InDel molecular marker related to size of lychee kernel and application of InDel molecular marker

Abstract

The invention relates to the technical field of molecular biology, and particularly provides an InDel molecular marker related to the size of a lychee kernel and application of the InDel molecular marker. The InDel molecular marker is located at a site 25610680 of a fifth chromosome of a reference genome GeneBank: GCA019925255.1 of the litchi, and the InDel molecular marker is used for detecting the DNA of the litchi. The genotype of the marker in a pyrokaryotic litchi material is homozygous' GAAAAA / GAAAAA ', and the genotype of the marker in a macrokaryotic litchi material is heterozygous' G---- / GAAAAA 'or homozygous' G---- / G----'. The invention further provides a primer group for detecting the molecular marker, an effective tool is provided for molecular marker-assisted breeding of the size of the lychee kernel, and the method is expected to assist in shortening the breeding period, improving the breeding efficiency and promoting the improvement work of the lychee variety.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology and relates to an InDel molecular marker related to litchi kernel size and an application thereof. Background Art

[0002] litchi( Litchi chinensis Sonn., belonging to the Sapindaceae family, is an important evergreen fruit crop grown in tropical and subtropical regions worldwide. Litchi has a history of over 3,500 years in China, making it the largest producer, with major growing areas located in provinces such as Guangdong, Guangxi, Fujian, Hainan, and Yunnan. To date, researchers have conducted in-depth molecular studies on numerous traits, including fruit size, seed development, fruit shedding, regulation of pericarp color, and flowering. Seed development and size are particularly important for lychee fruit quality. Small-seed lychees are preferred by consumers due to their high edible content. However, traditional lychee breeding methods primarily rely on phenotypic selection, which can lead to long breeding cycles and low efficiency. Molecular marker-assisted breeding technology enables early selection using molecular markers closely linked to target traits, significantly improving breeding efficiency and shortening the breeding cycle.

[0003] While researchers have investigated the molecular mechanisms underlying litchi traits such as fruit size and seed development, relatively little research has focused on molecular markers for litchi pit size, leading to a lack of stable and efficient molecular markers for assisted breeding. Block segregant analysis (BSA-seq) is a rapid method for identifying genetic markers associated with target traits. It has been successfully applied to QTL mapping for traits such as seed size in various crops, but its application in the development of molecular markers for litchi pit size has been limited. Therefore, developing molecular markers closely associated with litchi pit size is crucial for advancing marker-assisted breeding in litchi. Summary of the Invention

[0004] The purpose of the present invention is to overcome the deficiencies of the prior art, provide an InDel molecular marker related to litchi kernel size, as well as primers and methods for detecting the marker, and further provide its application in litchi molecular marker-assisted breeding.

[0005] To achieve the above object, the present invention adopts the following technical solutions: In a first aspect, the present invention provides an InDel marker significantly associated with litchi kernel size. This marker is located at locus 25610680 on chromosome 5 of the litchi reference genome (GeneBank: GCA_019925255.1). This marker is homozygous for "GAAAAA" in the genotype of pyrokeratinized litchis and heterozygous for "G---- / GAAAAA" or homozygous for "G----- / G-----" in the genotype of large-keratinized litchis.

[0006] A second aspect of the present invention provides a primer set for detecting the size of litchi kernels, comprising an upstream primer having a nucleotide sequence as shown in SEQ ID NO. 1, a downstream primer having a nucleotide sequence as shown in SEQ ID NO. 2, and a FAM fluorescent-labeled primer having a nucleotide sequence as shown in SEQ ID NO. 3.

[0007] The third aspect of the present invention provides the use of the primer in at least one of the following aspects: (1) Detect the size of litchi kernel; (2) Early prediction of litchi kernel size phenotype; (3) Molecular marker-assisted breeding of litchi.

