CN104004833A - EST-SSR core primer group developed on basis of transcriptome sequence of towel gourd and application thereof - Google Patents
EST-SSR core primer group developed on basis of transcriptome sequence of towel gourd and application thereof Download PDFInfo
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Abstract
The invention discloses an EST-SSR core primer group developed on the basis of the transcriptome sequence of towel gourd. The EST-SSR core primer group comprises 50 pairs of primers with nucleotide sequences as shown in SEQ ID No. 1-100 in a sequence table. The 50 pairs of EST-SSR core primers have the advantages of abundant polymorphism, stable amplification, good repeatability, convenience in statistics, etc.; meanwhile, since the primers are originated from the expressed gene of towel gourd and can reflect variation of functional sequences in a genome, so germplasm genetic diversity of towel gourd can be better distinguished. The core primer group can be used in fields like identification of the variety of towel gourd, genetic genealogical analysis of varieties and analysis of germplasm genetic diversity, is beneficial for protection of legal interests of breeders, producers and consumers and promotes sound development of genetic breeding and production of towel gourd.
Description
Technical field
The present invention relates to EST-SSR core primers group, be specifically related to a set of EST-SSR core primers group and application thereof of transcribing the exploitation of group sequence based on sponge gourd.
Background technology
Sponge gourd is annual the climbing up by holding on to property herbaceous plant of Curcurbitaceae (Cucurbitaceae) Luffa (Luffa spp.), and it all has cultivated area widely as a kind of important vegetable crop in the various places, north and south of China.Sponge gourd is except as vegetables, or a kind of important medicinal plant.Modern pharmacology proves, contains the plurality of active ingredients such as alkaloids, flavonoid, steroid, glucosides class in sponge gourd, has the effects such as antimycotic, antibacterium, anti-inflammatory.Nearest result of study proves, in sponge gourd seed, contains ribosome inactivating protein, can suppress the growth of hiv virus, is therefore the medicine of potential treatment acquired immune deficiency syndrome (AIDS).
In recent years, along with the pay attention to day by day of people to nutritive health-care, sponge gourd, as a kind of herbal cuisine dual-purpose and high temperature season market supply vegetables, is loved by the people, and demand constantly increases, and cultivated area is expansion trend.The research of China investigator aspect such as sponge gourd genetic breeding, hybrid vigour and hereditary property since the sixties in 20th century, has obtained certain progress, has cultivated a collection of improved Varieties.Due to the fundamental research weakness of sponge gourd, aspect sponge gourd breeding and kind protection, there are two outstanding problems at present: one, how the genetic diversity of sponge gourd germ plasm resource is carried out to precise Identification, thereby instruct efficiently breeding man to carry out parent's apolegamy; Two, how accurately, carry out fast the qualification of variety of luffa, thereby better promote the development of the aspects such as variety of luffa authorization, kind protection, true and false kind are distinguished, the solution of the property right dispute of kind.
Molecular marking technique based on DNA sequence polymorphism between individuality has many, the not affected by environment and season limit of short, potential marker number of test period, there is no tissue specificity, accuracy is high, be beneficial to the advantages such as high-throughput test analysis, has been widely used in during Genetic Diversity of Germplasm analysis, new variety qualification, seed purity detect.In sponge gourd, more existing investigators utilize molecule marker to carry out analysis of genetic diversity to sponge gourd germ plasm resource, but are generally all the random primer labelling such as RAPD, ISSR, SRAP adopting.These marks are all the labeling techniques based on without the exploitation of genome (or transcribing group) sequence information, although there is certain practicality, the randomness of mark is strong, poor stability, thereby have affected the accuracy of experimental result.
EST-SSR is a kind of SSR molecule marker based on est sequence or cDNA data mining.Because it is from expressing gene, thereby except possessing the institute of traditional genome source SSR mark has superiority, the advantage such as also have information content high (reflection expressing gene information), development cost are low, versatility is good, is marked at the application in genetic research thereby strengthened this.At present, this is marked at the research fields such as analysis of genetic diversity, cultivar identification, genetic map construction, gene (QTL) location and has obtained good application.At present, in the crops such as corn, wheat, millet, for improving the detection efficiency of SSR technology, carry out the correlative study of core primers group, obtained good effect.Core primers group refers to the full genome of uniform fold, a set of primer that polymorphism is high, stability is strong, reproducible, utilize its carry out analysis of genetic diversity, cultivar identification have advantages of quick, easy, accuracy is high.At present, sponge gourd there is no based on genome or transcribes the specific molecular marker that group sequence is developed, and therefore, exploitation sponge gourd EST-SSR core primers group, has become problem in the urgent need to address in sponge gourd breeding, production and kind protection field.
