CN116751879A - SSR microsatellite molecular marker of Morchella esculenta, and primer set and application thereof - Google Patents
SSR microsatellite molecular marker of Morchella esculenta, and primer set and application thereof Download PDFInfo
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- 108091092878 Microsatellite Proteins 0.000 title claims abstract description 101
- 240000002769 Morchella esculenta Species 0.000 title claims abstract description 61
- 235000002779 Morchella esculenta Nutrition 0.000 title claims abstract description 55
- 239000003147 molecular marker Substances 0.000 title claims abstract description 27
- 230000002068 genetic effect Effects 0.000 claims abstract description 34
- 238000004458 analytical method Methods 0.000 claims abstract description 28
- 108091028043 Nucleic acid sequence Proteins 0.000 claims abstract description 7
- 241000221638 Morchella Species 0.000 claims description 31
- 238000000034 method Methods 0.000 claims description 19
- 238000012408 PCR amplification Methods 0.000 claims description 16
- 244000271144 Crinum americanum Species 0.000 claims description 15
- 108700028369 Alleles Proteins 0.000 claims description 13
- 108020004414 DNA Proteins 0.000 claims description 11
- 230000003321 amplification Effects 0.000 claims description 11
- 238000001514 detection method Methods 0.000 claims description 11
- 238000003199 nucleic acid amplification method Methods 0.000 claims description 11
- 238000012216 screening Methods 0.000 claims description 10
- 238000000137 annealing Methods 0.000 claims description 5
- 238000006243 chemical reaction Methods 0.000 claims description 5
- 238000007621 cluster analysis Methods 0.000 claims description 4
- 238000004925 denaturation Methods 0.000 claims description 4
- 230000036425 denaturation Effects 0.000 claims description 4
- 239000002773 nucleotide Substances 0.000 claims description 4
- 125000003729 nucleotide group Chemical group 0.000 claims description 4
- 102000054765 polymorphisms of proteins Human genes 0.000 claims description 4
- 238000012257 pre-denaturation Methods 0.000 claims description 4
- 238000012163 sequencing technique Methods 0.000 claims description 4
- 238000001228 spectrum Methods 0.000 claims description 4
- 238000001962 electrophoresis Methods 0.000 claims description 3
- 238000011161 development Methods 0.000 abstract description 15
- 239000003550 marker Substances 0.000 abstract description 6
- 238000005516 engineering process Methods 0.000 abstract description 5
- 241000894007 species Species 0.000 abstract description 5
- 230000000052 comparative effect Effects 0.000 abstract description 2
- 241001303818 Morchella eximia Species 0.000 description 9
- 238000003752 polymerase chain reaction Methods 0.000 description 9
- 108091081062 Repeated sequence (DNA) Proteins 0.000 description 4
- 238000012512 characterization method Methods 0.000 description 4
- 239000011159 matrix material Substances 0.000 description 4
- 239000012634 fragment Substances 0.000 description 3
- 238000011160 research Methods 0.000 description 3
- 241000196324 Embryophyta Species 0.000 description 2
- 241000233866 Fungi Species 0.000 description 2
- 241000896101 Morchella crassipes Species 0.000 description 2
- 206010028980 Neoplasm Diseases 0.000 description 2
- 238000005251 capillar electrophoresis Methods 0.000 description 2
- 238000010276 construction Methods 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 241000894006 Bacteria Species 0.000 description 1
- 238000007400 DNA extraction Methods 0.000 description 1
- 244000153665 Ficus glomerata Species 0.000 description 1
- 235000012571 Ficus glomerata Nutrition 0.000 description 1
- 241000282414 Homo sapiens Species 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 241000221639 Morchella conica Species 0.000 description 1
- 241001303848 Morchella exuberans Species 0.000 description 1
- 241000488669 Morchella importuna Species 0.000 description 1
- 241000488673 Morchella sextelata Species 0.000 description 1
- 101100383903 Mus musculus Cisd3 gene Proteins 0.000 description 1
- 230000002159 abnormal effect Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000000975 bioactive effect Effects 0.000 description 1
- 238000010170 biological method Methods 0.000 description 1
- 238000009395 breeding Methods 0.000 description 1
- 230000001488 breeding effect Effects 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000012214 genetic breeding Methods 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 238000007689 inspection Methods 0.000 description 1
- 210000004185 liver Anatomy 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
- 238000011144 upstream manufacturing Methods 0.000 description 1
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- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
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Abstract
The invention relates to the technical field of molecular biology, in particular to an SSR microsatellite molecular marker of Morchella esculenta, a primer group and application thereof. The SSR microsatellite molecular marker disclosed by the invention comprises nucleotide sequences shown in SEQ ID NO. 1-40. The invention is based on high-quality genome data of the Morchella esculenta, and completes genome denovo assembly of 2 Morchella esculenta strains, SSR molecular markers are developed through a comparative genome technology, so that SSR locus information and polymorphism information shared among species can be directly obtained, further, SSR marker development is completed, a set of SSR microsatellite markers of the Morchella esculenta with good polymorphism and stability is obtained, and the primers designed by utilizing the SSR microsatellite molecular markers provided by the invention can be used for identification of the Morchella esculenta species, analysis of population genetic diversity and the like, and have good application value.
Description
Technical Field
The invention relates to the technical field of molecular biology, in particular to an SSR microsatellite molecular marker of Morchella esculenta, a primer group 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. The Morchella esculenta 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 molecular identity tag is formed by constructing a set of unique molecular markers for each strain through a DNA bar code technology so as to distinguish the abnormal bacteria and realize the purpose of strain protection.
SSR (Simple Sequence Repeats) molecular markers are molecular marker technologies with high intra-industry and intra-industry acceptance, SSR markers are designed based on repeated segment polymorphism of 1-6 nucleotides in genome, have the advantages of high sequence variation, high stability, co-dominance and the like, are widely used in animals, plants and microorganisms, and 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 have been analyzed based on their transcriptome data as per Liu Wei et al (Liu Wei et al SSR distribution and sequence characterization of Morchella crassipes transcriptome. Light industry journal 2017,32 (02): 33-39). Meng Qing equivalent SSR information analysis and molecular marker development are carried out based on the morchella hainanensis transcriptome data, wherein 4 pairs of primers in 12 pairs of SSR primers which are randomly screened show stable and repeatable polymorphism, and genetic diversity analysis is carried out on 19 collected morchella hainanensis (Meng Qing and the like. SSR information analysis and molecular marker development of morchella hainanensis M12-10 transcriptome, university of Qinghai, 2019,37 (06): 1-10). Ma Jie the equivalent utilizes transcriptome data to develop EST-SSR of Morchella, and screens to obtain 15 pairs of SSR primers with better stability, and performs genetic diversity analysis on 33 Morchella specimens (Ma Jie, etc.. Morchella EST-SSR marker development and genetic diversity analysis based on transcriptome sequences. Jiangsu agricultural journal, 2020,36 (05): 1282-1290). Du et al 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 characteristic analysis of genome data of Morchella, but the markers have lower polymorphism, 22 SSR markers only amplify to obtain 15-22 polymorphic sites in 6 species, and the possible reason for lower polymorphism of the universal SSR primers designed based on Morchella is that the SSR polymorphism between different species is different, and the high-polymorphic SSR sites in the species are lost when the strain is emphasized (Du XH et al hybrid, characterization and transferability of SSRs in the genus Morchella. Functional Biology,2019,123 (7): 528-538). Based on the above, a set of SSR microsatellite markers of Morchella esculenta with good polymorphism and stability is needed.
