A molecular marker combination related to litchi fruit size and application thereof

Abstract

The application provides a molecular marker combination related to litchi fruit size and application thereof, and belongs to the technical field of biotechnology. Based on whole genome association analysis, three molecular marker sites related to litchi single fruit weight are screened: an InDel marker (47 bp bases at the 16128136th base of Chr5 chromosome, in the intron region of LITCHI000636 gene), a T / G marker at the 8764522th base of two adjacent SNP markers of Chr4 chromosome and a T / A marker at the 8764809th base of the 3'UTR region of LITCHI030544 gene. The InDel and SNP molecular marker amplification primers are applied to litchi fruit size breeding material detection, can quickly screen large fruit excellent breeding material at the seedling stage, assist hybrid parent selection and match and early selection of seedling offspring, improve the selection efficiency of large fruit litchi, and the detection cost is low and the typing result is reliable.

Description

Technical Field

[0001] This invention relates to molecular marker-assisted selection breeding technology for litchi, belonging to the field of plant biotechnology. Specifically, it relates to a combination of molecular markers related to litchi fruit size and its application. Background Technology

[0002] Lychee (Litchi chinensis Sonn.) is a plant belonging to the genus Litchi in the family Sapindaceae. It is one of my country's most important economic evergreen fruit trees, native to the country. Due to its vibrant color, unique flavor, and rich nutritional value, it is one of my country's most internationally competitive fruits. In fruit production, fruit size is a core factor determining its commercial value and market competitiveness, directly influencing consumer purchasing intentions and market segmentation, and significantly impacting the economic benefits for fruit farmers.

[0003] Fruit size is a crucial phenotypic trait in the development of fruits in higher plants, and its final morphology is determined by the synergistic action of two core processes: cell division and cell enlargement. In the early stages of fruit development, cell division dominates, leading to a rapid increase in cell number. In the later stages, cell division gradually ceases, and cell enlargement becomes the dominant process, finely regulated by various factors such as plant hormones and carbohydrate supply and distribution. The edible part of the litchi is the aril. The growth space of the aril is limited by the framework formed by the early development of the pericarp, exhibiting a developmental characteristic of "pericarp growing first, then flesh growing." The final fruit size depends on the synergistic regulation of the pericarp, aril, and seed, resulting in significant differences in fruit size among different litchi varieties. In breeding practice, progeny selection is crucial. Traditional breeding methods suffer from limitations such as long cycles and low efficiency, while direct selection based on individual genotypes is more precise and efficient.

[0004] In recent years, significant progress has been made in elucidating the genetic basis of important plant traits, with a series of key genes controlling yield and quality being successfully cloned. Currently, the rapid development of biotechnology is propelling crop breeding into the era of molecular design, with marker-assisted selection (MAG) and gene editing becoming core drivers of variety improvement. In particular, with the reduction in the cost of SNP marker detection and the enhancement of bioinformatics analysis capabilities, the efficiency and accuracy of MAG in breeding practice have significantly improved. However, no usable molecular markers related to litchi fruit size have yet been developed or reported. Therefore, we propose a molecular marker combination related to litchi fruit size and its application. Summary of the Invention

[0005] The purpose of this invention is to address the problems raised in the existing background technology. To achieve the above-mentioned objective, this invention provides the following technical solution: a molecular marker combination related to the size of litchi fruit, wherein the molecular marker combination consists of one InDel molecular marker and two SNP molecular markers; (1) The nucleotide sequence of the InDel molecular marker is shown in SEQ ID NO.1. The 172nd base from the 5' end of the sequence shown in SEQ ID NO.1 is the InDel site, and the InDel site is an insertion of 47 bp bases. (2) The nucleotide sequence of the SNP molecular marker is shown in SEQ ID NO.2. The 245th base from the 5' end of the sequence shown in SEQ ID NO.2 is the SNP site, and the SNP site is T or G. (3) The nucleotide sequence of the SNP molecular marker is shown in SEQ ID NO.3. The 348th base from the 5' end of the sequence shown in SEQ ID NO.3 is the SNP site, and the SNP site is A or T.

