CN105087574A - Chenopodium quinoa willd EST-SSR molecular marker, development method of chenopodium quinoa willd EST-SSR molecular marker and application of chenopodium quinoa willd EST-SSR molecular marker - Google Patents
Chenopodium quinoa willd EST-SSR molecular marker, development method of chenopodium quinoa willd EST-SSR molecular marker and application of chenopodium quinoa willd EST-SSR molecular marker Download PDFInfo
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Abstract
The invention relates to the technical field of molecular markers, in particular to a chenopodium quinoa willd EST-SSR (Expressed Sequence Tag-Simple Sequence Repeat) molecular marker, a development method of the chenopodium quinoa willd EST-SSR molecular marker and application of the chenopodium quinoa willd EST-SSR molecular marker. A group of chenopodium quinoa willd EST-SSR molecular markers comprises forward primers and reverse primers corresponding to 66 sites. The development method of the chenopodium quinoa willd EST-SSR molecular marker comprises the following steps of: 1, obtaining RNA-seq and EST sequences from an NCBI (National Center of Biotechnology Information) database; 2, using bioinformatics software to process, assemble and splice sequences into unigenes, and performing SSR site searching on the unigenes to obtain the EST-SSR; and 3, designing EST-SSR primer amplification chenopodium quinoa willd genome DNA for verifying the EST-SSR, and using the verified EST-SSR molecular marker to realize application of genetic diversity analysis in chenopodiaceae germplasm. The EST-SSR molecular marker developed on the basis of a public database has the advantages of high efficiency, high success rate, rich polymorphism and high universality.
Description
Technical field
The invention belongs to molecular marking technique field, be specifically related to lamb's-quarters wheat EST-SSR molecule marker and development approach thereof and application.
Background technology
SSR (simplesequencerepeat) is also called microsatellite DNA, formed by the unit sequence tandem sequence repeats of 2 ~ 6 bases, there is the features such as polymorphism is high, codominance, quantity are abundant, be widely used in genetic map construction, analysis of genetic diversity etc.The method of early-stage development SSR marker adopts the mode building library to carry out usually, comprises genomic library and cDNA library.Usually the SSR developed based on EST (expressedsequencetag) is called EST-SSR; compared with the genome SSR developed by genome sequence; EST-SSR is comparatively strong because of the conservative property of its two ends flanking sequence, has good versatility between different plant species.In recent years, genome sequence abundant in public database and expressed sequence information development molecule marker is utilized to become the important channel of molecular markers development.
Lamb's-quarters wheat (ChenopodiumquinoaWilld.), originate in South America Andean region, that the dicotyledonous draft of a kind of annual Chenopodiaceae produces kind of plant, having the characteristic of outstanding drought-resistant, Salt And Alkali Tolerance, is the plant that the monomer of food and agricultural organization of unique a kind of united state accreditation can meet human body basic nutrition demand.It is reported, lamb's-quarters wheat Genome Size is about 967M, is allotrtraploid species (2n=4x=36), and genome sequence assembling difficulty, there is no gene order-checking relevant report so far, therefore, is difficult to large-scale development molecule marker.RAPD (randomamplifiedpolymorphicDNA) is the molecule marker based on lamb's-quarters wheat DNA first reported.This type of molecule marker can be used for the qualification of lamb's-quarters wheat interspecific hybrid and the Genetic Variation Analysis of lamb's-quarters wheat and other Chenopodiaceae species.Subsequently, by checking order to the clone of being rich in micro-satellite primitive, Masonetal. develops and demonstrates the codominance SSR molecular marker that 208 have higher polymorphism between lamb's-quarters wheat seeds.Research shows, compared to the SSR molecular marker that dinucleotides repeats, the SSR (> 20bp) of Trinucleotide repeats has more much higher state property.By being rich in GA, the library that CAA and AAT repeats and BES (bacterialartificialchromosome-endsequence), Jarvisetal. 216 novel multi-state SSR and 6 BES-SSR are developed, polymorphism PIC (polymorphisminformationcontent) between 0.12 ~ 0.90, and constructs the first genetic linkage maps based on SSR marker of lamb's-quarters wheat.This genetic linkage maps comprises 200 SSR, is made up of, covers the genetic distance of lamb's-quarters wheat 913cM 38 linkage groups.