Summary of the invention
One object of the present invention is to provide a set of EST-SSR core primers group of transcribing the exploitation of group sequence based on sponge gourd.
Another object of the present invention is to provide that above-mentioned EST-SSR core primers group of transcribing the exploitation of group sequence based on sponge gourd is analyzed at sponge gourd Genetic Diversity of Germplasm, application in variety of luffa Genetic lineages or variety of luffa qualification.
The technical solution used in the present invention is:
The EST-SSR core primers group of transcribing the exploitation of group sequence based on sponge gourd, it comprises 50 pairs of primers, its nucleotide sequence is as shown in sequence table SEQ ID NO.1～100.
Utilize and above-mentionedly transcribe based on sponge gourd the method that the EST-SSR core primers group of group sequence exploitation is carried out the analysis of sponge gourd Genetic Diversity of Germplasm, variety of luffa Genetic lineages or variety of luffa qualification, comprise the steps:
(1) extract sponge gourd sample gene group DNA to be measured;
(2) the testing sample DNA extracting taking step (1), as template, utilizes primer shown in sequence table SEQ ID NO.1～100 to carry out pcr amplification, obtains pcr amplification product;
(3) pcr amplification product of step (2) is adopted to 6% sex change polyacrylamine gel electrophoresis detection, silver dyes colour developing, statistics detected result;
(4) utilize the statistics of step (3) to carry out the analysis of sponge gourd Genetic Diversity of Germplasm, variety of luffa Genetic lineages or variety of luffa qualification.
Described pcr amplification reaction system (20 μ L) comprising: 2 μ L10 × buffer; 0.5 μ L Taq enzyme (2U μ L
-1); 0.4 μ L dNTP (10mmolL
-1); Each 0.1 μ L (the 50pmoL μ L of upstream and downstream primer
-1); 1 μ L template DNA (50ng μ L
-1); 15.9 μ L ddH2O.PCR program is Touch-down PCR:94 DEG C of denaturation 3min; 94 DEG C of 30S, 65 DEG C of (1 DEG C/cycle) 1min, 72 DEG C of 1min, 15 circulations; 94 DEG C of 30S, 50 DEG C of 1min, 72 DEG C of 1min, 25 circulations; 72 DEG C are extended 10min eventually.
The analysis of described sponge gourd Genetic Diversity of Germplasm or variety of luffa Genetic lineages analytical procedure are: use NTSYS-pc2.l0e software to carry out the cluster analysis based on UPGMA method; Use number of alleles (Na), gene diversity (h) and the Shannon value (I) of the each primer of POPGENE computed in software.
Described variety of luffa authentication method is: add up the amplification situation of each kind in primer, and constructed dna finger printing, more each primer sites difference one by one, difference primer number >=2, judge that two kinds are different varieties; Difference primer number=1, judges that two kinds are as approximate kind; Difference primer number=0, judges that two kinds are same breed.
The invention has the beneficial effects as follows: 50 pairs of EST-SSR core primers of the present invention have rich polymorphism, amplification stable, reproducible, be convenient to the advantages such as statistics, simultaneously because these primers are all from sponge gourd expressing gene, the variation of functional sequence in genome can be reflected, thereby the diversity of sponge gourd genetic germplasm can be better distinguished.This core primers group can be used for the fields such as variety of luffa qualification, the analysis of kind Genetic lineages and Genetic Diversity of Germplasm analysis; be conducive to protect breeder, producers and consumers's legitimate rights and interests, promote the sound development that sponge gourd genetic breeding and sponge gourd are produced.
Brief description of the drawings
Fig. 1 is to the cluster analysis of 46 parts of sponge gourd materials according to 50 pairs of sponge gourd EST-SSR core primers groups.
Embodiment
Below in conjunction with embodiment, the present invention is further illustrated, but be not limited to this.
