A method for fine mapping of qtl based on homologous hexaploid sweet potato

Abstract

The present application relates to the technical field of plant genetic engineering, in particular to a QTL fine mapping method based on homologous hexaploid sweet potato. The present application provides a molecular marker closely linked to the flesh color gene of sweet potato, including 14 InDel molecular markers and 1 dCAPS molecular marker. The molecular marker can be used to mine the genes related to the flesh color of sweet potato; at the same time, it can assist in breeding sweet potato varieties with different flesh colors, greatly reducing the number of breeding populations, shortening the breeding period, and realizing efficient breeding of excellent lines. At the same time, the present application first proposes a fine mapping method of QTL of the flesh color of sweet potato, realizes the fine mapping of QTL of sweet potato, and has important significance for mining functional genes related to the quality of sweet potato and assisting in breeding.

Description

Technical Field

[0001] This invention relates to the field of plant genetic engineering technology, and in particular to a method for fine mapping of QTLs based on homologous hexaploid sweet potato. Background Technology

[0002] Sweet potato is an important economic crop, characterized by its strong adaptability, high yield, and rich nutritional value. Quality traits such as flesh color are currently important goals in sweet potato breeding, and identifying key genes regulating these traits can provide excellent genetic resources for sweet potato breeding.

[0003] Sweet potato flesh color comes in white, yellow, purple, and orange-red varieties, influenced by various metabolites in the tuber. The factors affecting flesh color differ slightly among different sweet potato varieties, showing significant variations. Orange and purple sweet potato varieties primarily accumulate carotenoids and anthocyanins. The storage roots of sweet potatoes contain various carotenoids, including β-cryptoxanthin, β-carotene, lycopene, and zeaxanthin, with β-carotene being the most prevalent. The metabolic balance between β-carotene and its epoxide derivatives in sweet potato tubers regulates flesh color.

[0004] Constructing suitable sweet potato segregating populations for QTL mapping is crucial for identifying key functional genes. Factors such as population size, molecular marker type, and quantity affect the accuracy of QTL mapping and the discovery of functional genes. Sweet potato is a homohexaploid with high ploidy and heterozygosity. Currently, traditional sweet potato QTL mapping mostly uses markers such as AFLP, RAPD, and SSR, which suffer from low coverage, high cost, and difficulty in obtaining genotype data. Current sweet potato mapping studies mainly involve constructing F1 segregating populations through sexual hybridization, conducting relevant phenotypic surveys and identifications, constructing linkage maps, and then performing QTL mapping. This method is difficult to discover key sweet potato genes. There are currently few reports on the fine mapping of quality-related traits in sweet potato. In-depth exploration of loci controlling sweet potato flesh color quality traits is of great significance for optimizing sweet potato quality. This invention aims to develop molecular markers using simplex loci distributed on the sweet potato genome to map quality-related traits, thereby achieving precise utilization of target traits. Summary of the Invention

[0005] The purpose of this invention is to provide a QTL fine mapping method based on homologous hexaploid sweet potato to solve the problems existing in the prior art.

[0006] To achieve the above objectives, the present invention provides the following solution:

[0007] This invention provides molecular markers closely linked to the sweet potato flesh color gene, including 14 InDel molecular markers and 1 dCAPS molecular marker. The molecular markers and their amplification primers are shown in the table below:

[0008]

[0009] This invention provides the application of the above-mentioned molecular markers in the fine mapping of the sweet potato flesh color gene.

[0010] This invention provides the application of the above-mentioned molecular markers in the breeding of sweet potato varieties with different flesh colors.

[0011] This invention provides the application of the above-mentioned molecular markers in the preparation of a product with finely mapped sweet potato flesh color gene.

[0012] More preferably, the product includes reagents, reagent kits, and chips.

[0013] This invention provides a finely mapped product of the sweet potato flesh color gene, the product comprising the aforementioned molecular marker.