[0006] As a preferred technical solution of the present invention, when the litchi genome contains any two or more favorable genotypes from the molecular marker combination, it is identified as a candidate large-fruited litchi germplasm. (1) A 47 bp insertion / deletion marker located at 16128136 bases on chromosome 5 has an insert type as its preferred genotype; (2) The T / G SNP marker located at 8764522 bases on chromosome Chr4 has a favorable genotype of GG; (3) The T / A SNP marker located at base 8764809 on chromosome Chr4 has the favorable genotype AA.

[0007] As a preferred embodiment of the present invention, the primer pair consists of two single-stranded DNA molecules, including: (1) The primer pair used to amplify the 47 bp insertion / deletion marker has the upstream primer being 5'-GCCTGAATTGAGCTATGC-3', as shown in SEQ ID NO.4; and the downstream primer being 5'-ACCTCCTTTAGGCTCATAAG-3', as shown in SEQ ID NO.5; (2) The primer pair used to detect the Chr4-8764522 T / G SNP marker has an upstream primer of 5'-GCTGTTTATTCAATCTACCACC-3', as shown in SEQ ID NO.6; and a downstream primer of 5'-GTACTCTGGAAATTGTGCTAC-3', as shown in SEQ ID NO.7; (3) The primer pair used to detect the Chr4-8764809 T / A SNP marker has an upstream primer of 5'-GTGGTGGTTGTGGATTATG-3', as shown in SEQ ID NO.8; and a downstream primer of 5'-TTGGGTTTGTGGGTGATA-3', as shown in SEQ ID NO.9.

[0008] As a preferred technical solution of the present invention, the primer pair is used to identify high single fruit weight and low single fruit weight of litchi.

[0009] As a preferred embodiment of the present invention, the kit comprises the primer pair as described in claim 3.

[0010] As a preferred technical solution of the present invention, the kit is used to identify high single fruit weight and low single fruit weight of lychee.

[0011] As a preferred embodiment of the present invention, the kit further comprises one or more of PCR amplification buffer, dNTPs, thermostable DNA polymerase and ddH2O.

[0012] Application of molecular marker combinations in the kit for molecular marker-assisted selection breeding of litchi.

[0013] As a preferred technical solution of the present invention, the application is to predict the weight of a single fruit during the litchi seedling stage, or to screen breeding materials for large-fruited litchi, or to assist in the selection of hybrid parents and the early selection of seedling offspring.

[0014] A method for auxiliary identification of the size of litchi fruit, comprising the following steps: Step (1) Extract genomic DNA from the litchi to be tested; Step (2) Using the genomic DNA as a template, perform PCR amplification using the primer pair combination described in claim 4 or 5 to obtain the amplification products corresponding to the three molecular marker sites; Step (3) Sequencing the amplified product and comparing the sequencing results with the nucleotide sequences shown in SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3 respectively to determine the genotype of the litchi at the three molecular marker sites; Step (4) When the litchi genome to be tested contains any two or more favorable genotypes from the molecular marker combination described in claim 3, the litchi to be tested is identified as a candidate large-fruited litchi germplasm.

[0015] Compared with the prior art, the beneficial effects of the present invention are as follows: The present invention obtains a molecular marker combination that is highly significantly correlated with the size of litchi fruit. By using this molecular marker combination to detect litchi breeding materials, genotyping can be completed at the seedling stage, without waiting for the plants to flower and bear fruit for phenotypic screening. This allows for accurate and efficient prediction of fruit size, greatly improving the selection efficiency of litchi breeding. The primer pair of this invention is used to detect molecular marker combinations related to the size of litchi fruit. It has high specificity and can accurately amplify the sequence containing the InDel / SNP molecular marker site of this invention. The kit prepared using this primer pair can assist in selective breeding and efficiently identify high and low single fruit weight of litchi. The molecular marker combination related to litchi fruit size and its application in this invention can efficiently assist in litchi breeding and selection, and has extremely high economic value. Attached Figure Description

[0016] Figure 1 Manhattan plot for genome-wide association analysis of single fruit weight of litchi, where (A) 2022 and (B) 2023.

[0017] Figure 2 Manhattan plots for the transverse diameter (A) of 2021, the longitudinal diameter (B) of 2022, the longitudinal diameter (C) of 2021, and the longitudinal diameter (D) of 2023.

