CN117965787B - SNP (Single nucleotide polymorphism) marker and primer set for identifying authenticity of pineapple Josapine and MD2 hybrid and application of SNP marker and primer set - Google Patents
SNP (Single nucleotide polymorphism) marker and primer set for identifying authenticity of pineapple Josapine and MD2 hybrid and application of SNP marker and primer set Download PDFInfo
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- 235000007119 Ananas comosus Nutrition 0.000 title claims abstract description 43
- 239000003550 marker Substances 0.000 title claims abstract description 25
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- 238000005516 engineering process Methods 0.000 description 5
- 239000000463 material Substances 0.000 description 5
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- 244000141359 Malus pumila Species 0.000 description 1
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Abstract
The invention belongs to the technical field of pineapple hybridization breeding, and particularly relates to SNP markers and primer sets for identifying authenticity of pineapple Josapine and MD2 hybrids and application thereof. The SNP marker is characterized by comprising one or two groups of SNP markers: (a) 3376404 located on the LG6 chromosome, genotype G/C; (b) 5578047 located on the LG18 chromosome, genotype A/G. The primer group designed based on the SNP marker can rapidly and effectively identify the authenticity of the hybrid of the pineapple Josapine and the MD2, the identification result is accurate and reliable, and the genetic breeding of the pineapple is facilitated.
Description
Technical Field
The invention belongs to the technical field of pineapple hybridization breeding, and particularly relates to SNP markers and primer sets for identifying authenticity of pineapple Josapine and MD2 hybrids and application thereof.
Background
Pineapple is one of three tropical fruit trees in the world, china is one of ten major countries of pineapple, and is mainly distributed in provinces of Guangdong, hainan, yunnan, guangxi and Fujian, and is a prop industry of a hot area. However, the major cultivar in pineapple production is 'Bali', and the problems of concentrated harvest time, reduced quality and the like caused by single variety and degeneration are increasingly prominent in the history of nearly 100 years, and become one of the urgent problems of industry. The breeding and popularization of new varieties are effective means for adjusting the variety structure and improving the quality and efficiency of industry. At present, artificial hybridization is a main method for breeding new varieties of pineapple. But the breeding cycle is long, and false hybrids are easy to produce due to open pollination. The hybrid offspring is time-consuming and labor-consuming to identify the authenticity of the hybrid according to the phenotypic character of the field, is easily influenced by environment and human factors, and is more difficult to identify true and false hybrids by utilizing morphological characteristics as the morphological difference of the hybrid offspring is smaller and smaller along with the centralized use of parents.
The DNA molecular marker is used for identifying the identity of the variety, and has the advantages of accuracy, reliability, simplicity, rapidness, easiness in operation, good timeliness and the like. The method is widely applied to the identification of the authenticity of varieties of various fruit trees such as oranges, apples, grapes, cherries, peaches, kiwi fruits and the like. The international new plant variety protection consortium (UPOV) has recommended SSR marking technology and single nucleotide polymorphism technology (SNP) as two technologies for variety identification or seed purity identification in BMT molecular test guidelines. However, the SSR technology has the defects of fewer detection sites, low flux, poor result representativeness, difficult realization of data sharing and the like. SNP markers are distributed in the genome in high and uniform density, can realize high-throughput detection, and are more suitable for integration and sharing of databases. Therefore, the method is considered as the molecular marking technology with the most application prospect.
At present, the identification of the hybrid offspring of pineapple is mainly carried out by carrying out multi-point morphological characteristic observation on varieties for many years, and no report on the aspects of identification of the authenticity of hybrid of F 1 generation hybrid population by utilizing molecular markers exists.
Disclosure of Invention
Aiming at the problems, the invention provides a SNP marker for identifying the authenticity of the filial generation of 'Josapine' and 'MD2', which is used for identifying the authenticity of the hybrid of F 1 colony taking 'Josapine' and 'MD2' as parents, thereby laying a foundation for improving the efficiency of pineapple hybridization breeding and further developing genetic research.
In order to achieve the above purpose, the present invention may adopt the following technical scheme:
In one aspect, the invention provides a SNP marker for identifying the authenticity of pineapple Josapine and MD2 hybrids, comprising one or two sets of the following SNP markers: (a) 3376404 located on the LG6 chromosome, genotype G/C; (b) 5578047 located on the LG18 chromosome, genotype A/G.
In another aspect, the present invention provides a primer set for amplifying the above SNP markers for identifying the authenticity of hybrid pineapple Josapine and MD2, wherein the primer set for amplifying the above SNP marker (a) comprises sequences shown as SEQ ID No.1, SEQ ID No.2 and SEQ ID No. 3; the primer set for amplifying the SNP marker (a) comprises sequences shown as SEQ ID No.4, SEQ ID No.5 and SEQ ID No. 6.