Embodiment
One, the exploitation of sponge gourd EST-SSR core primers group
1, material
The sponge gourd germ plasm resource of preserving from our unit, filter out 46 parts of Core Germplasms resource materials (table 1) for the evaluation of sponge gourd EST-SSR core primers group and the qualification of resource genetic diversity.46 parts of sponge gourd resource materials comprise that 36 parts have rib sponge gourd, 10 parts of luffa-smooth loofahs; Wherein 25 parts of materials are from China, and 8 parts of materials are from Thailand, and 7 parts of materials, from Malaysia, separately have the unknown source of 6 parts of materials.
In table 1 the present invention for sponge gourd resource analysis of genetic diversity material information
Note :-represent unknown source.
2, the extraction of sponge gourd genomic dna
Adopt the simple and easy SDS method of improvement to extract sponge gourd leaves genomic DNA, step is as follows:
(1) get proper amount of fresh blade, put into 2ml centrifuge tube, add liquid nitrogen, blade is stampped to powdered with micro-grinding rod or cross screwdriver, add 600 microlitre 1.5%SDS trace extract (0.5%SDS, 250mmol/L NaCL, 25mmol/L EDTA, 200mmol/L Tris-HCL damping fluid, pH7.5);
(2) sample hose is placed in to 65 DEG C of water-baths and hatches 30 minutes, put upside down and mix once every 5-10 minute;
(3) take out sample, be cooled to room temperature, add the chloroform/primary isoamyl alcohol/ethanol (76:4:20) of equal-volume (600-700 microlitre), vibrate 10 minutes, fully mix;
(4), by centrifugal sample hose 12000rpm 12 minutes, carefully supernatant liquor is transferred in another 1.5ml centrifuge tube;
(5) add equal-volume Virahol or two volumes dehydrated alcohol precipitation, leave standstill 3-5 minute, centrifugal 5 minutes of 12000rpm;
(6) remove supernatant, by the ethanol rinsing precipitation of 0.5ml70%, air-dry, be dissolved in 50-100 μ l TE.
3, pcr amplification
The reaction system (20 μ L) of PCR comprising: 2 μ L10 × buffer, 0.5 μ L Taq enzyme (2U μ L
-1), 0.4 μ L dNTP (10mmolL
-1), each 0.1 μ L (the 50pmoL μ L of upstream and downstream primer
-1), 1 μ L template DNA (50ng μ L
-1), 15.9 μ L ddH2O.
PCR program is Touch-down PCR:94 DEG C of denaturation 3min; 94 DEG C of 30S, 65 DEG C of (1 DEG C/cycle) 1min, 72 DEG C of 1min, 15 circulations; 94 DEG C of 30S, 50 DEG C of 1min, 72 DEG C of 1min, 25 circulations; 72 DEG C are extended 10min eventually.
4, polyacrylamide gel electrophoresis detects
(1) configuration of reagent
1 × 6% acrylamide stock solution (100mL): 5.7g acrylamide, 0.3g Bis, 12g urea, 10mL10 × TBE, adds ddH
2o is settled to 100mL; Before encapsulating, every 10mL glue, adds 10% ammonium persulphate 70 μ L, TEMED3 μ L.
Staining fluid (1 ×): 0.1%AgNO
3
Developing solution (1 ×): 0.5g NaOH, 0.019g sodium tetraborate, 0.4mL formaldehyde (with front adding), ddH
2o is settled to 100ml.
(2) preparation of polyacrylamide gel
First sheet glass bottom end opening is sealed with sepharose, treat that it solidifies, 1 × 6% acrylamide soln preparing is stirred evenly, sheet glass is become to the angle of 30 ° of left and right with desktop, slowly injecting glue, avoids the generation of bubble.After filling, set level sheet glass and make it become the angle of 10 ° of left and right with desktop, insert suitable comb.After treating that it solidifies 0.5h, carefully extract comb.Repeatedly rinse point sample hole with distilled water, remove the unnecessary material not solidifying.
(3) electrophoresis
Sheet glass is fixed on Vertial electrophorestic tank, adds appropriate 1 × tbe buffer liquid.After PCR finishes, take out sample, every pipe adds the load sample damping fluid of 3 μ L containing bromjophenol blue and the blue or green two kinds of indicator of dimethylbenzene.Take out 3 μ L sample loadings with microsyringe.
(4) argentation dyeing
After electrophoresis, sheet glass is pulled down from electrophoresis chamber, taken out gel, rinsing twice in distilled water, each about 1min.Then proceed in staining fluid jog on shaking table, dyeing 10min.After dyeing finishes, gel is transferred to rinsing twice in distilled water, each about 1min.Then proceed in developing solution and develop the color, painted proceeding in clear water afterwards preserved, and gel imaging, records result.
