CN111304358A - EST-SSR primer developed based on wax gourd transcriptome sequence and application thereof - Google Patents
EST-SSR primer developed based on wax gourd transcriptome sequence and application thereof Download PDFInfo
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Abstract
The invention discloses an EST-SSR primer developed based on a wax gourd transcriptome sequence and application thereof. The method obtains the EST-SSR primer by screening SSR loci of wax gourd transcriptome sequences and designing primers according to nucleotide sequences at two sides of the SSR loci. The 3 pairs of EST-SSR primers are used for amplifying wax gourd resources, and the marking result verified by the full-automatic nucleic acid protein analyzer can be applied to the construction of manually drawing the wax gourd variety identification schematic diagram. According to the SSR amplification result, the wax gourd resources can be quickly and accurately distinguished, so that each resource is effectively identified, and the method can be used for the germplasm identification, the germplasm resource management, the variety right identification, the protection and the like of Chinese wax gourd plant varieties.
Description
Technical Field
The invention belongs to the technical field of EST-SSR (expressed sequence tag-simple sequence repeat) markers, and particularly relates to an EST-SSR primer developed based on a wax gourd transcriptome sequence and application thereof.
Background
Wax gourd belongs to the genus Benincasa (Benincasa Savi) of the family Cucurbitaceae (Cucurbitaceae), and is an annual sprawl herb. Wax gourd is a vegetable variety traditionally planted in China, and has been cultivated for over 2000 years. The white gourd breeding mode mainly depends on conventional cross breeding, and the breeding efficiency is greatly influenced because the new combination in the breeding has high randomness and narrow genetic basis among varieties in the cross combination preparation process, so that the problems of unobvious heterosis and the like occur. China has rich white gourd resources, and the phenomena of mutual introduction, cultivation and breeding among various regions and respective naming are frequently caused. In the process, the problem of foreign matters with the same name or the same name is easy to occur, so that the variety identification and the industrial application of the wax gourd are difficult. Therefore, the method has important significance for systematically identifying and evaluating the white gourd germplasm resources by using the DNA molecular markers.
A nucleotide repeat (SSR) is a DNA sequence formed by the tandem repeat of a gene sequence consisting of several basic groups. The SSRs are classified into genome SSRs and plant expression sequence tags-SSRs (EST-SSR) EST-SSRs according to their source. EST-SSR marker is characterized in that PCR technology is used as a key, and SSR is directly screened from the obtained EST sequence. Therefore, the EST-SSR has the advantages of co-dominant inheritance, random distribution trend, strong polymorphism and the like of genome SSR, and compared with the genome SSR, the SSR marker developed based on the EST sequence has higher universality and conservation than the genome SSR. In recent years, although markers such as ISSR, SRAP and gSSR are applied to the white gourd research, the markers mostly adopt universal primers, and the white gourd specific primers are fewer, so that the white gourd breeding progress is influenced. Therefore, the wax gourd specific primer becomes an important part for identifying wax gourd resources.
According to the EST-SSR specific primers and the characteristic spectral band after PCR amplification, a wax gourd variety identification schematic diagram can be drawn. The application of EST-SSR in the manual drawing of the variety identification schematic diagram method can intuitively display the identification process, draw the diagram relation among different varieties and accurately know the size (bp) of primers and polymorphic bands used for distinguishing the varieties. At present, the application of the SSR primer developed based on the wax gourd transcriptome sequence in the construction of the manual drawn variety identification schematic diagram of the wax gourd is not reported.
Disclosure of Invention
The invention discloses an EST-SSR primer developed based on a wax gourd transcriptome sequence and application thereof, which can screen out a primer for wax gourd EST-SSR marking and effectively improve the wax gourd germplasm resource identification process.