Summary of the invention
The object of the invention is the deficiency for current lamb's-quarters wheat molecule marker comparatively small amt, utilize lamb's-quarters wheat expressed sequence data in public database, lamb's-quarters wheat EST-SSR is excavated on a large scale by information biology means, checking and polymorphism analysis have been carried out to part lamb's-quarters wheat EST-SSR, and the genetic diversity of the EST-SSR of exploitation to Chenopodiaceae kind matter is evaluated.
To achieve these goals, the technical solution used in the present invention is: one group of lamb's-quarters wheat EST-SSR molecule marker, is characterized in that: include the forward corresponding to following 66 sites and reverse primer:
The development approach of lamb's-quarters wheat EST-SSR molecule marker of the present invention, comprises the following steps:
1) RNA-seq and est sequence is obtained from ncbi database;
2) utilize bioinformatics software to process sequence, assemble, be spliced into unigene, and the search of SSR site is carried out to unigene thus obtains EST-SSR;
3) design EST-SSR primer amplification lamb's-quarters wheat genomic dna, and EST-SSR is verified;
Further, in described step 3) in, EST-SSR design of primers adopts following optimum configurations: Tm is 58 DEG C ± 3 DEG C, and primer length is 20bp ± 3bp, and product expection length is 100bp ~ 450bp.
Further, described step 3) in pcr amplification program be 94 DEG C of 3min; 94 DEG C of 30s, 58 DEG C of 35s, 72 DEG C of 50s, 38 circulations; 72 DEG C of 3min.
Further, described step 3) in PCR amplification system be 25 μ l, containing 2mmol/LMgCl
2, 100 μm of ol/LdNTP, 0.2 μm of ol/L primer, 1UTaq enzyme and 50ngDNA.
The application of lamb's-quarters wheat EST-SSR molecule marker of the present invention in Chenopodiaceae germplasm genetic diversity is analyzed.
Further, described Chenopodiaceae kind matter refers to the chenopod comprising lamb's-quarters wheat.
Beneficial effect of the present invention:
1, the EST-SSR molecule marker that the present invention is based on public database exploitation has high efficiency.The present invention obtains 19,571 unigene according to the 14.0G lamb's-quarters wheat RNA-Seq data obtained and the splicing of EST data.Wherein, 16,854 sequences contain 20,338SSR site, comprise 1,862 non-mononucleotides repeat SSR (two, three, four, five, Hexanucleotide repeats).
2, high, the rich polymorphism of EST-SSR success ratio of the present invention's exploitation.Increase in random selecting 119 couples of EST-SSR primer pair lamb's-quarters wheat DNA and other Chenopodiaceae kind matter DNA.Result shows, and has 66 to obtaining amplified band clearly in lamb's-quarters wheat DNA cloning, success ratio 55.9%.In 66 pairs of primers, 58 pairs of primers can amplify the band of two or more type on the polyacrylamide gel of 10%, and wherein 5 pairs of primers can amplify 4 maximum type bands, and on average often pair of primer can amplify 2.2 kinds of bands.Calculated by PIC, 61 is polymorphism EST-SSR (PIC >=0.10), accounts for 92.4% of sum.
3, the EST-SSR versatility of the present invention's exploitation is stronger.The present invention have collected the different Chenopodiaceae kind matter in 8 parts of sources, comprises 4 parts of lamb's-quarters wheats, 2 parts of pale stem lamb's-quarters, 1 part of Taiwan lamb's-quarters and 1 part of cane lamb's-quarters, comes from 5 countries and regions in four continents respectively.66 pairs of EST-SSR primers all can amplify polytype band in 8 parts of Chenopodiaceae kind matter DNA, show good versatility.
Accompanying drawing explanation
Fig. 1 is the UPGMA dendrogram of 8 parts of Chenopodiaceae kind matter based on likeness coefficient structure.
Embodiment
Following embodiment is convenient to understand the present invention better, but does not limit the present invention.Experimental technique in following embodiment, if no special instructions, is ordinary method.Test materials used in following embodiment, if no special instructions, is and purchases available from routine biochemistry reagent shop.
