AFLP molecular markers, detection primers and their applications for sweet cherry fruit firmness

Abstract

The present invention discloses AFLP molecular markers, detection primers, and applications for sweet cherry fruit firmness. Using BSA-seq, the present invention preliminarily locates the target gene segment and performs fine mapping, determining that the key gene controlling sweet cherry fruit firmness is located within the 473Kb interval on chromosome 6. Combining gene expression analysis and gene cloning, the present invention identifies the gene PavSCPL controlling sweet cherry fruit firmness, and that a 5.2Kb transposon insertion allele is present in the coding region of the PavSCPL gene for firm-fleshed sweet cherries. This 5.2Kb transposon insertion allele inactivates the PavSCPL gene. Based on this, the present invention develops an AFLP molecular marker for sweet cherry fruit firmness. The AFLP molecular marker achieves 100% accuracy in detecting sweet cherry fruit firmness, demonstrating promising applications in assisting the genetic improvement of the firm flesh trait in sweet cherries and in the selection of storability-oriented sweet cherry germplasm.

Description

Technical Field

[0001] The invention relates to a molecular marker of fruit firmness, in particular to an AFLP molecular marker of sweet cherry fruit firmness, a detection primer and an application thereof, belonging to the field of molecular markers of sweet cherry fruit firmness, a detection primer and an application thereof. Background Art

[0002] Sweet cherry (Prunus avium L.), a species of the Rosaceae family, belongs to the subgenus Prunus. It is currently one of the most profitable deciduous fruit trees in northern China and the earliest to be marketed. Its high nutritional value and health benefits make it a popular choice among consumers. Fruit firmness is one of the most important quality attributes of sweet cherries. However, sweet cherry fruits tend to soften after ripening, significantly deteriorating in quality. This is a significant factor limiting the storage and shelf life of sweet cherry fruits, severely restricting the development of the sweet cherry industry. Currently, little research has been conducted on genes associated with sweet cherry fruit firmness, and the key mechanisms controlling sweet cherry fruit firmness remain unclear. Therefore, identifying genes or molecular markers associated with sweet cherry fruit firmness and analyzing their mechanisms are of great practical significance for improving the overall quality of sweet cherry fruits, enhancing market competitiveness, and selecting and cultivating new firm-fruited sweet cherry varieties. Summary of the Invention

[0003] One of the purposes of the present invention is to provide AFLP molecular markers for sweet cherry fruit firmness;

[0004] The second object of the present invention is to provide primers for detecting AFLP molecular markers of sweet cherry fruit firmness;

[0005] The third purpose of the present invention is to apply the molecular marker of sweet cherry fruit firmness or its detection primer to detect the firmness of sweet cherry fruit or to select and cultivate new hard sweet cherry varieties.

[0006] One aspect of the present invention is to provide a transposon insertion fragment having a nucleotide sequence shown in SEQ ID No. 1 as an AFLP molecular marker for sweet cherry fruit firmness.

[0007] In order to determine whether Pav_sc0000480.1_g920.1.mk is a candidate gene that controls the firmness of sweet cherry fruit, the present invention analyzed the sequence information differences of the coding region and promoter region of the Pav_sc0000480.1_g920.1.mk gene in the F1 individual plants of soft-fleshed, hard-fleshed and hard sweet cherries, respectively, considering that the Pav_sc0000480.1_g920.1.mk gene is not expressed in the F1 individual plants of hard-fleshed sweet cherries. The results showed that a 5.2 kb fragment was inserted in the second exon region of the coding region of the Pav_sc0000480.1_g920.1.mk gene in the F1 individual plants of hard-fleshed sweet cherries (the nucleotide sequence of the 5.2 kb fragment is shown in SEQ ID No. 1), and there were no sequence differences in other regions of the coding region and the promoter region. BLAST analysis of the 5.2 kb insert fragment in NCBI revealed a transposon insertion. The lack of expression of the Pav_sc0000480.1_g920.1.mk gene in the F1 plantlets of firm sweet cherry may be due to gene inactivation caused by the 5.2 kb transposon insertion. Therefore, the Pav_sc0000480.1_g920.1.mk gene is a candidate gene controlling sweet cherry fruit firmness. Based on gene annotation of the sweet cherry genome, the Pav_sc0000480.1_g920.1.mk gene is a member of the serine carboxypeptidase protein family and was named PavSCPL. The nucleotide sequence of its CDS is shown in SEQ ID No. 2.