The invention is realized by adopting the following technical scheme:
EST-SSR primers developed based on wax gourd transcriptome sequences comprise 3 pairs of EST-SSR primers, and the sequences of the EST-SSR primers are shown as follows:
EST-2: the sequence of the upstream primer is as follows: 5'-GAGGGCTGCACCTCTTCATA-3', respectively; the sequence of the downstream primer is as follows: 5'-CCAATTTGATACCTGCCAAAA-3', respectively;
EST-16: the sequence of the upstream primer is as follows: 5'-ATTGGAGTTCGATCCGAATG-3', respectively; the sequence of the downstream primer is as follows: 5'-CCCATTTCTCAATTCTTCTTCAA-3', respectively;
EST-62: the sequence of the upstream primer is as follows: 5'-TGCGACAAATGTGGAGAGAG-3', respectively; the sequence of the downstream primer is as follows: 5'-TTTTTGGAGTGGGTAAACCG-3' are provided.
The invention discloses application of an SSR primer developed based on a wax gourd transcriptome sequence in construction of a wax gourd variety identification schematic diagram.
The invention provides an SSR primer developed based on a wax gourd transcriptome sequence and a method for constructing the SSR primer in an artificial drawn variety identification schematic diagram, which comprises the following steps:
(1) obtaining a gene database of wax gourd samples;
(2) and (3) carrying out SSR locus search on the database in the step (1) by using MISA software. Screening criteria: the minimum number of times of repeat of mononucleotide is 10; dinucleotide is repeated for 6 times in a minimum; the minimum number of times of repetition of the three, four, five and six nucleotides is 5 times;
(3) designing SSR primers by using Primer 3.0, wherein each SSR Primer generates 3 groups of primers to generate candidate primers;
(4) randomly selecting 6 parts of wax gourd resources, extracting wax gourd genomic DNA by using a CTAB method, detecting the quality and concentration of the DNA by respectively adopting agarose gel electrophoresis with the concentration of 1% and a spectrophotometer, carrying out PCR amplification by using the candidate primers in the step (3), and preliminarily screening out primers containing target strips through the agarose gel electrophoresis;
(5) extracting 40 parts of wax gourd resource genome DNA as a template, carrying out PCR amplification by using the PCR primer preliminarily screened in the step (4), detecting by using a nucleic acid protein analyzer after amplification, and screening out a primer capable of amplifying the target sequence in the step (3), namely obtaining the primer for wax gourd EST-SSR marking;
(6) counting the sizes of characteristic bands and polymorphic bands amplified by each pair of primers according to the detection result of the nucleic acid protein analyzer in the step (5);
(7) constructing a wax gourd variety identification schematic diagram according to the statistical result in the step (6), wherein the specific method comprises the following steps: firstly, counting the existence of characteristic bands at the same electrophoretic mobility in a fingerprint chromatogram of 40 wax gourd varieties amplified by a certain primer, dividing variety resources with the same amplification band into a group, classifying the band-free group into a group, continuously adding the primer, gradually classifying the band-free group according to the same counting method until all the reference material varieties are clearly distinguished, finally drawing a wax gourd variety identification schematic diagram according to an identification result, and marking all the used primers and the size (bp) of the polymorphic band to the corresponding position of the wax gourd variety identification schematic diagram;
and (4) when the winter melon resource sample grows to three leaves and one heart, mixing 2 healthy tender leaves.
Diluting the DNA concentration in the step (4) to 50 ng. mu.L-1And storing at-20 ℃ for later use.
Designing primers in the step (3) and setting main parameters as follows: the length of the primer is generally 18-27 bp, the GC content is 45-55%, the annealing temperature is 55-63 ℃, and the difference between the upstream Tm value and the downstream Tm value is not more than 5 ℃. The problems of hairpin structure, dimer, mismatching, primer dimer and the like are avoided as much as possible.