1) acquisition of lamb's-quarters wheat expressed sequence data and the splicing of unigene.The RNA-Seq data (accession number: SRX257003 and SRX256971) of 11.9GIlluminaHiSeq2000 and the EST data (accession number: SRX084791) of 2.1GRoche454 are obtained altogether from the SRA database (http://www.ncbi.nlm.nih.gov/sra) of NCBI.Through changing raw data, clearing up, filter, utilize Trinity software to splice and obtain 19,571 unigene, total bases is 80,448,006bp, and average every bar unigene is about 4kb.Wherein 16,854 sequences contain SSR site.
2) qualification in lamb's-quarters wheat EST-SSR site.By MISA software (http://pgrc.ipk-gatersleben.de/misa/), the identification of SSR site is carried out to unigene sequence, the condition for identification in SSR site is: mononucleotide repeats to be not less than 10 times, dinucleotides repeats to be not less than 8 times, Trinucleotide repeats is not less than 7 times, tetranucleotide repeat is not less than 5 times, and pentanucleotide and Hexanucleotide repeat to be not less than 4 times; The condition for identification of composite S SR is that the distance between 2 SSR is no more than 50bp.Found that lamb's-quarters wheat EST-SSR repeat type enriches, repeat to Hexanucleotide from mononucleotide and repeat to occur.Non-mononucleotide has repeated 1,862.Wherein, it is maximum that dinucleotides repeats SSR, account for 38.3% (713) that non-mononucleotide repeats SSR sum, secondly be Trinucleotide repeats, account for 22.7% (423) that non-mononucleotide repeats SSR sum, minimum is Hexanucleotide repetition, accounts for 11.7% (217) that non-mononucleotide repeats SSR sum.
3) design of primers of lamb's-quarters wheat EST-SSR.Primer3.0 software is utilized to be greater than 200bp to flanking sequence and the non-mononucleotide that repeat length is greater than 16bp repeats SSR site carries out design of primers, design of primers adopts following optimum configurations: Tm is 58 DEG C ± 3 DEG C, primer length is 20bp ± 3bp, product expection length is 100bp ~ 450bp, and other parameter is set to acquiescence.Have 119 EST-SSR and devise primer pair.
4) checking of lamb's-quarters wheat EST-SSR and polymorphism analysis.Utilize KarrotenDNA to extract test kit and DNA extraction is carried out to the seedling of test materials.The DNA detected through 1% sepharose is used for pcr amplification.PCR reaction is totally 25 μ l, containing 2mmol/LMgCl
2, 100 μm of ol/LdNTP, 0.2 μm of ol/L primer, 1UTaq enzyme and 50ngDNA.PCR response procedures is: 94 DEG C of 3min; 94 DEG C of 30s, 58 DEG C of 35s, 72 DEG C of 50s, 38 circulations; 72 DEG C of 3min.Pcr amplification product on 10% polyacrylamide gel with 100V electrophoresis 120min, EB dyeing after on ultraviolet transilluminator observations.The polymorphism information content PIC of EST-SSR is by following formulae discovery:
p
ibe i-th allelic frequency, k is allelic quantity.PIC >=0.10 is polymorphism SSR, PIC >=0.70 is high polymorphism SSR.Have 119 pairs of EST-SSR primer pairs, 8 parts of Chenopodiaceae kind matter DNA to increase (see table 1), 66 pairs of primers can obtain amplified band clearly on the polyacrylamide gel of 10%, success ratio 55.9% (see table 2).Wherein, the success ratio that Hexanucleotide repeats SSR is the highest, is 74.3%, and the success ratio that pentanucleotide repeats SSR is minimum, is 31.8%.66 pairs of primer coamplifications go out 145 kinds of bands, and on average often pair of primer can amplify 2.2 kinds of bands.Calculated by PIC, 61 is polymorphism EST-SSR (PIC >=0.10), accounts for 92.4% of sum.
