A primer pair and kit for identifying or assisting in the identification of watermelon flesh color, and their applications.

Abstract

This invention belongs to the field of molecular marker-assisted breeding technology, specifically relating to a primer pair and kit for identifying or assisting in the identification of watermelon flesh color, and their applications. This invention provides a primer pair for identifying or assisting in the identification of watermelon flesh color, wherein the primer pair includes an upstream primer with the nucleotide sequence shown in SEQ ID No. 2 and a downstream primer with the nucleotide sequence shown in SEQ ID No. 3. Using the primer pair designed based on the InDel-P0152 molecular marker provided by this invention, the pink flesh color trait of watermelon can be accurately and rapidly identified.

Description

Technical Field

[0001] This invention belongs to the field of molecular marker-assisted breeding technology, specifically relating to a primer pair and kit for identifying or assisting in the identification of watermelon flesh color and its application. Background Technology

[0002] Watermelon (Citrullus lanatus) is a global horticultural crop, ranking fifth among the world's top ten fruits. With economic development, people's demands for watermelon quality are also increasing. Flesh color is an important component of watermelon quality, and selecting varieties with vibrant flesh colors is a priority for breeders. Watermelon flesh colors are relatively rich, with common colors including bright red, pink, yellow, and white. During watermelon development, lutein, xanthophyll, carotene, and lycopene in the flesh play important roles. Early on, xanthophyll content is higher, resulting in a yellow color; at maturity, lutein content exceeds xanthophyll content, resulting in a white color, and yellow is a recessive trait to white. Pink and bright red are determined by the content of carotene and lycopene. Research has found that watermelon flesh color is controlled by multiple genes. Based on map-based cloning, QTLs for white and red flesh were mapped to chromosomes 2 and 4, respectively. On chromosome 4, the candidate gene Cla005011 for lycopene β-cyclase (LCYB) (Zhang et al., Finemapping of the flesh color controlling genes in watermelon (Citrullus lanatus), Cucurbitaceae, 2014, 111-116) and the major QTL controlling red flesh content in tomatoes (Liu et al., Mapping of quantitative trait loci for lycopene content and fruit traits in Citrullus lanatus, Euhytica, 2015, 202:411-426; Liu et al., Development of cleaved amplified polymorphic sequence markers and a CAPS-based genetic linkage map in watermelon (Citrullus lanatus [Thunb.] Matsum. And Nakai) constructed using whole-genome re-sequencing data, Breeding Science, 2016, 66: 244-259.Branham et al. (Geneticmapping of a major codominant QTL associated with β-carotene accumulation in watermelon, Molecular Breeding, 2017, 37:146) located a major codominant QTL associated with β-carotene accumulation on chromosome 1, which provides a possibility for marker-assisted breeding of red-fleshed watermelon varieties rich in β-carotene. Fall et al. (QTLs associated with flesh quality traits in an elite×elite watermelon population, Euphaitica, 2019, 215:30) mapped QTLs controlling lycopene content in red and pink flesh, but did not find stable QTL loci. Li et al. (Discovery of the genomic region and candidate genes of the scarlet red flesh color (Y)) scr The study *Citrullus Lanatus L.* Frontiers in Plant Science, 2020, Article 116* used high-density genetic mapping and genome resequencing to locate the trait controlling bright red flesh color within a 40kb region on chromosome 6, and developed two InDel markers associated with this trait. The genetic basis of watermelon flesh color is complex, and the formation of this trait is controlled by multiple genes. However, research on pink flesh traits is relatively limited, and no InDel molecular markers for identifying pink flesh traits in watermelons have been reported. Summary of the Invention

[0003] The purpose of this invention is to provide a primer pair for identifying or assisting in the identification of watermelon flesh color. Using this primer pair, the pink flesh color trait of watermelon can be accurately and quickly identified.

[0004] This invention provides a primer pair for identifying or assisting in the identification of watermelon flesh color, the primer pair comprising an upstream primer with a nucleotide sequence as shown in SEQ ID No. 2 and a downstream primer with a nucleotide sequence as shown in SEQ ID No. 3.

