CN116855629A - SNP molecular marker for judging embryo coke nucleus character of litchi seeds and application thereof - Google Patents
SNP molecular marker for judging embryo coke nucleus character of litchi seeds and application thereof Download PDFInfo
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Abstract
The application belongs to the technical field of molecular biological engineering, and particularly relates to an SNP molecular marker for judging the scorched nuclear character of litchi seed embryos and application thereof. The SNP molecular marker is positioned at 17330407bp base of the No.3 litchi chromosome, the base is G or A, if the base is G and the genotype is GG, the variety with large seediness or low pyronuclear rate and unstable pyronuclear property of the litchi embryo is judged, and if the base is A and the genotype is GA/AA, the variety with high Jiao He rate and stable pyronuclear property of the litchi embryo is judged. The genome re-sequencing data (filtered SNPs) of litchi resources are utilized to carry out linkage disequilibrium and whole genome association analysis, and the SNP loci which are obviously related to the pyronuclear characters in the population are positioned by combining with field phenotype data, and then a KASP genotyping method is utilized to develop an efficient, universal and stable SNP molecular marker.
Description
Technical Field
The application belongs to the technical field of molecular biological engineering, and particularly relates to an SNP molecular marker for judging the scorched nuclear character of litchi seed embryos 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 (Litchi Chinensis sonn.) is a plant of the genus Litchi (Litchi sonn.) of the family Sapindaceae. The litchi is native in China and has the longest cultivation history in the world in China, so China is the world litchi genetic resource diversity center, and the collection and preservation of the national litchi germplasm resource nursery (Guangzhou) has 652 parts in total. The development degree and the seed size of the litchi embryo have rich genetic diversity, and can be classified into 3 types according to the embryo development condition of litchi resources (figure 1): (1) normal large karyotype: embryo development is normal, mature seeds are big and full, such as 'black leaf', 'big seed', 'arm', 'wild litchi No. 10', and the like; (2) stabilization of the focal karyotype: the seeds are almost completely aborted, embryo development is blocked, the embryo is stopped in the middle, mature seeds are tiny, and the aborting rate is very high, namely 'burnt nuclei', such as 'iced litchi', 'glutinous rice cake', 'Xiancheng Feng', 'guanyin green', and the like. The two types of characters are stable and are not easily influenced by environment. (3) Partial abortion (Jiao He)/large karyotype: the embryo development is different in different clusters of the same plant and even in different fruits of the same cluster, the fruits are continuously distributed from normal large-stone fruits to Jiao Heguo and even seedless fruits, such as 'cassia taste', 'seedless litchi', 'gramineous shrimp cluster', and the like, the variety characters are unstable and are easily influenced by environment, and the degree of fruit abortion and the proportion of the abortion are greatly different from pollen, temperature and year.
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 "burnt core" varieties with high embryo abortion rate and stable properties, such as 'nux-zil', 'XianchengFeng' with fruit edibility more than 80%, and 'nux-zil', 'XianchengFeng' with market price 4 to 10 times higher than '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
The application aims to provide an SNP molecular marker for judging the scorched nuclear character of litchi embryo and application thereof.
The technical content of the application is as follows:
the application provides an SNP molecular marker for judging the scorched nuclear character of litchi embryo, which is positioned at 17330407bp base of No.3 litchi chromosome, wherein the base is G or A;
if the base at the position is G and the genotype is GG, judging that the embryo character of the litchi seed is large nucleus or the coke nucleus rate is low and the coke nucleus/large nucleus character is unstable
If the base is A and the genotype is GA or AA, judging that the litchi embryo character is pyronuclear type with high pyronuclear rate and stable character.
The application also provides a primer pair for judging SNP molecular markers of the embryo pyronuclear character of litchi seeds, 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 application also provides application of the SNP molecular marker or the primer pair thereof for judging the embryo coke nucleus property of the litchi seeds, wherein the SNP molecular marker or the primer pair thereof is used for judging or assisting in judging the embryo property of the litchi seeds;
the embryo character includes stable large karyotype, unstable large karyotype/coke karyotype and stable coke karyotype.
The application also provides application of the SNP molecular marker for judging the embryo and core characteristics of the litchi seeds, and the SNP molecular marker is used for litchi breeding.
The application 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;
if the base at the position is G and the genotype is GG, judging that the embryo character of the litchi seed is large nucleus or the coke nucleus rate is low and the coke nucleus/large nucleus character is unstable
If the base is A and the genotype is GA or AA, judging that the litchi embryo character is pyronuclear type with high pyronuclear rate and stable character.