[0008] The present invention further designs amplified fragment length polymorphism (AFLP) markers PavSCPL-1-F / R (SEQ ID No. 3 and SEQ ID No. 4), PavSCPL-2-life-F / R (SEQ ID No. 5 and SEQ ID No. 6), and PavSCPL-3-right-F / R (SEQ ID No. 7 and SEQ ID No. 8) that are associated with the fruit firmness phenotype of sweet cherry based on the DNA polymorphism of the PavSCPL gene (5.2 kb TE insertion). 8) was used to detect co-segregation of fruit firmness traits and genotypes in selected natural and hybrid populations. Using the AFLP marker PavSCPL-1-F / R, only one large band was amplified in firm-fleshed sweet cherry fruit, suggesting a homozygous insertion of a 5.2kb transposon in the PavSCPL genotype of the firm type. Two bands (one large and one small) were amplified in firm-fleshed fruit, suggesting a heterozygous insertion in the PavSCPL genotype of the firm type. Only one short fragment was amplified in soft-fleshed sweet cherry fruit, suggesting the absence of a 5.2kb transposon insertion in the PavSCPL of the soft type. Further testing using the AFLP markers PavSCPL-2-life-F / R and PavSCPL-3-right-F / R revealed similar identification results: both firm-fleshed and firm-fleshed varieties amplified the 5.2kb transposon insertion, while the soft-fleshed variety did not. Therefore, the accuracy of the detection results of the AFLP markers related to the PavSCPL gene provided by the present invention reached 100%, and the results of the sweet cherry fruit firmness trait identified by the PavSCPL gene and alleles were consistent with the firmness phenotype results, indicating that the genotype of the PavSCPL gene co-segregated with the fruit firmness phenotype.

[0009] Another aspect of the present invention provides an application of a transposon insertion fragment having a nucleotide sequence as shown in SEQ ID No. 1 in detecting the firmness of sweet cherry fruits or in selecting and breeding hard-fleshed sweet cherry varieties, comprising: (1) designing AFLP marker primers using the transposon insertion fragment having a nucleotide sequence as shown in SEQ ID No. 1 as a target gene; (2) establishing a PCR amplification system using DNA of a sweet cherry sample to be detected as template DNA and the AFLP marker primers as amplification primers to perform PCR amplification; (3) if the transposon insertion fragment shown in SEQ ID No. 1 is amplified in the amplified product, the sweet cherry fruit to be detected is a hard-fleshed type or a hard-fleshed type; if the transposon insertion fragment shown in SEQ ID No. 1 is not amplified in the amplified product, the sweet cherry fruit to be detected is a soft-fleshed type.

[0010] In a preferred embodiment of the present invention, the PCR reaction amplification system is as follows: KOD One™ PCR Master Mix 12.5 μL, forward primer 0.75 μL, reverse primer 0.75 μL, ddH2O 9 μL, DNA 2 μL, total system 25 μL.

[0011] In a preferred embodiment of the present invention, the PCR amplification procedure is as follows: pre-denaturation at 95°C for 5 min; denaturation at 95°C for 15 s, annealing at 57°C for 15 s, extension at 72°C for 1 or 3 min, 32 amplification cycles; extension at 72°C for 10 min.

[0012] As a preferred specific embodiment of the present invention, the present invention designed three pairs of AFLP marker primers using the transposon insert fragment shown in SEQ ID No. 1 as the target gene, namely: PavSCPL-1-F / R, PavSCPL-2-life-F / R and PavSCPL-3-right-F / R; wherein the nucleotide sequences of the forward primer and reverse primer of the AFLP marker primer PavSCPL-1-F / R are shown in SEQ ID No. 3 and SEQ ID No. 4, respectively, the nucleotide sequences of the forward primer and reverse primer of the AFLP marker primer PavSCPL-2-life-F / R are shown in SEQ ID No. 5 and SEQ ID No. 6, respectively, and the nucleotide sequences of the forward primer and reverse primer of the AFLP marker primer PavSCPL-3-right-F / R are shown in SEQ ID No. 7 and SEQ ID No. 8, respectively.

