Molecular marker for identifying color of actinidia arguta fruit and application of molecular marker
By constructing a three-dimensional genome map of Actinidia chinensis, the InDel variation in the AaCBP60B-like promoter region was identified, and molecular markers were developed for breeding of red-skinned Actinidia chinensis, which solved the problem of uneven skin coloring and achieved efficient breeding and improved industrialization process.
Patent Information
- Application Number
- CN202510966862.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-07-14
- Publication Date
- 2025-09-23
AI Technical Summary
The existing technology lacks specific molecular markers for regulating the color of the red-skinned soft-fruited kiwifruit peel, resulting in low breeding efficiency and uneven peel coloring, which affects industrial promotion.
By constructing a three-dimensional genome map of Actinidia arguta, the calmodulin-binding protein gene AaCBP60B-like on chromosome 16 and a 346bp InDel mutation in its promoter region were identified. Molecular markers for identifying fruit color were developed and applied to the efficient breeding of red-skinned Actinidia arguta.
The precise and targeted breeding of the skin color of red-skinned soft-fleshed kiwifruit has been achieved, which has improved breeding efficiency and selection accuracy and provided a genetic basis for industrialization.
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Abstract
Description
Technical Field
[0001] The present application relates to the technical field of artificial cultivation and molecular markers of kiwifruit, and in particular to a molecular marker, primers and their application and a detection kit for identifying the fruit color of Actinidia arguta. Background Art
[0002] Fruit appearance quality is one of the core traits for evaluating the commercial viability of fruit varieties. With evolving consumer demands, new-generation fruit varieties that combine attractive appearance, excellent flavor, and healthy nutrition are becoming key breeding targets. Actinidia arguta, a key cultivated species in the genus Actinidia, has peel color as a key indicator of appearance quality, primarily exhibiting red and green phenotypes. Red-skinned kiwifruit, with its vibrant peel color and rich content of antioxidants such as anthocyanins, holds significant market advantages in both fresh and processed markets, making it highly sought after by consumers. However, currently cultivated red-skinned kiwifruit varieties suffer from uneven peel coloring and poor appearance quality, severely hindering their commercialization and market competitiveness. Therefore, systematically analyzing the mechanisms regulating peel color in kiwifruit and developing efficient breeding tools are crucial for promoting red-skinned kiwifruit cultivars and improving the quality and efficiency of the industry.
[0003] Marker-assisted selection (MAS), one of the core technologies of modern crop breeding, can significantly improve breeding efficiency and selection accuracy by constructing molecular markers closely linked to target traits. However, the discovery of key genes and the development of molecular markers for regulating the peel color of Actinidia chinensis are still in their infancy, and their genetic basis and molecular regulatory network are still unclear. This leads to a lack of specific molecular markers that can be used for early screening, which greatly limits the molecular breeding process of red-skinned Actinidia chinensis. On the other hand, from the perspective of functional genomics, identifying negative regulatory factors that regulate peel color and functionally verifying or creating allelic mutations through gene editing technology is an important way to obtain novel peel color germplasm resources, which can provide a genetic basis for subsequent variety improvement.
[0004] In view of the above situation, developing molecular markers related to the red skin trait of Actinidia arguta and using them for early identification and selective breeding of red skin Actinidia arguta will have great promoting significance for kiwifruit germplasm improvement and enhancing fruit value. Summary of the Invention
[0005] In order to fill the above-mentioned gaps in existing research applications, this application has developed a new and effective molecular marker and related primers and detection kit for identifying the fruit color of soft-skinned kiwifruit. The molecular marker and detection kit of this application can be used for the precise, targeted and efficient breeding of red-skinned soft-skinned kiwifruit.
[0006] Based on the above research background and industry needs, the present invention focuses on two main goals: one is to systematically analyze the genetic regulatory mechanism of the fruit skin color of soft-fleshed kiwifruit, focusing on discovering the key negative regulatory genes and their core genetic variations that control the fruit skin color of soft-fleshed kiwifruit; the second is to develop molecular markers that are co-segregated or tightly linked with these key genes and can be used to identify the fruit skin color of soft-fleshed kiwifruit, and apply them to the efficient breeding practice of red-skinned soft-fleshed kiwifruit, thereby providing technical support for the early and accurate identification of red-skinned soft-fleshed kiwifruit, molecular marker-assisted selection breeding and creation of excellent germplasm, and ultimately promoting the development of the soft-fleshed kiwifruit industry towards high quality and high efficiency.