[0005] Preferably, the primer pair uses the InDel-P0152 molecular marker as the detection target.

[0006] Preferably, the InDel-P0152 molecular marker is located at the 2100152 bp position on chromosome 2 of watermelon, and the InDel-P0152 molecular marker insertion / deletion nucleotide sequence is shown in SEQ ID No. 1, with the reference genome being Watermelon(97103)v2.5Genome.

[0007] This invention provides a kit for identifying or assisting in the identification of watermelon flesh color, comprising the primer pair and PCR amplification reagents described in the above technical solution.

[0008] Preferably, the upstream primer in the primer pair has a concentration of 9–11 μmol / L, and the downstream primer has a concentration of 9–11 μmol / L.

[0009] This invention provides an application of the primer pair or reagent kit described in the above-mentioned technical solution in identifying or assisting in the identification of watermelon flesh color.

[0010] This invention provides a method for identifying or assisting in the identification of watermelon flesh color, comprising the following steps:

[0011] 1) Extract genomic DNA from the watermelon to be identified;

[0012] 2) Using the watermelon genomic DNA to be identified as a template, PCR amplification was performed using the primer pair described in the above technical solution to obtain PCR amplification products;

[0013] 3) Perform polyacrylamide gel electrophoresis on the PCR amplification products. If the molecular weight of the PCR amplification product is 217 bp, the watermelon flesh is determined to be pink. If the molecular weight of the PCR amplification product is 228 bp, the watermelon flesh is determined to be red.

[0014] Preferably, the PCR amplification system, in 15 μL, comprises the following components: 7.5 μL 2×T5PCRMix, 4.5 μL ddH2O, 1 μL forward and reverse primers, 1 μL reverse primer, and 1 μL DNA template.

[0015] Preferably, the PCR amplification program is as follows: 98℃ pre-denaturation for 2 min; 98℃ denaturation for 10 s, 58℃ annealing for 10 s, 72℃ extension for 10 s, 35 cycles; 72℃ extension for 5 min.

[0016] The beneficial effects of the present invention are as follows: The present invention provides a primer pair for identifying or assisting in the identification of watermelon flesh color. The primer pair includes an upstream primer with a nucleotide sequence as shown in SEQ ID No. 2 and a downstream primer with a nucleotide sequence as shown in SEQ ID No. 3. The primer pair designed using the InDel-P0152 molecular marker provided by the present invention can accurately and quickly identify the pink flesh color trait of watermelon. Attached Figure Description

[0017] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the embodiments will be briefly described below.

[0018] Figure 1 The X-axis represents the physical location of the chromosome (Mb); the Y-axis represents the ΔSNP-index of each chromosome; the black line is the fitted line after sliding the ΔSNP-index window; the blue line is the 95% confidence line; and the red line is the 99% confidence line.

[0019] Figure 2 The results of amplification of the molecular marker InDel-P0152 in the F2 population are shown. Lane 1 is the molecular marker; lane 2 is the PCR product of the paternal parent; lane 3 is the PCR product of the maternal parent; lanes 4-12 are the PCR products of red-fleshed plants; lanes 13-23 are the PCR products of pink-fleshed plants.

[0020] Figure 3 The results show the amplification of the molecular marker InDel-P0152 in the natural population; lane 1 is the molecular marker; lane 2 is the PCR product of red pulp; lane 3 is the PCR product of pink pulp; lanes 4-39 are the PCR products of the natural population material; lanes 4-39 are, from left to right, Z1, Z24, Z25, Z2, Z6, Z23, Z5, Z26, Z4, Z20, Z21, Z22, Z10, Z13, Z14, Z15, Z18, Z34, Z3, Z27, Z7, Z28, Z8, Z9, Z32, Z33, Z11, Z12, Z16, Z19, Z17, Z35, Z36, Z29, Z30, Z31. Detailed Implementation

[0021] This invention provides a primer pair for identifying or assisting in the identification of watermelon flesh color. The primer pair includes an upstream primer with the nucleotide sequence shown in SEQ ID No. 2 and a downstream primer with the nucleotide sequence shown in SEQ ID No. 3. The primer pair for identifying or assisting in the identification of watermelon flesh color described in this invention is used to amplify the InDel-P0152 molecular marker.

