Indel marker for identifying peach fruit flesh color and related applications thereof

Abstract

The application belongs to the technical field of molecular breeding, and particularly relates to an Indel marker for identifying peach fruit flesh color and a related application. The Indel marker provided in the application is located at Prunus_persica_v2.0Chr4:4,329,938-4,329,947. The Indel marker provided in the application can increase the application of the type of red-fleshed peach in breeding, and can provide consumers with a variety of red-fleshed peach products.

Description

Technical Field

[0001] This invention belongs to the field of molecular breeding technology, and in particular relates to an Indel marker for identifying the color of peach flesh and its related applications. Background Technology

[0002] Peach germplasm resources are mainly classified into white-fleshed, yellow-fleshed, and red-fleshed varieties based on flesh color. Among these, red-fleshed peaches are rich in anthocyanins, which not only give them their vibrant color but also play a vital role in regulating human health through their strong antioxidant capacity, including scavenging free radicals, anti-tumor activity, and prevention of cardiovascular sclerosis. Therefore, red-fleshed peaches have become a favorite among breeders and consumers. However, red-fleshed peaches currently account for a relatively small proportion of production. This is related to both inherent defects in appearance and flavor, as well as the complexity of the genetic makeup of red-fleshed traits.

[0003] The inheritance of the red flesh trait in peaches is mainly divided into dominant red flesh trait (DBF_, dominant blood-flesh) and recessive red flesh trait (bf, blood-flesh). Current technology utilizes SSR markers to construct genetic linkage maps, locating the dominant red flesh gene DBF at the top of linkage group 5, between SSR markers AMP-PG157 and AMPPG178, matching the peach genome scaffold_5 (442159-947234). Subsequently, other researchers, using a hybrid population of "Da Hong Pao" and "Shu Guang" (yellow flesh), located the dominant locus controlling red flesh within a 3.2 cM region between WPS19 and WPS32 on LG5, and combined this with transcriptome data to identify the key gene PpBL controlling the red flesh trait in peaches. This gene can bind to PpNAC1 to form a dimer, upregulating PpMYB10.1 expression, leading to an increase in anthocyanin content in the flesh, resulting in a red flesh color. Subsequent research, through genome assembly and annotation of the red-fleshed peach variety "Tianjin Shuimi," identified a transposon inserted into the blood-TE region in the PpBL promoter region at the genomic level, and this transposon co-segregates with the red-fleshed trait. Other researchers, using the S1, S2, F1, F2F3, BC1p1, and BC1p2 populations of "HarrowBlood," analyzed the genetic patterns of the red-fleshed trait, finding that it is controlled by the recessive gene bf, and that this type of red-fleshed trait is linked to the trait of red midrib on the underside of leaves, as well as weaker tree vigor. Still others, using the F2 population of 'HarrowBlood' × 'Okinawa', located the bf locus from "HarrowBlood" at the upper end of linkage group 4 (10.3 cM above marker C41H). In existing technologies, the F2 population of “SanguineChanas” × “O'Henry” has been used to construct a genetic linkage map of the F2 single plant '1173', and the bf gene has been located between SNP_IGA_386619 and SNP_IGA_387198 in linkage group 4, matching the peach genome Scaffold_4 (4212145-4523432). Furthermore, these two types of red-fleshed peaches also differ in the timing of anthocyanin accumulation. In the DBF type, anthocyanins begin to increase rapidly before fruit maturity, reaching their maximum at maturity. Conversely, in the bf type, anthocyanins begin to accumulate in the mid-development stage and decrease before fruit maturity.

[0004] Currently, the breeding materials used for red-fleshed peaches in production are mainly of the DBF type. However, the transcription factor PpBL, which regulates the fruit color of this type of peach, can also promote early ripening, so the ripening period of this type of peach is concentrated from mid-June to early July. In contrast, the ripening period of the BF type of red-fleshed peach can extend to the end of July to early August. Increasing the application of this type of red-fleshed peach in breeding can not only provide consumers with a wider variety of red-fleshed peach products, but more importantly, it can help extend the supply cycle of red-fleshed peaches. However, there is currently a lack of molecular markers to identify the flesh color of BF type red-fleshed peaches. Summary of the Invention

[0005] To address the shortcomings of existing technologies, the Indel marker provided by this invention, which can identify the color of peach flesh, will help the development of selective breeding for red-fleshed peaches. It can quickly and accurately select according to breeding objectives and effectively regulate the supply cycle of red-fleshed peaches.