[0014] More preferably, the product includes reagents, reagent kits, and chips.

[0015] This invention provides the application of the above-mentioned finely mapped product in the fine mapping of the sweet potato flesh color gene.

[0016] This invention provides a method for finely mapping the QTL of sweet potato flesh color, comprising the following steps:

[0017] (1) Using white-fleshed sweet potato varieties and orange-red-fleshed sweet potato varieties as parents, reciprocal crosses were performed to obtain the F1 generation segregating population;

[0018] (2) Identify the potato flesh color phenotype of the F1 generation segregating population;

[0019] (3) Resequencing was performed on the parents and the F1 generation segregating population, and genome-wide association analysis was performed in conjunction with the potato flesh color phenotype to obtain the initial QTL localization interval for potato flesh color;

[0020] (4) Screen the simplex variation types between the parents in the initial localization interval of the potato flesh color QTL, and develop InDel molecular markers and dCAPS molecular markers using the InDel and SNP variation sites therein.

[0021] (5) Screening the InDel molecular markers and dCAPS molecular markers between the parents, and using the screened InDel molecular markers and dCAPS molecular markers to perform genotyping on the parents and the F1 segregating population, and obtaining genotype data of the parents and the F1 segregating population;

[0022] (6) Based on the genotype data of the parents and the F1 generation segregating population, exchanged single plants are screened, and the initial positioning interval obtained in step (3) is finely positioned in combination with the potato flesh color phenotype.

[0023] Preferably, in step (4), the primers used to amplify the InDel and dCAPS molecular markers are shown in the table below:

[0024]

[0025] Preferably, the white-fleshed sweet potato variety includes Xushu 18; the orange-red-fleshed sweet potato variety includes Pushu 32.

[0026] The F1 generation segregating population included 769 strains;

[0027] The potato flesh color phenotypes include white, pale yellow, yellow, orange-yellow, and orange-red;

[0028] The simplex variant types include ATTTTT×TTTTTT and TAAAAA×AAAAAA variant types on the six chromosome sets of the sweet potato parents, respectively.

[0029] Based on actual field growth conditions and experimental requirements, this invention classifies potato flesh color phenotypes into five categories, which are further divided into five color grades: white (1), light yellow (2), yellow (3), orange-yellow (4), and orange-red (5). The average color grade of each exchange recombination type is used to represent the potato flesh color.

[0030] In this invention, the InDel and dCAPS molecular markers were developed using DNA samples randomly selected from both parents and two individual strains from the F1 segregating population as templates. PCR amplification was performed using designed primers, and sites exhibiting polymorphic bands between the parents were screened and developed into InDel and dCAPS molecular markers. The dCAPS molecular marker introduced new restriction enzyme sites by introducing mismatched bases through primer design. The PCR amplification products were then digested with the corresponding restriction endonuclease, and the electrophoresis results between the parents showed polymorphism through length differences.

[0031] Preferably, the development of the InDel molecular marker and dCAPS molecular marker in step (3) and the genotyping in step (5) are both performed using non-denaturing polyacrylamide gel electrophoresis.

[0032] In this invention, the steps of the non-denaturing polyacrylamide gel electrophoresis method include: preparation of non-denaturing polyacrylamide gel stock solution, preparation of non-denaturing polyacrylamide gel electrophoresis plate, sample spotting and electrophoresis (electrophoresis conditions: 200V voltage, 250 mA current), silver nitrate staining (1g / L) and development (sodium hydroxide 15g / L, formaldehyde solution 4mL / L).

[0033] The present invention discloses the following technical effects:

[0034] This invention provides molecular markers closely linked to sweet potato flesh color genes, including 14 InDel molecular markers and 1 dCAPS molecular marker. These molecular markers can be used to discover genes related to sweet potato flesh color; at the same time, they can assist in the breeding of sweet potato varieties with different flesh colors, greatly reducing the number of breeding populations, shortening the breeding period, and achieving efficient breeding of superior lines.