[0022] Table 1. Nucleotide sequences of primer pairs

[0023]

[0024] In this invention, the primer pair for identifying or assisting in the identification of watermelon flesh color preferably uses the InDel-P0152 molecular marker as the detection target. The InDel-P0152 molecular marker of this invention is preferably located at the 2100152 bp position on chromosome 2 of watermelon, and the insertion / deletion nucleotide sequence is shown in SEQ ID No.1, which is determined with reference to the Watermelon (97103) v2.5 Genome.

[0025] In this invention, the design concept of the InDel-P0152 molecular marker is as follows: Using the Watermelon (97103) v2.5 Genome genome as a reference, association analysis was performed on the parental and offspring mixed-pool sequencing data for red and pink flesh color using the BSA pooled sequencing method. The target gene was located within a 3.3 Mb interval of 1040001-4340000 bp on Watermelon chromosome 2. An InDel molecular marker was designed targeting the InDel site within this interval. The InDel gene site was validated in the paternal, maternal, and F2 populations to obtain the InDel-P0152 molecular marker linked to the Watermelon flesh color gene. This InDel molecular marker is located at base position 2100152 bp on Watermelon chromosome 2, with a length of 217 bp and / or 228 bp, i.e., a polymorphism of 11 bp. The insertion / deletion nucleotide sequence is shown in SEQ ID No. 1, which is AATATATATAT.

[0026] The InDel-P0152 molecular marker of this invention is simple and quick to operate, achieving accurate and rapid identification of pink flesh in watermelons during the seedling stage with only a single PCR amplification. Using the InDel-P0152 molecular marker-assisted selection of this invention, the color of pink flesh in watermelons can be screened during the seedling stage, accelerating the improvement of the pink flesh trait and increasing breeding selection efficiency.

[0027] In the present invention, the method for developing the InDel-P0152 molecular marker preferably includes the following steps: individual plants of the F2 population obtained by crossing a watermelon with pink flesh as the female parent and a watermelon with red flesh as the male parent are phenotypically identified for the flesh color. Illumina HiSeq resequencing is used to locate the candidate region related to the pink flesh trait of watermelon to obtain the major QTL for the pink flesh trait of watermelon. The QTL candidate region is detected to obtain InDel sites, primers are designed for the InDel sites, and after polymorphism verification, InDel molecular markers are obtained. In the present invention, the female parent is preferably watermelon MW119 with pink flesh color, and the male parent is preferably watermelon MW120 with red flesh color. In the present invention, there are no other special requirements for the hybridization method, and the conventional hybridization method in the art can be adopted. The present invention preferably uses the SNP-index algorithm for trait association to obtain the major QTL for the pink flesh trait of watermelon.

[0028] In the present invention, the major QTL is preferably detected on chromosome 2 of watermelon. The SNP-Index in the 1-4.3 Mb segment of chromosome 2 of the present invention exceeds 0.99 confidence level and is the major QTL for the pink flesh trait of watermelon.

[0029] In the present invention, the InDel sites within the QTL candidate interval are preferably determined, primers are designed for the InDel sites, and after polymorphism verification, InDel molecular markers are obtained. The InDel sites in the present invention are preferably obtained by the following method: 1) There should be differences between the parents and the segregation form conforms to the population type; 2) The sequencing depth of both parents is ≥5×; 3) Neither of the two offspring pools is missing, the sequencing depth of each offspring pool is greater than 10× and less than 500×, and 0.3 < SNP-index < 0.7 in at least one offspring pool. The present invention preferably designs primers according to the differences of the InDel sites, screens the designed primers for polymorphism in the two parents, and the primers of the markers with polymorphism screened are the primer pairs of the InDel sites. In the present invention, the method for polymorphism screening can adopt the conventional primer polymorphism screening method in the art.

