CN110079628B - EST-SSR primer information and application thereof in bletilla striata strain identification - Google Patents
EST-SSR primer information and application thereof in bletilla striata strain identification Download PDFInfo
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- CN110079628B CN110079628B CN201910364964.7A CN201910364964A CN110079628B CN 110079628 B CN110079628 B CN 110079628B CN 201910364964 A CN201910364964 A CN 201910364964A CN 110079628 B CN110079628 B CN 110079628B
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Abstract
The scheme discloses the technical field of molecular markers, and a group of EST-SSR primer information comprises 25 pairs of EST-SSR primers and sequence information thereof, through the application of the group of markers, different rhizoma bletillae strains can be accurately identified, meanwhile, the genetic diversity information of the rhizoma bletillae strains can be read, and the genetic diversity caused by regional differences can be explained. Based on the EST-SSR primer information and the technical process, the method can be applied to bletilla striata strain identification, genetic diversity analysis and molecular marker breeding auxiliary selection, and has higher reliability.
Description
Technical Field
The invention relates to the technical field of molecular markers, in particular to a group of EST-SSR primer information and application thereof in identification of rhizoma bletillae strains.
Background
The traditional Chinese medicinal materials are national treasures, the genuine property is the inherent property of the traditional Chinese medicinal materials, however, along with ecological destruction and excessive excavation of original land, a plurality of rare traditional Chinese medicinal materials are endangered and extincted, so that counterfeit and shoddy products impact the market in the circulation of the traditional Chinese medicinal materials. At present, the authenticity of the traditional Chinese medicinal materials is primarily identified through apparent morphology and histology, but the morphological or histology method cannot identify and distinguish the difference between the traditional Chinese medicinal materials and genuine medicinal materials due to different geographical distributions, so that the gene or an expression product thereof is taken as an identification basis, namely, a technology for identifying germplasm resources and analyzing genetic diversity based on a molecular identification technology is very necessary, and the development of reliable molecular markers is urgent for the authenticity of the traditional Chinese medicinal materials and the identification of dominant populations thereof.
The DNA molecular marking technology, also called molecular identification technology, is a marking mode based on genetic material, and has the advantages of no influence of external environment, high detection precision, good reproducibility and the like. With the continuous application and innovation of DNA molecular marking technology, a large number of molecular marking technologies based on PCR amplification technology have emerged, and the technologies commonly used at present comprise EST-SSR, ISSR, SNPs and marking technologies. Recently, molecular marker technology has been widely and successfully applied to medicinal plants including resource identification, genetic evolution, functional gene assisted localization and separation, and the like. The ISSR and EST-SSR are both derived from SSR markers, and the SSR markers have the advantages of co-dominance, multiple alleles, high sensitivity, good reproducibility, simplicity and convenience in operation, low requirements on the quality of template DNA and the like, and have the characteristics. Such as ISSR, has higher inter-species versatility due to differences in marker design principles (anchored SSR sequences), but also increases the difficulty of developing specific markers. The EST-SSR labeling technology based on the EST database or the cDNA library has the characteristics of the traditional SSR labeling, is a labeling method capable of efficiently revealing gene capacity, and can specifically show the expression condition of a certain gene locus, so that the biological information of a functional gene is directly reflected. Therefore, the EST-SSR marker has more advantages in the aspects of population genetic diversity research, species identification, particularly functional gene related research and the like.
Rhizoma bletillae (Bletillae striata (Thunb.) Rchb.f.) is a traditional Chinese medicinal material widely used in China. The tuber is mainly used as a medicine, and the medicine has the properties of stopping bleeding, promoting tissue regeneration, resisting bacteria and tumors and the like, and the research in recent years shows that the tuber has important medicinal values of plasma substitutes, media for mediating carrying medicines and the like, and has great ornamental value due to the bright color. The wild resources are excessively harvested and dug, so that the wild resources are extremely deficient and even endangered to be extinct due to market stimulation in recent years, and meanwhile, the market fishes and dragons are mixed, so that fake and shoddy products are wantonly transverse.
Disclosure of Invention
The invention aims to provide a group of EST-SSR primer information, which comprises 25 pairs of EST-SSR primers, namely ZYBS-1, ZYBS-6, ZYBS-14, ZYBS-17, ZYBS-20, ZYBS-22, ZYBS-28, ZYBS-29, ZYBS-36, ZYBS-43, ZYBS-45, ZYBS-46, ZYBS-48, ZYBS-54, ZYBS-55, ZYBS-57, ZYBS-59, ZYBS-62, ZYBS-64, ZYBS-67, ZYBS-69, ZYBS-71, ZYBS-76, ZYBS-88 and ZYBS-89; the primer sequences for each of the EST-SSR primers are shown in the following table:
ZYBS is abbreviated Zunyi Bletilla striata, and the numbers followed are the numbers given to distinguish different primers when designing the primers, and do not have any meaning related to the substance of the protocol.
