CN114540530B - Hainan province flammulina SSR molecular marker primer group and application thereof - Google Patents
Hainan province flammulina SSR molecular marker primer group and application thereof Download PDFInfo
- Publication number
- CN114540530B CN114540530B CN202210145000.5A CN202210145000A CN114540530B CN 114540530 B CN114540530 B CN 114540530B CN 202210145000 A CN202210145000 A CN 202210145000A CN 114540530 B CN114540530 B CN 114540530B
- Authority
- CN
- China
- Prior art keywords
- amplification
- primers
- ttg
- molecular marker
- ssr
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- 239000003147 molecular marker Substances 0.000 title claims abstract description 36
- 241001537207 Flammulina Species 0.000 title description 2
- 241001032110 Ornithidium coccineum Species 0.000 claims abstract description 32
- 240000006499 Flammulina velutipes Species 0.000 claims abstract description 27
- 235000016640 Flammulina velutipes Nutrition 0.000 claims abstract description 27
- 230000002068 genetic effect Effects 0.000 claims abstract description 17
- 230000003321 amplification Effects 0.000 claims description 85
- 238000003199 nucleic acid amplification method Methods 0.000 claims description 85
- 238000001514 detection method Methods 0.000 claims description 2
- 108091092878 Microsatellite Proteins 0.000 abstract description 55
- 238000012163 sequencing technique Methods 0.000 abstract description 8
- 238000011156 evaluation Methods 0.000 abstract description 2
- 230000007614 genetic variation Effects 0.000 abstract description 2
- 238000000034 method Methods 0.000 description 20
- 238000002264 polyacrylamide gel electrophoresis Methods 0.000 description 16
- 241000894007 species Species 0.000 description 10
- 238000004458 analytical method Methods 0.000 description 8
- 244000144980 herd Species 0.000 description 8
- 238000010276 construction Methods 0.000 description 6
- 238000012216 screening Methods 0.000 description 6
- 241000196324 Embryophyta Species 0.000 description 5
- 239000003550 marker Substances 0.000 description 5
- 108090000623 proteins and genes Proteins 0.000 description 5
- 238000011161 development Methods 0.000 description 4
- 230000018109 developmental process Effects 0.000 description 4
- 108020004414 DNA Proteins 0.000 description 3
- 241000233855 Orchidaceae Species 0.000 description 3
- 244000040442 Renanthera coccinea Species 0.000 description 3
- 238000000137 annealing Methods 0.000 description 3
- 230000009286 beneficial effect Effects 0.000 description 3
- 238000009395 breeding Methods 0.000 description 3
- 230000001488 breeding effect Effects 0.000 description 3
- 238000013461 design Methods 0.000 description 3
- 238000011835 investigation Methods 0.000 description 3
- 238000005070 sampling Methods 0.000 description 3
- LZZYPRNAOMGNLH-UHFFFAOYSA-M Cetrimonium bromide Chemical compound [Br-].CCCCCCCCCCCCCCCC[N+](C)(C)C LZZYPRNAOMGNLH-UHFFFAOYSA-M 0.000 description 2
- 108091026890 Coding region Proteins 0.000 description 2
- 101100136092 Drosophila melanogaster peng gene Proteins 0.000 description 2
- 240000007228 Mangifera indica Species 0.000 description 2
- 235000014826 Mangifera indica Nutrition 0.000 description 2
- 235000011611 Pinus yunnanensis Nutrition 0.000 description 2
- 241000018652 Pinus yunnanensis Species 0.000 description 2
- 241001534096 Psammosilene tunicoides Species 0.000 description 2
- 240000008254 Rosa chinensis Species 0.000 description 2
- 102100023706 Steroid receptor RNA activator 1 Human genes 0.000 description 2
- 101710187693 Steroid receptor RNA activator 1 Proteins 0.000 description 2
- 240000001417 Vigna umbellata Species 0.000 description 2
- 235000011453 Vigna umbellata Nutrition 0.000 description 2
- 238000003766 bioinformatics method Methods 0.000 description 2
- 102000054765 polymorphisms of proteins Human genes 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 235000004936 Bromus mango Nutrition 0.000 description 1
- 241001328795 Camellia fascicularis Species 0.000 description 1
- 240000001548 Camellia japonica Species 0.000 description 1
- 241000652836 Camellia tetracocca Species 0.000 description 1
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 description 1
- 230000004544 DNA amplification Effects 0.000 description 1
- 238000007400 DNA extraction Methods 0.000 description 1
- 238000007399 DNA isolation Methods 0.000 description 1
- 108091092195 Intron Proteins 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 240000002853 Nelumbo nucifera Species 0.000 description 1
- 235000006508 Nelumbo nucifera Nutrition 0.000 description 1
- 235000006510 Nelumbo pentapetala Nutrition 0.000 description 1
- 244000170916 Paeonia officinalis Species 0.000 description 1
- 235000006484 Paeonia officinalis Nutrition 0.000 description 1
- 244000131316 Panax pseudoginseng Species 0.000 description 1
- 235000003181 Panax pseudoginseng Nutrition 0.000 description 1
- 235000012874 Phaseolus calcaratus Nutrition 0.000 description 1
- 108020005120 Plant DNA Proteins 0.000 description 1
- 238000002123 RNA extraction Methods 0.000 description 1
- 238000003559 RNA-seq method Methods 0.000 description 1
- 108091081062 Repeated sequence (DNA) Proteins 0.000 description 1
- 241000610442 Schima superba Species 0.000 description 1
- 235000009184 Spondias indica Nutrition 0.000 description 1
- 241001122767 Theaceae Species 0.000 description 1
- 241000045717 Vandeae Species 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 235000011089 carbon dioxide Nutrition 0.000 description 1
- 235000018597 common camellia Nutrition 0.000 description 1
- 238000009402 cross-breeding Methods 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 235000013399 edible fruits Nutrition 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 238000012268 genome sequencing Methods 0.000 description 1
