InDel molecular marker for identifying longan peel color and application of InDel molecular marker

Abstract

The invention discloses an InDel molecular marker for identifying the color of longan pericarp and application of the InDel molecular marker, and belongs to the field of plant molecular breeding. The molecular marker is located in a cis-regulatory region of 177bp upstream of an initiation codon ATG of a tenth chromosome DlMYB1 gene and is insertion / deletion of 91bp, and the nucleotide sequence of the molecular marker is shown as SEQ ID NO.3. The molecular marker affects the color of longan pericarp. After insertion, the promoter has transcriptional activity and can promote anthocyanin accumulation to make pericarp red; after deletion, no transcriptional activity exists, and the pericarp is non-red. The method can be successfully applied to longan germplasm resource peel color identification and filial generation peel color prediction. The molecular marker is derived from a natural longan population and is stable in heredity, and the detection method is easy and convenient to operate, high in accuracy and suitable for early screening of the pericarp color in longan crossbreeding and has important application value in genetic improvement of the pericarp color of the cultivated longan.

Description

Technical Field

[0001] This invention relates to the field of plant molecular breeding technology, and in particular to an InDel molecular marker for identifying the color of longan peel and its application. Background Technology

[0002] Peel color is an important fruit quality trait that greatly influences consumer choice. Currently, commercially available longan peels are mainly yellowish-brown, with some being bluish-brown, grayish-brown, or brownish-red, exhibiting a dull and monotonous color that limits the competitiveness of longan in the fresh fruit market. Red-skinned longan, a tropical ecotype, has a bright purplish-red peel and is currently the only known longan germplasm resource with a red peel. Although it is less commonly cultivated due to factors such as flavor, yield, climate, and cultivation, its vibrant peel color provides an irreplaceable genetic resource for improving longan peel color, making it of significant research and application value as a breeding parent material.

[0003] Currently, longan breeding mainly employs hybridization breeding. However, longan is a perennial tall tree that requires a large area, and it typically takes 6-8 years from sowing to the first flowering and fruiting. This results in a long breeding cycle and high costs. Moreover, color is a complex trait, and blindly conducting hybridization breeding without understanding its genetic laws is not only time-consuming and laborious but also may not achieve the desired results.

[0004] Therefore, discovering the key genetic factors controlling the color of red-skinned longan peel, developing molecular markers closely related to peel color, and applying them to molecular marker-assisted breeding to achieve early, precise, and efficient selection of red peel traits is an inevitable development direction for cultivating high-quality commercially cultivated red-skinned longan varieties. Summary of the Invention

[0005] In view of this, the purpose of this invention is to provide an InDel molecular marker for identifying the color of longan peel and its application. This InDel molecular marker can affect the transcriptional activity of DlMYB1 in the later stages of longan fruit development, thereby affecting the synthesis and accumulation of anthocyanins in longan peel.

[0006] In a first aspect, the present invention provides an InDel molecular marker for identifying the color of longan peel, the molecular marker being located upstream of the ATG start codon of the DlMYB1 gene on chromosome 10 of longan. It contains or omits a nucleotide fragment of 50–120 bp in length; The nucleotide fragment is a tandem repeat sequence region; The molecular markers can affect the color of longan peel.

[0007] The molecular marker described in this invention is located in the cis-regulatory region upstream of the start codon ATG of the DlMYB1 gene (NCBI accession: OR473701) on chromosome 10. Figure 1 The gene, consisting of two repeating units with a length of 30–150 bp, exists in two genotypes in longan populations: type P1, containing three TR1s (5'-GGTTTGTTGGACTGGTAGATGACTTATAATTGAGA-3', SEQ ID NO.4) and two TR2s (5'-GGTTCTGTTGTTAAATTT-3', SEQ ID NO.5); and type P2, containing one TR1 and one TR2. Luciferase assays show that the P1 promoter has transcriptional activity, while the P2 promoter does not. DlMYB1 is a positive regulator of anthocyanins in longan pericarp, and its transcription directly affects anthocyanin accumulation. This invention utilizes the differences in this functional region to develop an InDel marker, which has been successfully applied to the identification of longan pericarp color and the prediction of pericarp color in hybrid offspring. The molecular marker described in this invention is derived from a natural population of longan, is genetically stable, and has a simple and accurate detection method. It is suitable for early screening of peel color in longan hybridization breeding and has important application value in the genetic improvement of peel color in cultivated longan.

[0008] Furthermore, the molecular marker includes or omits a nucleotide fragment of 91 bp in length, the nucleotide sequence of which is shown in SEQ ID NO.3.

