CN116694663A - Gene related to litchi pulp sugar accumulation type, molecular marker, primer pair and application thereof - Google Patents
Gene related to litchi pulp sugar accumulation type, molecular marker, primer pair and application thereof Download PDFInfo
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- CN116694663A CN116694663A CN202310657296.3A CN202310657296A CN116694663A CN 116694663 A CN116694663 A CN 116694663A CN 202310657296 A CN202310657296 A CN 202310657296A CN 116694663 A CN116694663 A CN 116694663A
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- 229930006000 Sucrose Natural products 0.000 claims abstract description 25
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- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 claims abstract description 22
- 210000000349 chromosome Anatomy 0.000 claims abstract description 7
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- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The application belongs to the technical field of biological genetic engineering, and particularly relates to a gene related to litchi pulp sugar accumulation type, a molecular marker, a primer pair and application thereof. The gene is a gene LITCHI009111 capable of hydrolyzing sucrose into reducing sugar on a chromosome 7 of LITCHI, and the nucleic acid sequence of the gene is shown as SEQ ID NO. 1; the molecular marker Chr7:22496380G:A is positioned on the 2 nd intron of a gene LITCHI009111 which can hydrolyze sucrose into reducing sugar on a chromosome 7 of LITCHI; the nucleic acid sequences of the primer pair of the molecular marker are respectively shown as SEQ ID NO.2 and SEQ ID NO.3, so that candidate litchi varieties can be rapidly screened, the sugar accumulation type of litchi pulp can be predicted, the sugar accumulation type of edible parts can be judged or assisted to be judged, the varieties containing target characters can be screened, the breeding period can be shortened, and the breeding efficiency of high-quality varieties can be improved.
Description
Technical Field
The application belongs to the technical field of biological genetic engineering, and particularly relates to a gene related to litchi pulp sugar accumulation type, a molecular marker, a primer pair and application thereof.
Background
Litchi (Lichi chinensis Sonn) is a evergreen fruit tree widely cultivated in subtropical areas, has abundant germplasm resources and has high use value and nutritive value. Litchi fruits lack storable carbohydrates (such as starch), and sugar content is increased mainly by the entry of soluble sugar during ripening. The litchi sugar component and the proportion thereof are one of important factors influencing the flavor of the edible part of litchi. The litchi aril, as the edible part of litchi, stores a large amount of soluble sugar, mainly in the form of sucrose and reducing sugar (fructose+glucose). Sugar components and proportions of the sugar components of different litchi varieties have larger difference, and litchi resources can be divided into three types of sucrose accumulation type, intermediate type and reducing sugar accumulation type according to the ratio of sucrose to reducing sugar. However, so far, little is known about the mechanism involved in the conversion of the sugar component of the edible part of litchi. Therefore, the related genes influencing sugar conversion in litchi fruits are further explored, the sugar conversion mechanism is analyzed, and the method has important application value for genetic improvement of litchi quality.
The sweetness of litchi greatly influences the flavor and taste of litchi, and is an index which is concerned by consumers and breeders. The sugar components and proportion in the artificial seed coats determine the unique sweetness and flavor of litchi in the development and storage processes of litchi fruits, and the sugar, fructose and glucose contents of different litchi varieties are different, so that certain difference exists between sweetness and taste. In the process of collecting litchi quality resources and collecting data, the relevance R of reducing sugar in fruits and sweetness of the reducing sugar is found 2 =0.4739 (Li Wensheng, 2012), wherein fructose in reducing sugars is more highly correlated with fruit sweetness, and the sweetness contribution rate of fructose to fruit can reach more than 50% (Shan Fucheng, 1991). Whereas correlation of sucrose and sweetness in fruit R 2 = 0.1867 (Li Wensheng, 2012), reflects poor reliability of sucrose and sweetness in fruit. The results show that the content of the reducing sugar in the litchi reflects the main character of the flavor quality of the litchi.
Disclosure of Invention
In view of the above problems, the present application aims to provide a gene related to the accumulation type of litchi pulp sugar, and a molecular marker, a primer pair and application thereof.
The technical content of the application is as follows:
the application provides a gene related to LITCHI pulp sugar accumulation type, which is a gene LITCHI009111 capable of hydrolyzing sucrose into reducing sugar on a No. 7 LITCHI chromosome, and the nucleic acid sequence of the gene is shown as SEQ ID No. 1.