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

[0007] This invention provides an Indel marker for identifying the color of peach flesh, wherein the Indel marker is located at position 4,329,938 to 4,329,947 in Prunus_persica_v2.0 Chr4.

[0008] In this invention, the sequence information at both ends of the deletion site is as follows:

[0009]

[0010] The italicized and underlined part represents the missing 9bp in the red peach.

[0011] The present invention also provides a primer for detecting the Indel label of claim 1, wherein the nucleotide sequence of the Indel label primer is shown in SEQ ID No. 1 and SEQ ID No. 2;

[0012]

[0013] The present invention also provides a kit for detecting the above-described Indel markers, the kit comprising the above-described primers.

[0014] The present invention also provides the application of the above-described Indel marker, primers, or kits in peach breeding.

[0015] The present invention also provides the application of the above-described Indel marker, the above-described primer, or the above-described kit in identifying the color of peach pulp.

[0016] The present invention also provides a method for identifying the color of peach flesh, comprising the following steps: using the genomic DNA of the peach to be tested as a template, performing PCR amplification using the above-mentioned primers to obtain the amplification product;

[0017] When both bands of the amplified product are 121bp in length, or when one band is less than 121bp and the other is 121bp in length, the peach flesh is non-red.

[0018] When both bands of the amplified product are less than 121 bp, the peach flesh is red. Attached Figure Description

[0019] Figure 1 These are two different types of red-fleshed peaches; the top image shows the DBF type, and the bottom image shows the bf type.

[0020] Figure 2 BSA-seq of the F2 populations of black chicken peach and white stone peach;

[0021] Figure 3 Genotyping results of the F2 population of black chicken peach and white peach (hybridized in 2021);

[0022] Figure 4 Genotyping results of the F2 population of black chicken peach and white peach (hybridized in 2024);

[0023] Figure 5 Genotyping results of the BC population of black chicken peach and white stone peach (backcross in 2022);

[0024] Figure 6 Genotyping results of the BC population of black chicken peach and white stone peach (backcross in 2024);

[0025] Figure 7 Genotyping results of the hybrid population of 07-4-28 and Rugao purple peach F2 (hybridization in 2022). Detailed Implementation

[0026] For reagents or instruments used in the following text where specific technical or conditional specifications are not given, standard experimental conditions shall apply. If no reagent company instructions are explicitly provided, the conditions recommended in those instructions shall be followed. Reagents or instruments whose manufacturers are not specified are all commercially available standard products.

[0027] Example

[0028] 1. Obtaining the missing insertion marker (InDel)

[0029] This invention uses two peach germplasm resources from the Zhengzhou Fruit Research Institute of the Chinese Academy of Agricultural Sciences, "Wuhei Chicken Peach (BF type red flesh)" and "Shiyu White Peach (white flesh)," as parents to construct an F1 population. The F1 population consists of 251 plants, all of which exhibit white flesh and non-red leaf veins (on the back).

[0030] In 2021, individual plants from the F1 population were propagated by seed, yielding 115 F2 plants. In 2022, these F1 plants were backcrossed with the black-fleshed chicken peach, resulting in 98 BC1 plants. This indicates that the red-fleshed trait from the black-fleshed chicken peach is a recessive trait controlled by a single gene. (Two different types of red-fleshed peaches, DBF and bf, are mentioned...) Figure 1 As shown in the figure, to locate the key genes controlling the red-fleshed trait, 23 red-fleshed and 23 white-fleshed single plants were selected from the F2 population for pooled sequencing. Subsequently, using Prunus_persica_v2.0 as the reference genome (Verde, 2017), bioinformatics analysis was performed on the sequencing data (BSA-seq of the F2 populations of Wuhei Chicken Peach and Shiyu White Peach is shown in the figure). Figure 2 (As shown). The sequencing depth of the parental and progeny pools was 35-fold, and the genome coverage reached over 90%. A significant signal was identified in the Chr4 region (0.00-7.01 Mb) using BSA-seq. Due to the large localization range, 85 individual plants (red:non-red = 60:25) were selected from the F2 and BC populations for resequencing, and their SNP and Indel information was obtained by alignment with the reference genome Prunus_persica_v2.0. The obtained variant information was then genotyped, and combined with the phenotypic information of individual plants, it was finally found that the red-fleshed individual plants had a 9 bp homozygous deletion at positions 4,329,938–4,329,947 on chromosome 4, while the white-fleshed individual plants showed heterozygous deletion or no deletion. Subsequently, primers were designed based on the upstream and downstream reference sequences of the deletion site as molecular markers for identifying the flesh color (red / non-red).

