands.
Preferably, the PCR amplification reaction system consists of Mix 17. Mu.L, 10. Mu.M forward primer 1. Mu.L, 10. Mu.M reverse primer 1. Mu.L and 25-50 ng/. Mu.L Morchella genome DNA 1. Mu.L in 20. Mu.L.
Preferably, the PCR amplification procedure is: pre-denaturation at 98℃for 2min; denaturation at 98℃for 10s, annealing at 60℃for 10s, extension at 72℃for 10s,35 cycles; finally, the extension is carried out for 5min at 72 ℃.
Preferably, the genetic diversity analysis comprises calculating a genetic diversity index.
Preferably, the genetic diversity indicator comprises one or more of an allele, an effective allele, a private allele, and shannon index.
The invention provides an SSR molecular marker of morchella conica, which specifically comprises 40 SSR molecular markers; the nucleotide sequences of the SSR molecular markers are respectively shown in SEQ ID NO. 1-35. According to the invention, based on genome data of the morchella terraced M04M24 (https:// www.ncbi.nlm.nih.gov/assambly/GCA_ 003444645.1) and the morchella terraced M04M26 (https:// www.ncbi.nlm.nih.gov/assambly/GCA_ 003444635.2), 4 new morchella terraced strains 20D11A (https:// www.ncbi.nlm.nih.gov/sra/SRR 16686487), 20D13A (https:// www.ncbi.nlm.nih.gov/sra/SRR 16686505), 20D21A (https:// www.ncbi.nlm.nih.gov/sra/SRR 16686497) and 20D24A (https:// www.ncbi.nlm.nih.gov/sra/SRR 16686494) are integrated, and through comparative analysis of genome level of the morchella terraced strains, 35 polymorphic sites with high stability are finally obtained, and the method can be used for identification of morchella terraced species, genetic diversity analysis and the like.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are required to be used in the embodiments will be briefly described below.
FIG. 1 is a partial amplification capillary electrophoresis detection peak diagram of an SSR molecular marker Mimp_692 primer set, wherein 3 small diagrams from top to bottom respectively represent detection peak diagrams of Mimp_692 on a total of 3 samples of SC_ B, ZY _ZNO.1 and ZY_No. 4;
FIG. 2 is a 35 pairs of SSR primer amplification fingerprints of 24 Morchella conica populations;
FIG. 3 is a chart showing the results of SSR genetic diversity STRUCTURE analysis of Morchella conica;
fig. 4 shows the results of SSR genetic diversity analysis of 24 strains of morchella conica.
Detailed Description
The invention provides a Morchella esculenta SSR molecular marker, which is characterized by comprising SSR01, SSR02, SSR03, SSR04, SSR05, SSR06, SSR07, SSR08, SSR09, SSR10, SSR11, SSR12, SSR13, SSR14, SSR15, SSR16, SSR17, SSR18, SSR19, SSR20, SSR21, SSR22, SSR23, SSR24, SSR25, SSR26, SSR27, SSR28, SSR29, SSR30, SSR31, SSR32, SSR33, SSR34 and SSR35 which have nucleotide sequences shown in SEQ ID NO. 1-35 respectively.
The nucleotide sequences shown in SEQ ID NO. 1-35 of the present invention are specifically as follows:
SEQ ID NO.1(SSR01)：5’-TTGCACGGCATTGAGGAGAAGAAGAAGAAGGAGGAGGAGGAGGAGGA GGAGGAGATGGGAGGATTGGTTGGCCGTTGTTAATTCTTGCTTTCCTTCCCTCGCTCAATGTTGATACCGATT TGTTTTCTTTTGTGCGCGGGGCCGGAGGGGAAAAACGTACTGCTAGCTTTGTTTATCCTCTTTCCCCGCGTAT GTAGC-3’；