[0008] A fourth aspect of the present invention provides a method for detecting the size of a litchi pit, comprising the following steps: a. Extract total DNA from leaves of the material to be analyzed using the CTAB method: Extract total genomic DNA from the litchi germplasm material to be tested; b. PCR amplification reaction: using the DNA obtained in step a as a template, PCR amplification is performed using the primers to obtain an amplified fluorescently labeled product; c. Detection of PCR amplification products: 5 μL of PCR amplification product was electrophoresed on a 2% agarose gel containing Super Red nucleic acid dye in 1× TAE buffer at 120 V for 25 min. The gel run results were photographed and recorded using a gel imaging system. d. Genotyping of PCR amplification products: Fluorescently labeled PCR products were subjected to capillary electrophoresis, and different genotypes were distinguished based on band size and position. Macronuclei had an 184 bp band, indicating a genotype of G----- / GAAAAA or G----- / G-----; focal nuclei had only a 189 bp band, indicating a genotype of GAAAAA / GAAAAA.

[0009] Furthermore, the PCR amplification reaction system is: 15 μL, including 1.0 μL 50 ng / ul genomic DNA, 7.5 μL 2×Taq PCR Mix, 0.2 μL 1 μM upstream primer, 1.2 μL 1 μM downstream primer, 1.2 μL 1 μM FAM fluorescent labeled primer, and 3.9 μL ddH2O.

[0010] Furthermore, the PCR amplification program is as follows: pre-denaturation at 94°C for 5 min; 30 cycles of denaturation at 94°C for 30 s, annealing at 58°C for 30 s, and extension at 72°C for 60 s; 13 cycles of denaturation at 94°C for 30 s, annealing at 53°C for 30 s, and extension at 72°C for 60 s; and extension at 72°C for 10 min.

[0011] Beneficial effects of the present invention: This study, using BSA-seq technology, has for the first time identified an InDel molecular marker significantly associated with litchi pit size. This marker is tightly linked to the pit size trait and has a high phenotypic explanatory rate. Using the specific primer set and detection method provided by this invention, early prediction and selection of pit size traits can be performed in litchi seedlings without waiting for plants to bear fruit, significantly improving breeding efficiency, shortening the breeding cycle, and reducing breeding costs. This study provides an effective molecular tool for targeted improvement of litchi pit size and has significant application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 The results of agarose gel electrophoresis of the amplified products of 87 litchi materials using the primers of the present invention are shown.

[0013] Figure 2 The results of capillary electrophoresis detection of the macronucleus (homozygous) litchi material LZ0092 using the primers of the present invention are shown.

[0014] Figure 3 The results of capillary electrophoresis detection of the macronucleus (homozygous) litchi material LZ0676 using the primers of the present invention are shown.

[0015] Figure 4 The results of capillary electrophoresis of the primers of the present invention on the large-nucleus (homozygous) litchi material LZ1712 are shown.

[0016] Figure 5 The results of capillary electrophoresis detection of the macronucleus (heterozygous) litchi material LZ0617 using the primers of the present invention are shown.

[0017] Figure 6 The results of capillary electrophoresis detection of the macronucleus (heterozygous) litchi material LZ1008 using the primers of the present invention are shown.

[0018] Figure 7 The results of capillary electrophoresis detection of the macronucleus (heterozygous) litchi material LZ1018 using the primers of the present invention are shown.

[0019] Figure 8 The results of capillary electrophoresis of the primers of the present invention on the large-core (heterozygous) litchi material LZ1442 are shown.

[0020] Figure 9The results of capillary electrophoresis detection of the macronucleus (heterozygous) litchi material LZ1407 using the primers of the present invention are shown.

[0021] Figure 10 The results of capillary electrophoresis detection of the macronucleus (heterozygous) litchi material LZ1501 using the primers of the present invention are shown.

[0022] Figure 11 This is the capillary electrophoresis detection result of the focused core type litchi material LZ0053 using the primers of the present invention.

[0023] Figure 12 This is the capillary electrophoresis detection result of the primers of the present invention on the focused core type litchi material LZ0223.

