CN114525345B - Castor silkworm SSR molecular marker and application thereof - Google Patents
Castor silkworm SSR molecular marker and application thereof Download PDFInfo
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Abstract
The invention discloses a castor-silkworm SSR molecular marker and application thereof, belonging to the technical fields of molecular biology and silkworm variety identification. The invention obtains genome SSR molecular marker locus information by analyzing the genome sequence of the castor silkworms, and designs and synthesizes 15 pairs of SSR primers with high amplification efficiency and abundant polymorphism. 20 parts of castor silkworm germplasm is used as a material to construct the castor silkworm SSR fingerprint. According to the method established by the invention, the detection of the SSR markers can be completed by utilizing PCR amplification and electrophoresis detection technology, the method can be used for identifying the germplasm resources of castor silkworms, revealing the genetic variation of each strain at the DNA level, and developing and applying specific markers.
Description
Technical Field
The invention belongs to the technical field of molecular biology and silkworm variety identification, and in particular relates to a castor silkworm SSR molecular marker and application thereof, which are suitable for genetic diversity research, germplasm identification and genetic relationship analysis of castor silkworms.
Background
The SSR molecular marker technology has been the most popular and mature molecular marker technology to date by virtue of high polymorphism, abundant marker quantity and low cost. It is widely used in many research fields such as biological genetic diversity, genetic map construction, genetic analysis, molecular marker assisted breeding, and the like. Traditional methods for constructing SSR markers by constructing genomic libraries are limited in application of SSR markers due to time and labor consumption and low efficiency. At present, searching SSR sites in a large number of DNA sequences obtained by gene sequencing by using a microsatellite search tool has become the most convenient and effective way for developing microsatellite markers.
Castor silkworms (Philosamia cynthia ricini), also known as cassava silkworms and Indian silkworms, are economical insects with multiple properties and without diapause under proper environmental conditions. The 16 th century is domesticated and raised by Indian, the 20 th century is introduced into more than 20 countries and regions for raising and breeding, and the 40 th century is introduced into northeast, eastern and south China. Castor silkworms are widely-feeding insects, and can eat leaves of cassava, ailanthus altissima, coriaria sinensis and the like besides eating castor leaves. Castor silkworms have the advantages of faster development, easy breeding, strong disease resistance, strong silkworm bodies, backlight clusters and the like, become the third largest economic insect inferior to silkworms and tussahs in China, and can also be used as a good genetic research material. The research on the castor silkworm is relatively few, and only analysis of detecting genetic diversity in several varieties by adopting an ISSR method is reported, but ISSR markers have the defects of poor repeatability and the like, and no report on developing SSR markers by using the castor silkworm DNA sequence is available at present. The method is particularly urgent for the application of carrying out marker selection on castor silkworms and being used for variety molecular screening, especially for producing excellent varieties.
Disclosure of Invention
The invention aims to: the invention aims to solve the technical problem of screening SSR molecular markers on the genome level based on complete sequence information of castor silkworm genome, and providing technical support for germplasm resource preservation and molecular marker assisted breeding. The invention firstly utilizes a bioinformatics method to detect SSR sites of the castor silkworms, develops SSR molecular markers of the castor silkworms with good amplification and rich polymorphism, establishes a SSR amplification technical system of the castor silkworms, and applies the SSR molecular markers to genetic diversity research and DNA fingerprint construction of germplasm resources of the castor silkworms.
The technical scheme is as follows: in order to solve the technical problems, the invention provides the following technical scheme:
the SSR molecular marker of castor silkworms has a nucleotide sequence shown in SEQ ID NO. 1-15.
Further, the primer of the SSR molecular marker is shown as SEQ ID NO. 16-45.
The SSR molecular marker of the castor silkworms is applied to variety identification, germplasm resource diversity analysis, core germplasm establishment and molecular marker assisted breeding of the castor silkworms.
The PCR reaction system and the PCR reaction program of the castor-silkworm SSR molecular marker in application are as follows:
25. Mu.L of the total PCR reaction system: 2.5. Mu.L of 10 XPCR buffer, 2. Mu.L of dNTPs (2.5 mmol/L), 1. Mu.L of each of the upstream and downstream primers (10 mmol/L), 0.3. Mu.L of DNA polymerase, 1. Mu.L of DNA template (500 ng/. Mu.L), and ddH2O was added to fix the volume to 25. Mu.L.