- 230000035784 germination Effects 0.000 description 1
- 244000237330 gutta percha tree Species 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 238000011451 sequencing strategy Methods 0.000 description 1
- 230000017260 vegetative to reproductive phase transition of meristem Effects 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
- 239000002023 wood Substances 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
- C12Q1/6895—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for plants, fungi or algae
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6869—Methods for sequencing
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/156—Polymorphic or mutational markers
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Engineering & Computer Science (AREA)
- Health & Medical Sciences (AREA)
- Analytical Chemistry (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Biotechnology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Immunology (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- Biophysics (AREA)
- Physics & Mathematics (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Botany (AREA)
- Mycology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention belongs to the technical field of molecular biology, and particularly discloses a Hainan flame orchid SSR molecular marker primer group, wherein the SSR molecular marker comprises one or more Than Two of (TTG) 6-26, (TTG) 6-27, (TAA) 19-45, (AAT) 16-47, (AAC) 14-68, (CTC) 10-81, (TTG) 10-96, (TTA) 11-106, (TAT) 10-114, (AAT) 13-117, (CTT) 10-119, (CTT) 6-141, (CAT) 6-159, (GAA) 6-184, (ATA) 6-198, (TTA) 6-218 and (ATA) 6-242; primers for amplifying the SSR molecular markers are also included. The invention also discloses application of the flame orchid SSR molecular marker primer group in Hainan province. According to the invention, the third generation transcriptome sequencing is carried out on the flammulina velutipes in Hainan province, SSR (simple sequence repeat) information is mined, a batch of SSR molecular marker primers with high polymorphism are developed, the genetic variation and the population structure of the flammulina velutipes are revealed for carrying out the genetic diversity evaluation of the flammulina velutipes population in Hainan province, and important scientific basis is provided for the protection and the innovative utilization of the flammulina velutipes germplasm resources in Hainan province.
Description
Technical Field
The invention belongs to the technical field of molecular biology, and particularly relates to a flame orchid SSR molecular marker primer group in Hainan province and application thereof.
Background
The flame orchid (Renanthera coccinea) is a plant of the genus flame orchid (renanthla) of the subfamily of the vandymia (Vandeae) of the orchid family (aeeridine), named as the gorgeous color of fire, and is a tropical rare orchid with great characteristics and ornamental value. The flaming orchid has the characteristics of large conical inflorescence, small flower quantity, long flowering period, long pedicel, high temperature resistance, high humidity resistance, happiness and the like, and can be widely used for cut flowers, garden scenery, hedgerow, potted plants and the like. The genus flaccida has about 20 species, and is predominantly distributed in tropical regions of asia and in pacific islands. The Chinese medicine has 3 kinds of flammulina velutipes (R.imschooliana), flammulina velutipes (R.sinica) and flammulina velutipes (R.sinica), and is mainly distributed in regions of Yunnan, guizhou, hainan and the like.
Investigation shows that the population quantity of the flammulina velutipes in Hainan province is drastically reduced, and in some areas, the flammulina velutipes are difficult to find out traces, and the flammulina velutipes are urgent to protect, such as the wild flammulina velutipes has narrow distribution range and extremely small quantity, and is seriously excavated in recent years, so that wild germplasm resources of the flammulina velutipes are endangered, and the flammulina velutipes are listed in an extremely dangerous species of China biodiversity red directory-higher plant rolls and an annex I species of endangered wild animal species International trade convention.
At present, research on the flammulina velutipes at home and abroad is mainly focused on the aspects of in-vitro rapid propagation of the flammulina velutipes (Lin Danni and the like, 2006), sterile germination of seeds, tube seedling raising (Lin Danni and the like, 2008), crossbreeding (Caojing and the like, 2014; chen Heming and the like, 2019), analysis on the population status of the flammulina velutipes (Li Juan and the like, 2018) and the like by a method combining reference, specimen record, field community and population status investigation. Researchers may have studied the flammulina velutipes resources later, and only found that it was reported in 2015, for example Yao, genetic diversity analysis (Yao, 2015) was performed on 8 flammulina velutipes cultivated resources by a related sequence amplification polymorphism (sequence-related amplified polymorphism, SRAP) technology, but the sample size of the study is small, and the sample is not wild resources in Hainan province, and the species specificity of the SRAP marker is not advantageous, so that the situation of the germplasm resources of the whole flammulina velutipes in Hainan province cannot be reflected. The genetic diversity analysis of the flame orchid resources in Hainan province is urgent by adopting a molecular marker with high accuracy and high polymorphism.