[0030] The present invention provides a kit for identifying or assisting in identifying the flesh color trait of watermelon, including the primer pair and PCR amplification reagents described in the above technical solution.

[0031] In the primer pair of the present invention, the concentration of the upstream primer is preferably 9-11 μmol / L, and the concentration of the downstream primer is preferably 9-11 μmol / L. More preferably, the concentration of the upstream primer in the primer pair is 10 μmol / L, and the concentration of the downstream primer is 10 μmol / L.

[0032] The present invention does not have any special limitations on the composition and quantity of each component of the PCR amplification reagent in the kit; the composition and quantity of the components of a conventional kit can be used.

[0033] This invention provides the application of the primer pairs or kits described in the above-mentioned technical solutions in the identification or auxiliary identification of molecular marker-assisted selection of watermelon pulp color.

[0034] This invention provides a method for identifying or assisting in the identification of watermelon flesh color, comprising the following steps:

[0035] 1) Extract genomic DNA from the watermelon to be identified;

[0036] 2) Using the watermelon genomic DNA to be identified as a template, PCR amplification was performed using the primer pair for amplifying the InDel-P0152 molecular marker as described in the above technical solution to obtain the PCR amplification product;

[0037] 3) Perform polyacrylamide gel electrophoresis on the PCR amplification products. If the molecular weight of the PCR amplification product is 217 bp, the watermelon flesh is determined to be pink. If the molecular weight of the PCR amplification product is 228 bp, the watermelon flesh is determined to be red.

[0038] The PCR amplification system described in this invention is preferably 15 μL and includes the following components: 7.5 μL 2×T5PCRMix, 4.5 μL ddH2O, 1 μL forward primer, 1 μL reverse primer, and 1 μL DNA template.

[0039] The preferred PCR amplification program of the present invention is as follows: 98℃ pre-denaturation for 2 min; 98℃ denaturation for 10 s, 58℃ annealing for 10 s, 72℃ extension for 10 s, 35 cycles; 72℃ extension for 5 min, and storage at 12℃.

[0040] To further illustrate the present invention, the technical solutions provided by the present invention will be described in detail below with reference to the accompanying drawings and embodiments, but these should not be construed as limiting the scope of protection of the present invention.

[0041] Example 1: Development of InDel molecular markers for identifying the pink flesh color of watermelon

[0042] (1) Genome-wide QTL preliminary mapping of watermelon pink flesh trait: 108 individual plants from the F2 population obtained by crossing the female parent watermelon MW119 (flesh color is pink) and the male parent watermelon MW120 (flesh color is red) were used as experimental materials for phenotypic identification of flesh color. High-throughput sequencing was performed on the red flesh pool and pink flesh pool constructed from 20 red flesh individual plants and 20 pink flesh individual plants in the two parents and the F2 population using Illumina HiSeq resequencing. According to the sequencing results, there were 193,187 SNPs between the red flesh pool and the pink flesh pool. The SNP-index algorithm was used for trait association. First, the sites with SNP-index less than 0.3 in both the red flesh and pink flesh mixed pools were filtered. The SNP-index of each mixed pool and the Δ(SNP-index) between the mixed pools were calculated. Then, the distribution of Δ(SNP-index) on each chromosome was mapped by the sliding window method (window size is 2000 kb, step size is 10 kb), as shown in Figure 1 . The candidate regions related to the watermelon pink flesh trait were located on chromosome 2 of watermelon at 1 - 4.3 Mb, determined by referring to the reference genome Watermelon(97103)v2.5 Genome; on chromosome 4 at 3.7 - 16.8 Mb; on chromosome 6 at 1 - 6.3 Mb and on chromosome 10 at 14.1 - 18 Mb genomic regions. Among them, the 1 - 4.3 Mb segment on chromosome 2 (SNP-Index) exceeded a confidence level of 0.99 and was the major QTL for the watermelon pink flesh trait.