PCR amplification was performed on a BIO RAD T100 Thermal Cycler under reaction conditions of 95℃for 3min,95℃for 30s, annealing for 30s (annealing temperature depends on the primer) and 72℃for 30s; for a total of 35 cycles, the extension was carried out at 72℃for a further 5min, and the reaction was finally terminated at 12 ℃. mu.L of the PCR amplification product was subjected to 1% agarose gel electrophoresis as a primary screen.
The primer sequences, repeat motifs, fragment sizes and annealing temperatures are detailed in Table 1.
TABLE 1 SSR molecular marker primer sequences, annealing temperatures and amplified bands
The genome of the castor silkworms has 155 Scaffolds, the total base length is 450 479 495bp, the maximum Scaffold length is 33 970 1599bp, the average length is 2 906 319bp, and the GC content is 34.3%. The presence of 186 790 SSR sites was found in the genome sequence by searching with the MISA software, the total frequency of gsr sites in the genome was 0.04%, and 1 gsr occurred every 2.4kb on average. In order to improve the accuracy and the universality of gSSR marker development, the retrieved sequence containing gSSR sites is subjected to homologous comparison with the castor silkworm variety B7 genome data, and 7 036 polymorphic gSSR sites are screened. Segment random selection 28 SSR the sequences before and after the SSR repeat motif were evaluated and conserved primers were designed using Primer Premier 6.0 software. The primers were synthesized by Shanghai, inc. of biological engineering, and amplified in 20 parts of the germplasm resources of Ricinus communis. Agarose gel electrophoresis detection shows that 3 pairs of primers in the SSR have no amplified bands or non-specific bands, and the other 25 pairs of polymerase chain reaction (polymerase chain reaction, PCR) amplified products are consistent with the target bands, so that the primers can be used as candidate markers for further experiments, and the effective amplification rate reaches 89.3%. Selecting a specific primer for stable amplification, adding a fluorescent marker, and carrying out polymorphism detection in 20 parts of castor silkworm germplasm resources by using a full-automatic capillary nucleic acid sequencer to finally obtain 15 pairs of SSR primers with high amplification efficiency and abundant polymorphism, wherein the SSR primers comprise gSSR_P61, gSSR_P173, gSSR_P3547, gSSR_P507, gSSR_P3446, gSSR_P1556, gSSR_P3555, gSSR_P3410, gSSR_P3729, gSSR_P3411, gSSR_P3888, gSSR_P3840, gSSR_P6165, gSSR_P3765 and gSSR_P6101.
The beneficial effects are that:
the 15 pairs of SSR primers disclosed by the invention have the advantages of high amplification efficiency and rich polymorphism, and are beneficial to the application of germplasm resource identification, gene marker development and the like.
Drawings
Fig. 1:6101 amplification peak plot in B1;
fig. 2:6101 amplification peak plot in B13;
fig. 3: an amplification peak plot of 3888 in B13;
fig. 4: an amplification peak plot of 3888 in B1;
fig. 5: amplification peak plot of 6165 in B1;
fig. 6: amplification peak plot of 6165 in B2.
The specific embodiment is as follows:
the above-described aspects of the present invention will be described in further detail below with reference to specific embodiments in the form of examples. It should not be understood that the scope of the above subject matter of the present invention is limited to the following examples only. All techniques implemented based on the above description of the invention are within the scope of the invention.
For the purpose of describing the technical content and the structural characteristics of the SSR marker of the castor silkworms, the invention is further described with reference to the following embodiments.
1. Development method of molecular marker of castor silkworms
1.1 screening of SSR sites in the Castor silkworm DNA sequence
The SSR sites were searched using MISA software, and the search criteria parameters were set to mononucleotide, dinucleotide, trinucleotide, tetranucleotide, pentanucleotide and hexanucleotide, with minimum number of repetitions of 10, 6, 5 and 5, respectively. By searching, sequences containing SSR sites were obtained.