Disclosure of Invention
In order to solve the problems, the invention provides an SSR microsatellite molecular marker of Morchella esculenta, a primer group and application thereof. The SSR microsatellite molecular marker provided by the invention has the advantages of good polymorphism and stability, and the primer designed by utilizing the SSR microsatellite molecular marker provided by the invention can be used for identification of Morchella esculenta species and analysis of population genetic diversity, and has good application value.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides an SSR microsatellite molecular marker of Morchella esculenta, which comprises nucleotide sequences shown in SEQ ID NO. 1-40.
The invention also provides a screening method of the SSR microsatellite molecular marker, which comprises the following steps: through the denovo sequencing and assembly of 2 sets of genome, the polymorphism of SSR sites and SSR is determined by comparing with a reference genome, and then 40 SSR sites with the best polymorphism are obtained through screening.
The invention also provides a primer group for amplifying the SSR microsatellite molecular marker in the technical scheme, wherein the primer group comprises 40 pairs of primer pairs, and the nucleotide sequence of each primer is shown as SEQ ID NO. 41-120.
The invention also provides a kit for amplifying the SSR microsatellite molecular marker according to the technical scheme, and the kit comprises the primer set according to the technical scheme.
The invention also provides a molecular fingerprint of the Morchella esculenta population, which is obtained by constructing the primer group in the technical scheme after PCR amplification.
The invention also provides application of the primer group or the kit in the technical scheme in identifying Morchella esculenta.
The invention also provides application of the primer group or the kit in the technical scheme in genetic diversity analysis of Morchella esculenta.
The invention also provides a method for analyzing genetic diversity of Morchella esculenta, which comprises the following steps:
taking genome DNA of the Morchella esculenta to be detected as a template, and carrying out PCR amplification by using the primer group in the technical scheme to obtain an amplification product;
performing electrophoresis detection on the amplification product to obtain an amplification strip;
analyzing the amplified bands, wherein the same amplified bands of the to-be-detected seven-sister morchella at each SSR site are respectively marked as 1, and the non-same amplified bands are respectively marked as 0, so as to obtain the molecular fingerprint of the to-be-detected seven-sister morchella;
and (3) respectively calculating each genetic diversity index of the SSR locus based on the molecular fingerprint spectrum and obtaining a cluster analysis chart.
Preferably, each genetic diversity indicator of the SSR site comprises one or more of an allele, a useful allele, a private allele, and a shannon index.
Preferably, the PCR amplification reaction system comprises, in 20. Mu.L, 10. Mu.L of 2 XTaq PCRMastermix, 1. Mu.L of forward primer, 1. Mu.L of reverse primer, 1. Mu.L of genomic DNA and the balance of ddH 2 O; the PCR amplification reaction program is as follows: pre-denaturation at 98℃for 2min; denaturation at 98℃for 10s, annealing at 60℃for 10s, elongation at 72℃for 10s,35 cycles; extending at 72℃for 5min.
The beneficial effects are that:
the invention provides an SSR microsatellite molecular marker of Morchella esculenta, which comprises nucleotide sequences shown in SEQ ID NO. 1-40. The invention is based on high-quality genome data of the Morchella esculenta, and completes genome de novo assembly of 2 Morchella esculenta strains, SSR molecular markers are developed through a comparative genome technology, SSR locus information and polymorphism information shared among species are directly obtained, further, SSR marker development is completed, a set of SSR microsatellite markers of the Morchella esculenta with good polymorphism and stability is obtained, and the primers designed by utilizing the SSR microsatellite molecular markers provided by the invention can be used for identification of the Morchella esculenta species, analysis of population genetic diversity and the like, and have good application value.
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 diagram showing the result of capillary electrophoresis of a Morchella SSR primer of the present invention;
FIG. 2 is an SSR fingerprint of 8 strains of Morchella esculenta;
FIG. 3 shows the result of SSR genetic diversity analysis of 8 strains of Morchella esculenta in the present invention.
