CN114182034B - SSR molecular marker of apocarya variety McMillian and application thereof - Google Patents
SSR molecular marker of apocarya variety McMillian and application thereof Download PDFInfo
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Abstract
The invention relates to the technical field of apocarya variety identification, in particular to an SSR molecular marker of an apocarya variety McMillian and application thereof. The SSR molecular marker for identifying the apocarya variety McMillan is amplified by a primer shown in SEQ ID No. 1-2. The SSR molecular marker can be used for identifying the apocarya variety McMillan, the SSR molecular marker is used for identifying the McMillan, the identification cost can be effectively reduced, the efficiency is improved, the operation is simple and convenient, the identification result is accurate, and the SSR molecular marker and the identification method have wide application prospects.
Description
Technical Field
The invention relates to the technical field of apocarya variety identification, in particular to an SSR molecular marker of an apocarya variety McMillian and application thereof.
Background
Carya illinoensis (Carya illinoinensis), a plant belonging to the genus Carya (Juglandaceae), is an important dried fruit and woody oil tree species. The apocarya is also called as pecan in China, and has high oil content of nuts, wherein unsaturated fatty acid accounts for more than 90% of the total fat content, and meanwhile, the apocarya contains rich phenolic substances, mineral elements, amino acids, vitamins and the like, is rich in nutrition and has high economic value. In recent years, the planting of apocarya is continuously promoted, and meanwhile, the demand for improved variety seedlings is rapidly increased, so that how to ensure the authenticity of the variety of the seedlings becomes a problem to be solved urgently.
The apocarya variety McMillan, the single fruit weight is about 8 g, the stamen is mature type variety, the seed selection is carried out in real time, and the black spot disease is resisted. The apocarya is hermaphrodite and abnormal in flowers, and different varieties are required to be configured for full pollination in the female and male flowering periods, so that high quality and high yield are realized. There are also differences in regional adaptability and cultivation management between different varieties. However, in the seedling stage, the phenotype difference between different apocarya varieties is not obvious, and effective distinction is difficult to realize. If the planted variety is not in accordance with the target, the planted variety can be found after 4-6 years of flowering and fruiting, and great manpower, time and economic losses are caused for breeding units and farmers. Therefore, in the initial stage of the construction of the apocarya garden, the determination of the authenticity of the variety is particularly important. In production practice, the variety identification is usually realized by means of phenotype characteristics such as leaves, fruits, flowers, tree poses and the like by means of experienced experts, but the phenotype characteristics of the same variety under different climatic region conditions, different growth and development stages and different cultivation conditions have larger differences, so that the success rate of the variety identification method depending on experience is low.
The molecular marker can directly detect the difference of DNA level, does not depend on phenotypic character, has strong stability and good repeatability, and is widely applied to variety identification and kindred relation research of animals and plants. Molecular marker-based genetic relationship research exists in apocarya, but the problems of low variety coverage, difficulty in distinguishing varieties with blood relationship, complex operation and the like exist. Therefore, the establishment of the identification method of the apocarya variety with high accuracy, simple and convenient operation and stable identification result is needed.
Disclosure of Invention
The invention aims to provide an SSR molecular marker of a apocarya variety 'McMillan' and application thereof.
In order to achieve the aim, the invention adopts a bioinformatics method to discover more than 14 ten thousand SSR sites from the whole genome sequence of the apocarya, and designs more than 6 ten thousand pairs of specific primers in batches. These primers were further screened, with the following main screening criteria: 1) The SSR locus is 2-4 base repeats; 2) The annealing temperatures of the upstream primer and the downstream primer differ by not more than 1 ℃; 3) The primer does not contain unknown bases; 4) The target product is 100 bp-300 bp. 300 pairs of primers were initially screened and synthesized. Collecting tender leaves of 36 apocarya varieties which are applied in China, extracting genome DNA, and optimizing PCR reaction conditions and amplified product detection conditions. The optimized program is used for amplifying and detecting 36 varieties, and an SSR primer which has good amplification effect and strong stability and has specific bands in the common variety 'McMillan' of the apocarya is obtained by screening, so that the accurate identification of 'McMillan' by using 1SSR mark is realized.
Specifically, the invention provides the following technical scheme:
in a first aspect, the present invention provides an SSR (Simple Sequence Repeat, SSR) molecular marker for identifying the apocarya variety 'McMillan', amplified from the primers shown in SEQ ID No. 1-2.
The primer sequences described above are specifically as follows:
SEQ ID NO.1(5’-3’)：CTTAAGTGGAACGGCATCGT；
SEQ ID NO.2(5’-3’)：ATGTCTGTTAAACCGCGACC。
in a second aspect, the present invention provides primers for identifying the apocarya variety 'McMillan', the nucleotide sequence of which is shown in SEQ ID NO. 1-2.
In a third aspect, the invention provides a kit comprising the primers shown as SEQ ID NO. 1-2.
Preferably, the kit further comprises other components for PCR amplification including, but not limited to, PCR reaction buffers, dntps, DNA polymerase, negative controls, positive controls, and the like.
In a fourth aspect, the present invention provides a DNA chip comprising the primers shown in SEQ ID NO. 1-2.
In a fifth aspect, the invention provides the use of said SSR molecular markers or said primers or said kit or said DNA chip for identifying the apocarya variety 'McMillan'.
The invention also provides application of the SSR molecular marker or the primer or the kit or the DNA chip in constructing a DNA fingerprint database of the apocarya variety 'McMillan'.
The invention also provides application of the SSR molecular marker or the primer or the kit or the DNA chip in molecular marker assisted breeding of apocarya variety 'McMillan'.
The invention also provides application of the SSR molecular marker or the primer or the kit or the DNA chip in germplasm resource identification of a apocarya variety 'McMillan'.
The invention also provides application of the SSR molecular marker or the primer or the kit or the DNA chip in seedling quality detection of the apocarya variety 'McMillan'.
In a sixth aspect, the present invention provides a method of identifying the apocarya variety 'McMillan', the method comprising the steps of: and (3) taking the DNA of the apocarya to be identified as a template, adopting a primer with a nucleotide sequence shown as SEQ ID NO.1-2 to carry out PCR amplification, and judging the apocarya to be identified as 'McMillan' if the length of the obtained amplification product is 112 bp.
Specifically, the method for identifying the apocarya variety 'McMilan' comprises the following steps:
(1) Extracting genome DNA of the apocarya to be identified;
(2) Using the DNA extracted in the step (1) as a template, and adopting a primer with a nucleotide sequence shown as SEQ ID NO.1-2 for PCR amplification;
(3) Judging whether the apocarya to be identified is the apocarya variety McMillan according to the strip type of the PCR amplification product.
The invention has the beneficial effects that: the SSR molecular marker provided by the invention can realize the identification of the apocarya variety 'McMillan', and the SSR molecular marker is used for identifying the McMillan, so that the identification cost can be effectively reduced, the efficiency is improved, the operation is simple and convenient, the identification result is accurate, and the SSR molecular marker and the identification method have wide application prospects.
Drawings
FIG. 1 is an electrophoresis chart of amplification products obtained by PCR amplification of 45 samples of 36 apocarya varieties by using primers P1 and P2 (SEQ ID NO. 1-2) in example 2 of the present invention, wherein lane M is a DNA Marker, and the sizes of the bands from bottom to top are respectively: 100. 150, 200, 250, 300, 400, 500bp; each of the other lanes 1 to 45 is a sample, and samples of lanes 1 to 45 are in turn: 'Kanza', 'Mohawk', 'Shawnee', 'Mahan' (biological repetition), 'Osage', 'Pawnee', 'Mcmullan', 'Lakota', 'Silver back', 'Carter', 'Colby', 'Stuart', 'Greenriver', 'Waco', 'Major', 'Ocone', 'Ocon' (biological repetition), 'Navaho', 'Gloria Grande', 'forker', 'Choctaw', 'Creek', 'Mohawk' (biological repetition), 'Elliott'; 'Barton', 'Creek' (biological repetition), 'desired', 'Graking', 'Greenriver' (biological repetition), 'Hopi', 'Jayhawk', 'Kiowa', 'Lakota' (biological repetition), 'Maramec', 'Mohawk' (biological repetition), 'Nacon', 'Navaho' (biological repetition), 'Ofree' (biological repetition), 'Posey', 'Houma', 'Yates68', 'Shepherd', 'Deerstand', and 'Chetopa'.
FIG. 2 is a capillary electrophoresis chart of PCR amplification products of the apocarya 'McMillan', 'Pawnee' and 'Mahan' amplified using the primers P1 and P2 (SEQ ID NO. 1-2) in example 2 of the present invention.
Detailed Description
The following examples are illustrative of the invention and are not intended to limit the scope of the invention.
The 36 apocarya varieties used in the examples below are commercially available or are obtained from the subtropical forestry institute germplasm resource library of the national institute of forestry, wherein the varieties of 'McMillan', 'Mahan', 'payee', 'Greenriver', 'Mohawk', 'Osage', 'Lakota', 'Carter', 'Colby', 'Stuart' and the like are disclosed in literature (Zhang c.c., yan x.h., ren h.d., chang j, wu j, shao w.z., fang q.Characterification and Development of Genomic SSRs in Pecan (Carya illinoinensis) forest.2020, 11 (1), 61).
EXAMPLE 1 development of SSR molecular markers
More than 14 ten thousand SSR sites are discovered from the whole genome sequence of apocarya, and more than 6 ten thousand pairs of specific primers are designed in batches. These primers were further screened, with the following main screening criteria: 1) The SSR locus is 2-4 base repeats; 2) The annealing temperatures of the upstream primer and the downstream primer differ by not more than 1 ℃; 3) The primer does not contain unknown bases; 4) The target product is 100 bp-300 bp. 300 pairs of primers were initially screened and synthesized.
Selecting 36 apocarya varieties which are more applied in China, respectively collecting tender leaves, respectively extracting genome DNA of each sample by adopting at least 3 samples of each variety, and optimizing PCR reaction conditions and amplified product detection conditions. The optimized program is used for amplifying and detecting each sample of 36 varieties, and an SSR primer which has good amplification effect, strong stability and specific bands in the common variety 'McMillan' of the apocarya is obtained by screening, so that the accurate identification of 'McMillan' by using 1SSR mark can be realized. The sequence of the SSR primer is as follows:
P1：SEQ ID NO.1(5’-3’)：CTTAAGTGGAACGGCATCGT；
P2：SEQ ID NO.2(5’-3’)：ATGTCTGTTAAACCGCGACC。
PCR amplification was performed using the SSR primers described above, with the apocarya variety 'McMillan' having a single specific band at 112 bp.
Example 2 identification of apocarya variety ' McMillan ' Using SSR molecular markers '
The SSR molecular marker obtained in the example 1 and the amplification primer thereof are used for identifying the apocarya varieties, and the specific method is as follows:
1. extraction and detection of genomic DNA
Collecting tender leaves of the apocarya to be identified, and extracting leaf DNA of the apocarya to be identified by using a TSINGKE plant DNA extraction kit (general type), wherein the specific steps are as follows:
(1) Placing Spin Column in a Collection Tube, adding 250 μl Buffer BL, and centrifuging at 12000rpm/min for 1min to activate silica gel film;
(2) Sample tissue (no more than 100 mg) was taken, and fully ground by adding liquid nitrogen. Grinding, placing into a 1.5ml centrifuge tube, adding 400 μl Buffer gP1, vortex oscillating for 1min, and water-bathing at 65deg.C for 10-30 min, and taking out, and mixing for full lysis;
(3) Adding 150 μl Buffer gP2, vortex oscillating for 1min, and ice-bathing for 5min;
(4) Centrifuging at 12000rpm/min for 5min, and transferring the supernatant into a new centrifuge tube;
(5) Adding absolute ethyl alcohol with the same volume as the supernatant, immediately and fully oscillating and uniformly mixing, transferring all the liquid into Spin Column, centrifuging at 12,000rpm/min for 30s, and discarding waste liquid;
(6) 500 μl Buffer Pw (absolute ethanol is added before use) is added into Spin Column, and the mixture is centrifuged at 12000rpm/min for 30s, and the waste liquid is discarded;
(7) 500 μl Wash Buffer (absolute ethanol is added before use) is added into Spin Column, and the mixture is centrifuged at 12000rpm/min for 30s, and the waste liquid is discarded;
(8) Repeating the operation step 7;
(9) Putting Spin Column back into Collection Tube, centrifuging at 12,000rpm/min for 2min, uncovering, and air drying for 1min;
(10) Taking out Spin Column, placing into a clean centrifuge tube, adding 50-100 μl TE Buffer (preheated TE Buffer at 65deg.C) at the center of the adsorption film, standing at 20-25deg.C for 2min, and centrifuging at 12,000rpm/min for 2min.
(11) Genomic DNA quality was detected using 1% agarose gel electrophoresis and genomic DNA concentration was detected using an ultraviolet spectrophotometer.
2. PCR amplification of SSR molecular markers
The different samples were PCR amplified using the primers P1 and P2 (SEQ ID NO. 1-2) using the extracted genomic DNAs of the different samples as templates, and the PCR reaction system is shown in Table 1.
TABLE 1 reaction System for PCR amplification
The PCR reaction conditions are shown in Table 2.
TABLE 2 reaction conditions for PCR amplification
3. Polyacrylamide gel electrophoresis detection of PCR amplified products
The PCR amplification products were detected by polyacrylamide gel electrophoresis, and the formulation of the non-denaturing polyacrylamide gel is shown in Table 3.
TABLE 3 non-denaturing polyacrylamide gel formulations
The electrophoresis method is as follows:
the electrophoresis buffer solution is 0.5 XTBE, the left and right end blank spaces avoid edge effect, the electrophoresis sample loading amount is 1 mu l, and two ends are respectively provided with a lane sample loading 50bp Marker. The electrophoresis conditions were: 180V,400mA, electrophoresis for 180min.
Taking out the gel after electrophoresis, punching and marking with gun tip, agNO 3 Solution (1.0 gAgNO) 3 Dissolved in 1L of water) silver staining for 10-15min; developing solution (20 g NaOH is dissolved in 1L water, 10ml formaldehyde is added) for developing for 5-8min; rinsing with water for 2 times, and photographing on a lamp box. The stripe size of each sample is manually determined.
The sample was determined to be 'McMillan' when the amplification product had a single band at only 112 bp.
The identification method is used for identifying 36 samples of apocarya varieties collected by the apocarya germplasm resource library, the detection results of polyacrylamide gel electrophoresis of partial samples are shown in figure 1, the figure 1 comprises biological repeats of partial varieties such as 36 varieties, including 'Mahan', 'Ocone', 'Greenver', 'Navaho', 'Creek', 'Mohawk', etc., 45 samples are all obtained, and all other biological repeats have the same detection results, but not all the biological repeats are listed.
4. Capillary electrophoresis detection of PCR amplified products
The different samples were PCR amplified using primers P1 and P2 (SEQ ID NO. 1-2) using the genomic DNAs of the different samples as templates, and the PCR reaction system is shown in Table 4.
TABLE 4 reaction System for PCR amplification
The PCR reaction conditions are shown in Table 5.