1.2 design and Synthesis of SSR primer of Castor silkworm
The Primer Premier 6.0 software is utilized to evaluate the sequence of about 100bp before and after the SSR repeat motif and design the Primer. The distance between the SSR locus and the flanking sequence is about 50-300bp, the length of the primer sequence is 18-27bp, the GC content is 40-60%, the annealing temperature is 50-65 ℃, the length of the amplified product is 80-300bp, and dimers, hairpin structures, mismatches and the like are avoided as much as possible. After the primer design is completed, the engineering and bioengineering (Shanghai) Limited liability company is entrusted to synthesis.
1.3 screening of SSR primers of Castor silkworms
1.3.1 ordinary PCR reactions
25. Mu.L of the total PCR reaction system: 2.5. Mu.L of 10 XPCR buffer, 2. Mu.L of dNTPs (2.5 mmol/L), 1. Mu.L of each of the upstream and downstream primers (10 mmol/L), 0.3. Mu.L of DNA polymerase, 1. Mu.L of DNA template (500 ng/. Mu.L), and ddH2O was added to fix the volume to 25. Mu.L. PCR amplification was performed on BIO RAD T100 Thermal Cycler. The reaction conditions were set to 95℃for 3min,95℃for 30s, and 72℃for 30s (annealing temperature depending on the primer); for a total of 35 cycles, the extension was carried out at 72℃for a further 5min, and the reaction was finally terminated at 12 ℃. mu.L of the PCR amplification product was subjected to 1% agarose gel electrophoresis as a primary screen.
1.3.2 fluorescent primer PCR amplification
The total PCR reaction system was 25. Mu.L: 2.5. Mu.L of 10 XTaq Buffer (with MgCl 2), 1. Mu.L of dNTPs (mix, 10. Mu.M), 1. Mu.L of each of the upstream and downstream primers (10. Mu.M with HEX or 6-FAM fluorescein at the 5' end of the primer), 0.5. Mu.L of Taq enzyme (5U/. Mu.L), 1. Mu.L of DNA template (20-50 ng/. Mu.L), and ddH2O was added to fix the volume to a final reaction volume of 25. Mu.L. PCR amplification was performed on a Veriti 96well PCR apparatus, ABI, under conditions of 95℃for 3min,94℃for 30s,60℃for 30s (annealing temperature depending on the primer), 72℃for 30s, and 10 cycles were performed; denaturation at 94℃for 30s, annealing at 55℃for 30s (annealing temperature depending on the primer), extension at 72℃for 30s, further 35 cycles, further extension at 72℃for 5min, and finally the reaction was terminated at 12 ℃.
1.3.3 full-automatic capillary accounting Analyzer detection
A96-well reaction plate is taken, a marker pen is used for marking the plate name and the experimental date, an electronic short segment repeated sequence (Short tandem repeat, STR) detection table is manufactured, and an on-machine table is automatically generated. Using a continuous applicator, 990. Mu.L of a mixture of HIDI and 10. Mu.L of LIZ500 was pipetted into a 96-well reaction plate, 10. Mu.L per well, and the 96-well plate was placed in a plate centrifuge and centrifuged at 1200rmp for 15s. Using a 12-row 10. Mu.L gun, 1. Mu.L of sample was added to the corresponding well of the 96-well plate against the STR assay table, and the 96-well plate was placed in a plate centrifuge and centrifuged at 1200rmp for 15s. Sealing the 96-well plate by using a sealing plate film, oscillating, placing the 96-well plate in a flat plate centrifuge, centrifuging for 30s at 1200rmp, and placing in a PCR instrument. The denaturation procedure was 98 ℃,5min without heating the hot lid, and immediately after the procedure was completed, the 96-well plate was placed on an ice-water mixture and rapidly cooled. 96-well plates were placed in a plate centrifuge, centrifuged at 1200rmp for 15s, and STR samples were tested using the American ABI 3730xl apparatus. And automatically generating a map file of each site on each sample by GeneMapper v3.7 software, and obtaining the size of the product fragments according to the peak value diagram.