SEQ ID NO.2(SSR02)：5’-TAGCACTTGTTGGCGAGCTCGAAGGCGAGCTCTGTGCGGATGCGGTTTGGCCCATGGCTTCTTCTTTCTTTTCTTTCTTTCTTTCTTTCTTTCTATCTTTCTAAATGTGTAGTCTTTTATTTTTTTATGTAGGATAGGTTAAGGTCGAGGTTGAAGCTCAAGTCGGTGGTAGTGGTAGTGGT-3’；
SEQ ID NO.3(SSR03)：5’-CTGTTGCTGTTGCTGTTGCTTCGGCTGTTGTTGCTGCTGCTGCTGCTGCTGCTGCTGCTGCTGCTGCTGGGCTGGTACGGGCTCGGGGTAGGATTGTTGATGCGGAATAGGAGGTTCCTGGTGCTCATACGGAGA-3’；
SEQ ID NO.4(SSR04)：5’-TGGGTTGGACTAGAGGGAGTTTGGGAGATATGAGATTCTAGATATGGCCATGAGCTTATGTACATGTAGGCAGCTCTTCTTCTTCTTCTTCTTCTTAATGATCCGGACTGCATGAGC-3’；
SEQ ID NO.5(SSR05)：5’-GCCGCCGTTATTAGGACCTTTCCCTTCCCTCCCTTCCCTTCCCTTCCCTTCCCTTCCCTCCCCTACCCCATATCCCGAAGAAGGGAAAGAAAATCAAATCACAAAGATGATCAGTGCTCAACAGGATCTATGGGATCTGGCTGGCAG-3’；
SEQ ID NO.6(SSR06)：5’-CTAACGCACCCATCATCCCATCCCATCCCCTCCCCTCCCCTCCCCTCCCCTCCCCTCCCCTCCCCACAATCCCCCACAAAAGCACACACCCCAGCACACTACCGCATCCCCCAACCCCTCCCACCAAAACCGCACACTACATAAACTCCCTCAACCTCCTCAACTTCTCCTTGGCCG-3’；
SEQ ID NO.7(SSR07)：5’-TTCTGAAGCGGTACCGAGTGCTCTGAGAGCAATCTAGGAGTTTGATTCGACTTTGAAATAAAAGCAATGCAATTACTACCTGGAATAATCTAATCTAATCTAATCTAATCTAATCCTATCCGATATAACCCATCCATTGATATACTCTCGCAAGACAAAGGGA-3’；
SEQ ID NO.8(SSR08)：5’-TGTACTAGCTGGCTGGGACTCTTTTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTGTCAACGGGGCTTCTCTCCTCTCCTCTCTTCTCCTCTCCAACTGGCCCGACTAACGAAAATGTCAATGGGAAACAGGACCAGCTAGCGT-3’；
SEQ ID NO.9(SSR09)：5’-GTCAGGCCGATCAAATCCCAATCGTGTTACTTAAATTATAACTCAGTACGGAAGCCGCTATATATATATATATATATATATATAAAAAGAAAAAAAACTAGCTCCTCACGAATTTACACCAGTCCTTAATTAGTGAGAGATCACCTCGTCATGCAATGGACA-3’；
SEQ ID NO.10(SSR10)：5’-CCCGTCAGCTAAACCTGGAATTTGGCTGTTTTTGGGGGGAAACAAAAATTTTGGGTATGAAAAAGGGGAGTGAGTGAAGAAATCTATATATATATATATATATATATAAATTCCAAACACGCACACACACAGGACGTACCTTCCACTCTTCAACTTCGTACCGCAACGCCTCATTCTT-3’；
SEQ ID NO.11(SSR11)：5’-AGCAGAGAAGGGGAGAGAGGATGAGAAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGATGGGGCTGTAGAAGAAGAATCCTTCTGGAAGAACACCCTGCAACA-3’；
SEQ ID NO.12(SSR12)：5’-AGGTCTGGCGCTTGTTGAGCGCCAGGGCGGGGGTCCCGCTGCGGCGTTTGAGCTCTTGGAGCGTGGGGAGGTTTGTGGGGAGCGGGGCGTCGGCGTTCGGCGGGGATGAGGCGGAGGCGGCGGCGGCGGCGGCGGCGGAGGGTGTGGGTGCGCTGGGAGAGGCGGCGGAGGCGGTGAGCGCTAGCAACGCTATGGAGGGG-3’；
SEQ ID NO.13(SSR13)：5’-GCCATCTGCTAGTCCTGTCGGTGCCAAATCGGGAGGAGGAGGAGGAGGAGGAGGTGGAGGAGGAGGACATCAAGACGCCCCAACAATCGGACGGGGTGGTGGTAGAACTCCGGAACTGTGTGGAGAGCTT-3’；
SEQ ID NO.14(SSR14)：5’-CAAATCAATCTCACCATGTGAGCATATATTATAACTTGGGATGTGAGTAATTAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCACCGGTGGTACATGTAACTCTACCCTACATGTAATGTAGATAATGTATGTGATAGGTACATCGTACCTACATACATGAGCAACGTCCCCCCTAAATAGACGTCCGACA-3’；
SEQ ID NO.15(SSR15)：5’-TGGAGACTGGTGGGAGTAGGTGGAGTTGTGGTTCTGGGGAGAGGGAGGGAGGGAGGGAGGGAGGGAGGGAGGGAGGGAGACGGGGGGATAGAGAGACCCGAAGGAACAAGAAGAGCCAAAGGTCGAGAAGGGTTGATGCACAGCCAGCACCCCGAGTGACAATCCATGCACCACAGC-3’；
SEQ ID NO.16(SSR16)：5’-GGGAACGGGTTGGCTGATAAGGTGGGGAAGATGAGAGTGGAAGAAAAGGAAGAAGAAGAAGAAGAAGAAGAAGAAGAAGAAGAGAGCTGTGAGAAGCTAACAGCCCAAGGTGTTTTGGCCCGCCAGGTACCAACCAGCACTTGG-3’；
SEQ ID NO.17(SSR17)：5’-AGTGAGGGCGTGAGTGATTGAGGGAGTAGTGAGTGAGTGATAGAGAACCTTGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGATAGAACGCCGCGCCGCACAACCACCAAGCAGGCACACTCACTCGTTCACGCTT-3’；