[0013] The present invention further provides a PCR detection kit for detecting the firmness of sweet cherry fruit, comprising: 12.5 μL of KOD One™ PCR Master Mix, a forward primer, a reverse primer and ddH2O; wherein the forward primer and the reverse primer are selected from any one of the following three pairs of AFLP marker primers: (1) AFLP marker primer PavSCPL-1-F / R, the nucleotide sequences of the forward primer and the reverse primer are shown in SEQ ID No. 3 and SEQ ID No. 4, respectively; (2) AFLP marker primer PavSCPL-2-life-F / R, the nucleotide sequences of the forward primer and the reverse primer are shown in SEQ ID No. 5 and SEQ ID No. 6, respectively; and (3) AFLP marker primer PavSCPL-3-right-F / R, the nucleotide sequences of the forward primer and the reverse primer are shown in SEQ ID No. 7 and SEQ ID No. 8, respectively.

[0014] The present invention uses a 'Raney'×'Samido' F1 hybrid population as material and preliminarily locates the target gene segment using BSA-seq. Based on this, fine positioning is performed, and the key gene controlling the fruit firmness of sweet cherry is determined to be located in the 473Kb interval on chromosome 6. Combined with gene expression analysis and gene cloning, the gene PavSCPL controlling the fruit firmness of sweet cherry is identified. In addition, a 5.2Kb transposon insertion allele is present in the coding region of the PavSCPL gene of hard-fruited sweet cherry. The 5.2Kb transposon insertion allele causes the PavSCPL gene to be inactivated. The present invention further develops AFLP molecular markers for fruit firmness based on the diversity of the PavSCPL gene. The accuracy rate of sweet cherry fruit firmness detection using the AFLP markers reaches 100%. The sweet cherry fruit firmness trait results from the PavSCPL gene and allele identification are consistent with the firmness phenotypic results. The present invention has application prospects in assisting the genetic improvement of the hard flesh trait of sweet cherry and in the breeding of storability-type sweet cherry germplasm.

[0015] Definitions of terms used in this invention

[0016] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods, devices, and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods, devices, and materials are now described.

[0017] The term "soft flesh type" in the present invention refers to sweet cherry fruit with a hardness of 0-3.5 kg / cm 2 The term "hard flesh type" refers to the hardness of sweet cherry fruit is 3.5-6.5Kg / cm 2 The term "hard type" refers to the hardness of sweet cherry fruit is 6.5Kg / cm 2 above.

[0018] The term "AFLP" refers to amplified restriction fragment polymorphism.

[0019] The term "polynucleotide" or "nucleotide" means deoxyribonucleotides, deoxyribonucleosides, ribonucleosides or ribonucleotides and polymers thereof in single-stranded or double-stranded form. Unless specifically limited, the term encompasses nucleic acids containing known analogs of natural nucleotides that have binding properties similar to the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise specifically limited, the term also means oligonucleotide analogs, which include PNA (peptide nucleic acid), DNA analogs used in antisense technology (phosphorothioate, phosphoramidate, etc.). Unless otherwise specified, a specific nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (including but not limited to degenerate codon substitutions) and complementary sequences as well as explicitly specified sequences. In particular, degenerate codon substitutions can be achieved by generating a sequence in which the 3rd position of one or more selected (or all) codons is substituted with mixed bases and / or deoxyinosine residues. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 Statistics and genetic analysis of fruit hardness of paternal, maternal and hybrid populations; A. Fruit hardness statistics of paternal and maternal parents; BC. Frequency statistical analysis of fruit hardness of hybrid populations in 2018 (B) and 2019 (C).

[0021] Figure 2 These are the BSA positioning analysis results for the sweet cherry fruit firmness trait; A and B were positioned using the △(SNP-index) and △(InDel-index) methods, respectively.