Detailed Description
The invention provides an SSR microsatellite molecular marker of Morchella esculenta, which comprises nucleotide sequences shown in SEQ ID NO. 1-40, wherein specific information is as follows:
SEQ ID NO.1：GGGAGTCATATCGGCACTCGAGTGATTAGACGCATCAGATATCTAGAGAAA GTGAGAGGGTGAGTGAGTGAGTGAGTGAGAGAGTGAGAGTGGCTGCTGCAGTAATGACATGAAGGGGGAAGGGAAGAGGAGTAGTACGTATGTTTCGCGGCGCTCACAGATTAACTATACGAGAATTCGAAGCATGCACAGCCCCGCGACGTCTGGATGAGTACGTAATGCGAGTGAGGCTTTGCAGGATGAGGATGAGGATGAGGATGAGGATGAGGAGGAGGAGGAAGAGGAGGAGACCGT；
SEQ ID NO.2：TGGCAGTAATCGTGGAGAGCTCGCCCGGGGGTGGTATGAGCGCGCCGTGA AGGAGTATTCTTCTTCTTCTAACCCTCCCCCTACCTCTTCTTCTACCGGCAACCCTCCCGCCGCCG CCGCCGCCGCCGCCGCCGCCGCCATCGCCACCGGCACAGGGGAGCCAAAACGGAAGCAGTCCC CAGAGACTGGAGATAGCAGTGAAGACGAGATGGTGGGCCCTTTCCTAC；
SEQ ID NO.3：GGTGAGGAGGTCTACCCCATCTTGTTTCTAAAGACCGGTAAAATGGGATTG AGGCCTGTGATCAGTTTGGTTTGTGGTTGAATACTTTGTGAGCGGGTTTGAGTTGTTTTGGGGAG AAAGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGTAGTAGTAGAGGAGGAGCTCTG GATCGAGTGTCG；
SEQ ID NO.4：ACGCCAGTTCAAGCACTCTGCGATACCGCAGAGCGGCGGAGGCGGCCTGC CAAACGCAAAGAAGCGCCCCCGCCCCGAGCAGCAGCACAAGCACTCGAGCTCCGAGAGCAATGCGGGCGGCGCCGGCTCGTACAAGCGCTCCGGCAGCGTTAGCGGCGAGAGCTCCTCGGCGATGGGGAGTGCGATGAGGGCGGAGCAGTCGCAGTCGCAGTCGCAGTCGCAGTCGCAGTCGCAGTCGCAGCAGCCCAGGTCGACAATGTATGCGGCGTGTC；
SEQ ID NO.5：CCCAGAGGCTGAGAACACAGAGCCCGGGGATCCGTATCGAAATTAGCCCTAAATGGAGCACTGGACCGAACCTGATCTGAAGGAGCCAGGGCGGAGAGGAGGAGAAGGAGGCCTCATTAGAGAAGAGGAGATCGACGAGGAGGAGGAGGAGGAGGAGGAGGAGGTTGCTTAGTCTTGGCGAAATGAAGCTGAAGAAAAGTCTTTGGAGAGAAGTGACCCCGCACTTCACGAACTCTTGGTGGAGTTGAGAAGGGGCTTGTCAAGGACA；
SEQ ID NO.6：TCAAGCACAAGCACAAGCACAAGCCCAGGCTCAGGCTCAGGCCCAGGCTCAAGCCCAGGCTCAGGCTCAGGCCGAGGCTCAAGCACAGGCTCAGGCTCAGGCTCAAGCACAGGCCCAAGCCCAAGCCCAAGCCCAAGCCCAAGCCCAAGCCCAAGCCCAAGCCCAAGCCCAAGCCCAAGCCCAAGCTCAAGCTCAACATCAACAGCAACAGCAACAGC；
SEQ ID NO.7：TGTAAGTGGAGAGGGACGGACGAGGGAAATGATTGTTTGGGCAGGTACTGCTTGTTACTACTACTCTACCACTACTACTACTACTACTACTACTACTACTACTACTACTACGACGGATAAACCGGCGCTTAATCATTACCAATTATAACTTCTACAACAACTAAACAAAACAAAACCAACAGCAGCAATTAACAGTACCCATATACCTACAACCTAACCCAACCCATGACACGCTCAATTAAACCCCATATCCACACCCTCATCACACCCACAAAACCCCTC；
SEQ ID NO.8：ATCCTCGGCGATCTGTATGCGCAGAGGAGCCAGATTGAGAATACGCATAATACGCTCAATGATAGTGAGCGCTACTTGGATAGGAGTATCAAGACGCTGAGGGGGATGGCGCGTAGGTGAGATTCTATCCTTTTGTGGTGGTGGTGGTGGTGGTGGTGGGGATTGCAGGGAGAGGGGATTGGGGATTGGGGATTGGGGGGCTAATAGTGTGTGTGGGGGATAGGATGGCTACAAATCGGATGATTACGATTGCAATCATAACGGTGCTCGTGTTGCTGATTATGGCGG；
SEQ ID NO.9：AGTTCATTGCTCAGCGTCGAATGTTTCTTTCCTTCCTCTTCTTCCTCTTCTTCCTCCTCCTCTTCCTCCTCCTCCTCCTTCTCCTCGTCCCATCATTCTCCCTCATCACTCTTATCGTTCGTTATCCCTCATCGTCAATTTCTTTATCTCTACACTGATTTTCATAACCTCGAAAGTGCCTAAAATGGAGATGCATACTTATGAAGTCCTTGCGTACTTTCTACCCGCATTCCCTCC；
SEQ ID NO.10：GGTGCCGTCATCACTGAAGATGGCCATCTCGTATTCGGTGATGTGAACCCAGCCACCGGGCTTAGTGTGTCTGCACCCACCGCTATCAGAGCTGTGTTTGAAAGGGGAGGAGGAGGAGGAGGAGGAGGAGGAGGATTCTCACTCATAACACTGATCAATGACATTCCGCCAGTTGCAAACGCCACCCATGAGATTGCGGATGTGGATCAAGTCGAAGTGATTCTTGCTGAACGTCCAATCCTGCTCC；
SEQ ID NO.11：TTTCGGGTTCGGTGTCTACGGCGGGGACGGGGACGGGGACGGGGACGGGGACGGGGACGGGTGCAGGCTCAGGCTCAAGGTCAGGATCAGGGCCTTCTGCAGCGTCACCACCGACAATATCATGGGGGCCTGCTGGCGACACGCGGAGGAACACTGTTGCTGGCCCTCCTCCGCCCGGACAGGATGCGGCGCCGGCGAGCACGAGGCGCAAGTTGGTGGTGTCGTCGTC；
SEQ ID NO.12：TGGGACGGACGCTCAATTACGTTTTTGCAGATGTCACTCCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTTTGGTTGTGAAATATGGGTGGATAGACAGACGGGCCCCAAAGAGATAAAAATGTTTCCTTCATGAACGGACCGG；
SEQ ID NO.13：GAACCGGCCAAGTAATCCCACGATCCCTTCCACAGAAAACAGAACAAAGAAAGGAAGAACGAAAGAAAGAAAGAAAGAAAGAAAGAAAGAAAGAAATAAGGGAAGCCACGAGCTCTTAAGAGCCATTCTCATACCCGAGAAACTCCCGCGGAATTCCCAATATGTTCATACGATAACTAACACTCGCACCACGT；