SEQ ID NO.18(SSR18)：5’-CCGCCTCGATCTTTCTTCCAACAGCATACTGCGACCACCACCACCACCACTTGAGATACCAACCATTCCTTCAAAGTCGTTCTTTCAAATACATCCACAATGTCTGACCAATTCGCACAATTCGCCGAG-3’；
SEQ ID NO.19(SSR19)：5’-ACTCTACCGTCCTGTCGACAGGTGAGACAGCCGATAAGAGTAGAGTAGAGTATGTAGGTAGGTACATGTATGTTCTAAGCTACTGTCTGTCTGTCTGTCTGTCTAGCGAGTAGCCAGAGTCTCGCGAGCGGAAACAGAAACAGAAACAGAAACAGAAAAAGAACATCCGCACTTGAGTTCTCGG-3’；
SEQ ID NO.20(SSR20)：5’-CAGGGGTTGGCGAATAAGGTTTGACTGAGTCACTGCTACACACATACATACATACATACATACATACATACATACATACATACATACATACATACTCGTTTACCACGAAAAGAACCAGAAAATGCAAGTGACGGAAACAGGGATAGTTTGTATGAGAGATCGCCGCC-3’；
SEQ ID NO.21(SSR21)：5’-CGAAAGCGGTGTCGAAATGGTATCGTCGGCTGGCAGGATGGTTGAGGCGGCCGGTGACGGCGACGGCGACCGTGAACGAGTGTGTGGGAGCGACATTAAGAAATGGGTTTAATATGATCCGGTGGGGATTCGAGAGCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTTTTGTTTTGGCCCTGGAAGTGGAG-3’；
SEQ ID NO.22(SSR22)：5’-CTCGACACCACCACCATGAAAACAAACTTTGTCTGTTTGTAAATACCTACATGCTTAAGATAGTGAATATTCCAGGATACCATTCAGCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCGTTGCCCTAGGTATGTAGTACTTCTTCACCAACATGCACCAAACAAGCGAC-3’；
SEQ ID NO.23(SSR23)：5’-TCCGAGTCGACTCCTGCATAATATTGTAGATGTTCCTCTACGATTCCTTCCGCGATGCCGCGCAGTTCCGAATAGAACTCTCTCTCTCTCTCTCTCTCTCTCTCTCCCCGCGAGAAGTCTTGAACGGATATACACACCTTTTGATCTTATTTCGAGGATTTCGGCACTCTGGCTA-3’；
SEQ ID NO.24(SSR24)：5’-CACCATCCATCGTTCCTGCTAACGGATTCTTCCTCTCTCTCTCTCTCTCTCTCTCTCTCTCCTCGCACTCCCAACTCACTCTAGACCATTTGCGAGCATGCAT-3’；
SEQ ID NO.25(SSR25)：5’-CTAGGCAGATCACGCAGGTGTCGGCGGCGGCGGCGGCGGCGGCGGCGGCGGGCTCTGCGGTTTGGTCCTCGTTCGTGGGAGTGGCGCTCATTTGCCTGGCGAGGTGTGGAGGTGGAGGGAGTGGCGTGCGGGAGCTCTCTGTGCTTGTGTTACAGGGGATTGTTAGTTGGGGGCCCCTAACCCTTGCCAAGACGT-3’；
SEQ ID NO.26(SSR26)：5’-ATCCACGCTCAGGGCTGATGGGGTGCGTGGCCTTCTCGGTCGTCTCCGTGAGCGCCTACACCGCCGCCGCCGCCGCAGACGCCACAGGCACGGCGGCGGGCGCGGCAAGGATTGCGTGGACGCGGGGCGCGGCGCTGATTGTGGGCATTGTGAGCG-3’；
SEQ ID NO.27(SSR27)：5’-CCCATTCTCGTGCCTCTCTGTCCCTCTCTTTGAATTTTACACCTCTATTATGTACTCTATGTAGAGTAGCCCAGCCAGCCAGCTGCTGCCAGCCCAGCCCAGCCCAGCCCAGCCCAGCCCAGCCAGCCACCGCCCAAAAGAACATCCTCTTTGTTCCCTTTGACATTGTCGATTGAAAAGCACACTCCTCCCC-3’；
SEQ ID NO.28(SSR28)：5’-GGAGAAACCCGGTAACTGCAACTTCTAATATCTACTTCATCACCACCACCTCCACCACCACCACCACCACCACCACCTCCTCCTCCTCCCAGAGCTGGAACCTCAGGAAATTCTCATAGTCAGCGCTCACGAC-3’；
SEQ ID NO.29(SSR29)：5’-GCTCTGGGGCAAAAGCTTGCTAGGCAATCTACATTTTGTCTCACTCCCCT AAAAAAAAATAAAGAAAGAAAAGAAAAGAAAAGAAAAGAAAAGAAAAGAAAAGAAAAGAAAAGAAA AGAAAAGAAAAGAAAAGAAAAGAAAAGAAAAGATGCAATGCAACAAATTTGAGTTCATAAATATTTAACC AACGCCTGCCTACA-3’；
SEQ ID NO.30(SSR30)：5’-CCGCCGCCATTCACAATAAACCATACCTCCCAAATTTAAAAAACAGAAG AAAAAAAGAGAATCACGAACTGAGCAGATCCATCACACTCATCCCCCGCCCCTTCTTCTCTGGTATTACACC ACCACCACCACCACCACCACCACCACCACCACTACCCTGCCTACTACTACTCCCCGCACCC-3’；
SEQ ID NO.31(SSR31)：5’-GCAATTGCCTGAGCTAGACGAATATTACAGAAATTAATACAATCTCGCAT ATACATAACCTTTAATTGATGTTTAGGTGTAGCAGCTGTTAAGCCCTGGTTAATGCCTAACCCACACACACAC ACACACACCCCCCCCAGGGGGCCCCCCTTCCCCTTGTTTCCGTT-3’；
SEQ ID NO.32(SSR32)：5’-TTGCCGGCTTTAGGTGCATACCGGAATCGAAGGTGGATAGACGATCGAG GCCCATATATATATATATATATATATATATATATTTTCAATTTTCAATCTCTACGGGGCCAAAAACAGCCAAGC ACAGGGCAGTAGTACAGACTACTAAAGCCCTATACCAGCCCAGTCCACTAAGTCTTGGTCCGCAGC-3’；
SEQ ID NO.33(SSR33)：5’-CCACGCAGGTTTTGGTTTGTAGATACATACACAAGTAAATACTCAGTCTC ATGATTACAAAACGCATATATATATATATATATATATATTACTGGCATGTGAGTGCTGTTTGTCCGA-3’；