The beneficial effects of the application are as follows:
the SNP molecular marker for judging the embryo and core characteristics of the litchi seeds is positioned at 17330407bp basic group of chromosome 3 of litchi, wherein the basic group is G or A; if the base at the position is G and the genotype is GG, judging that the embryo character of the litchi seed is large or the coke nucleus rate is low and the coke nucleus/large nucleus character is unstable, and if the base at the position is A and the genotype is GA or AA, judging that the embryo character of the litchi seed is high and the character is stable; the SNP molecular marker is a universal stable SNP molecular marker which is developed by utilizing genome resequencing data (filtered SNPs) of litchi resources, carrying out Linkage Disequilibrium (LD) analysis and whole genome association analysis (GWAS), combining field phenotype data, positioning SNP loci with remarkable correlation of pyronuclear characters in a population, and then utilizing a KASP genotyping method. The SNP molecular marker successfully carries out genotyping on different 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 development type character of the target litchi embryo in the seedling stage, and screening out the target excellent and heterogeneous germplasm with high coke nucleus rate and stable character. The application provides effective technical support for litchi molecular breeding and improves breeding efficiency.
Drawings
FIG. 1 is a schematic diagram of a litchi seed and coke seed type material in an embodiment of the application;
FIG. 2 is a genome-wide association analysis of litchi embryo pyronuclear trait related phenotype data in an embodiment of the present application;
FIG. 3 statistical analysis of phenotype data associated with different embryo type pyronuclear traits;
FIG. 4 shows genotyping detection of litchi samples using SNP molecular markers based on KSAP technology in an embodiment of the application.
Detailed Description
The application 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 application and not for the purpose of limiting the same, and that various modifications of the application, which are equivalent to those skilled in the art, will fall within the scope of the appended claims after reading the present application.
All materials and reagents of the application are materials and reagents of the conventional market unless specified otherwise.
Examples
Development and application of SNP molecular marker for judging embryo coke nucleus character of litchi seeds
1. Construction of litchi char nucleus character related phenotype database
276 parts of litchi core germplasm resource varieties are used as experimental materials. Are planted in the national fruit tree germplasm-Guangzhou litchi nursery (North latitude 23 DEG 09', east longitude 113 DEG 22', altitude 20 m) and the 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.
A batch of representative large-core or core-type unstable materials 38 parts, 9 parts of stable coke core type materials and 3 parts of relatively stable coke core type materials are screened out through carrying out correlation, frequency distribution and other statistical analysis on the germ plasm phenotype data of 276 parts of core germplasm, and the table 1 shows.
TABLE 1 seed weight, percentage of core weight, seed longitudinal diameter, seed transverse diameter and seed lateral diameter of large-core, coke-core and relatively stable coke-core type seeds, statistics of coke-core rate
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). Using BLINKThe method comprises the steps of simultaneously correlating the two algorithms with a FarmCPU, calculating a p-value, and respectively completing a Bayesian information and 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, 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 |
By using 7,246,647 high-quality SNP markers obtained by the above 276 core germplasm re-sequencing, as shown in a result of a multi-locus correlation analysis (Blink and FarmCPU algorithm model) method shown in a figure 2 (A, B, C, D, E, F is a single seed weight, a seed transverse diameter, a longitudinal diameter, a lateral diameter, a Jiao He rate and a nuclear weight percentage respectively), the above-mentioned scorched nuclear trait related phenotype data are subjected to GWAS analysis, and positioned to a 17330407bp base of a 3 rd chromosome, wherein the base is G or A, namely SNP Chr3_17330407 point genotype is GG, GA and AA, and further statistical analysis is performed on a litchi germplasm correlation phenotype containing three types of genotypes (GG/GA/AA), as shown in a figure 3, and the result shows that significant difference exists between the two types, the GG genotype is a large-sized nuclear or a germplasm with a low scorched nuclear rate and unstable trait, namely, the SNP molecular marker is positioned to a 17330407bp base of a 3 rd chromosome, the base is G or A, and if the genetic genotype is a large-sized nuclear embryo is a nuclear embryo, and the genetic embryo is a stable trait embryo is a high, and if the genetic embryo is a stable genetic embryo is a nuclear embryo is a high, and the genetic embryo is a stable trait embryo is a.
KASP genotyping detection of candidate region SNPs loci
3.1 Design of KASP primer
Selecting 300bp sequences on 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 50 parts of litchi DNA samples (containing germplasm of all embryo development types), performing typing 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 1 PCR 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 2 PCR reaction procedure
After the PCR reaction was completed, fluorescence data were read and analyzed by an IntelliQube machine.