[0035] Simultaneously, this invention proposes for the first time a method for fine mapping of QTLs for sweet potato flesh color. Specifically, this invention identifies the flesh color phenotype in a segregating F1 generation of sweet potatoes, initially locates the flesh color QTL using resequencing technology, develops polymorphic InDel and dCAPS molecular markers using simplex loci, performs genotyping using the developed polymorphic molecular markers, and finally maps the sweet potato flesh color QTL to InDel. 6.39 and SNP 6.51 The physical distance between the two molecular markers is 124.33 kb, and there are 11 genes in this interval. This enables the fine mapping of sweet potato QTLs, which is of great significance for discovering functional genes related to sweet potato quality and assisting in breeding. Attached Figure Description

[0036] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0037] Figure 1 Partial screening results for polymorphic InDel (InD) molecular markers;

[0038] Figure 2 Screening for polymorphism dCAPS (SNP) tags;

[0039] Figure 3 This is the result of polymorphic molecular marker genotyping; where A represents the polymorphism InDel 6.01Marker genotyping; B is polymorphic dCAPS (SNP) marker genotyping; M is Marker, P1 is P32, P2 is X18, and 1-64 are some segregating plants in the sweet potato F1 segregating population;

[0040] Figure 4 For the precise QTL positioning of sweet potato flesh color. Detailed Implementation

[0041] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0042] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0043] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0044] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0045] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0046] Unless otherwise specified, the experimental methods described in the following examples are conventional methods.

[0047] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0048] Example 1: Fine localization of QTL for sweet potato flesh color

[0049] I. Experimental Materials

[0050] The white-fleshed sweet potato variety Xushu 18 (X18, a low-carotenoid content variety) and the orange-red-fleshed sweet potato variety Pushu 32 (P32, a high-carotenoid content variety) were selected as parents. F1 segregating populations were constructed through reciprocal crosses, resulting in 769 F1 lines. In June 2024, the parents and F1 segregating populations were planted at the Miyun Base of China Agricultural University in Beijing (40.37°N, 116.84°E). The plants were raised on ridges, covered with plastic film, and planted in single rows with a plant spacing of 25 cm. Each line was planted with five replicates. Basic field management, including regular watering and weeding, was implemented. From June to August 2024, after the sweet potato vines closed the canopy, the aboveground phenotype was investigated. The underground phenotype and yield were assessed during the harvest period in October. The growth cycle was approximately 120 days.

[0051] II. Identification of Sweet Potato Flesh Color Phenotype

[0052] Based on actual field growth and experimental requirements, the potato flesh color phenotype was divided into five categories, which were further divided into five color grades: white (1), light yellow (2), yellow (3), orange-yellow (4), and orange-red (5). The average color grade of each recombination type was used to represent the potato flesh color (a color grade average of less than or equal to 3 was identified as the X18 potato flesh type, and a color grade average of greater than 3 was identified as the P32 potato flesh type). The number of lines in the segregating population at each of the five color grades is shown in Table 1.

[0053] Table 1. Statistical results of the number of strains of five color grades in the segregating population.

[0054]

[0055] III. Genomic DNA Extraction and Detection

[0056] Genomic DNA was extracted from both parents of sweet potato and the F1 segregating population using the CTAB method. The detailed steps are as follows:

[0057] (1) Prepare a CTAB solution containing 1%-2% β-mercaptoethanol and an equal volume of a mixture containing chloroform and isopentane (the volume ratio of chloroform to isopentane in the mixture is 24:1). Place the prepared CTAB in an oven at 65°C for preheating.

[0058] (2) Collect 10-15 mg of tender tissue from the unfolded leaves of sweet potato, put it into a 2 mL centrifuge tube, freeze the sample with liquid nitrogen, and grind it with a sample grinder (30-35 Hz, about 1 min).