[0022] Figure 3 These are the results of fine mapping analysis of the trait segment on sweet cherry chromosome 6 that controls the fruit firmness of sweet cherry.

[0023] Figure 4 To analyze the expression changes of genes in the fine-mapped interval that controls sweet cherry fruit firmness based on transcriptome data.

[0024] Figure 5 The results of screening of candidate genes controlling sweet cherry fruit firmness and analysis of the gene coding regions were presented.

[0025] Figure 6 Genotyping of molecular markers for 5.2kb transposon insertion in some natural and hybrid populations; A. Detection results of AFLP marker PavSCPL-1-F / R; B. Detection results of PavSCPL-2-life-F / R marker primers; C. Detection results of PavSCPL-3-right-F / R detection primers. DETAILED DESCRIPTION

[0026] The present invention will be further described below with reference to specific embodiments, and the advantages and features of the present invention will become clearer as the description proceeds. However, these embodiments are merely exemplary and do not limit the scope of the present invention in any way. It should be understood by those skilled in the art that the details and forms of the present invention may be modified or replaced without departing from the spirit and scope of the present invention, and such modifications and replacements fall within the scope of protection of the present invention.

[0027] 1. Biomaterials

[0028] This experiment is based on the genetic population of sweet cherry 'Rainier' as the female parent and 'Summit' as the male parent constructed by the research team in the early stage. It was planted in the sweet cherry breeding nursery of Xinxiang Comprehensive Experimental Base, Zhengzhou Fruit Research Institute, Chinese Academy of Agricultural Sciences in 2012. The hybrid population has a total of 204 F1 plants, and the hardness of its F1 offspring fruits was tested for two consecutive years in 2018 and 2019.

[0029] The materials used for genetic positioning analysis were collected from fresh leaves of the above population, and the leaves were stored at an ultra-low temperature of -80°C for subsequent DNA extraction.

[0030] 2. Primer information

[0031] Table 1 Primer information used in the present invention

[0032]

[0033]

[0034]

[0035] Experimental Example 1: Cloning of candidate genes controlling sweet cherry fruit firmness

[0036] 1. Test methods

[0037] (1) Determination of fruit firmness of F1 hybrid population.

[0038] Fruit firmness was measured using a TA-XT plus texture analyzer with a P / 2 probe and a 2 mm diameter. Specific parameters included compression mode, pre-test speed of 1.00 mm / sec, mid-test speed of 1.00 mm / sec, post-test speed of 2.00 mm / sec, displacement of 3.000 mm, and a trigger force of 5.0 g. Ten mature fruits from each individual plant in the F1 hybrid population were randomly selected for testing with the skin intact. Each fruit was tested on three different surfaces at the equatorial position.

[0039] (2) BSA-sequencing analysis.

[0040] Based on the results of a survey of fruit firmness in F1 hybrid populations, 20 individuals representing two extreme traits, hard and 20 individuals representing two extreme traits, were randomly selected from the F1 segregating population for DNA extraction. Using a total DNA volume of 500 ng per sample in the progeny pool as a guideline, the required DNA volume per sample was calculated. Equal amounts of DNA from individual plants of the two firmness types were mixed to form a hard pool and a soft pool. The mixed BSA-seq samples were sent to Tianjin Novogene Biotechnology Co., Ltd. for resequencing.

[0041] (3) Fine positioning analysis.

[0042] Fifteen single-nucleotide polymorphisms (SNPs) and indels (InDels) within the major QTL region identified by BSA-seq analysis were validated by polyacrylamide gel electrophoresis (PAGE) between the hard and soft pools. Recombinant individuals in the F1 hybrid population were screened for fine mapping. Primers were designed using Primer Premier 5.0 software. PCR amplification was performed using 2× Taq Master Mix (Vazyme Biotechnology Co., Ltd., Nanjing, China) in a 50 μL volume containing 1 μL of 0.5-μM forward and reverse primers and 50–100 ng of DNA. PCR products were analyzed by PAGE or sequencing by Shanghai Sangon Biotechnology Co., Ltd.