[0043] (2) Development of InDel molecular markers in the major QTL region: The following criteria were used for filtering Indel sites: 1) There should be differences between the parents and the segregation pattern should conform to the population type; 2) The sequencing depth of the two parents should be ≥5×; 3) Neither of the two offspring pools should be missing, the sequencing depth of each offspring pool should be greater than 10× and less than 500×, and 0.3 < SNP-index < 0.7 in at least one offspring pool. After filtering, 1140 InDel sites were detected in the candidate region on chromosome 2. Taking 50 kb as the stepwise segment, InDel sites with a base deletion of more than 10 bp were screened, and 23 pairs of InDel primers were designed and screened for polymorphism between the two parents. Among them, the InDel-P0152 molecular marker primer showed polymorphism, amplifying a 217 bp band in the pink flesh parent; a 228 bp band was obtained in the red flesh parent. This marker is located at the 2100152 bp site on chromosome 2. The BSA analysis result showed that there was a 11 bp (AATATATATAT) base insertion in the male parent, that is, the nucleotide sequence of SEQ ID No.1 is AATATATATAT.

[0044] Example 2: Verification of InDel-P0152 molecular marker in the F2 population

[0045] (1) Total DNA was extracted from the leaves of 108 individual plants of watermelon maternal parent MW119 (pink flesh), watermelon paternal parent MW120 (red flesh) and F2 population using the FlaPurePlantDNAExtractionKit (Jinsha Biotechnology).

[0046] (2) Using the DNA extracted from the two parents and the F2 population in step (1) as templates, PCR amplification was performed using InDel-P0152 molecular marker primers. PCR reaction system and procedure: The total PCR reaction system was 15 μl, and the components were as follows: 7.5 μl of 2×T5PCRMix, 1 μL of forward primer (primer concentration 10 μmol / L), 1 μL of reverse primer (primer concentration 10 μmol / L), 1 μl of watermelon leaf DNA (10 ng / μl), and 4.5 μl of ddH2O.

[0047] The PCR reaction program was as follows: 98℃ pre-denaturation for 2 min; 98℃ denaturation for 10 s, 58℃ annealing for 10 s, 72℃ extension for 10 s, 35 cycles; 72℃ extension for 5 min, and storage at 12℃.

[0048] (3) Take 2 μl of the PCR product from step (2) and perform electrophoresis using an 8% polyacrylamide gel. Figure 2 The results of InDel-P0152 marker detection are shown for the paternal and maternal parents and some F2 individual plants. Lane 1 represents the molecular weight marker; lane 2 represents the paternal PCR product; lane 3 represents the maternal PCR product; lanes 4-12 represent the PCR products of red-fleshed individual plants; and lanes 13-23 represent the PCR products of pink-fleshed individual plants. The results show that the InDel-P0152 molecular marker can amplify a 228 bp fragment in both the paternal and red-fleshed watermelon individual plants; and a 217 bp fragment in both the maternal and pink-fleshed watermelon individual plants. In the F2 population, 55 out of 67 pink-fleshed individual plants showed a genotype-phenotype match, with an accuracy of 82.09% for pink-fleshed identification.

[0049] Example 3: Validation of the InDel-P0152 molecular marker in a natural population

[0050] The linkage between the InDel-P0152 marker and the pink flesh trait of watermelon was further verified using natural populations. The specific steps are as follows:

[0051] (1) Genomic DNA extraction. Total DNA was extracted from 75 watermelon leaf samples from natural populations using the FlaPurePlant DNA Extraction Kit (DET711-50, Beijing Jinsha Biotechnology Co., Ltd.).

[0052] The natural population traits are shown in Table 1.

[0053] Table 1. Materials and phenotypes of watermelon from natural populations

[0054]

[0055]

[0056] (2) PCR reaction system and procedure. Using the natural population DNA extracted in step (1) as a template, PCR amplification was performed using primers InDel-P0152. The total PCR reaction system was 15 μl, and the components were as follows: 7.5 μl of 2×T5PCRMix, 1 μL of forward primer (primer concentration 10 μmol / L), 1 μL of reverse primer (primer concentration 10 μmol / L), 1 μl of watermelon leaf DNA (10 ng / μl), and 4.5 μl of ddH2O.