SEQ ID NO.34(SSR34)：5’-GGACGGATCTCTGACACGTGTTTTCCCCGGAGCACACACAGCTCTCTC TCTCTCTCTCTCTCTCTCTCTCTCTCCATGTTTGTGGTTTGCTCTCAGCTGCGAGCATGACTGTACTGCACT GCACTCCA-3’；
SEQ ID NO.35(SSR35)：5’-CCGAGTTCGAGCTTGACCTTAAATTCGGCATAATTCCATCCTCACCTAGC TAGCTCTATATATATATATATATATATATATACCCACCCATATCTCCACTCTAAATCATCACAAAGCCCACACA CG-3’。
the invention integrates 4 new genome of the morchella terrae strains 0D11A, 20D13A, 20D21A and 20D24A based on genome data of the morchella terrae M04M24 and the morchella terrae M04M26, develops the general SSR molecular markers of the morchella terrae, finally obtains the 35 SSR sites with high polymorphism and good stability, can be used for identification of the morchella terrae species, analysis of population genetic diversity and the like, and has good application value.
The invention also provides a primer combination for amplifying the Morchella terraced SSR molecular marker, which comprises a forward primer combination and a reverse primer combination; the nucleotide sequence of the forward primer combination is shown as SEQ ID NO. 36-70; the nucleotide sequence of the reverse primer combination is shown as SEQ ID NO. 71-105.
The correspondence between the nucleotide sequences shown in SEQ ID NO. 36-105 and SSR molecular markers is shown in the following Table 1.
Table 1 correspondence of primer nucleotide sequences shown in SEQ ID Nos. 36 to 105 to SSR molecular markers
The invention designs the amplification primer based on the Morchella esculenta SSR molecular marker, has the characteristics of good stability of PCR amplification result and high polymorphism, can be used for identification of Morchella esculenta species, analysis of population genetic diversity and the like, and has good application value.
The invention also provides application of the SSR molecular marker or primer combination in one or more of the following (a) - (d):
(a) Constructing a molecular fingerprint of Morchella conica;
(b) Screening Morchella conica;
(c) Identifying strain quality resources of the morchella;
(d) Analysis of genetic diversity of Morchella conica.
The invention also provides a molecular fingerprint of Morchella conica, which is constructed by the primer combination in the technical scheme. The specific mode for constructing the molecular fingerprint of the morchella conica is not strictly required, and the molecular fingerprint construction mode well known in the invention is adopted.
The invention also provides a morchella conica genetic diversity analysis kit, which comprises the primer combination in the technical scheme. The morchella conica genetic diversity analysis kit preferably further comprises PCR amplified MIX, dNTPs and Mg 2+ 。
The invention also provides an analysis method of morchella conica genetics, which comprises the following steps:
taking Morchella conica genome DNA as a template, and carrying out PCR amplification by using the primer combination in the technical scheme to obtain an amplification product;
detecting the amplified product by electrophoresis to obtain an SSR locus polymorphism amplified strip;
genetic diversity analysis was performed based on the amplified bands.