5, EST-SSR primer development and screening
Utilize MIcroSAtellite identification tool (MISA,
http:// pgrc.ipk-gatersleben.de/misa/)software scans SSR site from the sponge gourd Unigene having spliced, and screening criteria is that 2 bases at least repeat six times, and 3 bases at least repeat five times, and 4,5,6 bases at least repeat four times and are just identified as SSR site.Obtain altogether SSR site 12,932, utilize Primer premier6.0 (PREMIER Biosoft International, Palo Alto, CA) to carry out design of primers to these candidate locus, successfully design 8,523 pairs of primers.Synthetic 641 primers wherein, utilize and have rib sponge gourd material S1174, and the F1 hybrid of luffa-smooth loofah material 93075 and the two hybridization detects the polymorphism of primer.
6, data analysis
Use NTSYS-pc2.l0e software to carry out the cluster analysis based on UPGMA method, use number of alleles (Na), gene diversity (h) and the Shannon value (I) of the each primer of POPGENE computed in software.
7, determining of core EST-SSR primer sets
Choosing wherein 641 pairs of marks is synthesized, and carry out screening verification in the F1 that has rib variety of luffa S1174 and luffa-smooth loofah kind 93075 and the two hybridization, and result shows, 494 pairs are marked with amplified production, 201 pairs are marked between two parents and show polymorphism, are wherein codominantly marked with 126 pairs.The genetic linkage maps building according to us, from 126 pairs of codominant primer pairs, filters out primer that 50 pairs of amplifications stablize, are uniformly distributed each linkage group as core EST-SSR primer sets, in table 2.
Table 2 sponge gourd 50 is to core EST-SSR primer sets
8, the validity check of sponge gourd EST-SSR core primers group
Utilize 50 pairs of core EST-SSR primer sets to carry out diversity analysis to the 46 parts of sponge gourd Germplasms (in table 1) from domestic and international collection.Cluster analysis result is shown in Fig. 1, and result shows: 36 parts have rib sponge gourd to gather is a class, and it is a class that 10 parts of luffa-smooth loofahs gather.Wherein, 36 parts have rib sponge gourd to have the trend of carrying out cluster according to source place, two green grass or young crops, fill with village sponge gourd, in clear sponge gourd, S1174, S095, S155,113005, ten thousand precious sponge gourds, full green sponge gourd, S177, medium green sponge gourd, Heshan pork sponge gourd, 113012,113024,113025 is gathered is a large class, these materials are all economized from Chinese Guangdong; In another large class except the husky skin in refined hilllock sponge gourd and elegant field from Chinese Guangdong outside the province, all sub-national Malaysia and Thailand southeast of remaining I-01, BridgeGourd, M18, V-1052, V-1052, BlackHank, Abacus, Huaykeaw, Luffa833, I-23, RidgeGourd; In other groups, except Grand Prize and Wister Chaichan are from Malaysia and Thailand, all the other materials 113003,113008, Lianzhou City's perfume sponge gourd, Meizhou pork sponge gourd, magnificent crown wire melon S087,113073 are all from Chinese Guangdong.Distant with other 7 parts of materials from MinQty, the LEELA of Thailand and the materials A 1206 in unknown source in 10 parts of luffa-smooth loofahs, in other 7 parts of materials, except S15 is from Thailand, other materials is all from China.Have between rib sponge gourd and luffa-smooth loofah kind hereditary difference very large, and inbred genetic difference is very little.More existing research reports show, there are rib sponge gourd and luffa-smooth loofah genetic variation narrow, but utilize 50 pairs of core EST-SSR primer sets in the present invention 46 parts of resource materials can be done well to distinguish, this illustrates that core primers group of the present invention can be used for variety of luffa qualification, the analysis of variety of luffa Genetic lineages and the analysis of sponge gourd Genetic Diversity of Germplasm.