In still another aspect, the present invention provides a detection reagent for identifying the aforementioned SNP markers identifying the authenticity of hybrids of pineapple Josapine and MD2, which is characterized by comprising the aforementioned primer set.
In still another aspect, the present invention provides a kit for detecting SNP markers for identifying the authenticity of hybrids of pineapple Josapine and MD2 as described above, comprising the above primer set or any one of the above detection reagents.
In a further aspect, the invention provides an SNP marker for identifying the authenticity of pineapple Josapine and MD2 hybrids, or a primer set, a detection reagent or a detection kit for identifying the authenticity of pineapple hybrids, and application of the SNP marker or the primer set, or the detection reagent kit in pineapple genetic breeding.
The beneficial effects of the invention at least comprise: the primer set designed based on the SNP marker for identifying the authenticity of the hybrid of the pineapple Josapine and the MD2 provided by the invention can rapidly and effectively identify the authenticity of the hybrid of the pineapple Josapine and the MD2, has accurate and reliable identification result, and is beneficial to genetic breeding of the pineapple.
Drawings
FIG. 1 shows the typing results of SNP20777 primers;
FIG. 2 shows the typing results of SNP16328 primers;
FIG. 3 shows the typing results of SNP25886 primers;
FIG. 4 shows the typing results of SNP30159 primers;
FIG. 5 shows the typing results of SNP4010 primers;
FIG. 6 shows the typing results of SNP22550 primers;
FIG. 7 shows the typing result A of 451-strain SNP 4010;
FIG. 8 shows typing results B of 451-strain SNP 4010;
FIG. 9 shows the typing result A of 451-strain single-strain SNP 22550;
FIG. 10 shows the typing result B of 451-strain single-strain SNP 22550;
FIG. 11 is a blade morphology of MJ 11;
FIG. 12 is a blade morphology of MJ 270;
FIG. 13 shows the sequencing results of MJ 11;
FIG. 14 shows the sequencing results of MJ 270;
FIG. 15 is a leaf morphology of MJ 183;
FIG. 16 is a leaf morphology of MJ 227;
FIG. 17 shows the sequencing results of MJ 183;
FIG. 18 shows the sequencing results of MJ 227;
FIG. 19 is a blade morphology of MJ 129;
FIG. 20 is a leaf morphology of MJ 173;
FIG. 21 shows the sequencing results of MJ 129;
FIG. 22 shows leaf morphology and sequencing results for MJ 173;
In fig. 1 to 6, the blue circle represents the genotype of 'Josapine', the red circle represents the genotype of 'MD2', and the green circle represents the genotype of the hybrid F 1 generation.
Detailed Description
The examples are presented for better illustration of the invention, but the invention is not limited to the examples. Those skilled in the art will appreciate that various modifications and adaptations of the embodiments described above are possible in light of the above teachings and are intended to be within the scope of the invention.
The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the disclosure. Unless the context clearly differs, singular forms of expression include plural forms of expression. As used herein, it is understood that terms such as "comprising," "having," "including," and the like are intended to indicate the presence of a feature, number, operation, component, part, element, material, or combination. The terms of the present invention are disclosed in the specification and are not intended to exclude the possibility that one or more other features, numbers, operations, components, elements, materials or combinations thereof may be present or added. As used herein, "/" may be interpreted as "and" or "as appropriate.
The embodiment of the invention provides an SNP marker for identifying the authenticity of pineapple Josapine and MD2 hybrids, which comprises one or two groups of SNP markers: (a) 3376404 located on the LG6 chromosome, genotype G/C; (b) 5578047 located on the LG18 chromosome, genotype A/G.
It should be noted that pineapple Josapine and MD2 are two pineapple varieties known in the art, and have no specific meaning; the hybrid in the present invention is an F1 hybrid of pineapple Josapine and MD 2. In addition, pineapple genome GCA_902162155.1 (http:// plants. Ensembl. Org/Ananas_comosus/Info/Index) is used as a reference genome in the present invention.
Another embodiment of the present invention provides a primer set for amplifying the above SNP markers for identifying the authenticity of hybrid of pineapple Josapine and MD2, wherein the primer set for amplifying the above SNP marker (a) comprises sequences shown as SEQ ID No.1, SEQ ID No.2 and SEQ ID No. 3; the primer set for amplifying the SNP marker (a) comprises sequences shown as SEQ ID No.4, SEQ ID No.5 and SEQ ID No. 6.
In still another embodiment, the present invention provides a detection reagent for identifying the above SNP markers for identifying the authenticity of hybrid pineapple Josapine and MD2, comprising the above primer set.
It should be noted that the above-mentioned primer set may be added with a common detection auxiliary reagent, which is known in the art, such as a buffer solution, etc., to prepare a detection reagent.