SSR (simple sequence repeat) molecular markers have the advantages of high polymorphism, large number of markers, good repeatability, wide coverage and the like, and are widely applied to the fields of molecular marker auxiliary selection, genetic diversity analysis, genetic map construction and the like.
Currently, commonly used strategies for developing SSR markers include a library construction method, a microsatellite enrichment method, a kindred species primer screening method and a bioinformatics analysis method. The library construction method is complex, low in development efficiency, high in laboratory equipment and instrument requirements and long in time consumption, and is only suitable for marking and developing species with little knowledge on genome information; compared with a library method, the microsatellite enrichment method omits the step of constructing the library, but the method also needs to obtain flanking sequences of SSR molecular markers by a PCR method, so that the operation is more complicated; the method for screening the primer of the kindred species needs SSR information of the kindred species and has poor universality for the primer of the species with relatively far kindred, so that the application of the method is limited to a certain extent; the bioinformatics analysis method is to search SSR molecular markers in EST sequences in NCBI and EMBL databases by software or search species which have completed genome sequencing and transcriptome sequencing, find out the SSR molecular markers, design primers according to flanking sequences of the SSR molecular markers, and screen the designed primers by using samples so as to screen primers containing the SSR molecular markers and having polymorphism. Due to the large data volume of EST sequences and transcriptome sequencing sequences, this approach still has high development efficiency compared to other SSR development methods (Xia Fengmei, 2016). In addition, the SSR developed by the method is positioned in a coding region of a gene, the obtained SSR molecular marker is often tightly linked with the gene, gene positioning and associated analysis with the character can be effectively carried out, and functional SSR molecular markers (Chen Chunyan and the like, 202 (1)) are obtained, so that the SSR marker developed based on transcriptome data is not only beneficial to evaluating genetic diversity of germplasm resources, but also beneficial to associated analysis with important characters in the later period, and time and cost are saved for parent breeding process in molecular marker assisted breeding work.
The development of SSR molecular markers based on transcriptome data is an effective means of studying biological genetic diversity analysis. As in recent years, researchers sequenced transcriptome of plants such as medicinal plants psammosilene tunicoides (Psammosilene tunicoides) (She Peng et al, 2019 a) and Yunnan golden camellia (Camellia fascicularis) (She Peng et al, 2019 b), yunnan pine (Pinus yunnanensis) (Cai Nianhui et al, 2015), eucommia ulmoides (eucommiauroides) (Shen Xiangbao et al, 2016), red beans (Vigna umbellata l.) (chen et al, 2016), tetraball tea (Camellia tetracocca) (Li Ruiyuan et al, 2017), wood lotus (Schima superba) (Lin Yan et al, 2018), mango (Mangifera indica) (Tang Yujuan et al, 2018), pseudo-ginseng (panaxnoginesen) (tearing green et al, 2018), peony (He Dan et al, 2019) and the like, and analyzed molecular marker information, providing early basic data for developing deep plant genetic structure and genetic diversity and molecular marker assisted breeding.
The SSR molecular markers of the flame orchid of Hainan province are developed by adopting a transcriptome sequencing strategy and are effective means for evaluating genetic diversity of the flame orchid of Hainan province, but reports on genetic diversity of flame orchid resources of Hainan province are not yet seen at present.
Disclosure of Invention
The invention aims to provide a flame orchid SSR molecular marker primer group in Hainan province and application thereof, so as to at least solve one of the technical problems.