The application also provides an SNP molecular marker related to the accumulation type of LITCHI sucrose and reducing sugar, wherein the molecular marker Chr7:22496380G:A is positioned on the 2 nd intron of a gene LITCHI009111 which can hydrolyze the sucrose into the reducing sugar on a chromosome 7 of LITCHI;
the nucleic acid sequences of the primer pair of the molecular marker are respectively shown as SEQ ID NO.2 and SEQ ID NO. 3.
SEQ ID NO.2：CACGTAAATTAATCCAATCC；
SEQ ID NO.3：CAGACAACATTACATCACAG。
The application also provides a primer pair for screening litchi sugar accumulation types, and the nucleic acid sequences of the primer pair are respectively shown as SEQ ID NO.2 and SEQ ID NO. 3.
The application also provides application of the molecular marker related to the litchi pulp sugar accumulation type, which is used for judging or assisting in judging the litchi sugar accumulation type;
performing PCR amplification on the primer pair of the molecular marker, taking the DNA of the litchi to be detected as a template, and judging that the litchi sample is reducing sugar accumulation (GA) when the size of an amplified product is 1155 bp;
when the amplified product is a DNA fragment with the size of 600-700bp, judging that the litchi sample is sucrose accumulation type (AA);
when the amplified product has a DNA fragment of 600-700bp and 1155bp, the litchi sample is judged to be of an intermediate type.
The application also provides application of the molecular marker related to the litchi pulp sugar accumulation type, which is used for litchi breeding.
The application also provides a method for judging the accumulation type of litchi sugar, which comprises the following steps:
1) Extracting DNA of litchi variety to be detected;
2) Using the DNA extracted in the step 1) as a template, and carrying out PCR amplification by adopting the primer pair;
the reaction system for PCR amplification comprises: primeSTAR Max Premix (2X) 25. Mu.L, 1. Mu.L of upstream primer, 1. Mu.L of downstream primer, 2. Mu.L of Template (DNA), ddH 2 O 21μL；
The nucleotide sequence of the upstream primer is shown as SEQ ID NO. 2;
the nucleotide sequence of the downstream primer is shown as SEQ ID NO. 3;
the PCR amplification procedure was: 98℃for 10sec;55 ℃,5sec,72 ℃,1min, and the cycle number of the first 3 steps is 35; preserving at 12 ℃.
3) The litchi sugar accumulation type is judged according to the fragment size of the amplified product, and the judgment method is as described above.
The beneficial effects of the application are as follows:
the application relates to a LITCHI pulp sugar accumulation type related gene LITCHI009111, develops a corresponding molecular marker and a primer pair thereof, can predict the pulp sugar accumulation type in the LITCHI seedling stage, is used for marker assisted selection, greatly improves the breeding efficiency, solves the problem of long LITCHI planting period, is designed in such a way that a LITCHI resource population is utilized to construct a phenotype database of the sugar and reducing sugar content of the LITCHI edible part, an Illumina sequencing platform (Illumina Novaseq6000 sequencer) is utilized to carry out genome re-sequencing on 276 parts of core germplasm, the sequencing depth is 16 times, meanwhile, the whole genome association analysis of the LITCHI fruit sugar content is carried out, a gene LITCHI009111 which is highly related to the difference of the LITCHI sugar accumulation type is identified, and a corresponding molecular marker and a primer pair thereof are developed, so that the LITCHI sugar accumulation type can be rapidly screened and marked by using the molecular marker, the LITCHI sugar accumulation type is predicted, the edible part is used for judging or assisting in judging the sugar accumulation type, and screening the high-quality variety containing target characters is realized, and the selective marker assisted breeding period is greatly shortened.
Drawings
FIG. 1 is a graph showing the sequencing result of 276 parts of litchi core germplasm;
FIG. 2 is a graph of results of a sucrose reducing sugar GWAS-associated analysis;
FIG. 3 is a schematic representation of the position of the cognate site in the gene structure of the LITCHI009111 gene at Chr7:22496380G: A;
FIG. 4 shows the expression level of LITCHI009111 gene in fruits of reducing sugar accumulation type and sucrose accumulation type LITCHI resources;
FIG. 5 is a schematic representation of the differential behavior of deleted region segments in sucrose and reduced sugar materials;
FIG. 6 is a graph showing the amplification results of the molecular labeled primer pairs in different litchi resource varieties;
FIG. 7 is a graph showing the results of sucrose and reducing sugar content in litchi resource fruits of different genotypes of litchi.