[0043] (4) Analysis of gene expression within the fine-grained positioning interval.

[0044] Using the sweet cherry reference genome, a total of 72 genes were annotated within the finely mapped candidate interval. Three individual plants were randomly selected from the hard-pool and soft-pool, and total RNA was extracted from their leaves. This RNA was then reverse-transcribed into cDNA. The expression levels of the 72 annotated genes were analyzed using the sweet cherry Histone2 gene as an internal reference.

[0045] qPCR reactions were performed on an ABI7500 PCR thermal cycler (Applied Biosystems, Foster City, CA, United States) using the TransStart Top Green qPCR SuperMix kit (Beijing Quanshijin Biotechnology Co., Ltd., Beijing, China). The sweet cherry Histone2 gene was used as an internal reference. Three biological replicates were performed, and the average value was calculated.

[0046] (5) Cloning of candidate gene sequences.

[0047] Total DNA was extracted from each of the two pools to amplify candidate gene sequences. Primers PavSCPL-F / PavSCPL-R and PavSCPL-Pro-F / PavSCPL-Pro-R were designed using Primer Premier 5.0 software based on the sweet cherry genome database to amplify the candidate gene DNA sequence and promoter information. ExTaq enzyme (TaKaRa, Dalian, China) was used to amplify the PavSCPL candidate gene DNA sequence and promoter region, respectively. PCR products were sequenced and analyzed to identify DNA polymorphisms.

[0048] 2 Test results

[0049] 2.1 Investigation and analysis of genetic patterns of fruit hardness of individual plants in hybrid populations.

[0050] The inventors' team conducted a three-year investigation of the fruit hardness of 204 F1 hybrids of 'Rainier' and 'Samitol' starting in 2018. Fruits of uniform maturity (fully ripe) were selected and tested for fruit hardness using a texture analyzer (TPA method). Twenty fruits were randomly selected from each plant, and each fruit was tested on four different sides at the equator. The results showed that the fruit hardness of this hybrid population ranged from 0.5 to 12.5 kg / cm 2 The hardness of the male and female parent is 4.65Kg / cm 2 and 4.55Kg / cm 2 The fruit hardness of the F1 population is classified according to the hardness of the parents. The fruit hardness is 0-3.5Kg / cm 2 Soft meat type, 3.5-6.5Kg / cm 2 Hard meat type, 6.5Kg / cm 2 The above are hard types.

[0051] The frequency distribution of fruit firmness of F1 individual plants in two years (2018 and 2019) was plotted, and a Shapiro-Wilk test was performed. The results showed that fruit firmness was normally distributed and existed in a unimodal form. The separation of soft-fleshed and hard-fleshed traits was roughly 1:1 (in 2018, the number of soft-fleshed plants: the number of hard-fleshed plants = 52:46; in 2019, the number of soft-fleshed plants: the number of hard-fleshed plants = 48:53, Figure 1 ), indicating that the fruit firmness trait of the F1 population may be controlled by a single gene or a major gene.

[0052] 2.2 BSA localization analysis of sweet cherry fruit firmness traits.

[0053] According to the fruit hardness phenotype of F1 individual plants, two extreme phenotypes of fruit hardness, soft flesh type (fruit hardness 0-3.5Kg / cm 2) and hard type (fruit hardness 9Kg / cm 2 Equal amounts of DNA were evenly mixed from 21 individual plants of each of the above lines to construct two gene pools, one for soft flesh and one for firm flesh. High-throughput sequencing analysis was performed on the parents ('Rainier' and 'Samit') and the two pools. Using both △ (SNP-index) and △ (InDel-index) mapping analysis methods, the candidate segments for the fruit firmness trait were mapped to the region between the two molecular markers InDel-5.759971 and SNP-11.506215 on chromosome 6. The segment size was 5.74Mb, indicating that the candidate gene controlling the firmness of sweet cherry fruit may be located within a 5.74Mb candidate region on chromosome 6 ( Figure 2 ).

[0054] 2.3 Fine positioning analysis of sweet cherry fruit firmness traits.