1.4 data statistics and analysis
The data format was converted using CONVERT (version 1.31) software, and indexes such as POP-GENE (version 1.32) and PIC-CALC software were used to calculate the allele factors (Na), effective allele factors (Ne), observed heterozygosity (Ho), expected heterozygosity (He), polymorphism Information Content (PIC), inbred coefficients (Fis) and Shannon (Shannon) information index (I) for each locus, and to examine whether polymorphic locus allele frequencies deviate from Hash equilibrium (Hardy-Weinberg equilibrium, HWE). And (3) performing Bayesian cluster analysis on genotypes of the population by using Structrue 2.3.4 software, and detecting potential genetic structures of the castor silkworm population. Genetic distance (Nei, 1983) and cluster analysis (1 000 booth methods test) were performed on the germplasm of ricinus communis using PowerMarker V3.25 software.
The 15 SSR markers can be used for genetic diversity analysis, molecular fingerprint construction and the like of castor silkworm germplasm resources, have good polymorphism and repeatability, and are reliable and effective molecular markers.
The foregoing is merely an example of the best modes of the invention and is not intended to limit the scope of the invention so that equivalent variations according to the claims of the invention still fall within the scope of the invention.
TABLE 1.15 SSR primer polymorphism band amplification cases
TABLE 2 genetic diversity of microsatellite loci and Hardy-Welnberg equilibrium test
(1) An allelic factor was observed.
(2) Effective allelic factors.
(3) The degree of heterozygosity was observed.
(4) The degree of heterozygosity is desired.
(5) Polymorphism information content.
(6) Inbred coefficients.
(7) Shannon index.
(8) Hardy-Weinberg equilibrium chi-square test P value,: p < 0.050,: p < 0.010.
Sequence listing
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<120> castor silkworm SSR molecular marker and application thereof
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<212> DNA
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aaaaagcatg aggtcttttc gacctctccc attaaaggtt tcaaatttta atttgatttc 300
gtttactaaa gtaaaattta tattcactca ggtaccgcat cgcatttcgc gagaccctct 360
gctgcgagaa agttggaaag cggaagagcc agtataaaga ccattcgagc ataag 415
<210> 14
<211> 420
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 14
atgaacccgt tatctgcaat attagaggga gcttccttta tgaacaggga tcaaacttaa 60
tagtacgaac tttagaccgg tctctataat agacatgtct atactataca gcgatataag 120
tcgagtaact tggcttttta attaactaaa tattgtttta tgttttctac ctataacgtt 180
tatgttatct ttctatctcc tatatatata tatatatata tgtatatata aggaaagttt 240
gttatctttg tccgaggtaa actcgaaaac tactggaccg atcagcatga aaccacaacc 300
attcgacgcg gaattaatcg tagctggtta taggctataa attgttcaaa ttctattata 360
aacttttaat ataaagaatg aatatagtaa ataataatag aatggatcta ttataaagaa 420
<210> 15
<211> 421
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 15
ttttcacaat tttttattat atttgtctca gcacgcgcag actctgggtc tgcgtgaaat 60
ttaaaatggt ttacaatttc aatattatag ttttaatata accaccatat caggtggacc 120
attacaaaat atttgcgggc cgtggattga gtatcactga tttaaagcta cttattccga 180
aactatattc aatcttcgtg taataataat aataataata acctatctaa tcgtttagaa 240
ttaaacacag aatagtgaat gtatgtatat attcaaggta attctgaata tattgagtgt 300
gcgtcgacgt tataagcaac ttctgtctgg ccaataaact agaggtcaag taacagatag 360
attatgtggt cagcgtccga gtattgtgta ctgtacggac ttgagtatgt ttttgaattt 420
t 421
<210> 16
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 16
ggtaggtaga gtcgggcagt 20
<210> 17
<211> 22
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 17
ccatcatgtg ttggttgatc ga 22
<210> 18
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 18
gagaatcgtg cactttaact 20
<210> 19
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 19
acggttcttt cggattcgtc 20
<210> 20
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 20
gcagctcgac gactatcagg 20
<210> 21
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 21
ttcaccaggt ggagttgacg 20
<210> 22
<211> 21
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 22
caaccaggtg acgatgatgg a 21
<210> 23
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 23
acggctaggg atctccagat 20
<210> 24
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 24
