An InDel molecular marker related to litchi pit size and its application

Abstract

This invention relates to the field of molecular biology, specifically providing an InDel molecular marker related to litchi seed size and its application. The InDel molecular marker is located at locus 25610680 on chromosome 5 of the litchi reference genome (GeneBank:GCA_019925255.1). This marker is homozygous for "GAAAAA / GAAAAA" in pyrose litchi materials and heterozygous for "G--- / GAAAAA" or homozygous for "G--- / G---" in large-seeded litchi materials. This invention also provides a primer set for detecting this molecular marker, providing an effective tool for marker-assisted breeding of litchi seed size, which is expected to help shorten the breeding cycle, improve breeding efficiency, and promote litchi variety improvement.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology and relates to an InDel molecular marker related to the size of litchi fruit seeds and its application. Background Technology

[0002] litchi( Litchi chinensis Lychee (Sonn.) belongs to the Sapindaceae family and is an important evergreen fruit crop cultivated in tropical and subtropical regions worldwide. Lychee has been cultivated in China for over 3500 years, and China is the largest lychee producer, with major growing areas located in Guangdong, Guangxi, Fujian, Hainan, and Yunnan provinces. To date, researchers have conducted in-depth molecular mechanism studies on various traits of lychee, including fruit size, seed development, fruit abscission, regulation of peel color, and flowering. Seed development and size are particularly important for lychee fruit quality. Small-seeded lychees are more popular with consumers due to their higher edibility. However, traditional lychee breeding methods mainly rely on phenotypic selection, which suffers from long breeding cycles and low efficiency. Molecular marker-assisted breeding technology can significantly improve breeding efficiency and shorten the breeding cycle by using molecular markers closely linked to target traits for early selection.

[0003] Currently, although researchers have conducted molecular mechanism studies on traits such as fruit size and seed development in litchi, research on molecular markers related to litchi seed size is still limited, and stable and efficient molecular markers for assisted breeding are lacking. Mixed aggregate segregation analysis (BSA-seq) is a method for rapidly locating genetic markers associated with target traits and 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 related to litchi seed size is limited. Therefore, developing molecular markers closely related to litchi seed size is of great significance for promoting marker-assisted breeding of litchi. Summary of the Invention

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

[0005] To achieve the above object, the present invention adopts the following technical solutions:

[0006] The first aspect of this invention provides an InDel marker that is significantly associated with the size of the litchi seed, located at locus 25610680 on chromosome 5 of the litchi reference genome GeneBank: GCA_019925255.1. This marker is homozygous for "GAAAAA" in scorched-seed litchi materials and heterozygous for "G---- / GAAAAA" or homozygous for "G----- / G-----" in large-seed litchi materials.

[0007] The second aspect of the present invention provides a primer set for detecting the size of litchi seeds, comprising an upstream primer with the nucleotide sequence shown in SEQ ID NO. 1, a downstream primer with the nucleotide sequence shown in SEQ ID NO. 2, and a FAM fluorescently labeled primer with the nucleotide sequence shown in SEQ ID NO. 3.

[0008] The third aspect of the present invention provides the use of the primers in at least one of the following aspects:

[0009] (1) Detect the size of the litchi seed;

[0010] (2) Early prediction of litchi seed size phenotype;

[0011] (3) Litchi molecular marker-assisted breeding.

[0012] A fourth aspect of the present invention provides a method for detecting the size of a litchi seed, comprising the following steps:

[0013] a. Total DNA was extracted from the leaves of the materials to be analyzed using the CTAB method: Total genomic DNA was extracted from the litchi germplasm materials to be tested;

[0014] b. PCR amplification reaction: Using the DNA obtained in step a as a template, PCR amplification is performed using the primers described above to obtain the amplified fluorescently labeled product.

[0015] c. Detection of PCR amplification products: Take 5 μL of PCR amplification products and perform electrophoresis on a 2% agarose gel containing Super Red nucleic acid dye. Electrophoresis is performed at 120 V for 25 min in 1×TAE buffer. The gel running results are photographed and recorded in a gel imaging system.

[0016] d. Genotype detection of PCR amplification products: The fluorescently labeled PCR amplification products are subjected to capillary electrophoresis, and different genotypes are distinguished according to the size and position of the bands. The macronucleus has a 184 bp band, and the genotype is G----- / GAAAAA or G----- / G-----; the pygmy nucleus has only a 189 bp band, and the genotype is GAAAAA / GAAAAA.

[0017] Further, the PCR amplification reaction system is 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 fluorescently labeled primer, and 3.9 μL ddH2O.