The invention uses the morchella genome DNA as a template, and uses the primer combination in the technical scheme to carry out PCR amplification to obtain an amplification product. In the invention, the reaction system for PCR amplification is preferably 20 mu L, and consists of Mix17 mu L, 10 mu M forward primer 1 mu L, 10 mu M reverse primer 1 mu L and Morchella conica genome DNA1 mu L; the PCR amplification procedure is preferably: pre-denaturation at 98℃for 2min; denaturation at 98℃for 10s, annealing at 60℃for 10s, extension at 72℃for 10s,35 cycles; finally, the extension is carried out for 5min at 72 ℃. The concentration of the morchella conica genomic DNA is preferably 25-50 ng/. Mu.L, and more preferably 50 ng/. Mu.L. The source of the morchella conica genomic DNA is not strictly required, and the morchella conica genomic DNA can be obtained by adopting a mode well known in the art, such as a Tsingke TSP102-50 genomic DNA extraction kit (Beijing qingke biosciences, inc.), and the morchella conica genomic DNA can be obtained by extracting according to the specification.
After the amplification product is obtained, the invention detects the amplification product by electrophoresis to obtain SSR locus polymorphism amplification strips. In the present invention, the electrophoresis preferably includes capillary electrophoresis.
After the SSR locus polymorphism amplification band is obtained, the invention performs genetic diversity analysis according to the amplification band. In the present invention, the genetic diversity analysis preferably includes calculating a genetic diversity index; the genetic diversity index preferably comprises one or more of allele (Na), effective allele (Ne), private allele (Np), shannon index (I). The present invention preferably uses the GenAlEx version 6.501 and Popgen32 software for genetic diversity analysis.
The SSR molecular marker has good polymorphism and higher stability, the primer combination of the technical scheme is used for analyzing the morchella conica genetics, has the characteristics of good stability of PCR amplification results and high polymorphism, and is different from the traditional polyacrylamide gel electrophoresis detection technology, the capillary electrophoresis detection technology is adopted, the experimental result is accurate and reliable, the detection efficiency and accuracy are greatly improved, a foundation is laid for the identification of the morchella conica species, the analysis of population genetic diversity and the like, and the method has good application value.
For further explanation of the present invention, the morchella esculenta SSR molecular markers, primers and applications thereof provided by the present invention are described in detail below with reference to the accompanying drawings and examples, but they should not be construed as limiting the scope of the present invention.
Example 1
Detection and development of morchella conica genome level SSR molecular markers
(1) Four morchella genome assembly
Four morchella 20D11A, 20D13A, 20D21A and 20D24A strains were sequenced with a sequencing protocol of two generations Illumina HiSeq 4000 double-ended PE500, each strain sequenced with the rawdata of 4G, two strains were genome assembled with the genome assembly software SPAde (v.3.15.3), SPAde specific parameters: the parameters are set.py-isolate-pe-111. Fastq-pe-212. Fastq-t 80-K91-cov-cutoff auto-m 1000-o./K91.
The assembly result shows that the 4 new morchella strains are independently assembled to obtain 49.85Mb, 50.07Mb, 50.68Mb and 49.82Mb, and the sizes of two genomes of the M04M24 strain (50.77 Mb) and the M04M26 strain (51.08 Mb) which are published in the earlier stage are equivalent, so that the later comparative genome analysis can be satisfied;
(2) The genome of the Morchella conica M04M26 is used as a reference, and the Candii SSR software is used to integrate the genome of M04M24, 20D11A, 20D13A, 20D21A and 20D24A5 Morchella conica, so as to develop the Morchella conica universal SSR molecular marker, wherein specific parameters of the software are as follows: candriSSR.pl-i Morexi.ctl-o Morexi out-l 200-t 100.
The result shows that 783 high-quality candidate SSR sites are obtained by screening among 6 morchella conica genomes, and after deleting the SSR sites deleted in a certain strain, 649 SSR sites which are common to all 6 strains and have polymorphism are obtained and used as candidate SSR sites. Through SSR site heavy sequence types, repetition times and polymorphism in the population, 40 SSR sites with uniform distribution and higher polymorphism are obtained by screening and used for candidate polymorphism marker development, and specific 40 SSR site information is shown in the following table 2.