SEQ ID NO.14：CGGGCATGTGATTGTGGTTGTGGCTGTGTGTAGGGGGCTCAGGCGTAGTAGCCGTCAGATCAATCATCGCGCTAGCCGGGATCGGCACCGGAGCGTGTGCGGGAGCGGGAGCAGGTGGCTTTTTAACCTTTAGCTTGATGATGAGCTTTCCTTTTCCCTTTTCCTGTGCCTGGGCAGGGGCTGGTGGTGCAGATGCAGGTGCAGGTGCAGGTGCAGGTGCAGGTGCAGCGTCGATCATCTCAACATCCTGCTTGAGATCAACGACCATCCTCGG；
SEQ ID NO.15：AAATACACCGCCCTCACCTCTCCGTAGAATTCAAGCACAAAAAAATACATCTTGAAGGTGCCATAGTATGGACGGTGGTGGTGGTGGTGGTGGTGGTGGCTTGAAAGCTATGGCTGGTTCGGGGGAGTCTACTTAGAGCTCTGGTTGCTGCGAGAATCCAT；
SEQ ID NO.16：GGGCGGTGGAGATGTTAACATTACGGCAGACGAGGTTTTTAACAATGATGATGATGATGATGATGATGATGATGGTTTTATTAGAGATTAGAAGCACACCTTTATTGTCACTCCATTTGATATCACTTGCATCGTGAAGAGAAAACACCCCGACAGC；
SEQ ID NO.17：CACGAGTGCCATACCGTACATATAGATGGCCACAAATAGTAGAAAAGAATAGGAGTGGGTTATGCATAATCTGTTTTCTCTCTTCCGAGAGGAGGTGAGAATGCATGTGTAGATACGGGTAGTATAGGAATGAGAAGAGATGGGGGGAAGAAGAAGAAGAAAGAGTATGATGCTCTTGCTATTCACTACAATAGGTATCACTCTAGCTCTTTGACATGCACTTACTTACATCAAAGAACCCTTGAGACTGCACCA；
SEQ ID NO.18：TGTCCCCGACTGCAACTAACTGCAATGGAACCCTGGGGGACTGGGGGAGAGAAGTGTCAGCTACCTAGCTAGTCTGGCCGTTATAACGTTCGTTCCTAGACAGGCAGACAGACAGACAGACAGACAGACAGAGAGTCACGTACCGGTGGTAGTTATGTAGGAAGTTGGTACCAGGTGATGGTAGGTGTCAGCTTAACCTAAAAACTGAGCTGTACTGGACTGAAAGGCGAATTGAGGCGAATCAAAGATAACGGTCTGTAGCTGGTAGCTGGTAGAGTAGCCGGAA；
SEQ ID NO.19：GGAGGTATCTCTCGGCAAGCTTTCGACGTGAGCAAGGGGTGTGGGTGTGGAGGGGAGGGGGTTGGTTGGTTGGTTGGTTGATTGGTGGATCCACAGGGGGTTGTTGAGTCGAGAAGAGAGAAAGAGGGAATGGGAGGGAGAG；
SEQ ID NO.20：GGTTGCGGTGTGTGTGTTACTGTGATTTCACCCAAAGGAGTGAGCTATAGCAGTCAGTTTAAACATGGTCGATCCCTCTTGCGAGGGATGTGAAACGGTTTCCGCTTACGTGCATGGGCAAAAGCCTTATTGCCCATGTTCCACCCTGGCTGGCTGGCTGGCTGGCTGGCTGGCTGGTGGCTGGCTGGCATCCGGTTTAGCCATCA；
SEQ ID NO.21：TAGAAGGGCAAGGGGACAACGCAGTGCAGGCTGGTGTCGAAGCACTCAAAGGTGCCGTATATTCGTGTACGTACTGCTACTACTACTGCTACTACTACTGCTACTACTACTACTACTACTACATACTGCTATTACTGTGGTGCAATTGAGGAATGTCTTTGATCTAATTTCTGTGTTCGCAATGTATGTAGTGTTTCGACTGGTCGCCCACGCAGGAAGGTATATATATCTACATCCATCTACCTACCTACACACACCCTAGC；
SEQ ID NO.22：AAGCATGGGAAGACGCTGAAGGGTTGATGGTGATGGTGATGGTGATGGTGATGGTGATGGTGATGGTTTGGTTGGAGGTATTGTTATTGTTATTATTTTCTGTGCGGAAATGATGATTGGATTCTGTATGCTTCTTGTTTTTTTCTTTCTGTAGGATGTTTTTAGGTATCATAATGAGGTAGCATTTTTTTCTTCTTGTACAAATCAAAAAGTCATTCTTGTATTAAAGTAGATTATGCGGTTTAGCTGTGCCT；
SEQ ID NO.23：TGCTGATGTTGTTGCTGCTGGTGATATATCTGTTGCTGCTGCTGCTGCTGCTGCTGCTGCTGCTGCTGTTGCTGTTGCGCACGAACAGCAGGGTTCCATGGACATCGTGGCACACTATATTGGTTTGGGAGTGGGGTCTGTGCTGGATTATCTGGCGTAATATTCGTTGTTACGGAGGAATATCCTGAGGAAGGAAGCCCTGAGGGAGAAACACCGTACATGCCGATCATCGACTCTGGGGTG；
SEQ ID NO.24：TGGTGGTATTGGTGGTGGTGGGGTGGGATACTTTTTTACCTAGCTGGGCCGACTGTAAATGATGAACCCTACATATTTGCGCAAACTTAACCACCCACCACCACCACCACCACCACCACCACCACCCAATCCAAATCCATACCTGCATTTGCATCTGCATCTGGCGGGGGTGGTGGGCGTTAGAGTATTTGTATTTCCTTTCTCTACTTCGGAGATTCAACGATCACAGATGTCCGCCTCGTATTCTATTGCAGAGATCTAGAGTAAGTAGAAAGCTCAAGAGGGGCGAACGATT；
SEQ ID NO.25：CGTGATGGGCGTTGTTGTTTGTGATGGTGCAGATCGATGCTTGGGCTGTCGTGAGGATGTTTGTGGTGGTGCTTCATTGTCTTGTCTCTCTGTCTGCCTGTCTGATACTCTCATGTATCTGTAAATATCTACCCATCACTGCTACATGAATTCGTTAACATTTAAGCTTATCCCTCCCCGGCCCCCCTTCACTACAACTACATATATGCGACGATCATGTTAGCTGCTGCTGCTGCTGCTGCTGCTGCTACCAACTACACGTGCTGTTGC；
SEQ ID NO.26：ACGTGTAGCACTGGACCTCTTTTTTCTCTCTCTCTTTCTTGCCTTTTTGTTAGGACTTGATATTTGGACATTAGTGTGTTGTTTTATTCCATTTTTATTCTTTATATTTTTTTTATTTTTATCTTCAAGGACAAGGTTTCGGTTTCTTCTTCTTCTTCTTCTTTTCGTTTCTTCTACGGGAATGGGTAATGGGTCAGTTCTTTGCCGTT；
SEQ ID NO.27：CCCCGGTTGAAACGAGACAAACAATCTACAATCTACATAAATAAATGTCGTACATGAACAACCAAAAAAAACCCCGATCTACGCACTATAGAGTACAGTACTTTGCATAGGCAATGCAAGGCCGGCAGGCCGGTAATCTATCTGCTGCTGCTGCTGCTGCTGCTGCTCCCGGTTAAGTTAGCATGCACACACATTCCGTGCAGGAGGGGAGGCCCGGCGCCGGACATAACCTTCATGGCAGTTTGTAGATCGCAGCC；