In some embodiments, the detection reagent may further comprise a fluorescent reagent. The genotyping may be performed by adding a fluorescent reagent to the above-mentioned detection reagent, specifically, a difference between the last bases at the 3' -end of the primer and a fluorescent signal value, distinguishing and amplifying a DNA fragment of a specific allele, and detecting a fluorescent signal value of a site to be detected.
In still another embodiment, the present invention provides a detection kit for identifying the above SNP markers for identifying the authenticity of hybrids of pineapple Josapine and MD2, which comprises the above primer set or any one of the above detection reagents. The detection reagent can be prepared into a kit form, and is convenient to carry and practical. The form of the kit is known to the person skilled in the art.
In a further embodiment, the invention provides an application of the SNP marker for identifying the authenticity of the hybrid of pineapple Josapine and MD2, the primer set, the detection reagent or the detection kit in pineapple genetic breeding.
In some embodiments, the above-described applications include: the SNP marker or primer set or detection reagent or detection kit is applied to the identification of the authenticity of the hybrid of pineapple Josapine and MD 2.
In some embodiments, the above-described applications include: when the genotype of the sample to be detected is homozygous genotype, the genotype is pseudo hybrid; and when the genotype of the sample to be detected is a heterozygous genotype, the genotype is a true hybrid.
For a better understanding of the present invention, the content of the present invention is further elucidated below in connection with the specific examples, but the content of the present invention is not limited to the examples below.
In the embodiment of the invention, the 'Josapine' is taken as a male parent, the 'MD2' is taken as a female parent, the hybridization is carried out by adopting an artificial pollination method in 3-4 months in 2020, and the hybridized offspring (with the number of JM1-JM 451) of 451 strains 'Josapine' and 'MD2' are obtained and planted in the south and inferior tropical crop institute of the national academy of tropical agriculture.
1. SNP primer screening
The modified CTAB method is used for extracting DNA of the parent and offspring leaf bases of pineapple. Based on the resequencing data of 'Josapine' and 'MD2', SNP homozygous sites with difference in parents were selected, 26 sets of primers were designed (see Table 1) (3 specific primers were designed using NCBI-Primer3, designated as Primer X, primer Y and Primer C, respectively, and the primers were sent to Shanghai Biotechnology).
TABLE 1 SNP marker information of homozygous sites with differences of Josapine 'and' MD2
Distinguishing and amplifying DNA fragments of specific alleles according to the difference of the last base at the 3' end of the primer and the fluorescence signal value, and detecting the fluorescence signal value of a site to be detected, so as to genotype; screening primers by using Josapine, MD2 and 40F 1 generation hybrid single plants as materials and using a PARMS-SNP labeling method, wherein SNP typing reagent PARMS is purchased from the Wuhan cloisonne organism; the PCR amplification reaction system is shown in Table 2 below.
TABLE 2 PCR reaction system
| Composition of the components | Volume of |
| 2×PARMS | 3ul |
| Primer X | 0.1ul |
| Primer Y | 0.15ul |
| Primer C | 0.15ul |
| DNA template | 1ul |
| Double distilled water | 1.5ul |
The PCR reactions were performed on ABI QuantStudio QS6 instrument and genotyping was performed on the instrument, and the reaction procedure is shown in Table 3.
TABLE 3PCR thermal cycling program
| Step (a) | Temperature (temperature) | Duration of time |
| 1 | 94℃ | 15min |
| 2 | 94℃ | 20s |
| 3 | 65 ℃ (0.7 ℃ Per cycle reduction) | 1min |
| 4 | Returning to step 2, 10 cycles | |
| 5 | 94℃ | 20s |
| 6 | 57℃ | 1min |
The results showed that the 26-group primers were distributed on 15 total chromosomes of LG1, LG2, LG3, LG5, LG6, LG7, LG8, LG11, LG13, LG14, LG15, LG18, LG19, LG21 and LG24, respectively. After PCR amplification, the 24 sets of primers did not distinguish 42 parts of material, and only SNP4010 and SNP22550 could distinguish 42 parts of material into 3 types, where blue represents the homozygous genotype of 'Josapine', red represents the homozygous genotype of 'MD2', and green represents the heterozygous genotype of hybrid F 1 (FIGS. 1-6). It was shown that SNP4010 and SNP22550 can be used for the authenticity identification of the 'Josapine' and 'MD2' crossing offspring (Table 4).
TABLE 4 SNP markers for identification of authenticity of ` Josapine ` and `MD 2` hybrids
2. Molecular characterization of hybrid authenticity
Performing PCR amplification on the hybridized F1 generation (with the number of JM1-JM 451) by using the screened primer groups 'Josapine', 'MD2' and 451 so as to genotype the genome DNA; counting the genotype of each sample, and if the genotype of the sample is a homozygous genotype, obtaining a false hybrid; when the genotype of the sample is heterozygous genotype, the genotype is true hybrid.