[0007] Building on the previously assembled, first high-quality reference genome of the all-red Actinidia arguta (Actinidia arguta) variety, this study utilized high-throughput chromosome conformation capture (Hi-C) technology to generate three-dimensional genome data for the red-skinned Actinidia arguta 'Zhonghongbei' and the green-skinned Actinidia arguta 'Zhonglvbei'. Differential analysis of compartments, TADs, and loops at different levels of higher-order chromatin structure revealed that A / B compartmental shifts on chromosomes 7 and 16 were significantly associated with pericarp color. Comparative genomic, transcriptomic, and ATAC-seq analyses revealed that a 346-bp structural variation in the upstream promoter region of the calmodulin-binding protein gene AaCBP60B-like on chromosome 16 influences higher-order chromatin structure (TAD disruption and loop remodeling) and promoter activity, regulating AaCBP60B-like gene expression and, consequently, pericarp coloration. Further studies using gradient application, transient overexpression, and gene silencing confirmed the role of AaCBP60B-like in regulating anthocyanin biosynthesis.
[0008] Specifically, in the first aspect, the present application provides a molecular marker for identifying the fruit color of Actinidia arguta. The molecular marker is an InDel marker, which is located at 18134176-18134522bp of chromosome 16 of the Actinidia arguta genome, with a total length of 346bp. Its nucleotide sequence is shown in SEQ ID NO.2. The molecular marker is used to identify the fruit color of Actinidia arguta.
[0009] Furthermore, the above-mentioned molecular marker is located in the upstream promoter region of the calmodulin binding protein gene AaCBP60B-like on chromosome 16 of the Actinidia arguta genome. The nucleotide sequence of the calmodulin binding protein gene AaCBP60B-like is shown in SEQ ID NO.1, which affects the coloring of the Actinidia arguta peel by participating in the negative regulation process of anthocyanin synthesis.
[0010] Furthermore, in the present invention, overexpression of AaCBP60B-like can inhibit the coloring of the fruit skin of Actinidia arguta by reducing the expression of anthocyanin synthesis genes; silencing AaCBP60B-like can promote the coloring of the fruit skin of Actinidia arguta by increasing the expression of anthocyanin synthesis genes.
[0011] Furthermore, the above-mentioned molecular marker was developed based on a 346bp InDel mutation in the AaCBP60B-like promoter. This InDel mutation regulates the expression of the AaCBP60B-like gene by affecting the higher-order chromatin structure (TAD destruction and loop reconstruction), thereby regulating the coloration of the fruit skin.
[0012] Furthermore, in the present invention, the InDel mutation is a homozygous insertion in red-skinned Actinidia chinensis and a heterozygous deletion in green-skinned Actinidia chinensis.
[0013] Furthermore, the amplification primer sequences of the above molecular markers are as follows:
[0014] Upstream primer: 5′-AATGGTAGCTGGGCAATGCT-3′ (SEQ ID NO. 3);
[0015] Downstream primer: 5'-CATGTACCTGGTATGCATTTATAAGC-3' (SEQ ID NO. 4).
[0016] In a second aspect, the present application also relates to the application of the above-mentioned molecular marker detection reagent in the breeding of red-skinned soft-fleshed kiwifruit varieties.
[0017] In a third aspect, the present application provides a detection kit for identifying the color of Actinidia arguta fruit, wherein the detection kit comprises a detection reagent for the above-mentioned molecular marker.
[0018] Furthermore, the above detection kit contains the following primer pairs:
[0019] Upstream primer: 5′-AATGGTAGCTGGGCAATGCT-3′ (SEQ ID NO. 3);
[0020] Downstream primer: 5'-CATGTACCTGGTATGCATTTATAAGC-3' (SEQ ID NO. 4).
[0021] In a fourth aspect, the present application also provides a method for identifying the color of Actinidia arguta fruit, which includes the step of detecting the above-mentioned molecular markers.
[0022] In summary, the present invention reveals the three-dimensional genomic basis for the formation of different peel colors of Actinidia arguta by constructing a three-dimensional genome map of the Actinidia arguta, and identifies a key gene AaCBP60B-like that regulates the peel color of Actinidia arguta. The 346bp InDel mutation in its upstream promoter region can affect the higher-order chromatin structure and thus affect the peel coloring. The molecular markers developed based on this mutation can be used for accurate, targeted, and efficient breeding and improvement of the peel color of Actinidia arguta. The present invention provides new insights into epigenetic regulation of the color formation of Actinidia arguta, and also provides a target for color breeding mediated by gene editing, which has important theoretical significance and application value. BRIEF DESCRIPTION OF THE DRAWINGS
[0023] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the following briefly introduces the drawings required for use in the description of the embodiments. Obviously, the drawings described below are only some embodiments of the present application. For those skilled in the art, other drawings can be obtained based on these drawings without creative work.