In step (4), the total PCR reaction system is 25. mu.L, including 0.5. mu.L dNTP, 0.3. mu.L Taq enzyme, 1.5. mu.L DNA, 1. mu.L each of the upstream and downstream primers, and 2.5. mu.L 10 XBuffer (containing Mg)2+），18.2 μL ddH2O。
In step (4), the PCR amplification procedure: pre-denaturation at 94 ℃ for 5 min; denaturation at 94 ℃ for 30s, renaturation at 57 ℃ for 30s, and renaturation at 72 ℃ for 1 min for 35 cycles; extension at 72 ℃ for 10 min.
The invention has the beneficial effects that:
1. the traditional method for developing SSR markers has the disadvantages of complex steps, large workload and high development cost, and limits the application of SSR marker technology in the research of white gourd germplasm resources. Different from the genome SSR, the EST-SSR is derived from a transcription region, can directly reflect the diversity of related genes, has the advantages of co-dominant inheritance, random distribution trend, strong polymorphism and the like of the genome SSR, and compared with the genome SSR, the SSR marker developed based on the EST sequence has higher universality and conservation than the genome SSR.
2. The 3 pairs of EST-SSR primers can effectively construct a wax gourd variety identification schematic diagram, quickly and accurately distinguish wax gourd resources, can be used for wax gourd variety identification, solve the problems of synonyms, homonymy foreign matters, unclear relationship and the like in the variety, and have reference values in the aspects of later wax gourd variety seedling stage identification, variety right identification and protection, germplasm resource management and the like.
Drawings
The invention will be further described with reference to the following examples with reference to the accompanying drawings.
FIG. 1 Material No. 1 primer 2 nucleic acid polymorphism detection map.
FIG. 2 nucleic acid polymorphism detection map of primer 2 of Material No. 2.
FIG. 3 nucleic acid polymorphism detection map of primer 2 of Material No. 3.
FIG. 4 nucleic acid polymorphism detection map of primer 2, Material No. 10.
FIG. 540 shows the identification of wax gourd varieties.
Fig. 6 verifies the authentication results of material numbers 3 and 12.
Fig. 7 verifies the authentication results of material numbers 3 and 12.
Fig. 8 verifies the authentication results of material numbers 10 and 11.
Fig. 9 verifies the authentication results of material numbers 33 and 34.
FIG. 1040 cluster map of wax gourd resources.
Detailed Description
The present invention is further illustrated by the following examples, but the present invention is not limited to the following examples.
Example 1:
the invention provides an SSR primer developed based on a wax gourd transcriptome sequence and a method for constructing the SSR primer in an artificial drawn variety identification schematic diagram, which comprises the following steps:
(1) obtaining a gene database of wax gourd samples;
(2) and (3) carrying out SSR locus search on the database in the step (1) by using MISA software. Screening criteria: the minimum number of times of repeat of mononucleotide is 10; dinucleotide is repeated for 6 times in a minimum; the minimum number of times of repetition of the three, four, five and six nucleotides is 5 times;
(3) designing SSR primers by using Primer 3.0, wherein each SSR Primer generates 3 groups of primers to generate candidate primers;
(4) randomly selecting 6 parts of wax gourd resources, extracting wax gourd genomic DNA by using a CTAB method, detecting the quality and concentration of the DNA by respectively adopting agarose gel electrophoresis with the concentration of 1% and a spectrophotometer, carrying out PCR amplification by using the candidate primers in the step (3), and preliminarily screening out primers containing target strips through the agarose gel electrophoresis;
(5) extracting 40 parts of wax gourd resource genome DNA as a template, carrying out PCR amplification by using the PCR primer preliminarily screened in the step (4), detecting by using a nucleic acid protein analyzer after amplification, and screening out a primer capable of amplifying the target sequence in the step (3), namely obtaining the primer for wax gourd EST-SSR marking;
(6) counting the sizes of characteristic bands and polymorphic bands amplified by each pair of primers according to the detection result of the nucleic acid protein analyzer in the step (5);
(7) constructing a wax gourd variety identification schematic diagram according to the statistical result in the step (6), wherein the specific method comprises the following steps: firstly, counting the existence of characteristic bands at the same molecular weight segment and the existence of characteristic bands at the same electrophoretic mobility in 40 wax gourd variety fingerprint electrophoretograms amplified by a certain primer. The variety resources with the same amplification band are divided into one group, and the variety resources without the band are divided into one group. And then, continuously adding the primers, and gradually classifying according to the same statistical method until all the varieties of the test materials are clearly distinguished. Finally, drawing a wax gourd variety identification schematic diagram according to the identification result, and marking all used primers and polymorphism band sizes (bp) to the corresponding positions of the wax gourd variety identification schematic diagram;
the PCR amplification comprises the following steps: PCR reaction total 25. mu.L, including 0.5. mu.L dNTP, 0.3. mu.L Taq enzyme, 1.5. mu.L DNA, 1. mu.L each of upstream and downstream primers, and 2.5. mu.L 10 Xbuffer (containing Mg)2+），18.2 μL ddH2O, PCR amplification procedure: pre-denaturation at 94 ℃ for 5 min; denaturation at 94 ℃ for 30s, renaturation at 57 ℃ for 30s, and renaturation at 72 ℃ for 1 min for 35 cycles; extension at 72 ℃ for 10 min.
