CN116732223B - Litchi embryo character SNP molecular marker developed based on KASP technology and application thereof - Google Patents
Litchi embryo character SNP molecular marker developed based on KASP technology and application thereof Download PDFInfo
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- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
- C12Q1/6895—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for plants, fungi or algae
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Abstract
The invention belongs to the technical field of molecular biological engineering, and particularly relates to a litchi embryo character SNP molecular marker developed based on a KASP technology and application thereof. The SNP molecular marker is positioned at 41766403bp base of chromosome 1 of litchi, the base is G or C, if the base is C genotype, the litchi embryo character is judged to be large-core or low-pyronucleosis variety, and if the base is G genotype, the litchi embryo character is judged to be pyronucleosis or small-core type. Through utilizing genome resequencing data of litchi resources, linkage disequilibrium analysis and whole genome association analysis are carried out, and field phenotype data are combined, SNP loci with remarkable correlation to pyronuclear characters in a population are located, and then a KASP genotyping method is utilized to develop a universal stable SNP molecular marker. The SNP molecular marker can be used for rapidly and accurately judging the embryo character of the target litchi seeds in the seedling stage, and screening out the germplasm resources with the required character.
Description
Technical Field
The invention belongs to the technical field of molecular biological engineering, and particularly relates to a litchi embryo character SNP molecular marker developed based on a KASP technology and application thereof.
Background
The abortion of the embryo of the fruit tree refers to the phenomenon that the fertilized zygote embryo is influenced by the zygote embryo or other factors to stop developing midway, so that the seed is degenerated, dysplasia (small nucleus) and even complete abortion (no nucleus) are caused. Embryo abortion occurs naturally in many fruit trees, such as citrus, grape, loquat, pear, date, persimmon, etc. (Zhang Wenying, etc., 2018). The seedless, less-seedless, small-seedless and the like are good target characters pursued by fruit tree cultivation and breeding, and can greatly improve the edible rate of fruits, improve the palatability of the fruits and improve the processing performance. However, the phenomena of few nuclei and no nuclei caused after embryo abortion generally affect fruit development, such as decreasing fruit setting rate, decreasing fruit size, and the like, thereby affecting yield. In addition, embryo abortion limits the choice of fruit tree cross-breeding parents.
Litchi is a famous Ling nan jia fruit in China and is favored by fresh food consumption and drying industry. Most litchi germplasm fruits usually have a large inedible pit, so that photosynthetic products are wasted without end, and the edibility is seriously affected. The edibility rate of common large-core varieties such as black leaves, branches and the like is less than 65 percent. However, there are also "burnt core" varieties of embryo abortion in litchi, such as 'glutinous rice cake', 'XianchengFeng' and the like, with fruit edibility exceeding 80%, and the market price of 'glutinous rice cake', 'XianchengFeng' often being 4 to 10 times higher than that of 'black leaf' or 'Huai branch'. Undoubtedly, the burnt leechee is popular with fruit growers and consumers. Therefore, the molecular genetic basis formed by the scorched pits of the litchi fruits is analyzed, excellent mutation sites and key genes are discovered, related molecular markers are developed, and the molecular markers are applied to production and breeding practices, so that the molecular genetic marker has important theoretical and practical significance for improving litchi production benefits, promoting litchi consumption and the like.
Disclosure of Invention
Aiming at the problems, the invention aims to provide a litchi embryo character SNP molecular marker developed based on a KASP technology and application thereof.
The technical content of the invention is as follows:
the invention provides a litchi embryo character SNP molecular marker developed based on KASP technology, wherein the SNP molecular marker is positioned at 41766403bp base of No.1 litchi chromosome, and the base is G or C;
judging that the embryo character of the litchi seed is a variety with large nucleus or low pyronuclear rate if the base at the position is C genotype;
if the base at the position is G genotype, judging that the embryo character of the litchi seed is pyronuclear or small-sized variety.
The invention also provides a primer pair of the litchi embryo character SNP molecular marker developed based on the KASP technology, wherein the nucleic acid sequence of a forward primer of the primer pair is shown as SEQ ID NO.1 and SEQ ID NO.2, and the nucleic acid sequence of a reverse primer is shown as SEQ ID NO. 3;
the 3' end of the forward primer is linked with a fluorescent molecule FAM label;
the 5' end of the reverse primer is linked with a fluorescent molecule HEX label.
The invention also provides application of the SNP molecular marker or the primer pair thereof for judging or assisting in judging the embryo character of the litchi seeds, which is developed based on the KASP technology;
the embryo character comprises a large nucleus or a coke nucleus.
The invention also provides application of the litchi embryo character SNP molecular marker developed based on the KASP technology, wherein the SNP molecular marker is used for litchi breeding.