[0024] Figure 1 This is a comparison of the three-dimensional genome differences between red and green-skinned Actinidia arguta. Part a in the figure is a whole-genome comparison diagram, where A: genomic position, the black block represents the position of the centromere; BC: RNA FPKM value of red and green-skin samples; DE: A and B compartments of red and green-skin samples; FG: TAD comparison of red and green-skin samples; Part b in the figure is the A and B compartment conversion diagram of chromosomes 7 and 16.
[0025] Figure 2 This is the screening diagram for the candidate gene AaCBP60B-like.
[0026] Figure 3 Figure 3. AaCBP60B-like regulates anthocyanin synthesis in response to exogenous calcium chloride treatment. Figure a: Effect of different concentrations of calcium chloride on the peel color of 'Zhonghongbei'; b: Color of anthocyanin extracts from peel samples treated with different concentrations of calcium chloride; c: Anthocyanin content of peel samples treated with different concentrations of calcium chloride; d: Expression level of AaCBP60B-like in peel samples treated with different concentrations of calcium chloride; e: Expression level of anthocyanin synthesis genes in peel samples treated with different concentrations of calcium chloride.
[0027] Figure 4 Figure 3: Overexpression of AaCBP60B-like inhibits peel coloring. Figure 3: a: peel color of overexpression and control samples; b: peel anthocyanin extract of overexpression and control samples; c: peel anthocyanin content of overexpression and control samples; d: expression levels of anthocyanin-related genes and AaCBP60B-like in overexpression and control samples.
[0028] Figure 5 This is a diagram showing that silencing AaCBP60B-like promotes fruit peel coloring. In the figure, a: fruit peel color of silenced and control samples; b: fruit peel anthocyanin extract of silenced and control samples; c: fruit peel anthocyanin content of silenced and control samples; d: expression levels of anthocyanin-related genes and AaCBP60B-like in silenced and control samples.
[0029] Figure 6 This is a diagram of the AaCBP60B-like promoter variation identification and activity analysis. In the figure, a: Schematic diagram of the AaCBP60B-like promoter in red-skin and green-skin kiwifruit; b: Fluorescence intensity analysis of promoter activity; c: Relative enzyme activity analysis of promoter activity.
[0030] Figure 7 This is a diagram verifying the application effect of the InDel marker of the present invention, which shows that the InDel marker AaCBP60B-likeProInDel346 can completely distinguish between red-skinned and green-skinned soft-fruited kiwifruit resources. DETAILED DESCRIPTION
[0031] In order to make the purpose and technical solution of this application more clearly understood, the following further describes this application in detail with reference to specific embodiments. Those skilled in the art can easily understand other advantages of this application from the contents disclosed in this specification. This application can also be implemented or applied through other different specific embodiments, and the details in this specification can also be modified or changed in various ways based on different viewpoints and applications without departing from the spirit of this application.
[0032] Before further describing the specific embodiments of the present application, it should be understood that the scope of protection of the present application is not limited to the specific specific embodiments described below; it should also be understood that the terms used in the examples of the present application are for describing specific specific embodiments, rather than for limiting the scope of protection of the present application.
[0033] Unless otherwise defined, all technical and scientific terms used in this application have the same meanings as those commonly understood by those skilled in the art. In this application, unless otherwise specified, all instruments, reagents, and raw materials are commercially available or commonly used in the art. The methods in the following examples, unless otherwise specified, are conventional methods in the art.
[0034] Example 1: Screening of the key gene AaCBP60B-like that regulates the peel color of Actinidia arguta
[0035] (1) Constructing the first comparative three-dimensional genome map of different peel colors of Actinidia arguta ( Figure 1 )
[0036] Using the red-skinned Actinidia chinensis cultivar 'Zhonghongbei' and the green-skinned Actinidia chinensis cultivar 'Zhonglvbei' as materials, and building on the previously constructed Actinidia chinensis reference genome (Li et al., 2025, Molecular Horticulture), high-throughput chromosome conformation capture (Hi-C) technology was used to obtain three-dimensional genome data for 'Zhonghongbei' and 'Zhonglvbei'. Combined with transcriptome profiles, the researchers compared the differences in functional units of different chromatin levels between red-skin and green-skin varieties at the genome-wide level. The results showed that at the genome-wide level, the B compartment of most chromosomes played a dominant role, with only chromosomes 7 and 16 showing a different result, namely the A compartment played a dominant role, indicating that more genes on chromosomes 7 and 16 were activated and expressed ( Figure 1 a). From the results of the conversion between A and B compartments, it was found that on chromosomes 7 and 16, the B compartment of the red-skinned sample was converted to the A compartment in the green-skinned sample, suggesting that some of the same anthocyanin regulatory inhibitors were activated in the green-skinned sample ( Figure 1 b) It is worth mentioning that chromosome 16 contains anthocyanin synthesis-related genes F3GT and F3H (Table 1), indicating that chromosome 16 may have anthocyanin-related regulatory factors.