The following 3 primers were obtained by screening according to the method described in this example:
EST-2: the sequence of the upstream primer is as follows: 5'-GAGGGCTGCACCTCTTCATA-3', respectively; the sequence of the downstream primer is as follows: 5'-CCAATTTGATACCTGCCAAAA-3', respectively;
EST-16: the sequence of the upstream primer is as follows: 5'-ATTGGAGTTCGATCCGAATG-3', respectively; the sequence of the downstream primer is as follows: 5'-CCCATTTCTCAATTCTTCTTCAA-3', respectively;
EST-62: the sequence of the upstream primer is as follows: 5'-TGCGACAAATGTGGAGAGAG-3', respectively; the sequence of the downstream primer is as follows: 5'-TTTTTGGAGTGGGTAAACCG-3' are provided.
Example 2: wax gourd EST-SSR amplification and polymorphism detection
According to the SSR sequences of the wax gourd transcriptome, 40 parts of wax gourd resources (table 2) are subjected to PCR amplification by using EST-SSR primers (table 1), and finally 3 pairs of primers (table 2) with better specificity are screened out. The result shows that the bands amplified by the 3 pairs of primers are clear and distinguishable. Further using nucleic acid protein analyzer to detect, the screened primer can detect the peak diagram of the polymorphic nucleic acid. FIGS. 1 to 4 show the detection results of primer 2, and it can be seen that the lengths of the polymorphic fragments detected by primer 2 in resource 1, resource 2, resource 3 and resource 10 are respectively 216bp +304bp +333bp, 216bp +317bp +346bp, 218bp +322bp +351bp and 213bp +300bp +329bp, the peak map result of the polymorphic nucleic acid is clear and clear, and 4 resources can be distinguished. The length of the polymorphic fragment can be determined by a polymorphic nucleic acid peak map and is applied to variety identification.
TABLE 1 SSR primers and annealing temperatures
TABLE 2 SSR primers and annealing temperatures
TABLE 3 materials used in the tests
Example 3: wax gourd resource identification
According to the 3 pairs of SSR primers screened out, the wax gourd resources are identified by an MCID method, and finally 40 wax gourd resources can be distinguished one by one (figure 5), and the previous relation of most materials can be basically and correctly reflected, so that the 3 pairs of primers can be used as the EST-SSR primer combination of the wax gourd resources.
Example 4: verification of wax gourd resource identification result
And screening out a primer with clear PCR amplification bands to identify 40 parts of wax gourd resources according to the detection result of the nucleic acid protein analyzer. First, the presence or absence of a characteristic band at the same electrophoretic mobility in an electrophoretogram amplified by a certain primer is counted. Resources with the same amplification band are grouped together, and resources without the band are grouped together. And then, continuously adding the primers, and gradually classifying according to the same statistical method until all the reference materials are clearly distinguished. And finally, drawing a CID map according to the identification result, and marking all used primers and polymorphism band sizes (bp) to the corresponding positions of the CID map.