tgcaaacatg caaggctaca 20
<210> 25
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 25
attcactggt gctcggttcc 20
<210> 26
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 26
aaccagtcgc aacaagctct 20
<210> 27
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 27
gggctagtga ctgactgacg 20
<210> 28
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 28
gctgagtatc gcggtgtaca 20
<210> 29
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 29
ctgtatgcac gcgttgtagg 20
<210> 30
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 30
cacaatatgg cgtgggtcca 20
<210> 31
<211> 22
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 31
ccatccgcct tacaattttc gt 22
<210> 32
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 32
tcaaaacccg aggcggtatt 20
<210> 33
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 33
ttcgaagcga ggctcgttag 20
<210> 34
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 34
gattcccggg cgaatcagaa 20
<210> 35
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 35
cccgacaagg ctcactactg 20
<210> 36
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 36
tccgattcgt tgcacaatgc 20
<210> 37
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 37
gttccgggta atctgcggaa 20
<210> 38
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 38
tgacggtaaa agtcaagcat 20
<210> 39
<211> 21
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 39
acgtagtccc ctgtttcctc t 21
<210> 40
<211> 24
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 40
accttctact gtaatctgta ccgt 24
<210> 41
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 41
cgatgcggta cctgagtgaa 20
<210> 42
<211> 24
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 42
gcgatataag tcgagtaact tggc 24
<210> 43
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 43
gcgtcgaatg gttgtggttt 20
<210> 44
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 44
atatttgcgg gccgtggatt 20
<210> 45
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 45
taacgtcgac gcacactcaa 20
Claims (3)
1. The castor silkworm SSR molecular marker is characterized in that the nucleotide sequence of the SSR molecular marker is shown as SEQ ID NO. 1-15.
2. The castor-silkworm SSR molecular marker according to claim 1, wherein the primer of the SSR molecular marker is shown as SEQ ID NO. 16-45.
3. The application of the castor-silkworm SSR molecular marker according to claim 1 or 2 in variety identification, germplasm resource diversity analysis, core germplasm establishment and molecular marker assisted breeding of castor silkworms.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1970792A (en) * | 2005-10-14 | 2007-05-30 | 中国科学院上海生命科学研究院 | SSR marker for domestic silkworm and application thereof |
| CN104328197A (en) * | 2014-11-14 | 2015-02-04 | 福建农林大学 | Hibiscus cannabinus L. expression sequence tag SSR (Simple Sequence Repeat) DNA markers |
| CN106755328A (en) * | 2016-11-25 | 2017-05-31 | 中国农业科学院作物科学研究所 | A kind of construction method of broad bean SSR finger-prints |
| CN109517925A (en) * | 2019-01-24 | 2019-03-26 | 中国农业科学院麻类研究所 | Flax SSR molecular marker and its application |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2003085133A2 (en) * | 2002-04-08 | 2003-10-16 | Centre For Dna Fingerprinting And Diagnostics | Novel fissr-pcr primers and method of genotyping diverse genomes of plant and animal systems including rice varieties |
-
2022
- 2022-02-14 CN CN202210134182.6A patent/CN114525345B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1970792A (en) * | 2005-10-14 | 2007-05-30 | 中国科学院上海生命科学研究院 | SSR marker for domestic silkworm and application thereof |
| CN104328197A (en) * | 2014-11-14 | 2015-02-04 | 福建农林大学 | Hibiscus cannabinus L. expression sequence tag SSR (Simple Sequence Repeat) DNA markers |
| CN106755328A (en) * | 2016-11-25 | 2017-05-31 | 中国农业科学院作物科学研究所 | A kind of construction method of broad bean SSR finger-prints |
| CN109517925A (en) * | 2019-01-24 | 2019-03-26 | 中国农业科学院麻类研究所 | Flax SSR molecular marker and its application |
Non-Patent Citations (2)
| Title |
|---|
| 李刚等.基于16SrDNA序列对蓖麻蚕肠道细菌多样性的研究.《蚕业科学》.2018,第第44卷卷(第第6期期),第867-874页. * |
| 郭秋红等.利用SSR标记鉴定家蚕不同系统的品种初探.《蚕业科学》.2005,第第31卷卷(第第3期期),第257-260页. * |
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