One of the objects of the present invention is to provide: flame orchid SSR molecular marker primer group in Hainan province:
the SSR molecular markers comprise one or more Than Two of (TTG) 6-26, (TTG) 6-27, (TAA) 19-45, (AAT) 16-47, (AAC) 14-68, (CTC) 10-81, (TTG) 10-96, (TTA) 11-106, (TAT) 10-114, (AAT) 13-117, (CTT) 10-119, (CTT) 6-141, (CAT) 6-159, (GAA) 6-184, (ATA) 6-198, (TTA) 6-218 and (ATA) 6-242;
(1) The forward primers (TTG) 6-26F of the amplified (TTG) 6-26 are: 5'-CCCTCAAAGTTCACGAGCTCAG-3';
reverse primers (TTG) 6-26R for amplification (TTG) 6-26 are: 5'-GCCAAGGCAGAGTACGTTTAC-3';
(2) The forward primers (TTG) 6-27F of the amplified (TTG) 6-27 are: 5'-CATGTAGCCGACCCCACTTAG-3';
reverse primers (TTG) 6-27R for amplification (TTG) 6-27 are: 5'-GCAGTCCCTCCAAAGTCGATAG-3';
(3) Forward primers (TAA) 19-45F for amplification (TAA) 19-45 are: 5'-CTTCCCAAGAGCAGTTAATTGAG-3';
reverse primers (TAA) 19-45R for amplification (TAA) 19-45 are: 5'-CATATATACTTGCCACCTTAC-3';
(4) Forward primers (AAT) 16-47F for amplification (AAT) 16-4 are: 5'-GAAGGATGGTTTCACGCAAGATC-3';
reverse primer (AAT) 16-47R for amplification (AAT) 16-4 is: 5'-GGTTTGCATAAGGGAGGTGTCG-3';
(5) The forward primers (AAC) 14-68F for amplification (AAC) 14-68 were: 5'-GCAGTATATGGTGAGTACTAATC-3';
the reverse primers (AAC) 14-68R of amplification (AAC) 14-68 are: 5'-GTTACACTAGGCTAGTTACATTTC-3';
(6) The forward primers (CTC) 10-81F for amplification (CTC) 10-81 are: 5'-CACCCACTACTTCTTCCTTCG-3';
reverse primers (CTC) 10-81R for amplification (CTC) 10-81 are: 5'-GATAAGCTCCCCTTCGGTTAAG-3';
(7) The forward primers (TTG) 10-96F for amplification (TTG) 10-96 are: 5'-CCACACTCTTCAAATGAATAC-3';
reverse primers (TTG) 10-96R for amplification (TTG) 10-96 are: 5'-GAGGACTCGAACAAGTACGC-3';
(8) The forward primers (TTA) 11-106F for amplification (TTA) 11-106 are: 5'-GCCAACCATTACCTGTAGTG-3';
reverse primers (TTA) 11-106R for amplification (TTA) 11-106 are: 5'-GAAGTTGAGAGGTTAGATATG-3';
(9) Forward primers (TAT) 10-114F for amplification (TAT) 10-114 are: 5'-GACATCCGCTTGCTCTTGACGTC-3';
reverse primers (TAT) 10-114R for amplification (TAT) 10-114 are: 5'-CGCGTGGTGTAGGGCTTAAATCG-3';
(10) The forward primers (AAT) 13-117F for amplification (AAT) 13-117 are: 5'-GCCTCTATCCTGCAATCGAAG-3';
reverse primers (AAT) 13-117R for amplification (AAT) 13-117 are: 5'-GAGTGTGTACTGAATTCTCGCG-3';
(11) The forward primers (CTT) 10-119F of the amplification (CTT) 10-119 are: 5'-GGCTCACCGGTAAAGACTTCTTC-3';
reverse primer (CTT) 10-119R of amplification (CTT) 10-119 is: 5'-CATGGCTCACTAACCGTCAGATTG-3';
(12) The forward primers (CTT) 6-141F of amplification (CTT) 6-141 are: 5'-GACACTGCTTCCCCATCACAC-3';
reverse primer (CTT) 6-141R of amplification (CTT) 6-141 is: 5'-CGATGCTGACAGGATAAGACG-3';
(13) The forward primers (CAT) 6-159F for amplification (CAT) 6-159 were: 5'-CATCGGGCCCATCTGAATTGTG-3';
the reverse primer (CAT) 6-159R for amplification (CAT) 6-159 was: 5'-GATCTGTTGACCTTGTATGAC-3';
(14) The forward primers (GAA) 6-184F for amplification (GAA) 6-184 were: 5'-GCTCCATCTCCCAAGCTAGTAC-3';
reverse primers (GAA) 6-184R for amplification (GAA) 6-184 were: 5'-GAGCTTCTTCTTCTTCTCCGG-3';
(15) Forward primers (ATA) 6-198F for amplification (ATA) 6-198 are: 5'-CATAGATGTTGAAATTTGGGTG-3';
reverse primer (ATA) 6-198R for amplification (ATA) 6-198 is: 5'-CAGCTTAAGTGGTAGTTGGTTTTC-3';
(16) The forward primers (TTA) 6-218F for amplification (TTA) 6-218 are: 5'-CGACGGTTCAACTATCAATCTTCC-3';
reverse primers (TTA) 6-218R for amplification (TTA) 6-218 are: 5'-GGCAAACTGAAGGACAAACATG-3';
(17) Forward primers (ATA) 6-242F for amplification (ATA) 6-242 are: 5'-CATGGAGAATTATTAGAGCTC-3';
reverse primers (ATA) 6-242R for amplification (ATA) 6-242 are: 5'-CTAGGCGTTGAAAGTGGAAAAG-3'.
The second object of the present invention is to provide: the construction method of the flame orchid SSR molecular marker primer group in Hainan province comprises the following steps:
s1, selecting a flammulina velutipes, hainan province, and carrying out transcriptome sequencing and assembly to obtain unigene;
s2, designing an SSR molecular marker primer:
carrying out SSR molecular marker search on Unigenes, wherein parameters are set to be at least six times of dinucleotide repetition, at least five times of trinucleotide repetition and at least four times of tetranucleotide to hexanucleotide repetition;
the set standard of the primer is as follows: the length is 16-22bp, the GC content is 40-60%, the annealing temperature is 40-60 ℃, and the length of the PCR product is 100-300bp;
s3, performing verification and polymorphism screening on SSR marker primers to obtain an SSR molecular marker primer group with 17 pairs of better polymorphisms.