[0055] To further narrow down the chromosomal location of candidate genes controlling sweet cherry fruit firmness, this study used SNP and InDel markers obtained by BSA-seq to perform fine mapping analysis on the F1 population.

[0056] In order to find the individual plants with chromosomal recombination in the F1 population, this experiment used the two markers InDel-5.759971 and SNP-11.506215 to perform genotyping analysis on 204 F1 plants, and found 13 exchange plants. Based on the BSA-seq results, 15 molecular markers were designed within the two molecular markers InDel-5.759971 and SNP-11.506215, and the genotypes of the 13 exchange plants were analyzed. Combined with the phenotype of the fruit hardness of the exchange plants, it was determined that the candidate segment controlling the hardness of sweet cherry fruit was defined in the 473Kb region between the two molecular markers SNP-7.418778 and SNP-7.891914 on the sweet cherry genome ( Figure 3 ).

[0057] 2.4 Combine transcriptomics to screen candidate genes that control sweet cherry fruit hardness.

[0058] Based on gene annotation of the sweet cherry genome, a total of 72 genes were identified within the 473 kb region between molecular markers SNP-7.418778 and SNP-7.891914 on chromosome 6. To further identify candidate genes regulating sweet cherry fruit firmness, this study investigated transcriptome differences between soft-fleshed (three individual plants) and firm-fleshed (three individual plants) F1 populations.

[0059] Through differential gene expression analysis and functional annotation of differentially expressed genes, the focus was on the expression changes of 72 genes in a 473Kb region between the molecular markers SNP-7.418778 and SNP-7.891914 on chromosome 6. The results showed that the expression levels of most genes in the candidate region did not change significantly between the soft-fleshed and hard-fleshed F1 plants, with only five genes (Pav_sc0000480.1_g920.1.mk, P ... 1_g990.1.mk, Pav_sc0000480.1_g1030.1.mk, Pav_sc0000480.1_g1040.1.mk and Pav_sc0000480.1_g1090.1.mk) showed significant differences in expression between the soft-fleshed and hard-fleshed F1 plants, especially the Pav_sc0000480.1_g920.1.mk gene was not expressed in the hard-fleshed F1 plants but highly expressed in the soft-fleshed F1 plants ( Figure 4 ), it is speculated that the Pav_sc0000480.1_g920.1.mk gene is most likely to be the candidate gene controlling the firmness of sweet cherry fruit.

[0060] 2.5 Cloning of candidate genes controlling sweet cherry fruit firmness

[0061] In order to determine whether Pav_sc0000480.1_g920.1.mk is a candidate gene controlling the firmness of sweet cherry fruit, considering that the Pav_sc0000480.1_g920.1.mk gene is not expressed in the hard-fleshed sweet cherry F1 plants, the sequence information differences of the coding region and promoter region of the Pav_sc0000480.1_g920.1.mk gene in the F1 plants of soft-fleshed, hard-fleshed and hard-fleshed sweet cherries were analyzed respectively. The results showed that a 5.2 kb fragment was inserted in the second exon region of the coding region of the Pav_sc0000480.1_g920.1.mk gene in the F1 plants of hard-fleshed sweet cherries (the nucleotide sequence of the 5.2 kb fragment is shown in SEQ ID No. 1), and there were no sequence differences in other regions of the coding region and the promoter region. The 5.2 kb insert was imported into NCBI for BLAST analysis, and it was found to be a transposon insert ( Figure 5The lack of expression of the Pav_sc0000480.1_g920.1.mk gene in hard-fruited sweet cherry F1 plants may be due to gene inactivation caused by a 5.2 kb transposon insertion. Therefore, the Pav_sc0000480.1_g920.1.mk gene is considered a candidate gene for controlling sweet cherry fruit firmness. Based on gene annotation of the sweet cherry genome, the Pav_sc0000480.1_g920.1.mk gene is a member of the serine carboxypeptidase protein family and is named PavSCPL. Its nucleotide sequence is shown in SEQ ID No. 2.