[0018] Furthermore, the PCR amplification program is as follows: 94℃ pre-denaturation for 5 min; 94℃ denaturation for 30 s, 58℃ annealing for 30 s, 72℃ extension for 60 s, 30 cycles; 94℃ denaturation for 30 s, 53℃ annealing for 30 s, 72℃ extension for 60 s, 13 cycles; 72℃ extension for 10 min.

[0019] The beneficial effects of this invention are:

[0020] This invention, using BSA-seq technology, identifies for the first time an InDel molecular marker significantly correlated with litchi seed size. This marker is tightly linked to the seed size trait and exhibits a high phenotypic explanatory power. Utilizing the specific primer set and detection method provided by this invention, early prediction and selection of seed size traits can be performed on litchi seedlings without waiting for fruit production, significantly improving breeding efficiency, shortening the breeding cycle, and reducing breeding costs. This invention provides an effective molecular tool for the targeted improvement of litchi seed size and has significant application value. Attached Figure Description

[0021] Figure 1 The results of agarose gel electrophoresis of the amplification products of the primers of this invention in 87 litchi materials are shown.

[0022] Figure 2 This is the capillary electrophoresis detection result of the primer pair of the present invention on the macronucleus (homozygous) litchi material LZ0092.

[0023] Figure 3 This is the capillary electrophoresis detection result of the primer pair of the present invention on the macronucleus (homozygous) litchi material LZ0676.

[0024] Figure 4 This is the capillary electrophoresis detection result of the primer pair of the present invention on the macronucleus (homozygous) litchi material LZ1712.

[0025] Figure 5 This is the capillary electrophoresis detection result of the primer pair of the present invention on the macronucleus (hybrid) litchi material LZ0617.

[0026] Figure 6 This is the capillary electrophoresis detection result of the primer pair of the present invention on the macronucleus (hybrid) litchi material LZ1008.

[0027] Figure 7 This is the capillary electrophoresis detection result of the primer pair of the present invention on the macronucleus (hybrid) litchi material LZ1018.

[0028] Figure 8 This is the capillary electrophoresis detection result of the primer pair of the present invention on the macronucleus (hybrid) litchi material LZ1442.

[0029] Figure 9 The results of capillary electrophoresis detection of the primer pair of the present invention on the macronucleus (hybrid) litchi material LZ1407 are shown.

[0030] Figure 10 This is the capillary electrophoresis detection result of the primer pair of the present invention on the macronucleus (hybrid) litchi material LZ1501.

[0031] Figure 11 The results of capillary electrophoresis detection of the primers of this invention on the litchi material LZ0053 are shown.

[0032] Figure 12 The results of capillary electrophoresis detection of the primers of this invention on the litchi material LZ0223 are shown.

[0033] Figure 13 The results of capillary electrophoresis detection of the primers of this invention on the litchi material LZ0629 are shown. Detailed Implementation

[0034] The specific embodiments of the present invention are described below to enable those skilled in the art to understand the present invention. However, it should be understood that the present invention is not limited to the scope of the specific embodiments. For those skilled in the art, various changes are obvious as long as they are within the spirit and scope of the present invention as defined and determined by the appended claims. All inventions utilizing the concept of the present invention are protected.

[0035] Example 1: Screening and Identification of InDel Molecular Markers

[0036] 1. Material preparation: 130 early-maturing litchi varieties with similar genetic backgrounds were selected. Fruit traits such as single fruit weight, seed weight, edible rate, percentage of drupe fruit weight, and scorched seed rate were measured. 15 extreme materials were selected to construct a mixed pool of large-kernel NS (scorched seed rate of 0, average seed weight > 3.5 g, percentage of drupe fruit weight > 20%) and a mixed pool of scorched seed AS (scorched seed rate ≥ 75%, average seed weight < 2.5 g, percentage of drupe fruit weight < 15%).

[0037] 2. BSA-seq analysis: Genomic DNA was extracted from two pools and whole-genome resequencing was performed. The sequencing data were quality controlled, aligned, and variant detected. Combined with the Euclidean distance (ED) algorithm and Δindex analysis, the region of chromosome 5 from 24.93 Mb to 25.69 Mb was found to be significantly correlated with the size of the fruit pit.

[0038] 3. Screening and Validation of the InDel Marker: Differential InDel sites were screened within the localized region, ultimately yielding one stable and reliable InDel marker located at locus 25610680 on chromosome 5 of the litchi reference genome (GeneBank: GCA_019925255.1). This marker is homozygous for "GAAAAA / GAAAAA" in pyrogenic litchi materials and heterozygous for "G----- / GAAAAA" or homozygous for "G----- / G-----" in macrokeratogenic litchi materials.