Table 2 morchella 40 universal SSR site features
SSR bit, dot Chromosome location Repeated sequence Number of repetitions SSR site Chromosome location Repeated sequence Number of repetitions
SSR01 8 AGG 8 SSR21 6 CT 18
SSR02 5 TTTC 6 SSR22 2 CT 18
SSR03 4 TGC 12 SSR23 19 CT 14
SSR04 14 TCT 7 SSR24 19 CT 14
SSR05 11 TCCCT 6 SSR25 13 CGG 10
SSR06 9 TCCCC 8 SSR26 23 CCG 5
SSR07 12 TAATC 6 SSR27 21 CCAGC 7
SSR08 9 TC 20 SSR28 5 CCA 8
SSR09 16 TA 13 SSR29 2 AAAGA 17
SSR10 22 TA 12 SSR30 3 CAC 12
SSR11 15 AG 20 SSR31 4 CA 9
SSR12 21 GGC 7 SSR32 16 AT 15
SSR13 12 GGA 7 SSR33 6 AT 12
SSR14 5 GCA 11 SSR34 5 CT 8
SSR15 3 GAGG 9 SSR35 23 TA 13
SSR16 7 GAA 11 SSR36 4 GTG 17
SSR17 11 GA 21 SSR37 11 GATTA 6
SSR18 6 ACC 5 SSR38 2 CCCTC 7
SSR19 22 CTGT 5 SSR39 6 CAGG 15
SSR20 1 ACAT 13 SSR40 23 CA 11
The nucleotide sequences of SSRs 36 to 40 in Table 2 are shown as SEQ ID NO.106 to 110, and are specifically as follows:
SEQ ID NO.106(SSR36)：5’-CAGGGGAGGGGATTCGTTTTTTTACTTTCTTGTTTTGTTTCCGGAATCAATTGCAAGTGGTGGCAGTGGTGGTGGTGGTGGTGGTGGTGGTGGTGGTGGTGGTGGTGGTGGTGGTGGTCGTGAAAAAAGAGGCAACGCATATATCATCTATGGAGGAGGGCGT-3’；
SEQ ID NO.107(SSR37)：5’-TCCCTTTGTCTTGCGAGAGTATATCAATGGATGGGTTATATCGGATAGGATTAGATTAGATTAGATTAGATTAGATTATTCCAGGTAGTAATTGCATTGTTTTTATTTCAAAGTCGAATCAAACTCCTAGATCGCTCTCAGAGCAGTCGGTACCGCTTCAGAA-3’；
SEQ ID NO.108(SSR38)：5’-CCTCTTCCTCACTCCCTCCTGCCCTCCCCTCCCCTCCCCTCCCCTCCCCTCCCCTCCCCCGCCATCAATAACAAACCATCCCCTCTATATACACCATCTGCTTCAGCTTAGACAAAGTGTCTATACTTCCTCCTTGCATACGCGA-3’；
SEQ ID NO.109(SSR39)：5’-GGCCAGGCAGAAAGGATGATATATATGTCAGGCAGTCCAGGCAGGCAGGCAGGCAGGCAGGCAGGCAGGCAGGCAGGCAGGCAGGCAGGCAGGCAGGCAGTTAAGTTGGTTAGGTTGTGAAGAAGAAAATGAGTGAGTGCCATGCCCGACCCACCCAACAACAACCTT-3’：
SEQ ID NO.110(SSR40)：5’-GGAGGGATGATGAGGGGAGAGGGGAAATCAGAAAGCAAGAGGGTGATGGGGGCCTTTTAACGATTCCTGTCACACACACACACACACACACACGCACACACACACCACAGCGGCGAAGCTTCACAGAGAGGGGAAATGTCTGCTGC-3’。
(3) Extracting 250bp sequences at the upstream and downstream of a candidate SSR locus, developing SSR primers by using Primer3.0 software, setting the annealing temperature of the candidate primers to be 60+/-3 ℃ and the length of a PCR product to be 20+/-3 bp, combining the length of the PCR product, the difference of the polymorphism sizes, the matching degree of the primers and the standard deviation of the polymorphism of the candidate locus, screening the candidate SSR locus and the primers, and finally selecting 40 pairs of primers with highest polymorphism and best transferability, namely, a forward primer with nucleotide sequences shown as SEQ ID NO. 36-70 and SEQ ID NO. 111-115 and a reverse primer with nucleotide sequences shown as SEQ ID NO. 71-105 and SEQ ID NO. 116-120, as the universal SSR primer combinations of the morchella, wherein the primer information is shown in the following table 3.
Table 3 morchella primer information
Example 2
SSR genetic diversity analysis of 24 strain groups of Morchella conica
(1) 45 strains of Morchella conica are collected from a national Morchella mainplanting area, 24 strains with different names of non-homologs are screened as far as possible through tracing and preliminary genetic diversity analysis, and classification status of all strains is determined through ITS-RPB1-RPB2-EF1a-LUS polygenic system developmental analysis. The main information of the test strains is shown in Table 4.