[0018] Figure 3 To identify and analyze the fruit phenotype of tomato overexpressing LITCHI000636, the following data were collected: (A) Phenotypic diagram of mature fruit of wild type and overexpression line, scale bar = 1 cm; (B) PCR verification of target gene; (C) Gene expression level verification; (D) Box plot of fruit transverse diameter; (E) Box plot of fruit longitudinal diameter; (F) Box plot of fruit lateral diameter; (G) Box plot of single fruit weight. Different letters indicate significant differences (Duncan multiple comparisons, p < 0.05).

[0019] Figure 4 To identify and analyze the fruit phenotype of tomatoes overexpressing LITCHI030544, the following data were collected: (A) Phenotypic diagrams of mature fruits of wild-type and overexpressing lines (scale bar = 1 cm); (B) PCR verification of the target gene; (C) Verification of gene expression level; (D) Box plot of fruit transverse diameter; (E) Box plot of fruit longitudinal diameter; (F) Box plot of fruit lateral diameter; (G) Box plot of single fruit weight. Different letters indicate significant differences (Duncan multiple comparisons, p < 0.05).

[0020] Figure 5 Box plot of differences in single fruit weight of litchi at different genotypes (insertion / deletion) at the Chr5 chromosome LcChr5:16128136_InDel47 locus (2021-2025), ns indicates no significant difference. This indicates that p < 0.05. This indicates that p < 0.01.

[0021] Figure 6 Box plots showing the differences in single fruit weight among different genotypes of litchi at two adjacent SNP loci on chromosome Chr4 (2021-2025), where (A) represents the LcChr4:8764522 locus and (B) represents the LcChr4:8764809 locus. ns indicates no significant difference. This indicates that p < 0.05. This indicates that p < 0.01. This indicates that p < 0.001. This means p < 0.0001.

[0022] Figure 7 The agarose gel electrophoresis typing diagrams of the LcChr5:16128136_InDel47 site in the seedling population and the core population are shown. (A) Amplification results of 20 large fruits and 20 small fruits in the seedling population; (B) Amplification results of 16 large fruits and 17 small fruits in the core population. Detailed Implementation

[0023] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are specific implementations of the present invention and are not limited to all embodiments.

[0024] Therefore, the following detailed description of embodiments of the present invention is not intended to limit the scope of the claimed invention, but merely illustrates some embodiments of the invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention.

[0025] It should be noted that, in the absence of conflict, the embodiments and features and technical solutions in the embodiments of the present invention can be combined with each other. It should be noted that similar reference numerals and letters in the following figures indicate similar items. Therefore, once an item is defined in one figure, it does not need to be further defined and explained in subsequent figures.

[0026] Example 1: Phenotypic Data Collection: The litchi fruit resources used in this invention were all collected from the litchi resource nursery in Danzhou City, Hainan Province, with 70, 129, 192, 59, and 174 samples collected in 2021, 2022, 2023, and 2025, respectively. Fifteen mature fruits were randomly selected, with five fruits per group, and the results were repeated three times. The transverse diameter, longitudinal diameter, and lateral diameter of each fruit were measured using an electronic digital caliper (accuracy 0.01 mm), and the weight of each fruit was measured using an electronic balance (accuracy 0.01 g). The average value of each indicator for each group was taken, and the average value of the three replicates was used to represent the measurement value.

[0027] Example 2: Genome-wide association analysis of litchi fruit size-related traits: like Figure 1 As shown, based on the previously obtained SNP and InDels variation maps of litchi materials, this invention screened for missing rates (missing rate < 0.2), minimum allele frequency (MAF > 0.05), average sequencing depth (average depth < 80 and average depth > 20), and unique alignment, resulting in 13,603,741 high-quality SNPs and 1,528,220 InDels for subsequent genome-wide association studies (GWAS). The population structure Q-matrix generated by Admixture was used as the input file for GWAS analysis to control for fixed effects. The FarmCPU model was used to perform GWAS on the five consecutive years of litchi single-fruit weight phenotypic data from 2021 to 2025, with a resolution of -log10( P >6 (corresponding) P <1×10 -6 () is used as a significant association threshold.

[0028] In this analysis, SNP loci significantly associated with single fruit weight were detected at 16263301 bp and 15976653 bp on chromosome 5, and these two loci were repeatedly detected in different years (Table 1). Figure 1 SNP loci significantly associated with the transverse and longitudinal diameters of the fruit were detected on chromosome Chr4. Specifically, an SNP locus significantly associated with both transverse and longitudinal diameters was detected at 8638437 bp; an SNP locus significantly associated with transverse diameter was detected at 8708156 bp; and an SNP locus significantly associated with longitudinal diameter was detected at 8130729 bp (Table 2). Figure 2 ).