[0057] The PCR reaction program was as follows: 98℃ pre-denaturation for 2 min; 98℃ denaturation for 10 s, 58℃ annealing for 10 s, 72℃ extension for 10 s, 35 cycles; 72℃ extension for 5 min, and storage at 12℃.

[0058] (3) Take 2 μl of the above PCR product and perform electrophoresis detection using 8% polyacrylamide gel. Figure 3 The results show the detection results of the InDel-P0152 molecular marker in some natural population materials. Lane 1 represents the molecular weight marker; lane 2 represents the PCR product of the red paternal parent fruit pulp in Example 1; lane 3 represents the PCR product of the pink maternal parent fruit pulp in Example 1; and lanes 4-39 represent the PCR products of some natural population materials. The results show that the InDel-P0152 marker can amplify a 228 bp fragment in red-fleshed single plants; and the InDel-P0152 marker can amplify a 217 bp fragment in pink-fleshed single plants. Figure 3 The identification results for 36 out of 75 natural populations are presented. Within the natural populations, 36 out of 45 pink-fleshed individual plants showed a genotype-phenotype match, resulting in an accuracy of 80% in identifying the pink-fleshed trait.

[0059] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A primer pair for identifying or assisting in the identification of watermelon flesh color, characterized in that, The primer pair includes an upstream primer with a nucleotide sequence as shown in SEQ ID No. 2 and a downstream primer with a nucleotide sequence as shown in SEQ ID No. 3; The primer pair uses the InDel-P0152 molecular marker as the detection target; The InDel-P0152 molecular marker is located at the 2100152 bp base position on chromosome 2 of watermelon. The InDel-P0152 molecular marker has an insertion / deletion nucleotide sequence as shown in SEQ ID No.1, and the reference genome is Watermelon(97103)v2.5Genome.

2. A kit for identifying or assisting in the identification of watermelon flesh color, characterized in that, Includes the primer pair and PCR amplification reagents as described in claim 1.

3. The reagent kit according to claim 2, characterized in that, The upstream primer in the primer pair has a concentration of 9-11 μmol / L, and the downstream primer has a concentration of 9-11 μmol / L.

4. The application of the primer pair of claim 1 or the kit of claim 2 or 3 in identifying or assisting in the identification of watermelon flesh color, characterized in that, The application method includes the following steps: using the watermelon genomic DNA to be identified as a template, performing PCR amplification using the primer pair described in claim 1 to obtain PCR amplification products; The PCR amplification products were subjected to polyacrylamide gel electrophoresis. If the molecular weight of the PCR amplification product was 217 bp, the watermelon flesh was determined to be pink. If the molecular weight of the PCR amplification product was 228 bp, the watermelon flesh was determined to be red.

5. A method for identifying or assisting in the identification of watermelon flesh color, characterized in that, Includes the following steps: 1) Extract genomic DNA from the watermelon to be identified; 2) Using the watermelon genomic DNA to be identified as a template, PCR amplification was performed using the primer pair described in claim 1 to obtain the PCR amplification product; 3) Perform polyacrylamide gel electrophoresis on the PCR amplification products. If the molecular weight of the PCR amplification product is 217 bp, the watermelon flesh is determined to be pink. If the molecular weight of the PCR amplification product is 228 bp, the watermelon flesh is determined to be red.

6. The method according to claim 5, characterized in that, The PCR amplification system, in 15 μL units, includes the following components: 7.5 μL 2×T5PCRMix, 4.5 μL ddH2O, 1 μL upstream primer, 1 μL downstream primer, and 1 μL DNA template.

7. The method according to claim 5, characterized in that, The PCR amplification program was as follows: 98℃ pre-denaturation for 2 min; 98℃ denaturation for 10 s, 58℃ annealing for 10 s, 72℃ extension for 10 s, 35 cycles; 72℃ extension for 5 min.

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