The invention also provides a method for judging or assisting in judging the embryo character of litchi seeds by using the SNP molecular marker or the primer pair thereof, which comprises the following steps:
SNP genotyping detection based on KSAP technology is carried out by utilizing a high-throughput InteliQube genotyping detection platform, and the litchi embryo character is judged according to the obtained genotype, and the method specifically comprises the following steps:
1) Taking DNA of a litchi sample, and performing parting detection after quality inspection;
measuring the DNA concentration and quality of litchi samples, and uniformly diluting all the samples to be measured to a proper upper concentration (about 5-10 ng/. Mu.L) according to the measurement result;
and subpackaging the DNA sample;
2) Preparing KASP genotyping mixed solution
Comprises a DNA sample, 2 XKASP Master mix+assay;
3) Adding KASP genotyping mixture to array tape membrane containing DNA template
Programming by utilizing an SNP gene detection platform of an IntelliQube, sequentially placing 384Array tape and a KASP genotyping mixed solution of a DNA sample plate into a machine, operating the machine to execute the program, and respectively and automatically carrying out the process of split charging and film sealing of the DNA sample diluent and the KASP genotyping mixed solution into 384-hole Array tape;
4) Performing PCR circulation reaction
The reaction procedure was 94℃for 15min of pre-denaturation, 1 cycle; denaturation at 94℃for 20sec, renaturation/extension at 61-55℃for 60sec (-0.6 ℃/cycle), 10 cycles; denaturation at 94℃for 20sec, renaturation/extension at 55℃for 60sec,26 cycles;
after the PCR reaction is finished, reading and analyzing fluorescence data by using an IntelliQube machine;
in the SPN typing result, the litchi embryo character is judged to be large nucleus if the type is C genotype, and the litchi embryo character is judged to be pyronuclear or small nucleus if the type is G genotype according to the genotype difference (different fluorescence emission) of the amplified fragments of the PCR amplification and the primer.
The beneficial effects of the invention are as follows:
the invention discloses a SNP molecular marker related to litchi embryo traits developed based on KASP technology, which is a universal stable SNP molecular marker developed by utilizing genome re-sequencing data (filtered SNPs) of litchi resources, carrying out Linkage Disequilibrium (LD) analysis and whole genome association analysis (GWAS), combining field phenotype data, and positioning SNP loci with remarkable related coke nucleus traits in a population, and then utilizing a KASP genotyping method. The SNP molecular marker successfully carries out genotyping on litchi resource varieties, and the effectiveness of the molecular marker is verified. The SNP molecular marker can be used for rapidly and accurately judging the embryo character of the target litchi seeds in the seedling stage, and screening out the germplasm resources with the required character. The invention provides effective technical support for litchi molecular breeding and improves breeding efficiency.
Drawings
FIG. 1 is a schematic diagram of litchi seed and coke seed variety materials in an embodiment of the invention;
FIG. 2 is a full genome association analysis of litchi embryo traits in an embodiment of the invention;
FIG. 3 shows genotyping detection of litchi samples using SNP molecular markers based on KSAP technology in an embodiment of the invention;
FIG. 4 is a graph showing statistical results of the phenotype associated with the litchi germplasm in the embodiment of the invention.
Detailed Description
The invention is described in further detail below with reference to specific embodiments and the accompanying drawings, it being understood that these embodiments are only for the purpose of illustrating the invention and not for the purpose of limiting the same, and that various modifications of the invention, which are equivalent to those skilled in the art, will fall within the scope of the appended claims after reading the present invention.
All materials and reagents of the invention are materials and reagents of the conventional market unless specified otherwise.
Examples
Development and application of litchi embryo character SNP molecular marker developed based on KASP technology
1. Construction of litchi char nucleus character related phenotype database
276 parts of litchi core germplasm resource large-core type and burnt-core type varieties are used as experimental materials. Are planted in fruit tree germplasm-Guangzhou litchi nursery (North latitude 23 DEG 09', east longitude 113 DEG 22', altitude 20 m) and breeding nursery. The tree age of the germplasm resources is more than 15 years, the filial generation grafting (old tree) in the childhood period is partially finished for more than 5 years, the soil and fertilizer management is consistent, and the seedlings can bloom normally and bear fruits each year.
As shown in fig. 1, the upper panel is Jiao He trait and the lower panel is large nuclear trait;
a representative batch of relatively stable-trait coke-core material 26 parts and large-core material 23 parts was screened by performing statistical analysis of the correlation, frequency distribution, etc. of the germ-line phenotype data of 276 parts of core germplasm, as shown in Table 1.