The third object of the present invention is to provide: the application of the SSR molecular marker primer group of the flame orchid of Hainan province: is used for detecting the genetic diversity of different flame orchid (Renanthera coccinea) varieties.
The fourth object of the present invention is to provide: the application of the SSR molecular marker primer group of the flame orchid of Hainan province: the method is used for identifying different flame orchid (Renanthera coccinea) varieties.
The fifth object of the present invention is to provide: according to the kit of the primer group for SSR molecular marker of the flame orchid of Hainan province.
The invention has the beneficial effects that:
the invention relates to a flame orchid named as 'flame orchid of Hainan' according to flame orchid distributed in Hainan, which is not a generic concept, but the flame orchid growing in Hainan province, which is the first major province of China territory area (land area plus ocean area).
The Hainan province is one of three nature distribution areas, but how to distribute resources and genetic diversity of the Hainan province flame orchid has not been reported yet, which is extremely unfavorable for resource protection and innovative utilization of the Hainan province flame orchid. The invention evaluates the genetic diversity level of the existing population based on the early germplasm resource investigation, and the constructed flame orchid SSR molecular marker primer group of the invention fills the blank of the flame orchid research of the Hainan province, and is hopeful to provide strategic reserve for the protection and innovation utilization of the wild germplasm resource of the flame orchid of the Hainan province.
In order to better protect and utilize the genetic resources of the flammulina velutipes in Hainan province, the invention develops third generation transcriptome sequencing on the flammulina velutipes in Hainan province, excavates SSR (simple sequence repeat) information, develops a batch of SSR molecular marker primers with high polymorphism, reveals genetic variation and population residence structure of germplasm resources for developing genetic diversity evaluation of flammulina velutipes population in Hainan province, and provides important scientific basis for protecting and innovating the flammulina velutipes germplasm resources in Hainan province.
When constructing, according to the general sequence characteristics of SSR, the parameters of the invention are set to be at least six times of dinucleotide repetition times, at least five times of trinucleotide repetition times and at least four times of tetranucleotide to hexanucleotide repetition times when carrying out SSR molecular marker search on Unigenes.
In the construction process, the set standard of the primer is as follows: the length is 16-22bp, the GC content is 40-60%, the annealing temperature is 40-60 ℃, and the length of the PCR product is 100-300bp.
Drawings
FIG. 1 is a polyacrylamide gel electrophoresis of primer 26 and primer 27;
FIG. 2 is a polyacrylamide gel electrophoresis of primer 45;
FIG. 3 is a polyacrylamide gel electrophoresis of primer 47;
FIG. 4 is a polyacrylamide gel electrophoresis of primer 68;
FIG. 5 is a polyacrylamide gel electrophoresis of primer 81; the method comprises the steps of carrying out a first treatment on the surface of the
FIG. 6 is a polyacrylamide gel electrophoresis of primer 96;
FIG. 7 is a polyacrylamide gel electrophoresis of primer 106;
FIG. 8 is a polyacrylamide gel electrophoresis of primer 114;
FIG. 9 is a polyacrylamide gel electrophoresis of primer 117;
FIG. 10 is a polyacrylamide gel electrophoresis of primer 119;
FIG. 11 is a polyacrylamide gel electrophoresis of primer 141;
FIG. 12 is a polyacrylamide gel electrophoresis of primer 159;
FIG. 13 is a polyacrylamide gel electrophoresis of primer 184;
FIG. 14 is a polyacrylamide gel electrophoresis of primer 218;
FIG. 15 is a polyacrylamide gel electrophoresis of primer 242.
Detailed Description
The following is a further detailed description of the embodiments:
constructing a flame orchid SSR molecular marker primer group in Hainan province, which specifically comprises the following steps:
the sample of the Hainan large-dwelling Hainan flammulina velutipes is selected as a template for screening, and roots, stems, leaves, inflorescences (including flower buds in different stages) and fruits (only the transcriptome information of tender leaves is collected, the requirement of a reference transcriptome cannot be met, so that all parts of the flammulina velutipes and flower buds in different stages are collected here), wherein the sampling amount is 1g fresh weight. Sampling, storing by dry ice, and sending to Beijing NodeB source technology Co., ltd for DNA extraction, RNA extraction, library construction and sequencing, wherein the sequencing platform is Illumina Hiseq 4000.
The large group of kiosks is located in the Miao county of kiosks, of the Hainan province. Germplasm source: wild sources of Baotechniaria.
The sequencing results obtained were assembled with reference transcriptomes according to the following procedure:
(1) Assembling the ginseng-free transcriptome by using Trinity to obtain an initial transcript sequence; trinity is an RNA-Seq data transcriptome assembly tool developed by the Broad Institute and Hebrew University of Jerusalem.
(2) Predicting the coding region sequence and corresponding protein sequence of the initial transcript using an ANGEL software package;
(3) Using a CD-HIT software package, and taking 95% as a threshold value to remove redundancy of the predicted CDS sequence;
(4) The alternative spliced sequence in the CDS sequence after redundancy removal is removed by utilizing all-by-all BLASTNs, transcripts without introns are reserved as unigene, and a total of 47,343 genes are obtained by taking a collection of the unigene as a reference transcriptome.