[0031] 2. Method for identifying peach pulp color using nucleotide insertion (InDel) markers.

[0032] (1) DNA extraction

[0033] Extract DNA from the sample tissue (leaf) to be tested; the DNA sample volume should be no less than 30 μl. Measure the OD values ​​of the DNA sample at 260 nm and 280 nm using a UV spectrophotometer, and calculate the DNA content and OD value. 260 / 280 The ratio of DNA sample purity OD. 260 / 280 The value should be between 1.8 and 2.0, and the concentration should be diluted to about 10 ng / μl.

[0034] (2) Primer design

[0035] Primers were designed based on upstream and downstream reference sequences of the deletion site:

[0036]

[0037] The total length of the product is 121 bp, and the product sequence is shown below:

[0038]

[0039] (3) PCR amplification and banding analysis

[0040] The PCR amplification system is shown in Table 1.

[0041] Table 1 PCR amplification system

[0042] reagents Added volume (μl) 2×TaqPCRMix 5 Primer F 1 Primer R 1 DNA template 1 ddH2O 2

[0043] Transfer the prepared reagent mixture to a PCR tube or plate, place it in a PCR instrument, and perform PCR amplification. The amplification program is shown in Table 2.

[0044] Table 2 PCR amplification program

[0045]

[0046] The reaction products were detected by 6% polyacrylamide gel electrophoresis to observe the band distribution. When all amplified bands were 121 bp or one band was less than 121 bp and the other was equal to 121 bp (BFbf), the fruit was non-red flesh. If both bands were less than 121 bp (bfbf), the corresponding flesh was red flesh.

[0047] Application Example 1

[0048] Validation on seed population 1

[0049] In 2021, a seedling hybrid population of black-fleshed chicken peach and white-fleshed Shiyu peach was propagated, yielding 115 F2 individual plants, including 23 red-fleshed plants and 92 non-red-fleshed plants. Genotyping of this population was performed using the primers described above. The results are as follows: Figure 3 As shown in Table 3, BFBF indicates homozygous non-red meat, BFbf indicates heterozygous non-red meat, and bfbf indicates homozygous red meat. The identification results show that in the 115 F2 accessions, 23 red meat individuals had the genotype bfbf, and in the 92 non-red meat individuals, 35 were BFBF and 57 were BFbf. The accuracy of identifying red and non-red meat in this population was 100%.

[0050] Table 3. Genotypic identification and phenotype summary of F2 populations of Wuhei Chicken Peach and Shiyu White Peach (2021)

[0051]

[0052]

[0053] Application Example 2

[0054] Validation was performed on seed population 2.

[0055] In 2024, seedlings were again used to propagate the F1 hybrid population of Wuhei Chicken Peach and Shiyu White Peach, resulting in 131 F2 individual plants. Of these, 44 had red leaf veins and 87 did not. Genotyping of this population was performed using the primers described above. The results are as follows: Figure 4 As shown in Table 4, BFBF indicates homozygous non-red-fleshed, BFbf indicates heterozygous non-red-fleshed, and bfbf indicates homozygous red-fleshed. The identification results show that in the 131 F2 accessions, 44 individual plants with red veins had the genotype bfbf, and among the 87 non-red-veined individual plants, 22 were BFBF and 65 were BFbf. The accuracy of identifying red and non-red-fleshed plants in this population was 100%.