[0009] Specifically, the 91bp nucleotide fragment consists of two TR1s, one TR2, and three "AGA" bases.

[0010] In a second aspect, the present invention provides the application of the InDel molecular marker described above in the identification of color traits of longan peel.

[0011] In a third aspect, the present invention provides the application of the InDel molecular marker described above in molecular breeding of longan.

[0012] In a fourth aspect, the present invention provides a primer for identifying the InDel molecular marker described above, comprising a forward primer and a reverse primer, wherein the nucleotide sequence of the forward primer is shown in SEQ ID NO.1 and the nucleotide sequence of the reverse primer is shown in SEQ ID NO.2.

[0013] In a fifth aspect, the present invention provides a kit for identifying the InDel molecular markers described above, comprising the primers described above.

[0014] In a sixth aspect, the present invention provides the application of the primers or kits described above in identifying the color traits of longan peel or in longan molecular breeding.

[0015] Furthermore, if the longan genome lacks a nucleotide fragment as shown in SEQ ID NO.3, it is a yellow-skinned longan; if it contains a nucleotide fragment as shown in SEQ ID NO.3, it is a red-skinned longan.

[0016] In a seventh aspect, the present invention provides a method for identifying the color of longan peel, comprising the following steps: (1) Extract DNA from the longan sample to be tested; (2) Using the longan DNA extracted in step (1) as a template, and the primers described above as amplification primers, perform PCR amplification, and perform electrophoresis detection and / or sequencing on the PCR amplification products. (3) Based on the results of step (2), determine the genotype and the color of the longan peel.

[0017] Furthermore, in step (1), the longan sample includes seedlings, leaves, pericarps, flowers, or embryogenic tissues.

[0018] The beneficial effects of this invention include at least the following: (1) This invention first clarified the direct association between the tandem repeat sequence structural variation of the DlMYB1 gene promoter region and the synthesis of anthocyanins in longan peel. This variation, as a core functional marker, accurately revealed the genetic essence of the red peel trait, providing a new and highly targeted molecular genetic target for the improvement of longan peel color, and filling the gap in key functional markers in the genetic improvement of red-skinned longan.

[0019] (2) The promoter structural variation corresponding to the functional marker described in this invention comes from the natural population or breeding population of longan. It has been verified by experiments to be genetically stable and can be directly applied to breeding practice without complicated artificial modification. This avoids the genetic instability problem that may exist in exogenous markers and reduces the technical threshold and uncertainty of marker application.

[0020] (3) The InDel molecular marker developed based on this structural variation can be used for genotyping by PCR amplification with specific primers combined with gel electrophoresis. The operation process is simple, the detection cost is low, and the accuracy has been verified by a large number of germplasm resources (high accuracy in distinguishing between red and non-red longan). At the same time, the test samples can be obtained from early tissues such as seedlings and leaves, without waiting for the long growth cycle of longan (6-8 years) to the fruiting period. This completely solves the pain point of long peel color screening cycle and high cost in traditional hybridization breeding, and realizes early and accurate prediction of red peel trait.

[0021] (4) The molecular markers provided by this invention can be directly applied to the rapid identification of peel color in longan germplasm resources, early screening of hybrid offspring, and cultivation of high-quality red-skinned longan varieties, significantly improving breeding efficiency, shortening the breeding cycle, reducing breeding costs, and effectively avoiding the blindness of traditional hybridization breeding. Its application will accelerate the cultivation process of commercially cultivated red-skinned longan varieties, enrich the variety types in the fresh longan market, enhance the market competitiveness of longan products, and provide a replicable and scalable technical example for the fruit tree genetic improvement industry, with significant economic value and application prospects. Attached Figure Description

[0022] Figure 1 A schematic diagram of functional markers for molecular-assisted breeding of longan with 'red' peel.

[0023] Figure 2 The differences in phenotype and anthocyanin types and contents between red-skinned longan (RP) and green-skinned bud mutation longan (RPM). (a) Differences in the color of peel, flower and leaf of RP and RPM; (b) Differences in the types of anthocyanins in the peel of RP and RPM; (c) Comparison of anthocyanin content in the peel of RP and RPM.