S2, designing SSR molecular marker primers
Based on the assembled reference transcriptome, the design of SSR marker primers was performed according to the following procedure:
(1) Performing SSR molecular marker search on all Unigenes in a reference transcriptome by using MISA and SAMtools, setting parameters to be at least six times of dinucleotide repetition, at least five times of trinucleotide repetition, at least four times of tetranucleotide to hexanucleotide repetition, randomly selecting 400 SSR sites after confirming the positions of repeated sequences in the genome, and performing primer design of the sequences;
(2) The SSR primers are designed in batches by Primer5.0 software, and the set standard of the primers is as follows: the length is 16-22bp, the GC content is 40-60%, the annealing temperature is 40-60 ℃, and the length of the PCR product is 100-300bp.
S3, screening SSR molecular marker primer
First, using Hexadecyl trimethyl ammonium bromide (CTAB) method, the tweezers were used to extract the DNA of young leaves of 0.1g or so in 2mL EP tube, using kit Shanghai ancient Biotechnology Co., ltd., plant DNA isolation kit (100) DE-06112, using 1% agarsose gel to detect the effect of extraction (appearance of obvious DNA bands), and diluting the extracted DNA to 50 ng/. Mu.l for DNA amplification.
And secondly, randomly selecting 8 clusters, wherein 1 sample of each cluster is used as a template to carry out polymorphic primer screening, and the amplified product is subjected to polymorphism detection by further utilizing 8% polyacrylamide gel electrophoresis, so that 17 pairs of SSR molecular marker primers with higher polymorphism are finally screened out.
The 8 communities are respectively: oriental big herd, oriental small herd, le Dong herd, qiongzhong herd, changjiang herd, baoten big herd, baoten small herd, wanning herd.
Table 1:8 pieces of crowd sampling information
Specific primer information is shown in the following table 3, and the primers in fig. 1-15 all have multiple polymorphic bands, have higher polymorphism and have typical SSR marking characteristics. As in lanes A-H of primer 26, the two most obvious bands in the 250bp-350bp lanes have obvious polymorphisms in 8 samples; in lanes A-H of primer 47, the 100bp-250bp band also had significant polymorphism in 8 samples. These bands all have typical SSR marker characteristics.
Table 2:17 information of SSR molecular marker primer with higher polymorphism
The foregoing describes in detail preferred embodiments of the present invention. It should be understood that numerous modifications and variations can be made in accordance with the concepts of the invention by one of ordinary skill in the art without undue burden. Therefore, all technical solutions which can be obtained by logic analysis, reasoning or limited experiments based on the prior art by the person skilled in the art according to the inventive concept shall be within the scope of protection defined by the claims.
Claims (4)
1. An SSR molecular marker primer set of flammulina velutipes in Hainan province, which is characterized in that the SSR molecular marker comprises (TTG) 6-26, (TTG) 6-27, (TAA) 19-45, (AAT) 16-47, (AAC) 14-68, (CTC) 10-81, (TTG) 10-96, (TTA) 11-106, (TAT) 10-114, (AAT) 13-117, (CTT) 10-119, (CTT) 6-141, (CAT) 6-159, (GAA) 6-184, (ATA) 6-198, (TTA) 6-218 and (ATA) 6-242;
(1) The forward primers (TTG) 6-26F of the amplified (TTG) 6-26 are: 5'-CCCTCAAAGTTCACGAGCTCAG-3';
reverse primers (TTG) 6-26R for amplification (TTG) 6-26 are: 5'-GCCAAGGCAGAGTACGTTTAC-3';