All base sequences have a reading start end and a reading end, which are 5'-3', 5 'and 3' ends, respectively, as indicated in the scheme.
The EST-SSR primer information provided by the invention can be applied to identification of bletilla striata germplasm resources, genetic diversity analysis of bletilla striata and molecular assisted breeding of bletilla striata.
The application of a group of EST-SSR primer information in bletilla striata strain identification comprises the following steps:
step 1): extracting genome DNA of bletilla striata test material by using a CTAB method, and storing for later use;
step 2): performing PCR amplification by using the EST-SSR primer by using the genomic DNA extracted in the step 1) as a template;
step 3): and (3) carrying out electrophoretic separation on the PCR amplification product in the step 2) by using non-denatured polyacrylamide gel, developing by using sodium hydroxide and formaldehyde after conventional silver staining, washing by using clear water, and finally carrying out artificial band reading on the polymorphic marker of the amplification band by taking the occurrence frequency and the size of the amplification band as reference, wherein a band is marked as '1', and an unclear or missing band is marked as '0', so as to establish an original (0,1) matrix.
Drawings
FIG. 1 is a GS clustering result of 23 parts of bletilla striata material using a set of EST-SSR primer information of the present invention;
FIG. 2 is the principal coordinate analysis results of 23 parts of common bletilla pseudobulb material using a set of EST-SSR primer information of the present invention.
Detailed Description
The EST-SSR primer information and the application thereof in the identification of rhizoma bletillae strains are explained in detail in the following by combining the attached drawings. The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention. Unless otherwise specified, the examples follow conventional experimental conditions. Those skilled in the art will appreciate that the details of the present invention not described in detail herein are well within the skill of those in the art.
Unless otherwise specified, the biochemical reagents used in the examples of the present invention are commercially available, and the instruments used are conventional instruments.
And determining EST-SSR primer information. Firstly, SSRFinder is used for scanning EST-SSR of a constructed transcriptome database of multiple species such as bletilla striata and the like, labels with the consistency rate higher than 90% are screened out from SSR labels obtained by scanning, 100 pairs of labels are randomly selected for a subsequent verification test, and finally 25 pairs of EST-SSR primers with stable amplification and good polymorphism are selected. Subsequently, 23 parts of bletilla striata line materials from different areas of Guizhou are taken for amplification and polymorphism data collection, and the 23 parts of bletilla striata are distinguished and identified by the 25 pairs of primers. The 23 test materials include 7 parts of cultivated bletilla striata (number 1-7), 8 parts of Guizhou Zhengannan wild bletilla striata (number 8-15) and 8 parts of Guizhou repaint wild bletilla striata (number 16-23).
Example 1: EST-SSR primer information for plant strain identification totally comprises 25 pairs of EST-SSR primers including ZYBS-1, ZYBS-6, ZYBS-14, ZYBS-17, ZYBS-20, ZYBS-22, ZYBS-28, ZYBS-29, ZYBS-36, ZYBS-43, ZYBS-45, ZYBS-46, ZYBS-48, ZYBS-54, ZYBS-55, ZYBS-57, ZYBS-59, ZYBS-62, ZYBS-64, ZYBS-67, ZYBS-69, ZYBS-71, ZYBS-76, ZYBS-88 and ZYBS-89; the primer sequences for each pair of EST-SSR primers are shown in Table 1:
TABLE 1 primer sequences for each pair of EST-SSR primers
Example 2: the invention provides application of a group of EST-SSR primer information in 23 parts of test and test materials, which comprises the following specific steps:
step 1): extracting the genome DNA of the 23 test materials by using a CTAB method, and storing at-20 ℃ for later use;
step 2): using the genomic DNA provided in the step 1) as a template, performing PCR amplification in 23 parts of test bletilla striata material by using 25 pairs of EST-SSR primers in the example 1;
step 3): carrying out electrophoretic separation on the PCR amplification product in the step 2) for 2h by using 6% non-denatured polyacrylamide gel, developing color by using sodium hydroxide and formaldehyde after conventional silver staining, washing for 3 times by using tap water, and finally carrying out artificial band reading on polymorphism of an amplification band by taking the occurrence frequency and the size of the amplification band as reference, wherein a band is marked as '1', and a band is unclear or missing as '0', so as to establish an original (0,1) matrix, and finally analyzing by using NTsys 2.10e software.