[0037] Table 1 Anthocyanin synthesis-related genes on chromosome 16
[0038]
[0039] (2) Screening and sequencing of candidate genes AaCBP60B-like
[0040] By integrating genes from four aspects (all differentially expressed up-regulated genes, differentially expressed down-regulated genes, genes known to be involved in regulating anthocyanins, and genes located in the A / B compartment transition) and conducting Venn analysis, we found a key gene Aar11780 ( Figure 2 ), encoding calmodulin-binding protein 60B like (AaCBP60B-like). The coding region of this gene is 1917 bp long and encodes 639 amino acids. The specific sequence is shown in SEQ ID NO. 1.
[0041] (3) AaCBP60B-like response to exogenous calcium chloride treatment ( Figure 3 )
[0042] Considering that AaCBP60B-like is a calmodulin-binding protein-related gene, we selected the red-skinned kiwifruit variety 'Zhonghongbei' as the material and conducted an exogenous calcium chloride treatment experiment at four concentration gradients: 0.5%, 1%, 2%, and 4%. Compared with the control, all four concentrations of calcium chloride treatment significantly inhibited peel coloration, with the 2% calcium chloride treatment having the most significant inhibitory effect ( Figure 3 a), the results of anthocyanin extract and anthocyanin content were consistent with the phenotypic results ( Figure 3 bc). Gene expression analysis results showed that AaCBP60B-like was significantly induced in samples treated with different concentrations of calcium chloride, among which AaCBP60B-like was expressed at the highest level at 2% concentration ( Figure 3 d). The expression of anthocyanin synthesis-related genes AaPAL, AaC4H, AaCHS, and AaLDOX were significantly down-regulated in samples treated with different calcium chloride concentrations, among which the most significant down-regulation was observed in samples treated with 2% concentration ( Figure 3 e). The above research results indicate that AaCBP60B-like can participate in the negative regulation of anthocyanins under exogenous calcium chloride treatment, thereby affecting the coloration of the fruit skin of Actinidia arguta.
[0043] (4) Functional identification of AaCBP60B-like
[0044] To further investigate the function of AaCBP60B-like, we constructed an overexpression vector 35S::AaCBP60B-like and transiently transformed it into the peel of Actinidia chinensis ( Figure 4 Compared with the empty vector, the fruit peel injected with the overexpression vector cannot be colored normally ( Figure 4 a), anthocyanin extract and anthocyanin content are consistent with the color phenotype ( Figure 4 bc). Gene expression results showed that the expression of anthocyanin synthesis-related genes was significantly inhibited, and the expression of AaCBP60B-like was significantly increased ( Figure 4 d), indicating that overexpression of AaCBP60B-like can inhibit the skin coloration of Actinidia arguta by reducing the expression of anthocyanin synthesis genes.
[0045] In addition, the silencing vector TRV2-AaCBP60B-like was constructed and mixed with TRV1 at a volume ratio of 1:1 and injected into green-skinned kiwifruit. The empty vector TRV2+TRV1 was used as a control ( Figure 5 Compared with the empty vector, the fruit skin injected with the silencing vector was significantly colored ( Figure 5 a), anthocyanin extract and anthocyanin content are consistent with the color phenotype ( Figure 5bc). Gene expression results showed that the expression of anthocyanin synthesis-related genes was significantly promoted, and the expression of AaCBP60B-like was significantly reduced ( Figure 5 d), indicating that silencing AaCBP60B-like can promote the coloration of the fruit skin of Actinidia arguta by increasing the expression of anthocyanin synthesis genes.
[0046] Based on the results of AaCBP60B-like overexpression and silencing experiments, AaCBP60B-like has the function of negatively regulating anthocyanin synthesis and can inhibit the coloring of the kiwifruit peel.