(2) The forward primers (TTG) 6-27F of the amplified (TTG) 6-27 are: 5'-CATGTAGCCGACCCCACTTAG-3';
reverse primers (TTG) 6-27R for amplification (TTG) 6-27 are: 5'-GCAGTCCCTCCAAAGTCGATAG-3';
(3) Forward primers (TAA) 19-45F for amplification (TAA) 19-45 are: 5'-CTTCCCAAGAGCAGTTAATTGAG-3';
reverse primers (TAA) 19-45R for amplification (TAA) 19-45 are: 5'-CATATATACTTGCCACCTTAC-3';
(4) Forward primers (AAT) 16-47F for amplification (AAT) 16-47 are: 5'-GAAGGATGGTTTCACGCAAGATC-3';
reverse primers (AAT) 16-47R for amplification (AAT) 16-47 are: 5'-GGTTTGCATAAGGGAGGTGTCG-3';
(5) The forward primers (AAC) 14-68F for amplification (AAC) 14-68 were: 5'-GCAGTATATGGTGAGTACTAATC-3';
the reverse primers (AAC) 14-68R of amplification (AAC) 14-68 are: 5'-GTTACACTAGGCTAGTTACATTTC-3';
(6) The forward primers (CTC) 10-81F for amplification (CTC) 10-81 are: 5'-CACCCACTACTTCTTCCTTCG-3';
reverse primers (CTC) 10-81R for amplification (CTC) 10-81 are: 5'-GATAAGCTCCCCTTCGGTTAAG-3';
(7) The forward primers (TTG) 10-96F for amplification (TTG) 10-96 are: 5'-CCACACTCTTCAAATGAATAC-3';
reverse primers (TTG) 10-96R for amplification (TTG) 10-96 are: 5'-GAGGACTCGAACAAGTACGC-3';
(8) The forward primers (TTA) 11-106F for amplification (TTA) 11-106 are: 5'-GCCAACCATTACCTGTAGTG-3';
reverse primers (TTA) 11-106R for amplification (TTA) 11-106 are: 5'-GAAGTTGAGAGGTTAGATATG-3';
(9) Forward primers (TAT) 10-114F for amplification (TAT) 10-114 are: 5'-GACATCCGCTTGCTCTTGACGTC-3';
reverse primers (TAT) 10-114R for amplification (TAT) 10-114 are: 5'-CGCGTGGTGTAGGGCTTAAATCG-3';
(10) The forward primers (AAT) 13-117F for amplification (AAT) 13-117 are: 5'-GCCTCTATCCTGCAATCGAAG-3';
reverse primers (AAT) 13-117R for amplification (AAT) 13-117 are: 5'-GAGTGTGTACTGAATTCTCGCG-3';
(11) The forward primers (CTT) 10-119F of the amplification (CTT) 10-119 are: 5'-GGCTCACCGGTAAAGACTTCTTC-3';
reverse primer (CTT) 10-119R of amplification (CTT) 10-119 is: 5'-CATGGCTCACTAACCGTCAGATTG-3';
(12) The forward primers (CTT) 6-141F of amplification (CTT) 6-141 are: 5'-GACACTGCTTCCCCATCACAC-3';
reverse primer (CTT) 6-141R of amplification (CTT) 6-141 is: 5'-CGATGCTGACAGGATAAGACG-3';
(13) The forward primers (CAT) 6-159F for amplification (CAT) 6-159 were: 5'-CATCGGGCCCATCTGAATTGTG-3';
the reverse primer (CAT) 6-159R for amplification (CAT) 6-159 was: 5'-GATCTGTTGACCTTGTATGAC-3';
(14) The forward primers (GAA) 6-184F for amplification (GAA) 6-184 were: 5'-GCTCCATCTCCCAAGCTAGTAC-3';
reverse primers (GAA) 6-184R for amplification (GAA) 6-184 were: 5'-GAGCTTCTTCTTCTTCTCCGG-3';
(15) Forward primers (ATA) 6-198F for amplification (ATA) 6-198 are: 5'-CATAGATGTTGAAATTTGGGTG-3';
reverse primer (ATA) 6-198R for amplification (ATA) 6-198 is: 5'-CAGCTTAAGTGGTAGTTGGTTTTC-3';
(16) The forward primers (TTA) 6-218F for amplification (TTA) 6-218 are: 5'-CGACGGTTCAACTATCAATCTTCC-3';
reverse primers (TTA) 6-218R for amplification (TTA) 6-218 are: 5'-GGCAAACTGAAGGACAAACATG-3';
(17) Forward primers (ATA) 6-242F for amplification (ATA) 6-242 are: 5'-CATGGAGAATTATTAGAGCTC-3';
reverse primers (ATA) 6-242R for amplification (ATA) 6-242 are: 5'-CTAGGCGTTGAAAGTGGAAAAG-3'.
2. The use of a SSR molecular marker primer set of flame orchid of hainan province according to claim 1 for detecting genetic diversity of different flame orchid varieties (renanthacocina).
3. The application of the molecular marker primer group for the flame orchid SSR in Hainan province according to claim 2, which is used for identifying varieties of different flame orchids (Renantheracocinea).