<110> Vegetables Inst., Guangdong Academy of Agricultural Sciences
<120> transcribes EST-SSR core primers group and the application of group sequence exploitation based on sponge gourd
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<213> artificial sequence
<400> 64
tcttcatcca aatcaagggc 20
<210> 65
<211> 20
<212> DNA
<213> artificial sequence
<400> 65
ctctccctgt ttccttctcc 20
<210> 66
<211> 20
<212> DNA
<213> artificial sequence
<400> 66
tttggaacct atgtgcctcc 20
<210> 67
<211> 20
<212> DNA
<213> artificial sequence
<400> 67
ttgtcctctt gcttcccatc 20
<210> 68
<211> 20
<212> DNA
<213> artificial sequence
<400> 68
agctccccaa atggagtttg 20
<210> 69
<211> 20
<212> DNA
<213> artificial sequence
<400> 69
tttgcctcca ccatatgacg 20
<210> 70
<211> 20
<212> DNA
<213> artificial sequence
<400> 70
gcctctttgc caagtgctac 20
<210> 71
<211> 20
<212> DNA
<213> artificial sequence
<400> 71
tccatggccc atggttcatc 20
<210> 72
<211> 19
<212> DNA
<213> artificial sequence
<400> 72
accaggctgg aaaaagggc 19
<210> 73
<211> 22
<212> DNA
<213> artificial sequence
<400> 73
ccaattcacc tccgatccaa tc 22
<210> 74
<211> 20
<212> DNA
<213> artificial sequence
<400> 74
acgttggtta ggcactcgtg 20
<210> 75
<211> 20
<212> DNA
<213> artificial sequence
<400> 75
tgccagatcg aaaccttgtg 20
<210> 76
<211> 20
<212> DNA
<213> artificial sequence
<400> 76
acaattctgt ccacgggaag 20
<210> 77
<211> 20
<212> DNA
<213> artificial sequence
<400> 77
atccaccaac ttccaagcac 20
<210> 78
<211> 20
<212> DNA
<213> artificial sequence
<400> 78
cgaaagtgaa gagaaacccg 20
<210> 79
<211> 22
<212> DNA
<213> artificial sequence
<400> 79
cccatttgct ggtttcagat cg 22
<210> 80
<211> 22
<212> DNA
<213> artificial sequence
<400> 80
cgaaaggcag aatgggaatt cg 22
<210> 81
<211> 20
<212> DNA
<213> artificial sequence
<400> 81
tcctcctcct ttttcccttc 20
<210> 82
<211> 20
<212> DNA
<213> artificial sequence
<400> 82
tctgagacga gaatgccaac 20
<210> 83
<211> 20
<212> DNA
<213> artificial sequence
<400> 83
ttccgatcac tgtcagcttg 20
<210> 84
<211> 20
<212> DNA
<213> artificial sequence
<400> 84
tccacatccc ctttttccag 20
<210> 85
<211> 20
<212> DNA
<213> artificial sequence
<400> 85
atttactcac tctgcttcgc 20
<210> 86
<211> 22
<212> DNA
<213> artificial sequence
<400> 86
aggcaaattc taccaaaaac ac 22
<210> 87
<211> 20
<212> DNA
<213> artificial sequence
<400> 87
aactacttga agccaggccc 20
<210> 88
<211> 20
<212> DNA
<213> artificial sequence
<400> 88
tgatctggca ctagctgcac 20
<210> 89
<211> 20
<212> DNA
<213> artificial sequence
<400> 89
ttcttctcct caggcacagg 20
<210> 90
<211> 21
<212> DNA
<213> artificial sequence
<400> 90
ctgattcgga ggaattcgag g 21
<210> 91
<211> 20
<212> DNA
<213> artificial sequence
<400> 91
cccgtaactc ggttaattcg 20
<210> 92
<211> 20
<212> DNA
<213> artificial sequence
<400> 92
tctgatccgt cattgccatc 20
<210> 93
<211> 20
<212> DNA
<213> artificial sequence
<400> 93
ggtggacctg caagattatg 20
<210> 94
<211> 22
<212> DNA
<213> artificial sequence
<400> 94
attcttccat ctcatttgtt gg 22
<210> 95
<211> 20
<212> DNA
<213> artificial sequence
<400> 95
tctacccgga gcctctcttc 20
<210> 96
<211> 20
<212> DNA
<213> artificial sequence
<400> 96
aacatggagg tctggaggag 20
<210> 97
<211> 20
<212> DNA
<213> artificial sequence
<400> 97
cgctgttgga ctgacatacg 20
<210> 98
<211> 20
<212> DNA
<213> artificial sequence
<400> 98
cattggagga gatggaaggc 20
<210> 99
<211> 20
<212> DNA
<213> artificial sequence
<400> 99
cttggatgca aagtgcttgt 20
<210> 100
<211> 20
<212> DNA
<213> artificial sequence
<400> 100
atggaagcac ccattttgag 20
Claims (10)
1. the EST-SSR core primers group of transcribing the exploitation of group sequence based on sponge gourd, is characterized in that: described primer sets comprises 50 pairs of primers, its nucleotide sequence is as shown in sequence table SEQ ID NO.1～100.