SEQ ID NO.28：GGGGAGTTGGAGGGATTTGGATTGTATGTATGTATGTATGTATGTACGTAGCAGATCCACTATCCAAAGAGAAGAAGGGAAATGGGAAGGGAAAGGGAAAATAAAAGGGAAGACAGAGAGAGTAGGGGAGAGTGGGGGGGAGGAAGAGGGAGAGGGAGAGGGAGAGAAAGAGAGAGAAACGGAGGAATAAAAAGGGAAATAAGGGAGGAAGAAGAAGAAGAAGAAGAAGAAGAAGAAGAAGAAGAAGAAGTGTACTAGCACACACTTACTTGGGCA；
SEQ ID NO.29：CGTTTGTTGTGTGGCGGTTTAATGACGCAGGAAGGATTCGTGAGTGCGATT GTGATTGTGATTGTGACTGTGATTGTGATTGCGATTGTGATTGTGATTGTGATTGTGATTGTGGGA GGAGTTGGTGTGGTTGTGG；
SEQ ID NO.30：GCCGTAGCTCTCCTGATCACGAGCGTAGACGCACCCGATCGCCACCAAAT GGCCGTCGTAATGGATCGAGCTCACATCAGCGTCCACCTTCCCGTTCCCCTCGCCGGCGCTCCCCAGTCCCTCCACCACGCGATAGCGGGGACTATAGATCAAGGCCCCAACGACAGCCTTACGTAAATCGTTCTAATGGGAATGGGAATGAGAACAGAAACCGAAATGGAAATGGAAATGGAAATGGAAATGGAAATGGAAATGGAAATGGAAATGCCGAAGAAGAAAGGGCAAGAAAATTGGCAGCCA；
SEQ ID NO.31：GGAGCGGAGATAACGGGAAGAGAGATGCGGGTGTGGGGCAGGGATGAGA GGGGAGGGGATGGGTGCGGGGGGAGGTGGAAGTGAAGCTAAGGGCACAGAGTGAGGCGGGAGTGCGGTATGGGTGTGGCGTGGGGTTTGGTGCTTGTGCTTGTGCTTGTGCTTGTGCTTGTGCTTGTGCTGGTGGTGGTGCATGGTGGGGTTAGTGGGTGGGCAATATGGAGATAGTGCTTGTGCTCATACGTACCGCGGAGAGGGAGAGTCTGGGTGACATCTGGCTGGGTTTGAGGTTGGTGCTT；
SEQ ID NO.32：TATCCCCAGTGCAGGAAGGAACAACGAAGGCCAAGAGGTCAAAGAAGAA ATATCTGATGTCTTCCCACCCTCATGGTCCAAGGGAGTGAGATTATTTGCACTTGGTTCTTTTTCT TACATAACTAGTTGCCTCCTCCTCCTCCTCCTCCTCCTCCTCCTCTGTCAGTGCTCGAACTTACTC CTCCTCCCCTTCGAAT；
SEQ ID NO.33：CTGCACTTTCAGGAGGAGCACAGCGAGGAGTAGAGGAGGAGGAGGAGG AGGAGGAGGAGTTCTGAATACTCGTGTAAAATACGGTATATAAAGAAAGCCAATTGGCATTATTA GACTACTGCAGTTCACTTCCAGCAATAGCCAGTATTTTAATTAGTGTAGCAATGTACAAACCAAA CAATTATCTCCAATTCCTCCCGTGTGTG；
SEQ ID NO.34：CCCGGCCCTAATTGCTACATTGGGCCCTTGAAGGAAGGGGTGCTGGCGAA GGGGGGGTAGGAGAGGAGGAGGAGGAGGGGGAGGAGGAGGGGGAGGGGGTTAAGTTACAGT AAACCAGTAGGTAAACCGCCTTCACTTTCCCCTTCCCATGTTTCGACCTTCCCTTTAATTCGACTT CCAGGCGGG；
SEQ ID NO.35：GGGACAGGTTGGACAGTACGTAGCTAGGTCTGTATTGAGCGTTAATGTGGA TAGCGGTATATGTAGTGACTTCTTCCTTTTAAACTCACCTTTCTTTTGTCTGGGGTATGGCATCTTCCTTAGGGATAAATAAATATTTCTTTCTTTTCTTTCTTCTTTCTTTCTCCTTTACCTGGGCATTCGTTTTTCTCTTGTTGTTATATATATATATATATATATATATATATACCCTTGAATTCCTTTTGTTTTCCTCTTATCGGTGATGGGTTTTGGGCTGGGAGGATT；
SEQ ID NO.36：TGTACTCCAGGCTGGGAACAGTAATTTAACCTTCCCGTCAACTGCAAGAA CCCACCCACCACCCACCCACCCACCCACCCACCTGCTCCATCAACGCCTTGCCAAGAG；
SEQ ID NO.37：CGCACGCAGTATTTCAGAGCTCAACGTCCGAGACATAAGCTCCTAGAGCT ACCTACCTACCTACCTACCTACCTACTTACCTACCTACTCTCCCCAATCCACCACCTCCCAATATC ACCGTACCCCCAAGTCCGAAAAAAAAACAAAAAAGTGAACTCTCGGCACCCACTCGGCTGTTC CACCACCACCATGATCCGACACCTGCATATCTCCGAACCCTGGCC；
SEQ ID NO.38：CAGAGTTGAATGCGGCGATGGCGGCGGCGCGGGCGATGTCGTCTCGATTG TTGTTGTTTTTGTTTGTGTTTTCGGCGGCGGCGGCGGCGGCGGCGGAGGGCAGCAGCGCGGGGT CGTAGTGCAGTATCTGCCCGACAGCGCCGTGCCAGTCGCCGCGCAGCAGCTTGACGCCAACCTC CCACGTCCCGACGGTGTAGCTGCCAAAGCGCTGCAGGCCAAAATAGTTGACAAAGCCGCCCTC；
SEQ ID NO.39：ACGCCAGTTCAAGCACTCTGCGATACCGCAGAGCGGCGGAGGCGGCCTGC CAAACGCAAAGAAGCGCCCCCGCCCCGAGCAGCAGCACAAGCACTCGAGCTCCGAGAGCAAT GCGGGCGGCGCCGGCTCGTACAAGCGCTCCGGCAGCGTTAGCGGCGAGAGCTCCTCGGCGATG GGGAGTGCG；
SEQ ID NO.40：CGTGGCACAACTTTGCTGTTGAGACTGACTGGGACGAGAAGTATTTCTCC ACCCCAACCGGCCCTGGAAGAAGAAGAAGAAGAAGAAGAAGAAAGCCATGCTGACGGACCGG GGTGGTGGTTGTTAGCACGATTGCGGTGTACTACTCTGCCGACTATGCGCCTCTCAGGAAGG。
the invention also provides a screening method of the SSR microsatellite molecular marker, which comprises the following steps: and comparing the de novo sequencing and assembly of 2 sets of genome with a reference genome to determine SSR sites and SSR polymorphisms, and screening to obtain 40 SSR sites with the best polymorphisms.