4. Use of a molecular marker primer set of a flame orchid SSR in hainan province according to any one of claims 1-3 in preparing a detection kit.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210145000.5A CN114540530B (en) | 2022-02-17 | 2022-02-17 | Hainan province flammulina SSR molecular marker primer group and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210145000.5A CN114540530B (en) | 2022-02-17 | 2022-02-17 | Hainan province flammulina SSR molecular marker primer group and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN114540530A CN114540530A (en) | 2022-05-27 |
| CN114540530B true CN114540530B (en) | 2023-12-05 |
Family
ID=81674896
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202210145000.5A Active CN114540530B (en) | 2022-02-17 | 2022-02-17 | Hainan province flammulina SSR molecular marker primer group and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN114540530B (en) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114480711A (en) * | 2022-02-17 | 2022-05-13 | 海南省农业科学院热带园艺研究所 | SSR-PCR reaction system of flame orchid in Hainan province and application thereof |
| CN117721243B (en) * | 2024-02-07 | 2024-04-30 | 云南省林业和草原科学院 | SSR primer and method for identifying purity of Yunnan pine hybrid seeds |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108676907A (en) * | 2018-06-04 | 2018-10-19 | 贵州师范大学 | A method of it is sequenced based on transcript profile and obtains green hedge bavin SSR primers |
| CN112662806A (en) * | 2021-01-29 | 2021-04-16 | 广东省农业科学院环境园艺研究所 | Rhynchosia SSR molecular marker primer composition and application thereof |
| CN114480711A (en) * | 2022-02-17 | 2022-05-13 | 海南省农业科学院热带园艺研究所 | SSR-PCR reaction system of flame orchid in Hainan province and application thereof |
-
2022
- 2022-02-17 CN CN202210145000.5A patent/CN114540530B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108676907A (en) * | 2018-06-04 | 2018-10-19 | 贵州师范大学 | A method of it is sequenced based on transcript profile and obtains green hedge bavin SSR primers |
| CN112662806A (en) * | 2021-01-29 | 2021-04-16 | 广东省农业科学院环境园艺研究所 | Rhynchosia SSR molecular marker primer composition and application thereof |
| CN114480711A (en) * | 2022-02-17 | 2022-05-13 | 海南省农业科学院热带园艺研究所 | SSR-PCR reaction system of flame orchid in Hainan province and application thereof |
Non-Patent Citations (2)
| Title |
|---|
| 云南火焰兰转录组SSR分布及其序列特征分析;辛静;南方农业学报;第51卷(第7期);第1634-1641页 * |
| 海南岛火焰兰 SSR 标记开发、遗传多样性与群体结构分析;赵慧琪;分子植物育种;第21卷(第7期);第 2300-2310 页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN114540530A (en) | 2022-05-27 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Dhakshanamoorthy et al. | Induced mutagenesis in Jatropha curcas L. using gamma rays and detection of DNA polymorphism through RAPD marker | |
| CN114540530B (en) | Hainan province flammulina SSR molecular marker primer group and application thereof | |
| Zhang et al. | A novel strategy for identification of 47 pomegranate (Punica granatum) cultivars using RAPD markers | |
| CN107653335B (en) | Banana wilt resistance molecular marker and application thereof | |
| Lim et al. | Organellar genome copy number variations and integrity across different organs, growth stages, phenotypes and main localities of sago palm (Metroxylon sagu Rottboll) in Sarawak, Malaysia | |
| Baraket et al. | Combination of simple sequence repeat, S-locus polymorphism and phenotypic data for identification of Tunisian plum species (Prunus spp.) | |
| Beser et al. | Marker-assisted introgression of a broad-spectrum resistance gene, Pi40 improved blast resistance of two elite rice (Oryza sativa L.) cultivars of Turkey | |
| CN113604596A (en) | KASP primer for detecting cucumber small zucchini yellow mosaic virus disease resistance gene zym and application thereof | |
| CN106119360A (en) | A kind of SCAR molecular marker identifying banana blight resistance and authentication method thereof | |
| Chang et al. | Identification and characterization of Colletotrichum fioriniae and C. fructicola that cause anthracnose in pecan | |
| KR101783347B1 (en) | CAPS marker for discriminating presence or absence of pollen in pear and uses thereof | |
| Zendler et al. | Confirmation and fine mapping of the resistance locus Ren9 from the grapevine cultivar ‘Regent.’Plants. 10 (1): 1–20 | |
| KR101563351B1 (en) | New aloe cultivar Aloe vera Saengjang and molecular marker for discriminating the same | |
| Taheri et al. | Use of intersimple sequence repeat assay for detection of DNA polymorphism induced by gamma rays in Curcuma alismatifolia | |
| Dhakshanamoorthy et al. | Induced mutagenesis in Jatropha curcas L. using ethyl methanesulphonate (EMS) and assessment of DNA polymorphism through RAPD markers | |
| Gedil et al. | Development of molecular genomic tools for verification of intergeneric hybrids between castor bean (Ricinus communis L.) and cassava (Manihot esculenta Crantz) | |
| Jiang et al. | Analysis of genetic diversity of salt-tolerant alfalfa germplasms | |
| CN114990251B (en) | Molecular marker closely linked with rape methylselenocysteine content trait QTL and application thereof | |
| Morikawa et al. | Identifying, discriminating and isolating cultivars of ‘Marguerites’ originated from Argyranthemum frutescens parentages and their intergeneric and interspecific hybridities by DNA markers amplified by RAPD (Random Amplified Polymorphic DNA) and ISSR (Inter-Simple Sequence Repeat)∗ | |
| Campos | Marker assisted selection, fine mapping and identification of candidate genes for three major traits of Prunus persica L.(Batsh) | |
| Osawaru et al. | Comparative micro-anatomical studies of the wood of two species of Okra [Abelmoschus species] | |
| Kumar et al. | Characterization of chrysanthemum (Chrysanthemum grandiflorum) varieties using ISSR markers | |
| Miazzi et al. | The IVC breeding program: development of new seedless table grapevines for a sustainable viticulture | |
| Schaefer et al. | Differential RAPD fingerprints in carrot tissues | |
| Awomukwu et al. | Identification, Validation and Classification of the Genus Phyllanthus in Nigeria using ITS Genetic Marker and the Taxonomic Implication |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |