CN115820894A - InDel molecular marker for identifying single and double petal characters of plum blossom, primer and application thereof - Google Patents
InDel molecular marker for identifying single and double petal characters of plum blossom, primer and application thereof Download PDFInfo
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Abstract
The invention discloses an InDel molecular marker, a primer and application thereof for identifying plum blossom single and double petal traits, wherein the InDel molecular marker comprises two InDel molecular markers, namely InDel9 positioned at the bp position of a plum blossom chromosome No. 1 5549358-5549382 and InDel90 positioned at the bp position of a plum blossom chromosome No. 1 6309277-6309290. And primers for amplifying two InDel markers were developed. The molecular marker can rapidly and efficiently identify the single-petal and double-petal plum blossom varieties in the plum blossom seedling stage, the accuracy rate reaches 100%, and compared with the traditional breeding method that the flowering needs about four years, the marker has the advantages of early time, low cost, accuracy and rapidness when being applied to auxiliary breeding.
Description
Technical Field
The invention relates to the technical field of molecular biology, in particular to an InDel molecular marker for identifying single and double petal characters of plum blossom and an application thereof.
Background
Plum blossom (Prunus mume Sieb. Et Zucc.) belongs to Prunoideae of Rosaceae, is a traditional famous flower in China, has been cultivated for more than 3000 years till now, is popular with people due to its unique ornamental character, and has one of important ornamental characters except flower color, flower fragrance, petal shape and tree shape. The original plum blossom variety is a single-petal type, and then in the long-term cultivation and domestication process, there appear the variation varieties such as heavy petals, pavilions and the like which have good ornamental value and are popular with people. Plum blossom is used as a woody plant, the childhood is long, and the traditional breeding method for screening according to phenotypic characters greatly limits the breeding efficiency.
The molecular marker is a marker on the gene level, detects genetic variation directly on the DNA molecular level, is not influenced by various factors such as tissue and organ types, development stages, habitat conditions and the like, and has high polymorphism and genetic stability. An Insertion-Deletion Length Polymorphism (InDel) marker is Length Polymorphism variation generated by inserting or deleting a certain number of nucleotides on an allelic locus, belongs to a third-generation molecular marker, is developed mainly based on whole genome sequencing, and has the advantages of high marker specificity, good stability, simple detection method, economy and the like.
Zhangjie (Zhangjie plum blossom high-density genetic map construction and partial ornamental character QTL analysis [ D ]. Beijing forestry university, 2016.) utilizes Specific-Locus Amplified Fragment Sequencing (SLAF-seq, specific-Locus Amplified Fragment Sequencing) technology to develop molecular markers in the whole genome range of plum blossom, construct a genetic linkage map with maximum plum blossom marker density, locate the heavy-petal character to the position of 116.46Mb of plum blossom chromosome 2, and simultaneously obtain an SLAF molecular marker which can be closely linked with the plum blossom heavy-petal character. Zhengtangchun, etc. (Zhengtangchun, zhang Xiang, liu Wei super, cheng Tang ren, wang Jia. A gene controlling the characters of plum blossom single petal and heavy petal and its molecular marker and application) are analyzed by BSA to identify the PmAP2-like gene and InDel marker controlling plum blossom single petal and heavy petal, but only applied in 20 plum blossom varieties, and the application range is relatively small.
Because the prior plum blossom double petal sex research is not much, the molecular markers of the current double petal character are reported less, or large-scale verification is not carried out among varieties, and the early identification of the single petal and the double petal character of the plum blossom filial generation cannot be reliably carried out. Therefore, further development of molecular markers linked with the plum blossom single petal and double petals is needed to assist the breeding work of the plum blossom double petals.
Disclosure of Invention
The invention aims to provide an InDel molecular marker, a primer and application thereof for identifying the single and double petal traits of plum blossom.
In order to achieve the purpose of the invention, in a first aspect, the invention provides an InDel molecular marker for identifying the plum blossom single-petal and double-petal character, which is InDel9, and is located at the position of 5549358-5549382 bp of a plum blossom 1 chromosome with a Genome version of Prunus mumme Genome v1.0 (http:// Prunus mumemeome.bju.edu.cn /), and corresponds to the plum blossom single-petal character when 5549358-5549382 bp of the plum blossom 1 chromosome is ATTTTCAAACTTGTACTATCTAT, and corresponds to the plum blossom double-petal character when 5549358-5549382 bp of the plum blossom 1 chromosome does not contain ATTTTCAAACTTGTACTATCTAT.
In a second aspect, the invention provides primers for amplifying the molecular marker InDel9, which comprise an upstream primer shown as SEQ ID NO. 1 and a downstream primer shown as SEQ ID NO. 2.
In a third aspect, the invention provides a reagent or a kit for detecting the plum blossom single and double petal traits, which comprises a primer pair SEQ ID NO. 1-2.
In a fourth aspect, the invention provides any one of the following applications of the molecular marker InDel9, a primer for amplifying InDel9 or a detection reagent or kit comprising the primer:
1) The method is used for identifying the characters of the plum blossom single and double petals;
2) The method is used for early prediction of the plum blossom single and double petal flower types;
3) Is used for plum blossom molecular marker assisted breeding.
In a fifth aspect, the invention provides a method for identifying the plum blossom single and double petal traits, which comprises the following steps:
1) Extracting quincunx genome DNA to be detected;
2) Taking the DNA extracted in the step 1) as a template, and carrying out PCR amplification by using primers shown in SEQ ID NO. 1-2;
3) Analyzing the amplification product: if the size of the amplified product is 204bp, the plum blossom to be detected is the double-petal character.
In an eighth aspect, the invention provides an InDel molecular marker for identifying the plum blossom single-petal and double-petal traits, which is InDel90, is located at 6309277-6309290 bp of plum blossom chromosome 1 with a Genome version of Prunus mumme Genome v1.0 (http:// Prunus mumeme. Bjfu. Edu. Cn /), corresponds to the plum blossom single-petal traits when the 6309277-6309290 bp of the plum blossom chromosome 1 is TTGTATGATTGCA, and corresponds to the plum blossom double-petal traits when the 6309277-6309290 bp of the plum blossom chromosome 1 does not contain TTGTATGATTGCA.
In a seventh aspect, the invention provides primers for amplifying the molecular marker InDel9, which comprise an upstream primer shown as SEQ ID NO. 3 and a downstream primer shown as SEQ ID NO. 4.
In an eighth aspect, the invention provides a plum blossom single-petal and double-petal character detection reagent or a kit, which comprises a primer pair SEQ ID NO. 3-4.
In a ninth aspect, the invention provides any one of the following applications of the molecular marker InDel9, a primer for amplifying InDel9 or a detection reagent or kit comprising the primer:
1) The method is used for identifying the characters of the plum blossom single and double petals;
2) The method is used for early prediction of the plum blossom single and double petal flower types;
3) Is used for plum blossom molecular marker assisted breeding.
In a tenth aspect, the invention provides a method for identifying the plum blossom single and double petal traits, which comprises the following steps:
(1) Extracting the genome DNA of the plum blossom to be detected;
(2) Taking the DNA extracted in the step (1) as a template, and carrying out PCR amplification by using primers shown in SEQ ID NO. 3-4;
(3) Analyzing the amplification product: if the size of the amplified product is 234bp, the plum blossom to be detected is in a double-petal character.
In the invention, the plum blossom varieties/materials to be detected include but are not limited to 'snow plum', 'sheet jelly powder' and their filial generation.
Further, the PCR amplification system used in the present invention comprises: 50 ng/. Mu.L DNA template 1. Mu.L, 10. Mu.L 2 XTAQUAPCR mix, 10. Mu.M upstream and downstream primers 1. Mu.L each, ddH 2 The content of O is filled to 20 mu L.
The PCR reaction program is: pre-denaturation at 94 ℃ for 3min; denaturation at 94 ℃ for 30s, annealing at 51 ℃ for 30s, extension at 72 ℃ for 40-60 s (preferably extension at 72 ℃ for 60 s), 30 cycles; final extension at 72 ℃ for 5min and storage at 4 ℃.
By the technical scheme, the invention at least has the following advantages and beneficial effects:
the invention provides an InDel molecular marker capable of identifying the characters of single petals and double petals of plum blossom at an early stage, and by utilizing a marker primer InDel9F/R or InDel90F/R, the varieties of the single petals and the double petals of plum blossom can be quickly and efficiently identified in the seedling stage of the plum blossom, while the flowering waiting period of the traditional breeding needs about four years.
Compared with the identification methods of other marker types such as SNP and the like, the method for identifying the InDel molecular marker with the plum blossom single and double petal characters at the early stage provided by the invention can be used for detecting by utilizing common PCR and polyacrylamide gel electrophoresis, does not need sequencing, and has the advantages of simple and convenient operation, rapidness and low cost.
And thirdly, the molecular marker is verified in 69 plum blossom varieties with the accuracy rate of 100 percent, so that the molecular marker is used for identifying the plum blossom single-petal and double-petal varieties in early stage, and the result is reliable.
Drawings
FIG. 1 is a graph showing the frequency distribution of the number of each floral organ of the ` Chimonanthus nitens ` X ` Pimenta masceras ` F1 population in the preferred embodiment of the present invention.
FIG. 2 is a diagram showing the distribution of variation sites Δ (All-index) in the pool of mixed plum blossom single-petal and double-petal materials in the preferred embodiment of the present invention.
FIG. 3 is a graph showing the distribution of the density of the variation sites between 0.4 and 0.6 in delta (All-index) in the preferred embodiment of the present invention.
FIG. 4 is a diagram showing the phenotype of the plum blossom single-petal and double-petal cultivars in the preferred embodiment of the invention (the left is the single-petal cultivar, and the right is the double-petal cultivar).
FIG. 5 is a diagram showing the result of amplification of InDel9 in 69 plum blossom cultivars in a preferred embodiment of the invention.
FIG. 6 is a diagram showing the results of the amplification of InDel90 in 69 plum blossom cultivars in the preferred embodiment of the invention.
Detailed Description
The invention provides two InDel molecular markers for early identifying plum blossom single-petal and double-petal traits, wherein InDel9 is positioned at the position of chromosome No. 1 of plum blossom 5549358-5549382 bp, wherein the Genome version is Prunus mumome Genome v1.0 (http:// Prunus mumemeome Genome. Bjfu. Edu. Cn /). When the plum blossom chromosome No. 1 5549358-5549382 bp is ATTTTCAAACTTGTACTTATCTAT, the plum blossom chromosome 1 corresponds to the plum blossom single petal character, and when the plum blossom chromosome No. 1 5549358-5549382 bp does not contain ATTTTCAAACTTGTACTTATCTAT, the plum blossom double petal character corresponds to the plum blossom double petal character; inDel90 is located at 6309277-6309290 bp of plum blossom chromosome 1, corresponds to plum blossom single-petal character when 6309277-6309290 bp of plum blossom chromosome 1 is TTGTATGATTGCA, and corresponds to plum blossom double-petal character when 6309277-6309290 bp of plum blossom chromosome 1 does not contain TTGTATGATTGCA.
The method utilizes 69 plum blossom varieties for verification, and the accuracy rate is 100%.
The invention also provides two pairs of primers for amplifying the molecular marker, which are respectively as follows:
forward primer InDel 9F: 5 'GCAAGGTCTGTCGTGGA-3' (SEQ ID NO: 1)
Reverse primer InDel 9R: 5 'GCAAAACTAATATGGTTGTAGC-3' (SEQ ID NO: 2)
Forward primer InDel 90F: 5 'TATTGTGTGTTAGGTTTTCCCA-3' (SEQ ID NO: 3)
Reverse primer InDel 90R: 5 'CCAGTTTTGACTATTTCGG-3' (SEQ ID NO: 4)
Further, the invention provides a method for early identifying the plum blossom single-petal and double-petal traits, which comprises the following steps:
step 1: extracting the genome DNA of the variety or material to be identified;
step 2: taking the DNA extracted in the step 1 as a template, and carrying out PCR amplification on the DNA by using the molecular marker or the molecular marker primer pair;
and step 3: and (3) carrying out electrophoretic separation on the PCR amplification product obtained in the step (2) to obtain the banding pattern of each sample, wherein if an InDel9F/R primer pair is used for amplifying to obtain a 204bp fragment, the variety or the material to be identified is a double-petal plum blossom, and if an InDel90F/R primer pair is used for amplifying to obtain a 234bp fragment, the variety or the material to be identified is a double-petal plum blossom.
The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention. Unless otherwise indicated, the examples follow conventional experimental conditions, such as the Molecular Cloning handbook, sambrook et al (Sambrook J & Russell DW, molecular Cloning: a Laboratory Manual, 2001), or the conditions as recommended by the manufacturer's instructions.
Example 1 development of InDel molecular markers related to plum blossom single and double petal traits
1. Experimental Material
The test materials were F1 isolates obtained from 'snow plum', 'mealy' and 'snow plum' x 'mealy'. The parent has obvious difference in the number of petals, the female parent is a single-petal variety, the number of petals is 5, the male parent is a double-petal variety, and the average number of the petals is 23.5. All the materials were planted in the university of China agriculture university plum garden, wuhan City, hubei province.
2. Mixed population isolation (BSA) whole genome re-sequencing
Selecting 10 single-petal character filial generations and 10 double-petal character filial generations in the 'Xuemei' x 'sheet jelly palace powder' F1 population, extracting DNA, and mixing in equal amount to respectively construct a single-petal gene pool and a double-petal gene pool. The DNA samples qualified for the test were randomly fragmented into fragments of 350bp in length. The DNA fragment is subjected to end repair, ployA tail addition, sequencing joint addition, purification, PCR amplification and other steps to complete the preparation of the whole library. The constructed library was sequenced by illumina HiSeqTM PE 150.
3. BSA analysis
As shown in fig. 1, the frequency distribution result of the number of each floral organ of the 'snowflake' × 'sheet jelly uterine powder' F1 population shows that neither the number of petals nor the number of layers of petals accords with normal distribution, the segregation ratio of single-petal and double-petal offspring is about 1, and the plum double-petal is presumed to be the quality trait in combination with the genetic rule of peach and Chinese rose double-petal. In the analysis based on the dominant trait of the double-lobe, the Manhattan plot of Δ (All-index) between the pools of single-lobe and double-lobe progeny was found to show a distinct peak on chromosome 1 (FIG. 2). With further reference to the Dougherty et al (2018) method, the mutation sites with Δ (All-index) of 0.4-0.6 were screened and density maps were drawn, and the results showed that the mutation density in the region Chr 1. The candidate region contains 17490 variation sites with delta (all-index) of 0.4-0.6, including 15599 SNP sites and 1891 InDel sites.
4. Development of InDel markers within localization Interval
196 InDel sites with the length larger than 10bp are randomly selected for preliminary screening. According to the plum blossom reference genome information, the sites obtained by screening are positioned on a genome, 200bp base sequences of the upstream and downstream of the InDel site are taken, primers are designed by utilizing Primer Premier 5.0 software, the size of the product is 100-450bp, and 172 pairs of primers are successfully designed. The materials used for primary screening of the InDel primer are 'sheet jelly palace powder', 'snow plum' x 'sheet jelly palace powder' F1 generation heavy valve mixing pool and F1 generation single valve mixing pool, and DNA of the InDel primer is extracted for PCR amplification. During PCR amplification, the PCR amplification system comprises 1 mu L of 50 ng/mu LDNA template, 10 mu L of 2 xTaq PCR mix,1 mu L of each of 10 mu M upstream and downstream primers and ddH 2 The content of O is filled to 20 mu L. The PCR reaction program is: pre-denaturation at 94 ℃ for 3min; denaturation at 94 ℃ for 30s, annealing at 51 ℃ for 30s, extension at 72 ℃ for 1min,30 cycles; final extension at 72 ℃ for 5min and storage at 4 ℃. The amplification product is detected by 1% agarose gel electrophoresis, and after detection, 172 pairs of primers are successfully amplified. The amplification product was then detected by 8% polyacrylamide gel electrophoresis, and 52 pairs of primers were detected to produce a polymorphic band between the single and double lobes. And (3) verifying 52 InDel primers obtained by preliminary screening by taking 10F 1 generation single-petal individuals for constructing the single-petal mixed pool and 10F 1 generation double-petal individuals for constructing the mixed pool as materials, and obtaining two InDel molecular markers linked with the plum blossom single-petal and double-petal characters through verification.
Example 2 verification of InDel molecular marker related to plum blossom single and double petal traits
1. Experimental materials
69 plum blossom cultivars were used in this experiment, 47 heavy petaloid cultivars, 22 single petaloid cultivars (Table 1, FIG. 4), and all materials were stored in the Chinese plum blossom research center (Wuhan).