[0024] Figure 13 The results of capillary electrophoresis of the focused-core litchi material LZ0629 using the primers of the present invention are shown. DETAILED DESCRIPTION

[0025] The specific embodiments of the present invention are described below to facilitate understanding of the present invention by those skilled in the art. However, it should be clear that the present invention is not limited to the scope of the specific embodiments. For those skilled in the art, as long as various changes are within the spirit and scope of the present invention as defined and determined by the appended claims, these changes are obvious, and all inventions and creations utilizing the concepts of the present invention are protected.

[0026] Example 1 Screening and identification of InDel molecular markers 1. Material preparation: 130 early-maturing litchi cultivars with similar genetic backgrounds were selected. Fruit traits such as single fruit weight, seed weight, edible rate, percentage of drupe weight, and burnt kernel rate were measured. Fifteen extreme accessions were selected to construct the large-kernel mixed pool NS (burnt kernel rate 0, average seed weight >3.5 g, percentage of drupe weight >20%) and the burnt kernel mixed pool AS (burnt kernel rate ≥75%, average seed weight <2.5 g, percentage of drupe weight <15%).

[0027] 2. BSA-seq analysis: Genomic DNA was extracted from two mixed pools and whole-genome resequencing was performed. The sequencing data were quality controlled, aligned, and detected for variations. Combining the Euclidean distance (ED) algorithm and Δindex analysis, the 24.93Mb~25.69Mb region on chromosome 5 was located and was significantly correlated with kernel size.

[0028] 3. InDel Marker Screening and Validation: A differentially expressed InDel site was identified within the mapping region, ultimately identifying a stable and reliable InDel marker located at position 25610680 on chromosome 5 of the litchi reference genome (GeneBank: GCA_019925255.1). This marker is homozygous for the "GAAAAA / GAAAAA" genotype in pyrokeratin litchis and heterozygous for the "G------ / GAAAAA" or homozygous for the "G------ / G------" genotype in large-keratin litchis.

[0029] 4. Primer design: Based on the InDel molecular markers obtained above, design specific amplification primers and fluorescent labeling primers as follows: Forward primer (SEQ ID NO. 1): 5'-TGTAAAACGACGGCCAGTTCTTGCATGAACCCAGGAAAGA-3'; Reverse primer (SEQ ID NO. 2): 5′-TGAAGCTTTTCCAAAACATAGGGA-3′; FAM fluorescent labelled primer (SEQ ID NO. 3): 5'-TGTAAAACGACGGCCAGT-3' (6-FAM fluorescent label added at the 5' end).

[0030] Example 2 Molecular identification of different litchi germplasm resources (1) Selection of experimental materials: 87 early-maturing inbred population breeding materials were selected as test materials for the verification of InDel molecular markers; (2) Extracting genomic DNA from samples to be analyzed: Using the modified CTAB method, DNA from the leaves of the above-mentioned litchi breeding materials was extracted; (3) PCR amplification reaction: PCR amplification was performed using the extracted DNA as the template and three primers labeled with InDel molecules as the primers. The PCR reaction system was 15 μL, including 1.0 μL 50 ng / ul genomic DNA, 7.5 μL 2×Taq PCR Mix, 0.2 μL 1 μM upstream primer, 1.2 μL 1 μM downstream primer, 1.2 μL 1 μM FAM fluorescent labeled primer, and 3.9 μL ddH2O. The PCR amplification procedure was as follows: 94°C pre-denaturation for 5 min; 30 cycles of 94°C denaturation for 30 s, 58°C annealing for 30 s, and 72°C extension for 60 s; 13 cycles of 94°C denaturation for 30 s, 53°C annealing for 30 s, and 72°C extension for 60 s; and 72°C extension for 10 min. (4) Detection of PCR amplification products: 5 μL of PCR amplification products were electrophoresed on a 2% agarose gel containing Super Red nucleic acid dye in 1×TAE buffer at 120 V for 25 min. The gel run results were photographed and recorded in a gel imaging system. (5) Genotype detection of PCR amplification products: The fluorescently labeled PCR amplification products were subjected to capillary electrophoresis, and different genotypes were distinguished based on the size and position of the bands. The macronucleus had a 184 bp band, and the genotype was G- - - - - / GAAAAA or G - - -- - / G - - - - -; the focal nucleus had only a 189 bp band, and the genotype was GAAAAA / GAAAAA.