[0062] Experimental Example 2 Development and Application of AFLP Markers for Sweet Cherry Fruit Firmness

[0063] Based on the DNA polymorphism of the PavSCPL gene (5.2 kb TE insertion), the amplified fragment length polymorphism (AFLP) markers PavSCPL-1-F / R (SEQ ID No. 3 and SEQ ID No. 4), PavSCPL-2-life-F / R (SEQ ID No. 5 and SEQ ID No. 6), and PavSCPL-3-right-F / R (SEQ ID No. 7 and SEQ ID No. 8) associated with the sweet cherry fruit firmness phenotype were designed and used to detect the co-segregation of fruit firmness traits with genotypes in selected natural and hybrid populations.

[0064] The materials tested included 80 sweet cherry cultivars (2 hard-fleshed materials, 7 hard-fleshed materials, and 71 soft-fleshed materials), 2 wild sweet cherry species (soft-fleshed type), and 6 hard-fleshed sweet cherry superior lines obtained by the applicant team, for a total of 96 varieties, strains or germplasms tested.

[0065] The PCR reaction system was as follows: 12.5 μL of KOD One™ PCR Master Mix, 0.75 μL of forward primer, 0.75 μL of reverse primer, 9 μL of ddH2O, and 2 μL of DNA, for a total of 25 μL. The target band was amplified using a PCR instrument (Eppendorf). The PCR amplification program was as follows: 95°C initial denaturation for 5 min; 32 cycles of denaturation at 95°C for 15 s, annealing at 57°C for 15 s, and extension at 72°C for 1 or 3 min; and 10 min of extension at 72°C.

[0066] Detection using the AFLP marker PavSCPL-1-F / R revealed that only one large fragment band was amplified in the hard-fleshed sweet cherry fruit, suggesting that the PavSCPL genotype in the hard-fleshed type was a homozygous insertion of a 5.2 kb transposon; two bands (one large fragment and one small fragment) were amplified in the hard-fleshed fruit, suggesting that the PavSCPL genotype in the hard-fleshed type was a heterozygous insertion; only one short fragment was amplified in the soft-fleshed sweet cherry fruit, suggesting that the PavSCPL genotype in the soft-fleshed type did not have a 5.2 kb transposon insertion ( Figure 6 A). Further detection using AFLP markers PavSCPL-2-life-F / R and PavSCPL-3-right-F / R revealed similar identification results, i.e., a 5.2 kb transposon insert could be amplified in both the hard-fleshed and firm-fleshed types, but not in the soft-fleshed type ( Figure 6 BC). The accuracy of AFLP marker detection results related to the PavSCPL gene reached 100%. The results of sweet cherry fruit firmness trait identified by the PavSCPL gene and its alleles were consistent with the firmness phenotype results, indicating that the genotype of the PavSCPL gene co-segregates with the fruit firmness phenotype.

Claims

1. Use of a transposon insert fragment having the nucleotide sequence shown in SEQ ID No. 1 as an AFLP molecular marker for sweet cherry fruit firmness; the fruit firmness is classified as soft-fleshed fruit, hard-fleshed fruit, or firm fruit; the soft-fleshed fruit refers to a sweet cherry fruit with a firmness of 0-3.5 kg / cm 2 The hard-fleshed fruit refers to the sweet cherry fruit with a hardness of 3.5-6.5 kg / cm 2 The hard fruit refers to the sweet cherry fruit with a hardness of 6.5Kg / cm 2 above.

2. The use according to claim 1, characterized in that include: (1) Designing AFLP molecular marker primers using the transposon insertion fragment shown in SEQ ID No. 1 as the target gene; (2) Using the DNA of the sweet cherry sample to be tested as the template DNA and the AFLP molecular marker primers as the amplification primers to establish a PCR amplification system for PCR amplification; (3) If the transposon insertion fragment shown in SEQ ID No. 1 is amplified in the amplification product, the fruit of the sweet cherry to be tested is a hard-fleshed or hard-type fruit; if the transposon insertion fragment shown in SEQ ID No. 1 is not amplified in the amplification product, the fruit of the sweet cherry to be tested is a soft-fleshed fruit.