[0039] 4. Primer Design: Based on the InDel molecular markers obtained above, specific amplification primers and fluorescently labeled primers were designed as follows:

[0040] Forward primer (SEQ ID NO. 1):

[0041] 5'-TGTAAAACGACGGCCAGTTCTTGCATGAACCCAGGAAAGA-3';

[0042] Reverse primer (SEQ ID NO. 2): 5'-TGAAGCTTTTCCAAAACATAGGGA-3';

[0043] FAM fluorescently labeled primer (SEQ ID NO. 3): 5'-TGTAAAACGACGGCCAGT-3' (with a 6-FAM fluorescent tag added to 5').

[0044] Example 2 Molecular identification among different litchi germplasm resources

[0045] (1) Selection of experimental materials: 87 early-maturing self-pollinated breeding materials were selected as test materials for the verification of InDel molecular markers;

[0046] (2) Extraction of genomic DNA from the samples to be analyzed: DNA was extracted from the leaves of the litchi breeding materials described above using a modified CTAB method;

[0047] (3) PCR amplification reaction: Using the extracted DNA as the amplification template, and three primers labeled with InDel as amplification primers, PCR amplification was performed. The PCR reaction system consisted of 15 μL, including 1.0 μL of 50 ng / μL genomic DNA, 7.5 μL of 2×Taq PCR Mix, 0.2 μL of 1 μM upstream primer, 1.2 μL of 1 μM downstream primer, 1.2 μL of 1 μM FAM fluorescently labeled primer, and 3.9 μL of ddH2O. The PCR amplification program was as follows: 94℃ pre-denaturation for 5 min; 94℃ denaturation for 30 s, 58℃ annealing for 30 s, 72℃ extension for 60 s, 30 cycles; 94℃ denaturation for 30 s, 53℃ annealing for 30 s, 72℃ extension for 60 s, 13 cycles; 72℃ extension for 10 min.

[0048] (4) Detection of PCR amplification products: Take 5 μL of PCR amplification products and perform electrophoresis on a 2% agarose gel containing Super Red nucleic acid dye. Electrophoresis is performed in 1×TAE buffer at 120 V for 25 min. The gel running results are photographed and recorded in a gel imaging system.

[0049] (5) Genotype detection of PCR amplification products: The fluorescently labeled PCR amplification products were subjected to capillary electrophoresis, and different genotypes were distinguished according to the size and position of the bands. The macronucleus had a 184 bp band, and the genotype was G- - - - - / GAAAAA or G - - -- - / G - - - - -; the pygmy nucleus had only a 189 bp band, and the genotype was GAAAAA / GAAAAA.

[0050] The test results are as follows:

[0051] like Figure 1 As shown:

[0052] The primers of this invention amplified bands of approximately 200 bp in 87 litchi materials.

[0053] Table 1 shows the phenotypic data of some litchi samples, and Table 2 shows the genotyping results. The amplified band sizes using the primers of this invention show insertion / deletion differences. Figure 2-13 This is a diagram of the results of capillary electrophoresis.

[0054] Table 1. Phenotypic data of some sample fruits

[0055]

[0056] Table 2 Analysis of primer amplification product results for some samples

[0057]

[0058] In summary, this study successfully screened an InDel marker (Chr5_25610680_InDel) significantly associated with litchi seed size through relevant experiments. Its primers (forward primer 5'-TGTAAAACGACGGCCAGTTCTTGCATGAACCCAGGAAAGA-3', reverse primer 5'-TGAAGCTTTTCCAAAACATAGGGA-3', and fluorescent marker primer 5'-TGTAAAACGACGGCCAGT-3') can be used to detect this marker. The genotype of this marker is homozygous "GAAAAA / GAAAAA" in scorched-seed litchi materials and heterozygous "G -- - - - - / GAAAAA" or homozygous "G -- - - - / G -- - - - -" in large-seed litchi materials. This provides an effective tool for marker-assisted breeding of litchi seed size, and is expected to help shorten the breeding cycle, improve breeding efficiency, and promote litchi variety improvement.

Claims

1. A primer set for detecting the size of litchi seeds, characterized in that, It consists of an upstream primer with the nucleotide sequence shown in SEQ ID NO. 1, a downstream primer with the nucleotide sequence shown in SEQ ID NO. 2, and a FAM fluorescently labeled primer with the nucleotide sequence shown in SEQ ID NO. 3.

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