TABLE 1 information of 69 plum blossom varieties
2. Experimental method
Extraction of genomic DNA of the plum blossom cultivar was performed using the magenta HiPure SF Plant DNA Mini Kit. Performing PCR amplification by taking genomic DNA as a template and InDel9F/R or InDel90F/R as a primer, wherein the PCR amplification system adopts a 20 mu L amplification system and comprises the following steps: 50 ng/. Mu.L DNA template 1. Mu.L, 10. Mu.L 2 XTaq PCR mix, 10. Mu.M upstream and downstream primers 1. Mu.L each, ddH was added 2 O to 20. Mu.L. The PCR amplification reaction program is as follows: pre-denaturation at 94 ℃ for 3min; denaturation at 94 ℃ for 30s, annealing at 51 ℃ for 30s, extension at 72 ℃ for 1min, and 30 cycles; final extension at 72 ℃ for 5min. 8% polyacrylamide gel electrophoresis is used for detection, if a 204bp fragment is obtained by amplification of an InDel9F/R primer pair, the variety or the material to be identified is a double-petal plum blossom, and if a 234bp fragment is obtained by amplification of an InDel90F/R primer pair, the variety or the material to be identified is a double-petal plum blossom.
3. Results of the experiment
When the InDel9F/R primers are used for detecting the plum blossom, bands are amplified at the position of 204bp for 47 plum blossom double-petal varieties, and bands are not amplified at the position of 204bp for 22 plum blossom single-petal varieties (figure 5); when the InDel90F/R primers are used for detecting the plum blossom, bands are amplified at 234bp positions of 47 plum blossom double-petal varieties, and bands are not amplified at 234bp positions of 22 plum blossom single-petal varieties (figure 6). In conclusion, the accuracy rate of identifying the single-petal and double-petal characters in 69 plum blossom varieties by InDel9F/R and InDel90F/R is 100%, and the accuracy rate is higher.
Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.
Claims (7)
1. The InDel molecular marker for identifying the plum blossom single and double petal traits is characterized in that the InDel9 is positioned at the position of 5549358-5549382 bp of a plum blossom 1 chromosome with a Genome version of Prunus mume Genome v1.0 plum blossom 1, corresponds to the plum blossom single petal traits when the 5549358-5549382 bp of the plum blossom 1 chromosome is ATTTTCAAACTTGTACTTATCTAT, and corresponds to the plum blossom double petal traits when the 5549358-5549382 bp of the plum blossom 1 chromosome does not contain ATTTTCAAACTTGTACTTATCTAT.
2. The primer for amplifying the molecular marker of claim 1, which comprises an upstream primer shown as SEQ ID NO. 1 and a downstream primer shown as SEQ ID NO. 2.
3. The reagent or the kit for detecting the plum blossom single and double petal character is characterized by comprising the primer of claim 2.
4. Any one of the following uses of the molecular marker of claim 1, the primer of claim 2, or the detection reagent or kit of claim 3:
1) The method is used for identifying the characters of the plum blossom single and double petals;
2) The method is used for early prediction of the plum blossom single and double petal flower types;
3) Is used for plum blossom molecular marker assisted breeding.
5. The identification method of the plum blossom single and double petal character is characterized by comprising the following steps:
1) Extracting quincunx genome DNA to be detected;
2) Taking the DNA extracted in the step 1) as a template, and carrying out PCR amplification by using primers shown in SEQ ID NO. 1-2;
3) Analyzing the amplification product: if the size of the amplified product is 204bp, the plum blossom to be detected is the double-petal character.
6. The method of claim 5, wherein the PCR amplification system used in step (2) comprises: 50 ng/. Mu.L DNA template 1. Mu.L, 10. Mu.L 2 XTaq PCR mix, 10. Mu.M upstream and downstream primers 1. Mu.L each, ddH 2 The content of O is filled to 20 mu L.
7. The method of claim 5 or 6, wherein the PCR reaction procedure used in step (2) is: pre-denaturation at 94 ℃ for 3min; denaturation at 94 ℃ for 30s, annealing at 51 ℃ for 30s, extension at 72 ℃ for 40-60s, and 30 cycles; final extension at 72 ℃ for 5min and storage at 4 ℃.
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Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103184277A (en) * | 2011-12-30 | 2013-07-03 | 北京林业大学 | Plum blossom genetic map construction method |
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