Table 4 morchella population information
Strain numbering Acquisition ground Year of acquisition Phylogenetic name
CY_No.1 Sichuan Jintang (Sichuan gold hall) 2012 Morchella importuna
XS_M140 Sichuan Qingchuan tea 2014 Morchella importuna
SC_S3 Sichuan Mianyang 2015 Morchella importuna
16-19 Sichuan Mianyang 2015 Morchellaimportuna
SCBC_No.4 Sichuan North Chuan 2016 Morchella importuna
SC_A9 Sichuan Mianyang 2014 Morchella importuna
CY_No.3 Sichuan Jintang (Sichuan gold hall) 2014 Morchella importuna
TC17_LN Sichuan Qingchuan tea 2018 Morchella importuna
WH_M2 Hubei Wuhan 2017 Morchella importuna
SZL-1 Hubei pine cone 2016 Morchella importuna
SC_Z13 Sichuan Deyang 2016 Morchella importuna
T302 Sichuan Mianyang 2018 Morchella importuna
SC_9B All of Sichuan Cheng 2014 Morchella importuna
ZY_ZNO.1 Chongqing Peng Shui 2013 Morchella importuna
ZY_No.4 Chongqing Peng Shui 2012 Morchella importuna
HE_JS Henan Luoyang (Henan Luoyang) 2018 Morchella importuna
HE-MA Zhengzhou Henan 2019 Morchella importuna
HE_G6 Sichuan Mianyang 2021 Morchella importuna
HE_T11 Sichuan Mianyang 2021 Morchella importuna
16-117 Sichuan Ab dam 2016 Morchella importuna
2016-37 Sichuan Guangyuan 2016 Morchella importuna
MMS-5 Hubei pine cone 2016 Morchella importuna
MMS-lan Hubei pine cone 2016 Morchella importuna
HE_LUO Henan Luoyang (Henan Luoyang) 2018 Morchella importuna
Note that: the above strains were stored in the wild biomass resource pool of southwest China of Kunming plant institute of China academy of sciences.
(2) Synthesizing 40 pairs of primers screened in Table 3 (primer synthesis is carried out by Beijing qing department Biotechnology Co., ltd.) and adding FAM fluorescent signals at the 5' end of the primers while synthesizing, respectively extracting the genome DNA of 24 strains in the step (1) by using a Tsingke TSP102-50 genome DNA extraction kit (Beijing qing department Biotechnology Co., ltd.), and carrying out PCR amplification on the genome DNA of each strain by using 40 pairs of primers (each primer pair respectively carrying out PCR amplification on the genome DNA of each strain), thereby obtaining an amplification product. Wherein, the PCR amplification system is as follows: mix (Tsingke TSE101, china) 17. Mu.L, 10. Mu.M forward primer 1. Mu.L, 10. Mu.M reverse primer 1. Mu.L, genomic DNA (gDNA, 25-50 ng/. Mu.L) 1. Mu.L;
the PCR reaction procedure was: pre-denaturation at 98℃for 2min; denaturation at 98℃for 10s, annealing at 60℃for 10s, extension at 72℃for 10s,35 cycles; finally, the extension is carried out for 5min at 72 ℃.
(3) Site detection: the amplified product obtained in step (2) was subjected to capillary electrophoresis by a 3730xl sequencer.
According to capillary electrophoresis, the polymorphism of the SSR molecular marker primer amplified by the screening and related method is good, and the capillary electrophoresis detection peak diagram of a part of samples (SC_ B, ZY _ZNO.1 and ZY_No. 4) amplified by one of the Mimp_692 molecular marker primers is shown in figure 1, and the SSR molecular marker primer has good amplification effect, stable base line, sharp peak and good polymorphism.
(4) Site analysis: analyzing the accurate position of the data by using software Gene mapper 4.1, judging the polymorphism of the detection primer according to the analyzed position information, and marking 1 if the sample has fragments at the position and marking 0 if the sample does not have fragments at the position. The results are shown in FIG. 2.
As can be seen from fig. 2, 35 pairs of the 40 pairs of primers screened in example 1 can be amplified efficiently in 24 samples, wherein 32 pairs of primers have obvious polymorphisms, 112 allele loci are detected in total, wherein the minimum allele number is 1 (mip103, mip328, mip596), the maximum allele number is 7 (mip242), and the average allele number is 3.2857.
(5) Analysis of genetic diversity: in the GenAlEx version 6.501 and Popgen32 software, the genetic diversity index of each SSR site, including sequence polymorphism (PP), observed allele (Na), effective allele (Ne), private allele (Np), shannon index (I), was calculated, respectively, and the results are shown in table 5.
The total number of effective alleles (Ne, the more evenly distributed the alleles are in the population, the closer Ne is to the number of actual detected alleles) is 48.6427, the range of variation in the number is 1.0926 (mip167, mip593) -1.9395 (mip103), and the average effective allele number per locus is 1.3560. The number range of the diversity index (H) is 0.0822 (mip309) -0.4844 (mip103) and the average value is 0.2161. The shannon index (I) has a value ranging from 0.1664 (mip309) to 0.6775 (mip103) and an average value of 0.3420. The SSR primer obtained by screening has higher polymorphism, and can be used for population genetic analysis of the experimental material.