[0059] (3) Add 800 μL of CTAB solution preheated at 65℃ to the centrifuge tube, mix the sample and solution, and then incubate in a 65℃ oven for 30 min. Take it out and shake it every 5-10 min to mix it thoroughly.

[0060] (4) Add a mixture containing chloroform and isoamyl alcohol of equal volume to CTAB (the volume ratio of chloroform to isoamyl alcohol in the mixture is 24:1), gently invert and mix 100-400 times, then let stand in a fume hood for 5-10 minutes. After the supernatant and precipitate are clearly separated, put them in a centrifuge and balance them. Centrifuge at 13000 rpm at room temperature for 10 minutes.

[0061] (5) Take 300 μL of isopropanol into a 1.5 mL EP tube in advance and pre-cool it in a -20℃ refrigerator; take 400 μL of supernatant and add it to the pre-cooled isopropanol, mix thoroughly and then place it in a 4℃ refrigerator for 20-30 min.

[0062] (6) After standing, centrifuge at 13000 rpm for 5 min, use a pipette to remove the supernatant, add 600-800 μL of 70% (v / v) ethanol, centrifuge at 13000 rpm at room temperature for 1 min, and then discard the supernatant.

[0063] (7) Wash again with 70% (v / v) ethanol 2-3 times, discard the supernatant and place it on a clean bench to dry.

[0064] (8) Add 100 μL of sterilized deionized water. After the DNA is completely dissolved, use NanoDrop (Thermoscientific) to determine the concentration of extracted DNA.

[0065] (9) Take a small amount of DNA for 1% agarose gel electrophoresis to check its purity. Use 1×TAE as the electrophoresis buffer, 180 V voltage, 300 mA current for 10 min. Select strains with clear agarose gel electrophoresis bands and DNA concentrations greater than 150 ng / μL and store them in an ultra-low temperature freezer at -80℃ for later use. Low-quality DNA with unclear bands and concentrations less than 150 ng / μL needs to be extracted again.

[0066] IV. Initial QTL determination of potato flesh color

[0067] By resequencing the parents and 349 F1 segregating lines, variation information was obtained from these samples. Subsequently, genome-wide association analysis was performed using the potato flesh color phenotype to obtain a QTL related to potato flesh color. Based on a potato flesh color-related QTL obtained by the inventors, an initial localization interval was determined. This QTL is located between 5.39 and 7.99 Mb on chromosome 3 of the Beauregard_v1 reference genome.

[0068] V. Development of Polymorphic Molecular Markers and Genotyping

[0069] 1. InDel Marker Design: Due to the high heterozygosity and complex genetic background of sweet potato, simplex markers of type 0 / 0 / 0 / 0 / 0 / 1 and 0 / 0 / 0 / 0 / 0 / 0 on the six chromosome sets of the two parents (i.e., variation types of mutation sites (e.g., T changing to A) ATTTTT×TTTTTT and TAAAAA×AAAAAA respectively) are more conducive to subsequent genotyping. Therefore, simplex sites were screened within the initial localization interval to develop molecular markers. Based on the information of the screened InDel sites, the sequences 250 bp before and after the site on the reference genome (Beauregard_v1) were extracted for primer design. Primers were designed using Premier 5 software, with primer lengths of 18-24 bp, a Tm of around 56℃, and amplification products greater than 100 bp and less than 300 bp. The primers were synthesized by Sangon Biotech (Sanya) Co., Ltd., and detailed primer information is shown in Table 2.