The invention develops SSR molecular markers by comparing genome technologies, directly obtains SSR locus information and polymorphism information shared among Morchella esculenta species, further completes the development of SSR markers, has the advantages of higher specificity, stability and transferability, and specifically comprises the following steps:
genome assembly of two seven-sister Morchella strains (20D 15A and 20D 18A) using second generation genome assembly software SPade with reference to the seven-sister Morchella genome (https:// www.ncbi.nlm.nih.gov/nuccore/JAMGZG000000000.1 /); two new genomes of 20D15A and 20D18A and 2015-9 reference genomes are integrated by using Candii SSR software, and development of universal SSR molecular markers at the whole genome level of Morchella esculenta is carried out; combining the length of PCR products, the polymorphism size difference, the matching degree of the primers and the standard deviation of candidate site polymorphism, screening the developed SSR sites and primers, and finally selecting 40 pairs of primers with the highest polymorphism and the best transferability as universal SSR primers of the Morchella esculenta, thereby obtaining a set of SSR microsatellite markers of the Morchella esculenta with good polymorphism and stability.
The invention also provides a primer group for amplifying the SSR microsatellite molecular marker in the technical scheme, wherein the primer group comprises 40 pairs of primer pairs, the nucleotide sequence of each primer is shown as SEQ ID NO. 41-120, and SSR locus characteristics and primer information corresponding to the primer group are shown in Table 1:
TABLE 1 general SSR site characteristics and primer information of Morchella esculenta
Note that: the number of repeat sequences in Table 1 was determined by combining two sets of genome analyses, 20D15A and 20D18A, using the genome of Morchella esculenta No. 2015-9 as the reference genome.
The primer group provided by the invention has the advantages of high polymorphism and strong universality, can be used for identifying Morchella esculenta species, analyzing population genetic diversity and the like, and has good application value.
The invention also provides a kit for amplifying the SSR microsatellite molecular marker according to the technical scheme, and the kit comprises the primer set according to the technical scheme.
In the present invention, the kit preferably further comprises a reagent for PCR amplification, preferably comprising 2×Taq PCRMastermix.
The invention also provides a molecular fingerprint of the Morchella esculenta population, which is obtained by constructing the primer group in the technical scheme after PCR amplification. The method for constructing the molecular fingerprint spectrum has no special requirement, and the method for constructing the molecular fingerprint spectrum is well known to those skilled in the art.
The invention also provides application of the primer group or the kit in the technical scheme in identifying Morchella esculenta. The primer group provided by the invention can carry out PCR amplification on genome DNA of the strain to be detected to obtain polymorphism data, and a cluster tree is constructed, so that the primer group is used for identifying the identity of the strain.
The invention also provides application of the primer group or the kit in the technical scheme in genetic diversity analysis of Morchella esculenta.
The invention also provides a method for analyzing genetic diversity of Morchella esculenta, which comprises the following steps:
taking genome DNA of the Morchella esculenta to be detected as a template, and carrying out PCR amplification by using the primer group in the technical scheme to obtain an amplification product;
performing electrophoresis detection on the amplification product to obtain an amplification strip;
analyzing the amplified bands, wherein the same amplified bands of the to-be-detected seven-sister morchella at each SSR site are respectively marked as 1, and the non-same amplified bands are respectively marked as 0, so as to obtain the molecular fingerprint of the to-be-detected seven-sister morchella;
based on the molecular fingerprint, the analysis software is adopted to respectively calculate the genetic diversity index of each SSR locus, and then the related software is utilized to obtain a cluster analysis chart.
In the present invention, the reaction system for PCR amplification is preferably 20. Mu.L, preferably comprising 2×Taq PCRMastermix 10. Mu.L, forward primer 1. Mu.L, reverse primer 1. Mu.L, genomic DNA 1. Mu.L and the balance ddH 2 O; the concentration of both the forward primer and the reverse primer is preferably 10. Mu.M; the concentration of the genomic DNA is preferably 25-50 ng/. Mu.L; the reaction procedure for the PCR amplification is preferably: pre-denaturation at 98℃for 2min; denaturation at 98℃for 10s, annealing at 60℃for 10s, elongation at 72℃for 10s,35 cycles; extending at 72℃for 5min.
In the present invention, the analysis software preferably includes GenAlEx version 6.501 and Popgen32; the related software preferably includes NTSYS software; the individual genetic diversity indicator of the SSR locus preferably comprises one or more of an allele, an effective allele, a private allele, and shannon index.
The method for analyzing the genetic diversity of the Morchella esculenta can perform cluster analysis on the Morchella esculenta, and establishes a new method for variety identification and genetic diversity analysis of the Morchella esculenta.
For further explanation of the present invention, the SSR microsatellite molecular markers of morchella and their primer sets and applications 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 protection of the present invention.
Example 1 detection and characterization of full genome level SSR molecular markers of Morchella esculenta
SSR sequence feature analysis of Morchella esculenta reference genome
The invention uses 2015-9 seven sister morchella (Liu Wei, the growth and development process of the morchella and the group study of morchella [ D ]. The university of agriculture in China, 2020.DOI: 10.27158/d.cnki.ghznu.2020.000544.) genome as reference (https:// www.ncbi.nlm.nih.gov/nuccore/JAMGZG000000000.1 /), the genome size is 50.83Mb, the number of contis 26, the GC% is 47.18%, and the genome integrity is 98.6%, which indicates a high-quality seven sister morchella genome.
Analysis of the repeat sequence of the genome of strain 2015-9 based on the repeat mask (v.4.1.2-p 1) and the repeat model (v.2.0.2 a) showed that the repeat sequence in strain 2015-9 of Morchella esculenta was about 8.09Mb, accounting for 15.92% of the total genome length. Wherein 28489 Non-scattered repeats (Non-interspersed Repeats), total length of 1.26Mb, account for 2.48% of genome; scattered repeats (Non-interspersed Repeats) 14869, total length 6.92Mb, 13.61% of the total length of the genome. The non-scattered repeated sequence is mainly a simple repeated sequence, and 24385 sites are used for developing genome microsatellite markers.
Construction of two genome sketches of Morchella esculenta 20D15A and 20D18A
Two seven-sister morchella were sequenced separately using a sequencing protocol for the second generation Illumina HiSeq 4000 double-ended PE 500: 20D15A (http s:// www.ncbi.nlm.nih.gov/sra/SRR 16686503) and 20D18A (https:// www.ncbi.nlm.nih.gov/sra/SRR 16686500), each strain sequenced the rawdata of 4G separately, and two strains were genomically assembled using the second generation genome assembly software SPADE (v.3.15.3), SPADE specific parameters: the samples were 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 20D15A assembly results in 10236 contig sequences, the genome size is 48.51Mb, the GC% content is 47.15%, and the contig N50 is 47kb in length; 20D18A assembled 24437 contig sequences, genome size 50.42Mb, GC% content 47.25%, contig N50 length 35kb; compared with a reference genome, the integrity is 95.43% and 99.19%, and the SSR characteristic analysis on the whole genome level of Morchella esculenta and the development of polymorphic locus markers can be satisfied.