[0047] Example 2: Screening of AaCBP60B-like gene-related molecular markers
[0048] (1) Identification and activity analysis of AaCBP60B-like promoter mutations
[0049] Differential gene expression often stems from transcriptional regulation, that is, regulation of the promoter region. Therefore, we cloned the AaCBP60B-like promoter of red-skin and green-skin kiwifruit varieties and analyzed the sequences. We found a 346bp InDel variant (SEQ ID NO. 2) in both the red-skin and green-skin AaCBP60B-like promoters. This variant is a homozygous insertion in red-skin kiwifruit and a heterozygous deletion in green-skin kiwifruit. Figure 6 a). By constructing a dual-luciferase reporter system vector and transforming it into Nicotiana benthamiana leaves to detect promoter activity, it was found that the AaCBP60B-like promoter activity of green-skinned kiwifruit containing a deletion mutation was significantly stronger than that of red-skinned kiwifruit containing an insertion mutation ( Figure 6 bc). Therefore, this mutation can affect the activity of the AaCBP60B-like promoter and regulate its expression level.
[0050] (2) Development and application of AaCBP60B-like gene-related Indel markers
[0051] Based on the 346bp natural variant InDel of the AaCBP60B-like promoter discovered in this experiment, we developed a molecular marker targeting this variant, named: AaCBP60B-likeProInDel346. The specific primer sequences are:
[0052] Upstream primer: 5′-AATGGTAGCTGGGCAATGCT-3′ (SEQ ID NO. 3);
[0053] Downstream primer: 5'-CATGTACCTGGTATGCATTTATAAGC-3' (SEQ ID NO. 4).
[0054] We randomly selected 12 red-skinned and 12 green-skinned kiwifruit materials from the natural population and verified the accuracy of the markers by agarose gel electrophoresis. The results showed that the red-skinned ones were homozygous and the green-skinned ones were heterozygous, with an accuracy of 100% ( Figure 7 ).
[0055] Application: This marker can be used to subsequently identify the color of male Actinidia arguta plants and conduct early selection of hybrid offspring derived from this marker, thereby accelerating the color breeding process of Actinidia arguta.
[0056] The preferred specific implementation methods and embodiments of the present application are described in detail above, but the present application is not limited to the above implementation methods and embodiments. Various changes can be made within the scope of knowledge possessed by those skilled in the art without departing from the concept of the present application.
Claims
1. A molecular marker for identifying the color of Actinidia arguta fruit, characterized in that: The molecular marker is an InDel marker, which is located at 18134176-18134522bp of chromosome 16 of the Actinidia arguta genome, has a total length of 346bp, and its nucleotide sequence is shown in SEQ ID NO.
2. The molecular marker is used to identify the fruit color of Actinidia arguta.
2. The molecular marker according to claim 1, characterized in that The molecular marker is located in the upstream promoter region of the calmodulin binding protein gene AaCBP60B-like on chromosome 16 of the Actinidia arguta genome. The nucleotide sequence of the calmodulin binding protein gene AaCBP60B-like is shown in SEQ ID NO.
1. It affects the coloring of the Actinidia arguta peel by participating in the negative regulation process of anthocyanin synthesis.
3. The molecular marker according to claim 1, characterized in that Overexpression of AaCBP60B-like can inhibit the peel coloration of Actinidia arguta by reducing the expression of anthocyanin synthesis genes; silencing AaCBP60B-like can promote the peel coloration of Actinidia arguta by increasing the expression of anthocyanin synthesis genes.
4. The molecular marker according to claim 1, characterized in that The molecular marker was developed based on a 346bp InDel mutation in the AaCBP60B-like promoter. The InDel mutation regulates the expression of the AaCBP60B-like gene by affecting the higher-order structure of chromatin, thereby regulating the coloring of the fruit peel.
5. The molecular marker according to claim 4, characterized in that The InDel mutation is a homozygous insertion in red-skinned Actinidia chinensis and a heterozygous deletion in green-skinned Actinidia chinensis.
6. The molecular marker according to claim 1, characterized in that The sequences of the amplification primers for the molecular markers are as follows: Upstream primer: 5′-AATGGTAGCTGGGCAATGCT-3′; Downstream primer: 5′-CATGTACCTGGTATGCATTTATAAGC-3′.
7. Use of the molecular marker detection reagent according to any one of claims 1 to 6 in the breeding of red-skinned kiwifruit varieties.
8. A detection kit for identifying the color of Actinidia arguta fruit, characterized in that: The detection kit comprises a detection reagent for the molecular marker according to claim 1.
9. The detection kit according to claim 8, characterized in that The detection kit contains the following primer pairs: Upstream primer: 5′-AATGGTAGCTGGGCAATGCT-3′; Downstream primer: 5′-CATGTACCTGGTATGCATTTATAAGC-3′.
10. A method for identifying the color of Actinidia arguta fruit, characterized in that: The method comprises the step of detecting the molecular marker according to claim 1.