According to the constructed wax gourd CID map relation, randomly selecting a plurality of wax gourd resources from groups or in the groups, and verifying the reliability and the availability of the wax gourd resources by using corresponding primers. Such as resources # 3 and 12, # 10 and 11, and # 33 and 34, respectively. Based on the results of the CID diagram, primers No. 3 and No. 12 were completely identified using primers 16 and 2. As shown in FIG. 6, resources No. 3 and No. 12 can be divided into two groups according to the presence or absence of the amplified band 304bp, and then identified separately by the characteristic band 309 bp of primer 2 (FIG. 7). As shown in FIGS. 8 and 9, the primers No. 10 and 11 and No. 33 and 34 can be distinguished from each other by the primer 2, and the difference bands are 256 bp, 300bp and 314 bp, respectively. According to results, primers and genotypes of the primers required by the wax gourd resources identification can be visually seen by a CID map constructed by the EST-SSR primers, so that the wax gourd resources can be quickly identified.
Example 5 Cluster analysis
And (4) analyzing according to the obtained electropherogram. Each strip in the electrophoretogram represents a binding site between the template and the primer, clear and reliable strips on the electrophoretogram are calculated according to the amplification products of the SSR, the label of the strip appearing at the same position is '1', the label of the strip not appearing at the same position is '0', and Excel is input according to the calculated '0' and '1' to form a two-dimensional data matrix. The genetic diversity of 40 wax gourd resources was analyzed using Ntsys2.10e software. As can be seen from the cluster map (FIG. 10), the variation range of the genetic similarity coefficient is 0.73-0.95, the average similarity coefficient is 0.76, and all wax gourd resources can be distinguished by the mark. With the similarity coefficient at 0.77 as the threshold, 40 wax gourd parts can be divided into 3 groups. The group I mainly comprises 23 resources, mainly long cylindrical black white gourd resources, and most of the type of the group I is prismatic flat seeds; the second category comprises 4 parts of resources, mainly long cylindrical wax gourd resources; the third category includes 13 resources, mainly fruit-shaped short cylindrical seeds with edge and flat seeds.
The above examples are merely illustrative of several embodiments of the present invention, and the description thereof is more specific and detailed, but not to be construed as limiting the scope of the invention.
SEQUENCE LISTING
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Claims (8)
1. EST-SSR primers developed based on wax gourd transcriptome sequences are characterized in that the primers comprise 3 pairs of primers, and specifically comprise the following components:
EST-2: the sequence of the upstream primer is as follows: 5'-GAGGGCTGCACCTCTTCATA-3', downstream primer sequence: 5'-CCAATTTGATACCTGCCAAAA-3', respectively;
EST-16: the sequence of the upstream primer is as follows: 5'-ATTGGAGTTCGATCCGAATG-3', downstream primer sequence: 5'-CCCATTTCTCAATTCTTCTTCAA-3', respectively;
EST-62: the sequence of the upstream primer is as follows: 5'-TGCGACAAATGTGGAGAGAG-3', downstream primer sequence: 5'-TTTTTGGAGTGGGTAAACCG-3' are provided.
2. The use of the wax gourd transcriptome sequence-based EST-SSR primer developed according to claim 1 in construction of a wax gourd variety differential representation.