[0029] Table 1: Genome-wide association analysis of single fruit weight of litchi in 2022-2023

[0030] Table 2: Genome-wide association analysis of transverse and longitudinal diameters of litchi fruit from 2021 to 2023

[0031] Example 3: Candidate gene identification and functional verification: To further clarify the candidate genes corresponding to the stable genetic loci located by GWAS in Example 2, this invention performed gene annotation on the two associated regions of Chr5 and Chr4, and identified candidate genes within the Chr5 associated region. LITCHI000636 Candidate genes within the Chr4-associated region LITCHI030544 . Construct separately LITCHI000636 and LITCHI030544 The plant overexpression vector was transformed into 'Micro Tom' tomato, resulting in three independent transgenic lines from each vector. PCR identification results showed that each overexpression line could amplify the target gene band, while WT showed no corresponding band. Figure 3 B, Figure 4 B); qRT-PCR analysis further confirmed that both genes were significantly overexpressed in their respective overexpression lines (B). Figure 3 C, Figure 4 C). Phenotypic results show ( Figure 3 A, Figure 4 A) Compared to WT, overexpression LITCHI000636 and LITCHI030544 The transverse diameter, longitudinal diameter, lateral diameter, and single fruit weight of mature tomatoes are all significantly reduced. Figure 3 DG, Figure 4 The presence of DG indicates that both genes play a negative regulatory role in fruit size regulation.

[0032] Example 4: Identification of molecular markers related to fruit size: Given LITCHI000636 and LITCHI030544 Both genes negatively regulate fruit size. This invention further performs variation analysis on the genomic sequences of the two genes to develop molecular markers associated with fruit size.

[0033] right LITCHI000636Resequencing of the gene revealed a 47 bp insertion / deletion variant (Chr5:16128136), named LcChr5:16128136_InDel47. The nucleotide sequence of this marker is shown in SEQ ID NO.1, with the 47 bp insertion / deletion site located at position 172 from the 5' end. Resequencing of the LITCHI030544 gene identified two SNP sites (Chr4:8764522 and Chr4:8764809), named LcChr4:8764522 and LcChr4:8764809, respectively. The nucleotide sequence of LcChr4:8764522 is shown in SEQ ID NO.2, with the 245th base from the 5' end being a T / G polymorphism site; the nucleotide sequence of LcChr4:8764809 is shown in SEQ ID NO.3, with the 348th base from the 5' end being a T / A polymorphism site.

[0034] Genotyping of litchi germplasm resources from 2021 to 2025 was performed at the three loci mentioned above, and the differences in single fruit weight among different genotypes were compared. The results showed that at the LcChr5:16128136_InDel47 locus, the average single fruit weight of the insertion genotype was significantly higher than that of the deletion genotype in 2022, 2023, and 2025. Figure 5 At the LcChr4:8764522 locus, the average single fruit weight of the GG genotype was significantly higher than that of the TT and TG genotypes in 2022-2025. Figure 6 In the LcChr4:8764809 locus, the average single fruit weight of the AA genotype was significantly higher than that of the TT and TA genotypes from 2021 to 2025. Figure 6 B). The favorable genotypes for the three loci are insertion type, GG, and AA, respectively.

[0035] Example 5: Cloning and sequencing verification of molecular markers in litchi germplasm resources: To verify the reliability of the above SNP / InDel sites, specific primers were designed using the upstream and downstream sequences of each site as templates. The primer sequences are as follows: Primer pair used to amplify the LcChr5:16128136_InDel47 site: Upstream primer: 5'-GCCTGAATTGAGCTATGC-3' (SEQ ID NO.4); Downstream primer: 5'-ACCTCCTTTAGGCTCATAAG-3' (SEQ ID NO.5).

[0036] Primer pair used to amplify the LcChr4:8764522 site: Upstream primer: 5'-GCTGTTTATTCAATCTACCACC-3' (SEQ ID NO.6); Downstream primer: 5'-GTACTCTGGAAATTGTGCTAC-3' (SEQ ID NO.7).