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
Screening of molecular markers related to litchi pulp sugar accumulation type and application thereof
1. Litchi selection and determination of fructose content
276 parts of litchi core germplasm resources are selected as experimental materials and planted in a national fruit and vegetable center-Guangzhou litchi nursery (North latitude 23 DEG 09', east longitude 113 DEG 22', altitude 20 m). The orchard is in a micro-hilly land, the tree ages of germplasm resources are more than 15 years, the soil and fertilizer management is consistent, and the orchard can normally bloom and fruit each year.
Freezing 276 parts of litchi samples in a refrigerator at the temperature of minus 80 ℃, taking out the samples, grinding the litchi samples into powder by using a mortar under ice bath, adding 0.1g of the samples into 40% acetonitrile solution, carrying out metal bath at the temperature of 85-95 ℃ for 10min, carrying out ultrasonic treatment for 10min after cooling, centrifuging at 12000rpm for 10min, taking supernatant, passing through a 0.22 mu m water phase filter membrane (PES) for one to two times, taking 500 mu L of filtrate, uniformly mixing the filtrate with 500mL of acetonitrile, and then carrying out machine measurement on sugar content, wherein table 1 is litchi materials with higher reducing sugar content, table 2 is litchi materials with higher sucrose content, and representative materials are selected.
Litchi materials (from high to low) with higher reducing sugar content screened in Table 1
Table 2 shows that litchi materials with higher sucrose content (from high to low) are screened
2. Construction of litchi resource genotype database
The method comprises the steps of selecting 276 types of litchi samples, fully expanding green-turning litchi tender leaves, extracting DNA by using a plant genome DNA extraction kit (OMEGA), sending the extracted DNA to Beijing Baimaike biotechnology company, and carrying out genome re-sequencing on 276 parts of core germplasm by using an Illumina sequencing platform (Illumina Novaseq6000 sequencer), wherein the sequencing result is shown in figure 1, and the sequencing depth is 16 times. The short sequence of the resequenced raw data was aligned to the reference genome (Feizixiao litchi genome) using BWA software.
The results are shown in the following table:
TABLE 3 results of resequencing data for SNP sizing
| 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 |
Sucrose can be hydrolyzed to glucose and fructose using whole genome association analysis (GWAS) to map to one SNP site Chr7:22496380g: a (fig. 2) on chromosome 7, which is located in the second intron region of the lischi 009111 gene (fig. 3), which encodes β -fructofuranose synthase, and the gene structure of the gene lischhi 009111 is shown in fig. 2.
Expression analysis of LITCHI009111 Gene qRT-PCR
The first strand cDNA was synthesized using GoScriptTM Reverse Transcription System (Promega) using the EASYspin plant RNA rapid extraction kit (Aidlab Biotech, beijing) to extract litchi mature pulp RNA.
qRT-PCR analysis was performed using a fluorescent quantitative PCR instrument (ABI) with LcActin (GenBank Accession No. HQ615689) as reference gene.
The relative expression level is automatically calculated by using software according to the ratio of the target gene to the LcActin. Each sample was biologically replicated 3 times.
As shown in FIG. 4, the expression level of LITCHI009111 gene in LITCHI fruits shows that LITCHI009111 gene is expressed in different LITCHI resources, but the expression level of the LITCHI variety with accumulation of reducing sugar is obviously higher than that of the LITCHI variety with accumulation of sucrose.
4. Molecular marker selection and application
Based on the comparison of the re-sequencing results, it was found that in the sucrose accumulation-mode material, the 2 nd intron region of the LITCHI009111 gene contains a large fragment deletion (about 500-700 bp), two primers are designed upstream and downstream of the deletion region (the sequence of which is shown as SEQ ID NO. 4) as shown in FIG. 5, the nucleotide sequence of the upstream primer is shown as SEQ ID NO.2, the nucleotide sequence of the downstream primer is shown as SEQ ID NO.3, and PCR amplification is performed by using about 200 LITCHI resource varieties sequenced as templates:
PCR amplification system: primeSTAR Max Premix (2×): 25 μL, LITH5f:1 μl, LITH5r:1 μl, template (DNA): 2 mu L, ddH 2 O：21μL；
The nucleotide sequence of the upstream primer LITH5f is shown as SEQ ID NO. 2;
5'CACGTAAATTAATCCAATCC
the nucleotide sequence of the downstream primer LITH5r is shown as SEQ ID NO. 3;
3'CAGACAACATTACATCACAG
PCR amplification procedure: 98℃for 10sec;55 ℃,5sec,72 ℃,1min, and the cycle number of the first 3 steps is 35; preserving at 12 ℃.