Universal SSR locus detection in genome range of Morchella esculenta
The development of a universal SSR molecular marker for Morchella esculenta was performed using Candi SSR software (Xia E-H et al (2016) Candi SSR: an Efficient Pipeline used for Identifying Candidate Polymorphic SSRs Based on Multiple Assembled sequences. Front. Plant Sci.6:1171.doi: 10.3389/fpls.2015.01271) with reference to the high quality genome of 2015-9, integrating the two sets of 20D15A and 20D18A genomes, using the following parameters: candriSSR.pl-i Morexi.ctl-omorexi out-l 200-t 100.
Analysis results show that 2406 SSR loci in total have polymorphism between at least two of the seven sister Morchella candidate strains in the genome of the seven sister Morchella, wherein 1974 loci have polymorphism in three sets of genomes and are used as candidate loci for SSR marker development.
Example 2 development of Universal SSR markers within the genome of Morchella esculenta
Extracting 250bp sequences at the upstream and downstream of the candidate SSR locus, developing SSR primers by using Primer3.0 software, setting the annealing temperature of the candidate primers to 60+/-3 ℃, setting the PCR products to 125-300 bp, and setting the length of the primers to 20+/-3 bp, wherein the number of the candidate primers is 5. The result shows that all candidate SSR sites can be successfully designed to obtain corresponding SSR primers.
Combining the length of PCR products, the polymorphism size difference, the matching degree of the primers and the standard deviation of candidate site polymorphism, screening 1974 SSR sites, and finally selecting 40 pairs of primers with highest polymorphism and best transferability as universal SSR primers of Morchella esculenta, wherein 40 SSR sites are nucleotide sequences shown in SEQ ID NO. 1-40; the nucleotide sequences of the 40 pairs of primers are shown in SEQ ID NO. 41-120 (see Table 1).
Example 3 SSR genetic diversity analysis of Morchella populations
The seven sister Morchella strains with clear genetic background collected from the various places of the country are taken as samples to be tested, and the strain information is shown in Table 2:
table 2 seven sister morchella population information
| Strain numbering | Acquisition ground | Acquisition time | Phylogenetic name |
| 19-32-2 | All of Sichuan Cheng | 2019 | Morchella eximia |
| Q-No.9 | Sichuan Deyang | 2014 | Morchella eximia |
| H-G5 | Sichuan Mianyang | 2021 | Morchella eximia |
| H-M733 | Sichuan Mianyang | 2021 | Morchella eximia |
| H-M328-1 | Sichuan Mianyang | 2021 | Morchella eximia |
| H-M328-2 | Sichuan Mianyang | 2021 | Morchella eximia |
| H-7416 | Sichuan Mianyang | 2021 | Morchella eximia |
| H-7418 | Sichuan Mianyang | 2021 | Morchella eximia |
Wherein the H-M328 strain was repeated once, and the strain numbers were designated as H-M328-1 and H-M328-2, respectively, to determine the stability of the 40 pairs of SSR primers obtained by the screening in example 2.
The 8 Morchella esculenta strains in Table 2 are used as samples to be tested, the SSR marker (40 pairs of primers) adaptability detection and genetic diversity analysis developed in the embodiment 2 of the invention are carried out, and the specific method is as follows:
performing PCR (polymerase chain reaction) amplification by using the genome DNA of the Morchella esculenta to be detected as a template and using the primer set obtained in the example 2 to obtain an amplification product;
pure culture, DNA extraction and quality inspection of strains are conventional molecular biological methods, and specific references (Liu Wei, et al, morchella conica monospora and cultivation fruiting experiments and polarity analysis of hybridized populations, fungus research, 2019, 17 (01): 43-49.DOI: 10.13341/j.jfr.2018.1238);
the 40 pairs of primers obtained by screening in example 2 were synthesized by Beijing qing biotechnology limited company and FAM fluorescence signals were added;
detecting the PCR product by a 3730xl sequencer, analyzing the obtained data by using a genemap software, and judging whether different primers have fragment polymorphism according to analysis results;
PCR amplification system: 2X Taq PCR Mastermix (Green) 10. Mu.L, 10. Mu.M forward primer 1. Mu.L, 10. Mu.M reverse primer 1. Mu. L, ddH 2 O7. Mu.L and 1. Mu.L of genomic DNA;
PCR reaction procedure: pre-denaturation at 98℃for 2min; denaturation at 98℃for 10s, annealing at 60℃for 10s, elongation at 72℃for 10s,35 cycles; extending at 72 ℃ for 5min;
site detection: the PCR product is subjected to capillary electrophoresis by a 3730 sequencer, and partial detection examples are shown in figure 1, and the target strip peak diagram is sharp and clear, so that the specificity of the primer is higher;
site analysis: analyzing accurate sites of data by using software Gene mapper 4.1, judging polymorphism of the detection primer according to the analyzed site information, and marking 1 if fragments exist in the sites of the sample and 0 if fragments do not exist in the sites of the sample;
SSR fingerprints of 8 Morchella strains are shown in figure 2: comprises 40 pairs of SSR polymorphism primers and 121 polymorphism bands, wherein the strain numbers corresponding to the first row from left to right in the figure 2 are H-7418, H-7416, H-M328-2, H-M328-1, H-M7_33, H-G5, Q-No.9 and 19-32-2 respectively;
analysis of genetic diversity: in the GenAlEx version 6.501 and Popgen32 software, individual genetic diversity indicators for SSR sites were calculated, including the observed allele (Na), the effective allele (Ne), the proprietary allele (Np), shannon index (I), respectively. The results are shown in Table 3, wherein among 40 pairs of primers, the amplified polymorphism of 38 pairs of primers in 8 Morchella esculenta groups is more than or equal to 2, and the maximum number is 5; the polymorphism ratio is between 12.5% and 50%. The effective allele is between 1.28 and 2.00, on average 1.531; the shannon index is between 0.3768 and 0.6931, and the average 0.5004.