[0037] Primer pair used to amplify the LcChr4:8764809 site: Upstream primer: 5'-GTGGTGGTTGTGGATTATG-3' (SEQ ID NO.8); Downstream primer: 5'-TTGGGTTTGTGGGTGATA-3' (SEQ ID NO.9).

[0038] The three primer pairs mentioned above were used to perform PCR amplification on the DNA of the litchi germplasm to be tested. The amplification system is shown in the table below: Table 3: PCR reaction system

[0039] The amplification program employed a three-step cyclic method: pre-denaturation at 98℃ for 2 min; denaturation at 98℃ for 10 s, annealing at 54-56℃ for 5 s, extension at 68℃ for 5 s, for a total of 32 cycles; and a final extension at 72℃ for 5 min. The obtained DNA fragments were sequenced, and the sequencing results were compared with SEQ ID NO. 1-SEQ ID NO. 3 to detect the genotype at each target locus, thus enabling effective prediction of the individual fruit weight of the litchi. KOD Mix amplification buffer was purchased from Toyobo (Shanghai) Biotechnology Co., Ltd., and primers were synthesized by Sangon Biotech (Shanghai) Co., Ltd.

[0040] To verify the practicality of the aforementioned molecular markers, this invention utilizes two independent litchi germplasm resources: a seedling population and a core population. The seedling population comprises 20 large-fruited germplasm accessions (single fruit weight 35.24-54.89 g) and 20 small-fruited germplasm accessions (single fruit weight 8.55-24.02 g), while the core population comprises 16 large-fruited germplasm accessions (single fruit weight 39.19-58.90 g) and 17 small-fruited germplasm accessions (single fruit weight 7.97-18.53 g). Genotyping and single fruit weight were performed at three loci: LcChr4:8764522, LcChr4:8764809, and LcChr5:16128136_InDel47. The primers and amplification reaction system used for sequencing were the same as described above (the target fragment sequencing after PCR was outsourced to Sangon Biotech (Shanghai) Co., Ltd.). The sequencing results were analyzed using SnapGene 7.0.2 software for peak plotting and genotype reading, as shown below. Figure 7 As shown in Tables 4 and 5.

[0041] Table 4: Single fruit weight and target locus genotyping of litchi germplasm resources from seedling populations

[0042] Table 5: Single fruit weight and target locus genotyping of litchi germplasm resources in the core population

[0043] Table 6: Genotypic distribution of the LcChr5:16128136_InDel47 locus in two litchi populations

[0044] Table 7: Genotypic distribution of LcChr4:8764522 locus in two litchi populations

[0045] Table 8: Genotypic distribution of LcChr4:8764809 locus in two litchi populations

[0046] Tables 6 to 8 show that the three molecular markers of this invention have a good effect on distinguishing between high and low single fruit weight litchi germplasm. In the core population, the favorable genotypes LcChr5:16128136_InDel47, LcChr4:8764522, and LcChr4:8764809 accounted for 37.5%, 37.5%, and 31.3% of the large-fruited germplasm, respectively, while all were 0% in the small-fruited germplasm, and the differences were statistically significant. p <0.05). In the seedling population, the favorable genotypes LcChr4:8764522 and LcChr4:8764809 accounted for 25% of the large-fruited germplasm, which was also significantly higher than that of the small-fruited germplasm (0%). p <0.05). The above results indicate that this molecular marker combination can effectively distinguish between high and low single fruit weight germplasm in populations with different genetic backgrounds.

[0047] In summary, the molecular marker combination and primer pairs described in this invention can rapidly and accurately predict the weight of a single litchi fruit during the seedling stage, effectively improving breeding selection efficiency and making it suitable for large-scale application in litchi molecular marker-assisted selection breeding.

[0048] Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention.

[0049] Although the invention has been described in conjunction with specific features and embodiments, it is obvious that various modifications and combinations can be made therein without departing from the spirit and scope of the invention. Accordingly, this specification and drawings are merely exemplary descriptions of the invention as defined by the appended claims, and are considered to cover any and all modifications, variations, combinations, or equivalents within the scope of the invention. Clearly, those skilled in the art can make various alterations and modifications to the invention without departing from its spirit and scope. Thus, if such modifications and modifications of the invention fall within the scope of the claims and their equivalents, the invention is also intended to include such modifications and modifications.