[0070] Table 2 Basic Information on Polymorphic Primers

[0071]

[0072] 2. InDel molecular marker polymorphism screening: Using DNA from two randomly selected individual plants from the parents (P32, X18) and the F1 segregating population as templates, PCR amplification was performed using designed InDel primers. The reaction system and amplification reaction are shown in Tables 3 and 4. The PCR amplification products were electrophoresed on an 8%-10% non-denaturing polyacrylamide gel for polymorphic InDel primer screening. The electrophoresis apparatus (DYY-6C type) and electrophoresis tank (JY-SCZF-B) used were from Beijing Liuyi Biotechnology Co., Ltd. and Beijing Junyi Dongfang Electrophoresis Equipment Co., Ltd. The polyacrylamide gel formulation is shown in Table 5. The polymorphic InDel marker screening results are as follows: Figure 1 As shown. The results showed that the InDel molecular marker showed polymorphic bands in only one parent (P32, X18), and the banding patterns of two randomly selected lines from the two F1 segregating populations were consistent with one of the parents, such as InDel. 6.92 The marker showed a polymorphic band in the parental P32, but no polymorphic band was observed in X18. Both progeny lines showed polymorphic bands, similar to P32. Based on this screening method, a total of 16 pairs of polymorphic molecular markers were obtained, including InDel. 6.63Three polymorphic molecular markers were identified through location screening. Therefore, one of these markers was selected as a representative for subsequent fine localization work. The 14 polymorphic InDel molecular markers are shown in Table 2.

[0073] Table 3 PCR reaction system

[0074]

[0075] Table 4 PCR reaction procedure

[0076]

[0077] Table 5. Main reagents and preparation methods for electrophoresis

[0078]

[0079] 3. Non-denaturing polyacrylamide gel electrophoresis:

[0080] The preparation method and electrophoresis steps for 8% non-denaturing polyacrylamide gel electrophoresis plates are as follows:

[0081] (1) Glass plate pretreatment: Place the gel attachment plate and the gel peeling plate horizontally, and wipe the dirt and residue on the glass plate with 95% (v / v) alcohol; wipe the gel attachment plate and the gel peeling plate with the prepared affinity silane and peeling silane respectively, and let them air dry for 3-5 minutes before use.

[0082] (2) Gel preparation: Place the gel attachment plate horizontally, cover it with the gel peeling backing plate and align it with the attachment plate, and clamp it with a clamp. Prepare an 8% polyacrylamide gel solution according to Table 6, mix it thoroughly in a 100 mL Erlenmeyer flask and slowly pour it into the interlayer between the two glass plates. Slowly insert a comb into the gel solution between the two plates and let it solidify after being placed horizontally at room temperature for about 40 minutes.

[0083] Table 6 8% Non-denaturing Polyacrylamide Gel Formulation

[0084]

[0085] (3) Pre-electrophoresis: After the gel solidifies, install the gel plate vertically on the electrophoresis tank and fix it with a clamp. Add 1×TBE buffer to the electrophoresis tank. The buffer needs to cover the sample well. Gently pull out the comb and use 190 V voltage and 250 mA current for pre-electrophoresis for 20 min.

[0086] (4) Sample loading: Use a 200 μL pipette to blow out the air bubbles and residual gel in the sample well, and use a 10 μL pipette to slowly inject 2.5-3 μL of PCR product into the sample well.

[0087] (5) Electrophoresis: Electrophoresis is performed at 190 V voltage and 250 mA current. The electrophoresis time depends on the size of the amplified product and the position of the indicator, and is generally 2.5-3.5 h.

[0088] The solutions required for silver staining and development must be prepared fresh for each use. The specific steps for staining and development are as follows:

[0089] (1) Preparation of staining and developing solutions: The staining solution is prepared using silver nitrate. Add 1 g of silver nitrate powder to 800 mL of pure water. After the silver nitrate dissolves, add pure water to bring the total volume to 1 L. The developing solution is prepared using sodium hydroxide and formaldehyde. Add 15 g of sodium hydroxide to 800 mL of pure water. After it dissolves, add pure water to bring the total volume to 1 L. Finally, add 4 mL of formaldehyde solution to the fume hood, mix well, and set aside.