3. The use according to claim 2, characterized in that The PCR reaction amplification system is as follows: KOD One TM PCR Master Mix 12.5 μL, forward primer 0.75 μL, reverse primer 0.75 μL, ddH2O 9 μL, DNA 2 μL, total system 25 μL.

4. The use according to claim 2, characterized in that The PCR amplification procedure was as follows: pre-denaturation at 95°C for 5 min; denaturation at 95°C for 15 s, annealing at 57°C for 15 s, extension at 72°C for 1 or 3 min, 32 amplification cycles; and extension at 72°C for 10 min.

5. AFLP molecular marker detection primers for detecting sweet cherry fruit firmness, characterized in that: The AFLP molecular marker detection primers are selected from any one of the following three groups of primers: PavSCPL-1-F / R, PavSCPL-2-life-F / R, and PavSCPL-3-right-F / R; wherein the nucleotide sequences of the forward primer and the reverse primer of the AFLP molecular marker detection primer PavSCPL-1-F / R are shown in SEQ ID No.3 and SEQ ID No.4, respectively; the nucleotide sequences of the forward primer and the reverse primer of the AFLP molecular marker detection primer PavSCPL-2-life-F / R are shown in SEQ ID No.5 and SEQ ID No.6, respectively; and the nucleotide sequences of the forward primer and the reverse primer of the AFLP molecular marker detection primer PavSCPL-3-right-F / R are shown in SEQ ID No.7 and SEQ ID No.8, respectively; the fruit hardness is divided into soft-fleshed fruit, hard-fleshed fruit, or hard-type fruit; the soft-fleshed fruit refers to a sweet cherry fruit with a hardness of 0-3.5 kg / cm 2 The hard-fleshed fruit refers to the sweet cherry fruit with a hardness of 3.5-6.5 kg / cm 2 The hard fruit refers to the sweet cherry fruit with a hardness of 6.5Kg / cm 2 above.

6. Use of the AFLP molecular marker detection primers according to claim 5 in detecting the firmness of sweet cherry fruit or in selecting and breeding hard sweet cherry varieties.

7. The use according to claim 6, characterized in that include: (1) A PCR amplification system is established using the DNA of the sweet cherry sample to be tested as the template DNA and the AFLP molecular marker detection primers as the amplification primers to perform PCR amplification; (2) if a transposon insertion fragment having a nucleotide sequence as shown in SEQ ID No. 1 is amplified in the amplification product, the fruit of the sweet cherry to be tested is a hard-fleshed or hard-textured fruit; if a transposon insertion fragment having a nucleotide sequence as shown in SEQ ID No. 1 is not amplified in the amplification product, the fruit of the sweet cherry to be tested is a soft-fleshed fruit.

8. A PCR detection kit for detecting sweet cherry fruit firmness, comprising: KOD One™ PCR Master Mix 12.5 μL, forward primer, reverse primer and ddH2O; characterized in that the forward primer and reverse primer are selected from any one of the following three pairs of AFLP molecular marker detection primers: (1) AFLP molecular marker detection primer PavSCPL-1-F / R, the nucleotide sequences of the forward primer and reverse primer are shown in SEQ ID No.3 and SEQ ID No.4 respectively; (2) AFLP molecular marker detection primer PavSCPL-2-life-F / R, the nucleotide sequences of the forward primer and reverse primer are shown in SEQ ID No.5 and SEQ ID No.6 respectively; (3) AFLP molecular marker detection primer PavSCPL-3-right-F / R, the nucleotide sequences of the forward primer and reverse primer are shown in SEQ ID No.7 and SEQ ID No.8 respectively; the fruit hardness is divided into soft flesh type fruit, hard flesh type or hard type fruit; the soft flesh type fruit refers to the sweet cherry fruit with a hardness of 0-3.5 kg / cm 2 The hard-fleshed fruit refers to the sweet cherry fruit with a hardness of 3.5-6.5 kg / cm 2 The hard fruit refers to the sweet cherry fruit with a hardness of 6.5Kg / cm 2 above.

9. Use of the PCR detection kit according to claim 8 in detecting the firmness of sweet cherry fruits or in selecting and cultivating hard sweet cherry varieties.

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