TABLE 1 statistics of longitudinal diameter and Coke Nuclear Rate of seeds of large Nuclear and Coke Nuclear type materials
2. Mining litchi pyronuclear character excellent allelic variation point position based on resequencing and GWAS analysis
Deep resequencing is carried out on 276 parts of litchi core germplasm, feizixiao is taken as a reference genome (Feizixiao litchi genome), and SNP (single nucleotide polymorphism) tapering is carried out on resequencing data by using BWA software and GATK software packages, and the specific steps are as follows:
each mutation detection (variant rolling) is carried out by the biplotypeCaller in the GATK software, mainly comprising SNP, and further correction is carried out. Deleting SNPs site sequences larger than MAF < 0.005, retaining SNPs with deletion rate less than or equal to 0.2, converting into VCF file by using VCFtools software (Version 0.1.112a), and performing LD correlation (r) by using PLINK software (Version 1.07) 2 ) Calculating, wherein parameters are set as follows: -file-r 2-ld-window 99999-ld-window-kb 1000-out, obtaining r from between two single nucleotide polymorphisms 2 And the distance between two single nucleotide polymorphisms. SNPs with the deletion rate less than or equal to 0.2 obtained by the early screening are subjected to association analysis of phenotype data related to the pyronuclear character, and the obviously related SNP loci are screened according to the association value (p-value). UsingThe BLINK algorithm and the FarmCPU algorithm are simultaneously associated, a p-value is calculated, and the algorithm is completed by using Bayesian information, a linkage disequilibrium iteration nested key slot (Bayesian-information and Linkage-disequilibrium Iteratively Nested Keyway, BLINK) software package and a fixed and random model (Fixed and random model Circulating Probability Unification, farmCPU) software package with unified cyclic probability respectively, wherein the results are shown in the following table:
TABLE 2 sequencing data and SNPs classification evaluation Table for 276 parts of litchi germplasm resources
| Sequencing resource count | 276 |
| Sequencing depth | 16X |
| Sequencing data | 1.416T |
| Mutation site | 89,836,921 |
| SNP locus | 76,796,261 |
| Double allele SNP locus | 65,001,265 |
| High quality SNP loci | 7,246,647 |
According to 7,246,647 high-quality SNP markers obtained by the 276 core germplasm re-sequencing, the result is shown in figure 2, GWAS analysis is carried out on the relevant phenotype data of the pyronuclear character and positioned to 41766403bp basic group of a No.1 litchi chromosome, the basic group is G or C, namely SNP Chr1_41766403 point genotype is CC or CG, and statistical analysis is further carried out on the relevant phenotype of litchi germplasm containing two genotypes (CC/CG), as shown in figure 4, the result shows that significant difference exists between the two types, the CC genotype is a large-core type or a germplasm with lower pyronuclear rate, the CG genotype is a pyronuclear type or a small-core germplasm, and the fact that the genotypes and the phenotypes are consistent is also shown;
namely, the SNP molecular marker is positioned at 41766403bp basic group of the No.1 litchi chromosome, the basic group at the position is G or C, if the basic group at the position is C genotype, the litchi embryo character is judged to be large-core or low-scorched nuclear rate variety, and if the basic group at the position is G genotype, the litchi embryo character is judged to be scorched nuclear type or small-core type.
KASP genotyping detection of candidate region SNPs loci
3.1 Design of KASP primer
Selecting sequences of 50bp respectively at the upstream and downstream of SNP loci associated with significant signals and candidate gene regions as Primer synthesis reference templates, respectively synthesizing 2 allele specific competitive forward primers (the forward Primer nucleotide sequences are shown as SEQ ID NO.1 and SEQ ID NO. 2) and 1 common reverse Primer (the reverse Primer nucleotide sequences are shown as SEQ ID NO. 3) by using allele variation loci with two different terminal bases, and mixing to form a Primer Mix;
the 3' end of the forward primer is linked with a fluorescent molecule FAM label; the 5' end of the reverse primer is linked with a fluorescent molecule HEX label;
3.2 Application of KASP primer
a) DNA sample plate: taking 49 litchi DNA samples in Table 1, performing parting detection, performing DNA concentration measurement and quality measurement on the samples to be detected by using a Biodrop uLite nucleic acid microassay, and uniformly coefficient all the samples to be detected to about a proper upper concentration (5-10 ng/. Mu.L) according to detection results;
the sample to be measured is added to a 96-well PCR plate after being regulated to a proper upper concentration, and two negative controls are added to each plate;
b) Preparing KASP genotyping mixed liquor, and the following table is provided:
TABLE 1PCR reaction System
c) Adding KASP genotyping mixture to array tape membrane containing DNA template
Programming by utilizing an SNP gene detection platform of an IntelliQube, sequentially placing 384Array tape, a DNA sample plate and KASP genotyping mixed solution into a machine, operating the machine to execute the program, and respectively and automatically carrying out the processes of diluting the DNA sample and split charging and sealing the KASP genotyping mixed solution into 384-hole Array tape;
d) PCR cycle was performed
The PCR reaction can be performed in a water bath PCR mode of SNP genotyping inline (when single membranes) in an IntelliQube machine;
the program settings are shown in the following table:
TABLE 2PCR reaction procedure
After the PCR reaction was completed, fluorescence data were read and analyzed by an IntelliQube machine.