TABLE 5 reaction conditions for PCR amplification
The ABI3730 capillary electrophoresis detection method comprises the following steps:
the concentration of PCR products was estimated based on the detection result of agarose gel electrophoresis, and after 10-fold dilution of the products, the products were mixed with ROX 500 internal standards (70, 80,100,120,140,160,180,200,240,280,320,360,400,450,490,500base, respectively) to give a reaction system shown in Table 6, reacted at 95℃for 5min and then rapidly ice-bathed for 3min, and then placed on an ABI3730 sequencer sample rack for capillary electrophoresis detection, and the detection results of PCR amplification products of the varieties 'Mahan', 'Pawnee' and 'Mcmilan' are shown in Table 7, wherein the detection results of the PCR amplification products of the varieties are shown in FIG. 2.
TABLE 6 capillary electrophoresis reaction System
TABLE 7 band sizes of PCR amplified products of 36 apocarya varieties
The result shows that the banding patterns among biological repeats of 36 varieties are consistent, and the SSR molecular marker has better repeatability among different individuals of the same variety. The amplification product of `McMillan` has a single band at only 112 bp.
The result shows that the SSR molecular marker provided by the invention can accurately distinguish 'McMillan' from other 35 apocarya varieties, and the common variety 'McMillan' is identified from 36 apocarya varieties. The SSR molecular marker realizes that the identification of McMillan' can be carried out by using a single SSR molecular marker.
While the invention has been described in detail in the foregoing general description and with reference to specific embodiments thereof, it will be apparent to one skilled in the art that modifications and improvements can be made thereto. Accordingly, such modifications or improvements may be made without departing from the spirit of the invention and are intended to be within the scope of the invention as claimed.
Sequence listing
<110> China national institute of forestry science subtropical forestry institute
<120> SSR molecular marker of apocarya variety McMillian and application thereof
<130> KHP211121076.1
<160> 2
<170> SIPOSequenceListing 1.0
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<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 1
cttaagtgga acggcatcgt 20
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<212> DNA
<213> Artificial sequence (Artificial Sequence)
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atgtctgtta aaccgcgacc 20
Claims (2)
- Application of SSR molecular markers or primers or kits in identifying apocarya variety 'McMillan' from 36 apocarya varieties;the SSR molecular marker is amplified by a primer shown in SEQ ID NO. 1-2;the nucleotide sequence of the primer is shown as SEQ ID NO. 1-2;the kit comprises the primer;the 36 apocarya varieties are ' Carter ', ' chocolaw ', ' Colby ', ' Creek ', ' desicable ', ' Elliott ', ' forsert ', ' Gloria Grande ', ' Greenriver ', ' Kanza ', ' Lakota ', ' Mahan ', ' Major ', ' McMillan ', ' Mohawk ', ' navacho ', ' ocene ', oncone ', and a combination thereof ' Osage ', ' Pawnee ', ' Shawnee ', ' Stuart ', ' Waco ', ' Silver back ', ' Barton ', ' Graking ', ' Hopi ', ' Jayhawk ', ' Kiowa ', ' Maramec ', ' Nacon ', ' Posey ', ' Houma ', ' Yates68', ' Shepherd ', ' Deerstand ', and ' Chetopa '.
- 2. A method of identifying a apocarya variety 'McMillan' from 36 apocarya varieties, comprising: performing PCR amplification by using DNA of the apocarya to be identified as a template and adopting a primer with a nucleotide sequence shown as SEQ ID NO.1-2, and judging that the apocarya to be identified is 'McMillan' if the length of an obtained amplification product is 112 bp;the 36 apocarya varieties are ' Carter ', ' chocolaw ', ' Colby ', ' Creek ', ' desicable ', ' Elliott ', ' forsert ', ' Gloria Grande ', ' Greenriver ', ' Kanza ', ' Lakota ', ' Mahan ', ' Major ', ' McMillan ', ' Mohawk ', ' navacho ', ' ocene ', oncone ', and a combination thereof ' Osage ', ' Pawnee ', ' Shawnee ', ' Stuart ', ' Waco ', ' Silver back ', ' Barton ', ' Graking ', ' Hopi ', ' Jayhawk ', ' Kiowa ', ' Maramec ', ' Nacon ', ' Posey ', ' Houma ', ' Yates68', ' Shepherd ', ' Deerstand ', and ' Chetopa '.
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