[0031] The test results are as follows: like Figure 1 As shown: The size of the amplified bands of 87 litchi materials amplified by the primers of the present invention was about 200 bp.

[0032] Table 1 shows the phenotypic data of some litchi fruit samples, and Table 2 shows the genotypic detection results. The sizes of the amplified bands of the primers of the present invention show insertion / deletion differences. Figure 2-13 This is the capillary electrophoresis result.

[0033] Table 1. Phenotypic data of some sample fruits

[0034] Table 2 Analysis of primer amplification products of some samples

[0035] In summary, this study successfully identified an InDel marker (Chr5_25610680_InDel) significantly associated with litchi kernel size through related experiments. The primers for this marker (forward primer 5'-TGTAAAACGACGGCCAGTTCTTGCATGAACCCAGGAAAGA-3', reverse primer 5'-TGAAGCTTTTCCAAAACATAGGGA-3', and fluorescent marker primer 5'-TGTAAAACGACGGCCAGT-3') can be used for detection. This marker is homozygous for "GAAAAA / GAAAAA" in pyrokeratin litchi genotypes and heterozygous for "G - - - - - / GAAAAA" or homozygous for "G -- - - - / G - - - - -" in large-keratin litchi genotypes. This provides an effective tool for molecular marker-assisted breeding of litchi kernel size, potentially shortening breeding cycles, improving breeding efficiency, and promoting litchi variety improvement.

Claims

1. A primer set for detecting litchi pit size, characterized in that: The upstream primer is represented by the nucleotide sequence of SEQ ID NO. 1, the downstream primer is represented by the nucleotide sequence of SEQ ID NO. 2, and the FAM fluorescent-labeled primer is represented by the nucleotide sequence of SEQ ID NO.

3.

2. Use of the primer set according to claim 1 in at least one of the following aspects: (1) Detect the size of litchi kernel; (2) Early prediction of litchi kernel size phenotype; (3) Molecular marker-assisted breeding of litchi.

3. A method for detecting the size of litchi kernels, characterized in that: The following steps are involved: a. Extract total DNA from leaves of the material to be analyzed using the CTAB method: Extract total genomic DNA from the litchi germplasm material to be tested; b. PCR amplification reaction: using the DNA obtained in step a as a template, PCR amplification is performed using the primers described in claim 1 to obtain an amplified fluorescently labeled product; c. Detection of PCR amplification products: 5 μL of PCR amplification product was electrophoresed on a 2% agarose gel containing Super Red nucleic acid dye in 1× TAE buffer at 120 V for 25 min. The gel run results were photographed and recorded using a gel imaging system. d. Genotype detection of PCR amplification products: The fluorescent-labeled PCR amplification products were subjected to capillary electrophoresis, and different genotypes were distinguished according to the size and position of the bands: the macronuclei had a 184 bp band, and the genotype was G----- / GAAAAA or G----- / G-----; the focal nuclei had only a 189 bp band, and the genotype was GAAAAA / GAAAAA.

4. The method according to claim 3, characterized in that The PCR amplification reaction system was as follows: 15 μL, including 1.0 μL 50 ng / ul genomic DNA, 7.5 μL 2× Taq PCR Mix, 0.2 μL 1 μM upstream primer, 1.2 μL 1 μM downstream primer, 1.2 μL 1 μM FAM fluorescent-labeled primer, and 3.9 μL ddH2O.

5. The method according to claim 3, characterized in that The PCR amplification program was as follows: pre-denaturation at 94°C for 5 min; 30 cycles of denaturation at 94°C for 30 s, annealing at 58°C for 30 s, and extension at 72°C for 60 s; 13 cycles of denaturation at 94°C for 30 s, annealing at 53°C for 30 s, and extension at 72°C for 60 s; and extension at 72°C for 10 min.

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