The results showed that each of the 2 markers divided 451 individuals into 3 genotypes. Among them, 17 and 24 individuals identical to the ` Josapine ` genotype, 26 and 15 individuals identical to the `MD 2` genotype were identified by using SNP4010 and SNP22550, respectively (FIGS. 7 to 10). By combining the two markers, 395 true hybrids and 56 false hybrids were identified together, with a true hybrid rate of 87.58% (Table 5).
Table 5 statistical results of identification of authenticity of the `MD2` X ` Josapine ` hybrid
| Hybrid combination (female) | F 1 Total (strain) | True hybrid (plant) | False hybrid (strain) | True hybrid Rate (%) |
| ‘MD2’בJosapine’ | 451 | 395 | 56 | 87.58 |
3. Hybrid authenticity verification
At 150bp before and after the candidate marker SNP22550 locus of chromosome 5578047 of 18, a pair of specific primers, named F22550 and R22550 (see Table 6 below), are randomly selected to carry out specific PCR amplification by true hybrids and false hybrids, and the PCR products are sequenced; when the sequencing peak diagram of the SNP locus is single peak, the SNP locus is homozygote genotype; when the SNP locus sequencing peak diagram is a cover peak, the SNP locus sequencing peak diagram is a heterozygous genotype.
TABLE 6 specific primers at 150bp each before and after SNP22550 site
| Primer name | Primer sequence (5 '-3') |
| F22550 | ATCATTCTCGCTTGCCTCCG |
| R22550 | TCCATGTAACTCCAGCATTTCAGA |
The primers were used to amplify the primers for PCR of the pseudohybrid MJ83, MJ11, MJ227, MJ270 and the true hybrid MJ129, MJ173, respectively, and the PCR products were sent to Shanghai Biotechnology Co. The results show that MJ11 and MJ270 are unimodal at position 5578047, with base A; MJ83 and MJ227 are unimodal, and the base is G; MJ129 and MJ173 are bimodal, with bases A/G (FIGS. 11-22). It is shown that MJ11, MJ270, MJ83 and MJ227 are false hybrids and MJ129 and MJ173 are true hybrids. The sequencing result is consistent with the SNP typing result, which indicates that the SNP typing result is stable and reliable.
Finally, it is noted that the above embodiments are only for illustrating the technical solution of the present invention and not for limiting the same, and although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications and equivalents may be made thereto without departing from the spirit and scope of the technical solution of the present invention, which is intended to be covered by the scope of the claims of the present invention.
Claims (6)
1. A primer set for identifying SNP markers of pineapple Josapine and MD2 hybrid authenticity, characterized in that the primer set for amplifying SNP marker (a) comprises sequences shown as SEQ ID No.1, SEQ ID No.2 and SEQ ID No. 3; the primer group for amplifying the SNP marker (b) comprises sequences shown as SEQ ID No.4, SEQ ID No.5 and SEQ ID No. 6; SNP marker (a) is located at 3376404 of LG6 chromosome, and the genotype is G/C; SNP marker (b) is located at 5578047 of LG18 chromosome, and the genotype is A/G; the SNP markers were determined from the pineapple genome GCA_ 902162155.1.
2. A detection reagent for SNP markers for identifying the authenticity of hybrid of pineapple Josapine and MD2, comprising the primer set according to claim 1.
3. A kit for detecting SNP markers for identifying the authenticity of hybrids of pineapple Josapine and MD2, comprising the primer set as set forth in claim 1 or the detection reagent as set forth in claim 2.
4. Use of the primer set of claim 1 or the detection reagent of claim 2 or the detection kit of claim 3 in pineapple genetic breeding.
5. The application according to claim 4, wherein the application comprises: the SNP marker or primer set or detection reagent or detection kit is applied to the identification of the authenticity of the hybrid of pineapple Josapine and MD 2.
6. The use according to claim 5, wherein the genotype of the sample to be tested is a homozygous genotype, then a false hybrid; and when the genotype of the sample to be detected is a heterozygous genotype, the genotype is a true hybrid.
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| CN115807122A (en) * | 2022-10-25 | 2023-03-17 | 中国热带农业科学院南亚热带作物研究所 | SNP molecular marker for pineapple seed resource identification and application thereof |
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| CN117089649B (en) * | 2023-10-16 | 2024-04-30 | 中国热带农业科学院南亚热带作物研究所 | SNP molecular marker related to citric acid content of pineapple fruits and application thereof |
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| CN115807122A (en) * | 2022-10-25 | 2023-03-17 | 中国热带农业科学院南亚热带作物研究所 | SNP molecular marker for pineapple seed resource identification and application thereof |
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