3. Use according to claim 2, characterized in that the construction method comprises the following steps:
(1) obtaining a gene database of wax gourd samples;
(2) carrying out SSR locus search on the database in the step (1) by using MISA software;
(3) designing SET-SSR primers by using Primer 3.0, wherein each SSR generates 3 groups of primers to generate candidate primers;
(4) randomly selecting 6 parts of wax gourd resources, extracting wax gourd genomic DNA by using a CTAB method, detecting the quality and concentration of the DNA by respectively adopting agarose gel electrophoresis with the mass concentration of 1% and a spectrophotometer, carrying out PCR amplification by using the candidate primers in the step (3), and preliminarily screening out primers containing target strips through the agarose gel electrophoresis;
(5) extracting 40 parts of wax gourd resource genome DNA as a template, carrying out PCR amplification by using the PCR primer preliminarily screened in the step (4), detecting by using a nucleic acid protein analyzer after amplification, and screening out a primer capable of amplifying the target sequence in the step (3), namely obtaining the primer for wax gourd EST-SSR marking;
(6) counting the sizes of characteristic bands and polymorphic bands amplified by each pair of primers according to the detection result of the nucleic acid protein analyzer in the step (5);
(7) constructing a wax gourd variety identification schematic diagram according to the statistical result in the step (6), wherein the specific method comprises the following steps: firstly, counting the existence of characteristic bands at the same molecular weight segment and the existence of characteristic bands at the same electrophoretic mobility in a finger-print electrophoretogram of 40 wax gourd varieties amplified by a certain primer, dividing variety resources with the same amplification bands into a group, classifying the band-free group into a group, then continuously adding the primer to classify the materials step by step according to the same counting method until all the reference material varieties are clearly distinguished, finally drawing a wax gourd variety identification schematic diagram according to an identification result, and marking the sizes of all the used primers and polymorphism bands at the corresponding positions of the wax gourd variety identification schematic diagram.
4. The use as claimed in claim 3, wherein the winter melon resource sample in step (4) is a mixture of 2 healthy young leaves when the winter melon resource sample grows to three leaves and one heart.
5. The use of claim 3, wherein the DNA concentration of step (4) is diluted to 50 ng. mu.L-1And storing at-20 ℃ for later use.
6. The use of claim 3, wherein the primer setting parameters in step (3) are as follows: the length of the primer is 18-27 bp, the GC content is 45-55%, the annealing temperature is 55-63 ℃, the difference of Tm values of the upstream and the downstream is not more than 5 ℃, and the hairpin structure, dimer, mismatching and primer dimer are avoided.
7. The use of claim 3, wherein in step (4), the PCR reaction system comprises 25. mu.L of dNTP, 0.3. mu.L of Taq enzyme, 1.5. mu.L of DNA, 1. mu.L of upstream and downstream primers, and 2.5. mu.L of 10 XBuffer containing Mg2+，18.2 μL ddH2O。
8. Use according to claim 3, wherein in step (4) the PCR amplification procedure: pre-denaturation at 94 ℃ for 5 min; denaturation at 94 ℃ for 30s, renaturation at 57 ℃ for 30s, and renaturation at 72 ℃ for 1 min for 35 cycles; extension at 72 ℃ for 10 min.
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| WO2009031740A1 (en) * | 2007-09-04 | 2009-03-12 | Korea Research Institute Of Bioscience And Biotechnology | Functional data integrated genetic marker system, ssr-fmm, and improved genetic research and marker assisted selection with ssr-fmm |
| CN104004833A (en) * | 2014-05-09 | 2014-08-27 | 广东省农业科学院蔬菜研究所 | EST-SSR core primer group developed on basis of transcriptome sequence of towel gourd and application thereof |
| CN106434646A (en) * | 2016-11-30 | 2017-02-22 | 宁波城市职业技术学院 | Four pairs of EST-SSR (Expressed Sequence Tag-Simple Sequence Repeat) primers as well as preparation method thereof and application thereof to construction of fingerprint map of cerasus plants |
| CN107190097A (en) * | 2017-07-26 | 2017-09-22 | 贵州大学 | The method that the SSR molecular marker being sequenced using transcript profile identifies dragon fruit germplasm |
| CN107345256A (en) * | 2017-08-22 | 2017-11-14 | 山西省农业科学院农作物品种资源研究所 | One kind is based on transcript profile sequencing exploitation grass vetch EST SSR primer sets and methods and applications |
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