The GS clustering result (figure 1) shows that 7 parts of bletilla striata cultivated germplasm is singly clustered into a row, and in addition, 8 parts of wild bletilla striata of Guizhou Zhengan and 8 parts of wild bletilla striata of Guizhou repaint are also clustered into two major categories according to different origins, wherein 8 parts of wild bletilla striata of Guizhou Zheng an and 8 parts of wild bletilla striata of Guizhou repaint prove that EST-SSR primer information provided by the research can not only accurately identify the cultivated strains and the wild species of bletilla striata, but also can accurately distinguish the wild species of different origins. Meanwhile, further analysis shows that the variation range of the genetic similarity coefficient between the test materials in the part is 0.630-0.963, and the average value is 0.773. The genetic similarity coefficient between the two combinations of the material numbers 2 and 4, and the material numbers 5 and 6 is 0.963 at most, and the genetic similarity coefficient between the material numbers 3 and 5 is 0.954 at most, which indicates that the above 3 material combinations are not suitable for breeding cross parent combinations, and the above materials should be avoided as much as possible to become parent combinations in breeding combination matching. On the contrary, the genetic similarity coefficient of the materials between the three combinations numbered 1 and 22, 3 and 22, and 1 and 11 is the smallest, and the material can be used as a good combination for quality breeding and cross breeding. In addition, the main coordinate analysis result (fig. 2) further proves the GS clustering result, and also proves that the EST-SSR primer set provided by the research can accurately read the genetic information between different rhizoma bletillae strains and further accurately identify the rhizoma bletillae strains.
In conclusion, the EST-SSR primer information provided by the invention can be successfully applied to bletilla striata germ plasm line identification, genetic diversity analysis and molecular marker-assisted breeding selection, can not only lay a solid foundation for the technical requirements in the aspects of germ plasm resource identification, genetic diversity analysis, molecular-assisted selection, genetic map construction, functional gene positioning and the like, but also provide abundant marker resources for the bletilla striata and related species thereof based on the EST-SSR molecular marker, so that the application prospect is very wide.
The above is only a preferred embodiment of the present invention, and it should be noted that it is obvious to those skilled in the art that several variations and modifications can be made without departing from the EST-SSR molecular marker provided by the present invention, and these are all within the scope of the present invention.
Claims (5)
1. A group of EST-SSR primer groups are characterized in that: consists of 25 pairs of EST-SSR primers, namely ZYBS-1, ZYBS-6, ZYBS-14, ZYBS-17, ZYBS-20, ZYBS-22, ZYBS-28, ZYBS-29, ZYBS-36, ZYBS-43, ZYBS-45, ZYBS-46, ZYBS-48, ZYBS-54, ZYBS-55, ZYBS-57, ZYBS-59, ZYBS-62, ZYBS-64, ZYBS-67, ZYBS-69, ZYBS-71, ZYBS-76, ZYBS-88 and ZYBS-89; the primer sequences for each of the EST-SSR primers are shown in the following table:
2. the use of a set of EST-SSR primers in rhizoma bletillae strain identification according to claim 1, wherein the EST-SSR primers comprise: the EST-SSR primer information is applied to identification of rhizoma bletillae germplasm resources.
3. The use of a set of EST-SSR primers in rhizoma bletillae strain identification according to claim 2, wherein the EST-SSR primers comprise: the EST-SSR primer information is applied to genetic diversity analysis of the rhizoma bletillae.
4. The use of a set of EST-SSR primers in rhizoma bletillae strain identification according to claim 3, wherein the EST-SSR primers comprise: the EST-SSR primer information is applied to molecular assisted breeding of rhizoma bletillae.
5. The application of the EST-SSR primer group in bletilla striata strain identification according to any one of claims 2 to 4 is characterized by comprising the following steps:
step 1): extracting genome DNA of bletilla striata test material by using a CTAB method, and storing for later use;
step 2): performing PCR amplification by using the EST-SSR primer by using the genomic DNA extracted in the step 1) as a template;
and step 3): and (3) carrying out electrophoretic separation on the PCR amplification product in the step 2) by using non-denatured polyacrylamide gel, developing by using sodium hydroxide and formaldehyde after conventional silver staining, washing by using clear water, and finally carrying out artificial band reading on the polymorphic marker of the amplification band by taking the occurrence frequency and the size of the amplification band as reference, wherein a band is marked as '1', and an unclear or missing band is marked as '0', so as to establish an original (0,1) matrix.
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| AU2003298492A1 (en) * | 2002-12-18 | 2004-07-14 | Jsc Fermtech | Enzymatic method for the isolation of dna from plant tissue |
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| CN106591302A (en) * | 2017-01-24 | 2017-04-26 | 四川农业大学 | SSR core primer set based on Pennisetum purpureum Schum transcriptome sequence development and its application |
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