The results are shown in FIG. 4, in which the upper left pyronuclear stable AA genotype and the lower right pyronuclear GG genotype are relatively stable GA genotype and metakaryotic GG genotype, respectively, and the genotypes are consistent with the phenotypes and the different genotypes show significant differences according to the phenotype statistics of the transverse diameters and the pyronuclear rates of the metakaryotic germplasm and the metakaryotic germplasm which are detected correspondingly.
Claims (10)
1. The SNP molecular marker for judging the embryo and core characteristics of litchi seeds is characterized in that the SNP molecular marker is positioned at 17330407bp base of chromosome 3 of litchi, the base at the locus is G or A, and the corresponding genotypes are GG, GA and AA;
if the locus genotype is GG, judging that the embryo character of the litchi seed is large nucleus or the coke nucleus rate is low and the coke nucleus/large nucleus character is unstable;
if the locus genotype is GA or AA, judging that the lychee pyronuclear rate is high and the pyronuclear character is stable.
2. The application of the SNP molecular marker for judging the embryo coke nucleus character of the litchi seeds is characterized in that the SNP molecular marker is used for judging or assisting in judging the development type character of the litchi seeds;
the embryo character includes stable large karyotype, unstable large karyotype/coke karyotype and stable coke karyotype.
3. The application of the SNP molecular marker for judging the embryo pyronuclear character of the litchi seeds is characterized in that the SNP molecular marker is used for assisting litchi breeding, improving the breeding efficiency and shortening the breeding period.
4. A primer pair for judging SNP molecular markers of embryo pyronuclear characters of litchi seeds is characterized in that 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.
5. The primer pair for judging SNP molecular markers of litchi embryo pyronuclear properties according to claim 4, wherein the 3' end of the forward primer is linked with fluorescent molecular FAM markers.
6. The primer pair for judging SNP molecular markers of litchi embryo pyronuclear properties according to claim 4, wherein the 5' end of the reverse primer is linked with fluorescent molecular HEX markers.
7. The application of the primer of the SNP molecular marker for judging the embryo coke nucleus property of the litchi seeds is characterized in that the SNP molecular marker or the primer thereof is used for judging or assisting in judging the development type property of the litchi seeds;
the embryo character includes stable large karyotype, unstable large karyotype/coke karyotype and stable coke karyotype.
8. A method for judging or aiding in judging the type of a litchi seed embryo trait using the SNP molecular marker or primer pair thereof as set forth in claim 7, 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 method of claim 4, wherein the amplified fragments of the primer pair are different in fluorescence, and typing detection is completed.
9. The method for judging or assisting in judging litchi seed embryo characteristics by using the SNP molecular marker or the primer pair thereof according to claim 8, wherein the PCR reaction procedure in the step 4) is 94 ℃ pre-denaturation for 15min and 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.
10. The method for judging or assisting in judging the embryo properties of litchi seeds according to the SNP molecular marker or the primer pair thereof as set forth in claim 8, wherein in the SPN typing result obtained by the detection in the step 4), if the locus genotype is GG, judging that the embryo properties of litchi seeds are large nuclei or low in pyronuclear rate and unstable in pyronuclear/large nuclear properties;
if the locus genotype is GA or AA, judging that the litchi embryo character is the pyrokaryotype with high pyrokaryotype rate and stable character.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102174510A (en) * | 2011-01-06 | 2011-09-07 | 广东省农业科学院果树研究所 | Method for constructing lichi core collection by using EST-SSR (Expresses Sequence Tag-Simple Sequence Repeat) molecular marker |
| CN115927731A (en) * | 2022-12-15 | 2023-04-07 | 海南大学 | SNP (Single nucleotide polymorphism) site combination for constructing litchi SNP fingerprint, application and identification method |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102174510A (en) * | 2011-01-06 | 2011-09-07 | 广东省农业科学院果树研究所 | Method for constructing lichi core collection by using EST-SSR (Expresses Sequence Tag-Simple Sequence Repeat) molecular marker |
| CN115927731A (en) * | 2022-12-15 | 2023-04-07 | 海南大学 | SNP (Single nucleotide polymorphism) site combination for constructing litchi SNP fingerprint, application and identification method |
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| FENG, J等上传: "GenBank: CM034463.1，Litchi chinensis cultivar FZX ecotype Cultivated chromosome 3, whole genome shotgun sequence", 《NCBI GENBANK》, pages 1 - 2 * |
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