The amplified products were detected by agarose gel electrophoresis and the results recorded by a gel imaging system. The detection results are shown in figures 6 and 7, the content of reducing sugar and sucrose in litchi resource fruits with different genotypes of litchi is different, and if the electrophoresis product has a fragment with the size of 1155bp, the litchi sample is judged to be reducing sugar accumulation (GA); if the electrophoresis product has DNA fragments with the size of 600-700bp, judging that the litchi sample is sucrose accumulation type (AA); if the electrophoresis products have DNA fragments of 600-700bp and 1155bp at the same time, the litchi sample is judged to be of an intermediate type (GG). The molecular marker can effectively distinguish litchi varieties with high reducing sugar content from litchi varieties with low reducing sugar content.
Claims (8)
1. A gene related to the accumulation type of LITCHI pulp sugar, which is characterized in that the gene is LITCHI009111 on chromosome 7 of LITCHI, and the nucleic acid sequence of the gene is shown in SEQ ID NO.1, wherein the gene can hydrolyze sucrose into reducing sugar (glucose and fructose).
2. A SNP molecular marker related to LITCHI pulp sugar accumulation type, characterized in that the molecular marker Chr7:22496380g:a is located on the 2 nd intron of the gene LITCHI009111 on chromosome 7 of LITCHI, which can hydrolyze sucrose into reducing sugar.
3. The use of the SNP molecular marker related to litchi pulp sugar accumulation type as set forth in claim 2, wherein the SNP molecular marker is used for judging or assisting in judging litchi sugar accumulation type or used for litchi breeding.
4. A primer pair of SNP molecular markers associated with litchi pulp sugar accumulation type as set forth in claim 2, wherein the nucleic acid sequences of the primer pair are shown in SEQ ID No.2 and SEQ ID No.3, respectively.
5. The use of the primer pair of SNP molecular markers associated with litchi pulp sugar accumulation types as set forth in claim 4, wherein the primer pair is used for judging or assisting in judging the litchi sugar accumulation type;
performing PCR amplification on the primer pair of the molecular marker, taking the DNA of the litchi to be detected as a template, and judging that the litchi sample is reducing sugar accumulation (GA) when the size of an amplified product is 1155 bp;
when the amplified product is a DNA fragment with the size of 700bp, judging that the litchi sample is sucrose accumulation type (AA);
when the size of the amplified product is a DNA fragment between 700bp and 1155bp, the litchi sample is judged to be of an intermediate type.
6. The method for judging the accumulation type of the litchi sugar is characterized by comprising the following steps of:
1) Extracting DNA of litchi variety to be detected;
2) Performing PCR amplification by using the DNA extracted in the step 1) as a template and the primer pair of claim 5;
3) The litchi sugar accumulation type is judged according to the fragment size of the amplified product, and the judgment method is as claimed in claim 4.
7. The method according to claim 6, wherein the reaction system for PCR amplification in step 2) comprises: primeSTAR Max Premix (2X) 25. Mu.L, 1. Mu.L of upstream primer, 1. Mu.L of downstream primer, 2. Mu.L of Template (DNA), ddH 2 O 21μL。
8. The method according to claim 6, wherein the PCR amplification in step 2) is performed by: 98℃for 10sec;55 ℃,5sec,72 ℃,1min, and the cycle number of the first 3 steps is 35; preserving at 12 ℃.
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| CN117867158A (en) * | 2023-12-28 | 2024-04-12 | 中国热带农业科学院环境与植物保护研究所 | Molecular marker related to sucrose content of litchi pulp and application of molecular marker |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN117867158A (en) * | 2023-12-28 | 2024-04-12 | 中国热带农业科学院环境与植物保护研究所 | Molecular marker related to sucrose content of litchi pulp and application of molecular marker |
| CN117867158B (en) * | 2023-12-28 | 2024-07-02 | 中国热带农业科学院环境与植物保护研究所 | Molecular marker related to sucrose content of litchi pulp and application of molecular marker |
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