Table 5 SSR polymorphism characteristics of 35 pairs of general purpose type obtained by screening Morchella esculenta populations
(6) The population genetic structure of the experiment is carried out in STRUCTURE version 2.3.3 software based on a population clustering method of a Bayesian model. The Markov chain (Markov Chain Monte Carlo, MCMC) method employed may preset the population grouping (K) while individuals are calculated, sampled and grouped according to allele frequencies. Parameter setting: the K values were set to a range of 1-10, 10 independent runs were performed for each K value, and the number of resampling per cycle was set to 100,000. Finally, in a STRUCTURE HARVESTER (http:// taylor0.biology. Ucla. Edu/struct_harvest /) website, the optimal K value was calculated based on the method of Evanno et al (2005), and the results are shown in FIG. 3.
As can be seen from FIG. 3, the optimum deltaK value calculated in STRUCTURE HARVESTER according to Evano et al (Evano G., regnaut S., goudetJ.2005. Detection the number ofclusters ofindividuals using the software STRUCTURE: a-analysis student. Molecular biology, 14:2611-2620.) was 3, indicating the presence of 3 gene banks in 2 populations.
(7) After the similarity matrix file is calculated in NTSYS, individual clustering trees are respectively established based on the similarity matrix by adopting an unweighted group average method (UPGMA) of group method with arithmetic means. The specific method comprises the following steps: the similarity matrix or distance matrix is obtained in the similarity module of the NTSYS software, and the clustering tree is obtained in the clustering module, and the result is shown in FIG. 4.
As can be seen from fig. 4, the UPGMA plot based on individuals shows that there is more cross-mixing between groups and groups of individuals for 24 strains, indicating that there is more genetic variation in the groups. There are some synonyms such as WH-M2 and MMS-5, and TC17 LN and SZL-1 strains with 100% genetic similarity. The other 20 strains are clustered on different branches respectively, show obvious genetic diversity, are clustered together under 50% similarity, and can be divided into 3 groups under 60% similarity.
According to the content, the SSR molecular marker provided by the invention has high polymorphism and good stability, can be used for identification of Morchella conica species, analysis of population genetic diversity and the like, and has good application value.
Although the foregoing embodiments have been described in some, but not all, embodiments of the invention, it should be understood that other embodiments may be devised in accordance with the present embodiments without departing from the spirit and scope of the invention.

Claims (10)

1. The Morchella esculenta SSR molecular marker is characterized by comprising SSR01, SSR02, SSR03, SSR04, SSR05, SSR06, SSR07, SSR08, SSR09, SSR10, SSR11, SSR12, SSR13, SSR14, SSR15, SSR16, SSR17, SSR18, SSR19, SSR20, SSR21, SSR22, SSR23, SSR24, SSR25, SSR26, SSR27, SSR28, SSR29, SSR30, SSR31, SSR32, SSR33, SSR34 and SSR35 which have nucleotide sequences shown in SEQ ID NO. 1-35 respectively.
2. Amplifying the primer combination of the morchella SSR molecular marker of claim 1, wherein the primer combination comprises a forward primer combination and a reverse primer combination;
the nucleotide sequence of the forward primer combination is shown as SEQ ID NO. 36-70;
the nucleotide sequence of the reverse primer combination is shown as SEQ ID NO. 71-105.
3. Use of an SSR molecular marker according to claim 1 or a primer combination according to claim 2 in one or more of the following (a) - (d):
(a) Constructing a molecular fingerprint of Morchella conica;
(b) Screening Morchella conica;
(c) Identifying strain quality resources of the morchella;
(d) Analysis of genetic diversity of Morchella conica.
4. A molecular fingerprint of morchella conica, wherein the molecular fingerprint is constructed from the primer combination of claim 2.
5. A kit for analysis of genetic diversity of morchella conica, comprising the primer combination of claim 2.
6. The analysis method of morchella conica genetics is characterized by comprising the following steps:
PCR amplification is carried out by using the primer combination of claim 2 by taking the morchella genome DNA as a template to obtain an amplification product;
detecting the amplified product by electrophoresis to obtain an SSR locus polymorphism amplified strip;
genetic diversity analysis was performed based on the amplified bands.
7. The method according to claim 6, wherein the PCR amplification reaction system comprises, in terms of 20. Mu.L, mix 17. Mu.L, 10. Mu.M forward primer 1. Mu.L, 10. Mu.M reverse primer 1. Mu.L, and 25-50 ng/. Mu.L Morchella esculenta genomic DNA 1. Mu.L.
8. The method according to claim 6 or 7, wherein the PCR amplification procedure is: pre-denaturation at 98℃for 2min; denaturation at 98℃for 10s, annealing at 60℃for 10s, extension at 72℃for 10s,35 cycles; finally, the extension is carried out for 5min at 72 ℃.
9. The method of analysis according to claim 6, wherein the genetic diversity analysis comprises calculating a genetic diversity index.
10. The method of analysis of claim 9, wherein the genetic diversity indicator comprises one or more of an allele, a useful allele, a private allele, and a shannon index.
CN202310450501.9A 2023-04-25 2023-04-25 Morchella SSR molecular marker, primer and application thereof Pending CN116640871A (en)

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