[0090] (2) Staining: After electrophoresis, remove the glass plate and gently peel off the gel back plate with a knife. Place the plate with gel attached into the staining basin and put it into the fume hood. Add the prepared silver nitrate staining solution to cover the gel surface and stain for 20-30 minutes. During this period, shake the staining solution once every 3 minutes.

[0091] (3) Rinsing: After dyeing, rinse the glue surface with pure water for about 30 seconds.

[0092] (4) Development: Pour the prepared developer into the rinsed gel in a fume hood. After the developer covers the gel surface, shake it well and let it stand for 3-5 minutes. When the bands are clearly visible, the development is complete.

[0093] (5) Rinsing: After development, pour out the developer, rinse the gel with tap water, wash away the impurities and residual glue on the glass plate, and take out the plate with glue attached and let it dry for later use.

[0094] 4. dCAPS Molecular Marker Design: After pasting the wild type sequence and mutagenesis using the dCAPS Finder 2.0 Output website (http: / / helix.wustl.edu / dcaps / dcaps.html), the primer mismatch number was set, and primers containing the optimal restriction enzyme sites were selected. After obtaining the forward or reverse primers containing the restriction enzyme sites, the other end primer was designed using Premier 5. The PCR amplification fragment length was 100-200 bp. The primers were synthesized by Sangon Biotech (Sanya) Co., Ltd. Detailed primer information is shown in Table 2 (SNPs). 6.51 .

[0095] 5. dCAPS molecular marker polymorphism screening: Using DNA from two randomly selected single plants from the parents (P32, X18) and the F1 segregating population as templates, PCR amplification was performed using the dCAPS primers designed in Table 2. The reaction and amplification system are shown in Tables 3 and 4. After obtaining the amplification products, they were digested with the corresponding restriction endonucleases. This invention used two restriction sites, Rsa I and Taq I, with recognition sites “GTAC” and “TCGA”, respectively. The restriction reaction system is shown in Table 7. The screening results of polymorphic dCAPS (SNP) markers are as follows: Figure 2 As shown in the figure. The results showed that the dCAPS molecular marker showed polymorphic bands in only one parent (P32, X18), and the banding patterns of two randomly selected lines from the two F1 segregating populations were consistent with one of the parents, such as SNP. 6.51 The marker showed a polymorphic band in parental P32 but not in X18. Both offspring also showed no polymorphic band, similar to X18. Using this method, a total of SNPs were obtained. 6.51 SNP 6.61 SNP 6.63 SNP 6.70 SNP 6.73 Five pairs of polymorphic dCAPS molecular markers were selected, with SNPs chosen to avoid overlap with InDel markers. 6.51 Molecular markers are used for subsequent fine localization.

[0096] Table 7 Restriction endonuclease reaction system

[0097]

[0098] 6. Processing of Polymorphic Molecular Markers in Sweet Potato: After drying, photograph and preserve the developed gel for subsequent statistical analysis of genotype data. Using a 100-300 bp DNA marker as the standard, and DNA from both parents and 423 F1 isolates as templates, PCR amplification was performed using primers from Table 1. The reaction system and amplification reaction are shown in Tables 2 and 3. Polymorphic bands with the same amplification product size and variation type, and clear bands, were selected for reading. Based on the presence of polymorphic bands at the same position in the electrophoresis images of the parents and 423 F1 isolates, the bands were converted into genotype data. That is, a polymorphic band at the same position was recorded as 1, no polymorphic band as 0, and a missing or unclear band as N. Polymorphic bands appearing in X18 but not in P32 were recorded as X18 markers; polymorphic bands not appearing in X18 but appearing in P32 were recorded as P32 markers.

[0099] 7. Genotyping: Using the developed polymorphic InDel and dCAPS molecular markers, PCR amplification and polyacrylamide gel electrophoresis were performed on 423 F1 segregating lines from the segregating population. Combined with genotyping data from 349 resequencing F1 segregating lines, genotypic data for each individual segregator were obtained. Some molecular marker genotyping results are shown below. Figure 3 As shown in Table 8, the genotyping statistics of each polymorphic molecular marker are presented.