Table 18 part supplies examination material
5) application of lamb's-quarters wheat EST-SSR molecule marker in Chenopodiaceae germplasm genetic diversity is analyzed.66 EST-SSR molecule markers are used for the gene type assay of 8 parts of Chenopodiaceae kind matter.Cluster analysis adopts NTSYSpc2.1 software, and this analysis calculates genetic similarity based on UPGMA method (unweightedpairgroupmethodanalysis).Choose according to UPGMA cluster result, four parts of Chenopodiaceae kind matter from South America are polymerized to a class, comprise three parts of lamb's-quarters wheats and the pale stem lamb's-quarters of portion; Significantly separated from the cane lamb's-quarters of North America, the lamb's-quarters wheat in Europe and the Taiwan lamb's-quarters of China, be divided into three classes separately; Another part of pale stem lamb's-quarters from South America is not divided into a class with other four parts of Chenopodiaceae kind matter in South America, points out this part of pale stem lamb's-quarters and other four parts of Chenopodiaceae kind matter sibships from the same area (see Fig. 1) comparatively far away.This result shows that Chenopodiaceae kind matter can be separated according to region by EST-SSR, illustrates the versatility that lamb's-quarters wheat EST-SSR is good between different Chenopodiaceae species.
Claims (7)
1. one group of lamb's-quarters wheat EST-SSR molecule marker, is characterized in that: include the forward corresponding to following 66 sites and reverse primer:
2. the development approach of lamb's-quarters wheat EST-SSR molecule marker according to claim 1, is characterized in that: the method comprises the following steps:
1) RNA-seq and est sequence is obtained from ncbi database;
2) utilize bioinformatics software to process sequence, assemble, be spliced into unigene, and the search of SSR site is carried out to unigene thus obtains EST-SSR;
3) design EST-SSR primer pair lamb's-quarters wheat genomic dna and carry out pcr amplification, and EST-SSR is verified.
3. method according to claim 2, is characterized in that: in described step 3) in, EST-SSR design of primers adopts following optimum configurations: Tm is 58 DEG C ± 3 DEG C, and primer length is 20bp ± 3bp, and product expection length is 100bp ~ 450bp.
4. method according to claim 2, is characterized in that: described step 3) in pcr amplification program be 94 DEG C of 3min; 94 DEG C of 30s, 58 DEG C of 35s, 72 DEG C of 50s, 38 circulations; 72 DEG C of 3min.
5. method according to claim 2, is characterized in that: described step 3) in PCR amplification system be 25 μ l, containing 2mmol/LMgCl
2, 100 μm of ol/LdNTP, 0.2 μm of ol/L primer, 1UTaq enzyme and 50ngDNA.
6. the application of lamb's-quarters wheat EST-SSR molecule marker described in claim 1 in Chenopodiaceae germplasm genetic diversity is analyzed.
7. application according to claim 6, is characterized in that: described Chenopodiaceae kind matter refers to the chenopod comprising lamb's-quarters wheat.
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| CN108998553A (en) * | 2018-08-14 | 2018-12-14 | 西北大学 | The method and primer of a kind of quick screening polymorphic micro-satellite site target primer |
| CN112176085A (en) * | 2020-09-18 | 2021-01-05 | 山东师范大学 | SSR molecular markers of Chenopodium quinoa Hance No. 2 and application thereof |
| CN116904638A (en) * | 2023-09-08 | 2023-10-20 | 山西稼祺农业科技有限公司 | Kasp markers linked to early females of quinoa and uses thereof |
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| CN107022616A (en) * | 2017-04-26 | 2017-08-08 | 江苏省农业科学院 | Quinoa dimorphism InDel molecular labelings and its development approach and application |
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| CN112176085A (en) * | 2020-09-18 | 2021-01-05 | 山东师范大学 | SSR molecular markers of Chenopodium quinoa Hance No. 2 and application thereof |
| CN116904638A (en) * | 2023-09-08 | 2023-10-20 | 山西稼祺农业科技有限公司 | Kasp markers linked to early females of quinoa and uses thereof |
| CN116904638B (en) * | 2023-09-08 | 2024-05-07 | 山西稼祺农业科技有限公司 | Kasp markers linked to early females of quinoa and uses thereof |
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