2. the application of EST-SSR core primers group of transcribing group sequence exploitation based on sponge gourd according to claim 1 in sponge gourd Genetic Diversity of Germplasm is analyzed.
3. the application of EST-SSR core primers group of transcribing group sequence exploitation based on sponge gourd according to claim 1 in variety of luffa Genetic lineages.
4. the application of EST-SSR core primers group of transcribing group sequence exploitation based on sponge gourd according to claim 1 in variety of luffa qualification.
5. utilize described in claim 1 based on sponge gourd and transcribe the method that the EST-SSR core primers group of group sequence exploitation is carried out the analysis of sponge gourd Genetic Diversity of Germplasm, variety of luffa Genetic lineages or variety of luffa qualification, comprise the steps:
(1) extract sponge gourd sample gene group DNA to be measured;
(2) the testing sample DNA extracting taking step (1), as template, utilizes primer shown in sequence table SEQ ID NO.1～100 to carry out pcr amplification, obtains pcr amplification product;
(3) pcr amplification product of step (2) is adopted to detected through gel electrophoresis, statistics detected result;
(4) utilize the statistics of step (3) to carry out the analysis of sponge gourd Genetic Diversity of Germplasm, variety of luffa Genetic lineages or variety of luffa qualification.
6. method according to claim 5, is characterized in that, the analysis of described sponge gourd Genetic Diversity of Germplasm or variety of luffa Genetic lineages analytical procedure are: use NTSYS-pc 2.l0e software to carry out the cluster analysis based on UPGMA method; Use number of alleles (Na), gene diversity (h) and the Shannon value (I) of the each primer of POPGENE computed in software.
7. method according to claim 5, is characterized in that, described variety of luffa authentication method is: add up the amplification situation of each kind in primer, constructed dna finger printing, more each primer sites difference one by one, difference primer number >=2, judge that two kinds are different varieties; Difference primer number=1, judges that two kinds are as approximate kind; Difference primer number=0, judges that two kinds are same breed.
8. method according to claim 5, is characterized in that, step (3) adopts 6% sex change polyacrylamine gel electrophoresis detection, and silver dyes colour developing.
9. method according to claim 5, is characterized in that, 20 μ L pcr amplification reaction systems comprise: 2 μ L 10 × buffer; 0.5 μ L Taq enzyme (2U μ L
-1); 0.4 μ L dNTP(10mmol L
-1); The each 0.1 μ L(50 pmoL μ L of upstream and downstream primer
-1); 1 μ L template DNA (50 ng μ L
-1); 15.9 μ L ddH2O.
10. method according to claim 5, is characterized in that, pcr amplification program is Touch-down PCR:94 DEG C of denaturation 3min; 94 DEG C of 30S, 65 DEG C of (1 DEG C/cycle) 1min, 72 DEG C of 1min, 15 circulations; 94 DEG C of 30S, 50 DEG C of 1min, 72 DEG C of 1min, 25 circulations; 72 DEG C are extended 10min eventually.
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| CN105203670A (en) * | 2015-11-02 | 2015-12-30 | 黑龙江大学 | Establishing method of HPLC (high performance liquid chromatography) fingerprints of loofah sponge, standard fingerprints established with method and application |
| CN105755117A (en) * | 2016-03-03 | 2016-07-13 | 广东省农业科学院蔬菜研究所 | Nucleotide primer composition for quickly identifying purity of 'Yalv No.6' hybrid seeds of luffa acutangula roxb and detection method |
| CN105821154A (en) * | 2016-06-03 | 2016-08-03 | 福建省农业科学院作物研究所 | SSR primers and method for purity identification of luffa hybrid seeds |
| CN106011251A (en) * | 2016-06-07 | 2016-10-12 | 山西农业大学 | Primer group, kit and method for identifying population genetic structure of Hemerocallis L. plants |
| CN108359740A (en) * | 2018-04-28 | 2018-08-03 | 江苏省中国科学院植物研究所 | EST-SSR molecular labelings and its application are developed based on Lycoris aurea transcript profile sequence |
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