Claims

1. A molecular marker combination related to litchi fruit size, characterized in that, The molecular marker combination consists of one InDel molecular marker and two SNP molecular markers; (1) The nucleotide sequence of the InDel molecular marker is shown in SEQ ID NO.

1. The 172nd base from the 5' end of the sequence shown in SEQ ID NO.1 is the InDel site, and the InDel site is an insertion of 47 bp bases. (2) The nucleotide sequence of the SNP molecular marker is shown in SEQ ID NO.

2. The 245th base from the 5' end of the sequence shown in SEQ ID NO.2 is the SNP site, and the SNP site is T or G. (3) The nucleotide sequence of the SNP molecular marker is shown in SEQ ID NO.

3. The 348th base from the 5' end of the sequence shown in SEQ ID NO.3 is the SNP site, and the SNP site is A or T.

2. The molecular marker combination related to litchi fruit size according to claim 1, characterized in that, When the litchi genome contains any two or more favorable genotypes from the molecular marker combination, it is identified as a candidate large-fruited litchi germplasm. (1) A 47 bp insertion / deletion marker located at 16128136 bases on chromosome 5 has an insert type as its preferred genotype; (2) The T / G SNP marker located at 8764522 bases on chromosome Chr4 has a favorable genotype of GG; (3) The T / A SNP marker located at base 8764809 on chromosome Chr4 has the favorable genotype AA.

3. The method for detecting the molecular marker combination related to litchi fruit size as described in claim 1, characterized in that, The primer pair consists of two single-stranded DNA molecules, including: (1) The primer pair used to amplify the 47 bp insertion / deletion marker has the upstream primer being 5'-GCCTGAATTGAGCTATGC-3', as shown in SEQ ID NO.4; and the downstream primer being 5'-ACCTCCTTTAGGCTCATAAG-3', as shown in SEQ ID NO.5; (2) The primer pair used to detect the Chr4-8764522 T / G SNP marker has an upstream primer of 5'-GCTGTTTATTCAATCTACCACC-3', as shown in SEQ ID NO.6; and a downstream primer of 5'-GTACTCTGGAAATTGTGCTAC-3', as shown in SEQ ID NO.7; (3) The primer pair used to detect the Chr4-8764809 T / A SNP marker has an upstream primer of 5'-GTGGTGGTTGTGGATTATG-3', as shown in SEQ ID NO.8; and a downstream primer of 5'-TTGGGTTTGTGGGTGATA-3', as shown in SEQ ID NO.

9.

4. The molecular marker combination related to litchi fruit size according to claim 3, characterized in that, The primer pair was used to identify high and low single fruit weights of lychee.

5. The application of the kit for detecting the molecular marker combination described in claim 1 in marker-assisted selection breeding of litchi, characterized in that, The kit contains the primer pair as described in claim 3.

6. The application of the molecular marker combination kit according to claim 5 in marker-assisted selection breeding of litchi, characterized in that, The kit is used to identify high and low single fruit weights of lychee.

7. The application of the molecular marker combination kit according to claim 6 in marker-assisted selection breeding of litchi, characterized in that, The kit also contains one or more of the following: PCR amplification buffer, dNTPs, thermostable DNA polymerase, and ddH2O.

8. The use of the molecular marker combination according to any one of claims 1 to 3 in the kit for molecular marker-assisted selection breeding of litchi.

9. The application according to claim 8, characterized in that, The application is to predict the weight of a single fruit during the litchi seedling stage, or to screen breeding materials for large-fruited litchi, or to assist in the selection of hybrid parents and the early selection of seedling offspring.

10. A method for auxiliary identification of the size of litchi fruit, characterized in that, Includes the following steps: Step (1) Extract genomic DNA from the litchi to be tested; Step (2) Using the genomic DNA as a template, perform PCR amplification using the primer pair combination described in claim 4 or 5 to obtain the amplification products corresponding to the three molecular marker sites; Step (3) Sequencing the amplified product and comparing the sequencing results with the nucleotide sequences shown in SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3 respectively to determine the genotype of the litchi at the three molecular marker sites; Step (4) When the litchi genome to be tested contains any two or more favorable genotypes from the molecular marker combination described in claim 3, the litchi to be tested is identified as a candidate large-fruited litchi germplasm.

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