Table 3 SSR polymorphism characteristics of Morchella esculenta
| Primer(s) | Total number of sites | Polymorphic site count AP | PP(％) | Na | Ne | H | I |
| Mexi-1846 | 8 | 4 | 50.00％ | 2.0000 | 1.4306 | 0.2813 | 0.4480 |
| Mexi-856 | 8 | 3 | 37.50％ | 2.0000 | 1.5875 | 0.3542 | 0.5336 |
| Mexi-1400 | 8 | 3 | 37.50％ | 2.0000 | 1.4808 | 0.3021 | 0.4717 |
| Mexi-1520 | 8 | 2 | 25.00％ | 2.0000 | 1.7412 | 0.4219 | 0.6120 |
| Mexi-1458 | 8 | 3 | 37.50％ | 2.0000 | 1.4808 | 0.3021 | 0.4717 |
| Mexi-1534 | 8 | 2 | 25.00％ | 2.0000 | 1.2800 | 0.2188 | 0.3768 |
| Mexi-168 | 8 | 3 | 37.50％ | 2.0000 | 1.5875 | 0.3542 | 0.5336 |
| Mexi-1838 | 8 | 3 | 37.50％ | 2.0000 | 1.4808 | 0.3021 | 0.4717 |
| Mexi-2106 | 8 | 3 | 37.50％ | 2.0000 | 1.4808 | 0.3021 | 0.4717 |
| Mexi-1572 | 8 | 3 | 37.50％ | 2.0000 | 1.5875 | 0.3542 | 0.5336 |
| Mexi-1034 | 8 | 2 | 25.00％ | 2.0000 | 1.7412 | 0.4219 | 0.6120 |
| Mexi-1062 | 8 | 3 | 37.50％ | 2.0000 | 1.5875 | 03542 | 0.5336 |
| Mexi-1376 | 8 | 3 | 37.50％ | 2.0000 | 1.6267 | 0.3646 | 0.5441 |
| Mexi-2298 | 8 | 3 | 37.50％ | 2.0000 | 1.4808 | 0.3021 | 0.4717 |
| Mexi-1699 | 8 | 3 | 37.50％ | 2.0000 | 1.7333 | 0.4167 | 0.6059 |
| Mexi-510 | 8 | 3 | 37.50％ | 20000 | 1.6267 | 0.3646 | 0.5441 |
| Mexi-1358 | 8 | 2 | 25.00％ | 2.0000 | 1.7412 | 0.4219 | 0.6120 |
| Mexi-722 | 8 | 3 | 37.50％ | 2.0000 | 1.7333 | 0.4167 | 0.6059 |
| Mexi-1710 | 8 | 2 | 25.00％ | 2.0000 | 1.2800 | 0.2188 | 0.3768 |
| Mexi-1228 | 8 | 2 | 25.00％ | 2.0000 | 1.2800 | 0.2188 | 0.3768 |
| Mexi-1133 | 8 | 1 | 12.50％ | 2.0000 | 1.2800 | 02188 | 0.3768 |
| Mexi-2256 | 8 | 4 | 50.00％ | 2.0000 | 1.5106 | 0.3204 | 0.4944 |
| Mexi-1 192 | 8 | 3 | 37.50％ | 2.0000 | 1.4808 | 0.3021 | 0.4717 |
| Mexi-754 | 8 | 5 | 62.50％ | 2.0000 | 1.4645 | 0.3000 | 0.4709 |
| Mexi-1564 | 8 | 5 | 62.50％ | 2.0000 | 1.4240 | 0.2750 | 0.4401 |
| Mexi-2368 | 8 | 5 | 62.50％ | 2.0000 | 1.5400 | 0.3282 | 0.5023 |
| Mexi-1215 | 8 | 3 | 37.50％ | 2.0000 | 1.6267 | 0.3646 | 0.5441 |
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. As shown in FIG. 3, H-M238-1 and H-M238-2 in 8 strains are the same sample, and the results also show that there is no difference in detection of 40 pairs of SSR primers, and also show that the stability of the SSR designed by the invention is higher; the other 6 strains are clustered in different branches, and the whole strains are divided into two major categories on a genetic distance of 0.5, which shows that the strains have obvious genetic diversity and different genetic backgrounds.
In conclusion, the SSR microsatellite molecular marker provided by the invention has the advantages of good polymorphism and stability, and the primer designed by utilizing the SSR microsatellite molecular marker provided by the invention can be used for identification of Morchella esculenta 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 SSR microsatellite molecular marker of the Morchella esculenta is characterized in that the SSR microsatellite molecular marker comprises nucleotide sequences shown in SEQ ID NO. 1-40.
2. The method for screening SSR microsatellite molecular markers according to claim 1, comprising the steps of: and comparing the de novo sequencing and assembly of 2 sets of genome with a reference genome to determine SSR sites and SSR polymorphisms, and screening to obtain 40 SSR sites with the best polymorphisms.
3. The primer group for amplifying the SSR microsatellite molecular marker according to claim 1, wherein the primer group comprises 40 pairs of primer pairs, and the nucleotide sequence of each primer is shown as SEQ ID NO. 41-120.
4. A kit for amplifying an SSR microsatellite molecular marker according to claim 1, said kit comprising the primer set according to claim 3.
5. A molecular fingerprint of a group of morchella esculenta, wherein the molecular fingerprint is constructed by PCR amplification of the primer set of claim 3.
6. Use of the primer set of claim 3 or the kit of claim 4 for identifying morchella esculenta.
7. Use of the primer set of claim 3 or the kit of claim 4 in genetic diversity analysis of morchella esculenta.
8. A method for analyzing genetic diversity of Morchella esculenta, which is characterized by comprising the following steps:
performing PCR amplification by using the primer set of claim 3 with genome DNA of the Morchella esculenta to be detected as a template to obtain an amplification product;
performing electrophoresis detection on the amplification product to obtain an amplification strip;
analyzing the amplified bands, wherein the same amplified bands of the to-be-detected seven-sister morchella at each SSR site are respectively marked as 1, and the non-same amplified bands are respectively marked as 0, so as to obtain the molecular fingerprint of the to-be-detected seven-sister morchella;
and (3) respectively calculating each genetic diversity index of the SSR locus based on the molecular fingerprint spectrum and obtaining a cluster analysis chart.
9. A method according to claim 8, wherein each genetic diversity indicator of the SSR site comprises one or more of an allele, a useful allele, a private allele and a shannon index.
10. The method according to claim 8, wherein the PCR amplification reaction system comprises, in 20. Mu.L, 2×Taq PCRMastermix 10. Mu.L, forward primer 1. Mu.L, reverse primer 1. Mu.L, genomic DNA 1. Mu.L and the balance ddH 2 O; the PCR amplification reaction program is as follows: pre-denaturation at 98℃for 2min; denaturation at 98℃for 10s, annealing at 60℃for 10s, elongation at 72℃for 10s,35 cycles; extending at 72℃for 5min.
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| CN116751879B (en) | 2024-06-14 |
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