[0100] Table 8. Results of Polymorphic Molecular Marker Genotyping

[0101]

[0102] V. Precise Positioning of Sweet Potato Flesh Color QTL

[0103] Polymorphic molecular markers (InDel markers and dCAPS markers, Table 1) were designed using InDel and SNP variant sites within the localization interval to screen recombinant single plants from the F1 segregating population. Subsequently, 12 pairs of InDel molecular markers (InDel...) were used... 6.01 InDel 6.57 InDel 6.61 InDel 6.63 InDel 6.66 InDel 6.69 InDel 6.77 InDel 6.92 InDel 7.07 InDel 7.19 InDel 7.77 and InDel 7.93 Genotyping was performed on 769 lines from the F1 segregating population, and recombinant single plants were screened. A total of 14 single-crossover recombinant single plants were obtained. Based on the phenotypic data from previous investigations and the genotype data of each segregating single plant, the localization interval was narrowed down to InDel. 6.01 (6010477 bp) and InDel 6.57 The physical distance between the two molecular markers (6575627 bp) is 565.15 kb. To further narrow down the localization region, in InDel... 6.01 and InDel 6.57 Develop novel InDel and dCAPS molecular markers between two molecular markers, utilizing one dCAPS marker (SNP). 6.51 ) and 2 InDel (InDel 6.30 InDel 6.39Recombinant plants were screened from the F1 segregating population using markers, resulting in six recombinant plants with single crossover patterns. Combining phenotypic and genotypic data, the QTL related to potato flesh color was ultimately mapped to InDel. 6.39 (6392279 bp) and SNP 6.51 The physical distance between the two molecular markers (6516614 bp) is 124.33 kb, and there are 11 genes within this interval. Figure 4 Gene annotation information is shown in Table 9. Furthermore, the inclusion of IbPSY, a key gene regulating sweet potato carotenoid biosynthesis, among these 11 genes demonstrates the accuracy and application value of this method.

[0104] Table 9. Basic information on genes within the finely mapped regions

[0105]

[0106] In summary, this invention has achieved fine mapping of sweet potato flesh color QTLs through phenotypic identification, resequencing, association analysis, development of polymorphic molecular markers, and genotyping of the F1 generation segregating population of sweet potatoes. This is of great significance for discovering functional genes of traits related to sweet potato quality.

[0107] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A molecular marker closely linked to the sweet potato flesh color gene, characterized in that, The list includes 14 InDel molecular markers and 1 dCAPS molecular marker, and the molecular markers and their amplification primers are shown in the table below: The SNP 6.51 The restriction enzyme site is Rsa I; The gene ID of the sweet potato flesh color gene is: Ibat.Brg.03A_G008440 .

2. The application of the molecular marker described in claim 1 in the fine mapping of the sweet potato flesh color gene, characterized in that, The gene ID of the sweet potato flesh color gene is: Ibat.Brg.03A_G008440 .

3. The application of the molecular marker described in claim 1 in the breeding of sweet potato varieties with different flesh colors, characterized in that, The sweet potato variety accumulates carotenoids.

4. The application of the molecular marker according to claim 1 in the preparation of a product with finely mapped sweet potato flesh color gene, characterized in that, The gene ID of the sweet potato flesh color gene is: Ibat.Brg.03A_G008440 .

5. A product with finely mapped sweet potato flesh color gene, characterized in that, The product includes the molecular marker as described in claim 1, characterized in that the gene ID of the sweet potato flesh color gene is: Ibat.Brg.03A_G008440 .

6. The application of the finely mapped product according to claim 5 in the fine mapping of the sweet potato flesh color gene, characterized in that, The gene ID of the sweet potato flesh color gene is: Ibat.Brg.03A_G008440 .

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