[0056] Table 4. Genotypic identification and phenotype summary of F2 populations of Wuhei Chicken Peach and Shiyu White Peach (2024)

[0057]

[0058]

[0059]

[0060] Application Example 3

[0061] Validation on backcross population 1

[0062] In 2022, backcrossing was performed on the F1 hybrid population of the black-fleshed peach and its cross with the Shiyu white peach, yielding a total of 98 BC individual plants, including 47 red-fleshed plants and 51 non-red-fleshed plants. Genotyping of this population was performed using the primers described above. The results are as follows: Figure 5 As shown in Table 5, BFBF indicates homozygous non-red meat, BFbf indicates heterozygous non-red meat, and bfbf indicates homozygous red meat. The identification results show that in the 131 F2 accessions, 47 red meat individuals had the genotype bfbf, and in the 51 non-red meat individuals, 41 were BFbf and 10 were BFBF. The accuracy of identifying red and non-red meat in this population was 100%.

[0063] Table 5. Genotypic identification and phenotype summary of the BC populations of Wuhei Chicken Peach and Shiyu White Peach (backcross in 2022)

[0064]

[0065]

[0066]

[0067] Application Example 4

[0068] Validation was performed on backcross population 2.

[0069] In 2024, the F1 hybrids of the black-fleshed chicken peach and the white-fleshed Shiyu peach were backcrossed with the black-fleshed chicken peach, resulting in 109 BC individual plants. Among them, 50 plants had red leaf veins and 59 plants did not. Genotyping of this population was performed using the primers mentioned above. The results are as follows: Figure 6 As shown in Table 6, BFBF indicates homozygous non-red-fleshed, BFbf indicates heterozygous non-red-fleshed, and bfbf indicates homozygous red-fleshed. The identification results show that in the 109 BC accessions, 50 individual plants with red veins all had the genotype bfbf. Among the 59 non-red-veined individual plants, 57 were BFbf and 2 were BFBF. The accuracy of identifying red and non-red-fleshed plants in this population was 100%.

[0070] Table 6. Genotypic identification and phenotype summary of the BC populations of Wuhei Chicken Peach and Shiyu White Peach (backcross in 2024)

[0071]

[0072]

[0073]

[0074] Application Example 5

[0075] Validation was performed on seed population 3.

[0076] In 2022, a seedling experiment was conducted on the F1 hybrid population of 07-4-28 (DBF type red-fleshed peach) and Rugao purple peach (bf type red-fleshed peach). (The F1 plants did not exhibit leaf vein color separation, and the flesh did not show early coloring phenotypes.) A total of 133 F2 individual plants were obtained, including 28 plants with red leaf veins and 105 plants without red veins. The plants with red leaf veins accumulated anthocyanins in their fruits during early development, exhibiting characteristics of the bf type red-fleshed peach. Genotyping of this population was performed using the primers described above. The results are as follows: Figure 7 As shown in Table 7, BFBF indicates homozygous non-red-fleshed, BFbf indicates heterozygous non-red-fleshed, and bfbf indicates homozygous red-fleshed. The identification results show that in the 133 F2 populations, 28 individual plants with red veins had the genotype bfbf, and among the 105 non-red-veined individual plants, 64 were BFbf and 41 were BFBF. The accuracy of identifying red and non-red-fleshed plants in this population was 100%.

[0077] Table 7 summarizes the genotypic identification and phenotype of the 07-4-28 and Rugao purple peach populations (2022).

[0078]

[0079]

[0080]

[0081] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. An application of an Indel marker in identifying the color of peach flesh, characterized in that, The Indel marker is located at positions 4,329,938 to 4,329,947 in Prunus_persica_v2.0 Chr4.

2. A primer for detecting Indel markers, characterized in that, The nucleotide sequences of the Indel-labeled primers are shown in SEQ ID No. 1 and SEQ ID No. 2; The Indel marker is located at positions 4,329,938 to 4,329,947 in Prunus_persica_v2.0 Chr4.

3. A kit for detecting Indel markers, characterized in that, The kit contains the primers as described in claim 2; The Indel marker is located at positions 4,329,938 to 4,329,947 in Prunus_persica_v2.0 Chr4.

4. The application of the primers of claim 2 or the kit of claim 3 in peach breeding.

5. The application of the primers of claim 2 or the kit of claim 3 in identifying the color of peach pulp.

6. A method for identifying the color of peach flesh, characterized in that, The steps include: using the peach genomic DNA to be tested as a template, performing PCR amplification using the primers described in claim 2 to obtain the amplification product; When both bands of the amplified product are 121bp in length, or when one band is less than 121bp and the other is 121bp in length, the peach flesh is non-red. When both bands of the amplified product are less than 121 bp, the peach flesh is red.

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