The results are shown in FIG. 3, and the phenotype statistics of the transverse diameters and the pyronuclear rates of the large-core germplasm and the pyronuclear germplasm which are correspondingly detected show that the genotypes are consistent with the phenotypes, and different genotypes show obvious differences.
Claims (6)
1. The litchi embryo character SNP molecular marker developed based on the KASP technology is characterized in that the SNP molecular marker is positioned at 41766403bp basic group of chromosome 1 of litchi, wherein the basic group is G or C;
judging that the embryo character of the litchi seed is a variety with large nucleus or low pyronuclear rate if the base at the position is C genotype;
judging that the embryo character of the litchi seed is a pyronuclear type or a micronuclear type variety if the base at the position is G genotype;
the nucleic acid sequence of the forward primer of the primer pair based on the SNP molecular marker of the litchi embryo character developed by the KASP technology is shown as SEQ ID NO.1 and SEQ ID NO.2, and the nucleic acid sequence of the reverse primer is shown as SEQ ID NO. 3.
2. The application of the litchi seed embryo character SNP molecular marker developed based on the KASP technology as set forth in claim 1, wherein the SNP molecular marker is used for judging or assisting in judging the litchi seed embryo character, and the seed embryo character comprises a large nucleus or a coke nucleus.
3. The application of the primer pair of the SNP molecular marker for the litchi embryo character developed based on the KASP technology as set forth in claim 1, wherein the primer pair of the SNP molecular marker is used for judging or assisting in judging the litchi embryo character;
the embryo character comprises a large nucleus or a coke nucleus.
4. A method for determining or aiding in determining a litchi embryo trait using the primer set of claim 3, comprising the steps of:
1) Taking DNA of a litchi sample, and performing parting detection after quality inspection;
measuring the DNA concentration and quality of litchi samples, and uniformly diluting all the samples to be measured to a proper upper concentration according to the measurement result;
and subpackaging the DNA sample;
2) Preparing KASP genotyping mixed solution
Comprises a DNA sample, 2 XKASP Master mix+assay;
3) Adding KASP genotyping mixture to array tape membrane containing DNA template
Programming by utilizing an SNP gene detection platform of an IntelliQube, sequentially placing 384Array tape and a KASP genotyping mixed solution of a DNA sample plate into a machine, operating the machine to execute the program, and respectively and automatically carrying out the process of split charging and film sealing of the DNA sample diluent and the KASP genotyping mixed solution into 384-hole Array tape;
4) Performing PCR circulation reaction
After the PCR reaction is finished, reading and analyzing fluorescence data by using an IntelliQube machine;
the amplified fragment difference of the primer pair according to claim 1, and the fluorescence is different, thereby completing typing detection.
5. The method for judging or assisting in judging litchi embryo characteristics according to the primer pair of claim 4, wherein the PCR reaction procedure of the step 4) is 94 ℃ pre-denaturation for 15min and 1 cycle; denaturation at 94 ℃ for 20sec, renaturation/extension at 61-55 ℃ for 60sec, 10 cycles, -0.6 ℃/cycle; denaturation at 94℃for 20sec, renaturation/extension at 55℃for 60sec,26 cycles.
6. The method according to claim 4, wherein the step 4) is performed to determine the embryo shape of litchi seed as large nucleus if the embryo shape is C genotype, and the step G genotype if the embryo shape is C genotype.
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| CN113249510A (en) * | 2021-03-03 | 2021-08-13 | 北京农业生物技术研究中心 | Method for identifying authenticity of lettuce hybrid and KASP primer combination used by method |
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| Identifying Litchi (Litchi chinensis Sonn.) Cultivars and Their Genetic Relationships Using Single Nucleotide Polymorphism (SNP) Markers;Wei Liu;《PLoS One .》;第10卷(第8期);e0135390 * |
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| Two divergent haplotypes from a highly heterozygous lychee genome suggest independent domestication events for early and late-maturing cultivars;Guibing Hu;《Nat Genet》;第54卷(第1期);第73-83页 * |
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| CN116732223A (en) | 2023-09-12 |
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