[0024] Figure 3 Identification of functional marker regions. (a) Two genotypes were found in the DlMYB1 gene promoter region of the RP genome: the P1 allele containing 3 TR1 repeat units and 2 TR2 repeat units, and the P2 allele containing 1 TR1 repeat unit and 1 TR2 repeat unit; (b) PCR verification of DlMYB1 promoter tandem repeat polymorphism. M, DNA size marker; lane 1, RP; lane 2, RPM; lanes 3-24, representative self-pollinated RP progeny; lanes 25-96, other longan varieties. (c) Fluorescence images of transient transformation of tobacco luciferase activity. Each tobacco leaf was divided into three regions containing the following transformation vectors: empty pGreenII 0800-LUC vector (LUC), P1 promoter vector (P1::LUC), and P2 promoter vector (P2::LUC); (d) Statistical analysis of luciferase reporter gene activity, including 3 biological replicates and 3 technical replicates per biological replicate.

[0025] Figure 4Transcriptomic analysis of RP and RPM samples revealed that structural variations in the DlMYB1 promoter affected anthocyanin synthesis. (a) PCA analysis of RP and RPM samples; S1 (10 DAF), S2 (45 DAF), and S3 (90 DAF). (b) Number of differentially expressed genes between RP and RPM samples. (c) Relative expression levels of DlMYB1 in RP and RPM samples at three developmental stages. (d) WGCNA co-expression module. (e) Gene expression characteristics of the DlMYB1 co-expression module. (f) Co-expression network of the DlMYB1 co-expression module. (g) Differences in gene expression related to anthocyanin synthesis between RP and RPM samples.

[0026] Figure 5 The results of promoter structural variation marker detection were obtained from 120 self-crossed progeny of red-skinned longan. Genotypes with the DlMYB1 promoter tandem repeat sequence showed a 380 bp band, while genotypes without the DlMYB1 promoter tandem repeat sequence showed a 289 bp band. Heterozygous genotypes P1:P2 showed two bands, homozygous P1:P1 genotypes showed only a 380 bp band, and homozygous P2:P2 genotypes showed only a 289 bp band. Detailed Implementation

[0027] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of them. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0028] Furthermore, the technical solutions of the various embodiments of the present invention can be combined with each other, but only if they are based on the ability of those skilled in the art to implement them. When the combination of technical solutions is contradictory or cannot be implemented, it should be considered that such combination of technical solutions does not exist and is not within the scope of protection claimed by the present invention.

[0029] The following specific embodiments illustrate the solution proposed in this invention: Example 1: Identification of Functional Marker Regions Red-skinned longan is a tropical ecotype of longan, characterized by its red peel, red flowers, and red leaves. Within a red-skinned longan plant RP1901 (RP for short), a bud mutation shoot RPM1901 (RP for short) was discovered. This shoot possesses yellowish-green fruit, and unlike the outer and inner peels of RP red-skinned longan, RPM has a yellowish-green outer peel and a pale yellow inner peel. Furthermore, the colors of its flowers and leaves have also changed. Figure 2 a). This indicates that this branch tip is a bud mutation material whose color has changed.

[0030] To demonstrate the color difference between RP and RPM pericarps, anthocyanins were extracted from each sample using a hydrochloric acid / methanol solution (methanol:ddH2O: 1 M HCl = 2:1:1, v / v / v). Based on 15 anthocyanin standards (delphinidin-3-O-galactoside chloride, delphinidin-3-O-glucoside chloride, cyanidin-3-O-galactoside chloride, delphinidin-3-O-arabinoside chloride, cyanidin-3-O-glucoside chloride, and petunidin-3-O-galactoside chloride), anthocyanins were extracted using liquid chromatography. (Chlorinated petunidin-3-O-galactoside), cyanidin-3-O-arabinoside chloride, petunidin-3-O-glucoside chloride, peonidin chloride, petunidin-3-O-arabinoside chloride, peonidin-3-O-glucoside chloride, malvidin-3-O-galactoside chloride, peonidin-3-O-arabinoside chloride, malvidin-3-O-glucoside chloride, malvidin-3-O-arabinoside chloride. The types and contents of anthocyanins in the pericarp color of both fruits were analyzed. The results showed that the main anthocyanin components in the RP pericarp were Cy3G (Cyanidin-3-O-glucoside) and Pt3G (Petunidin-3-O-glucoside), with Pt3G accounting for 90% and containing 254.2 μg. g -1FW, Cy3G accounts for 10% (28.2 μg) g -1 In FW, the Pt3G content in RPM was only 9.2 μg. g -1 FW cannot detect Cy3G ( Figure 2 (b, 2c). This indicates that the type and content of anthocyanins are the main factors affecting the color difference of RP and RPM pericarps.

[0031] To identify the variations in bud mutant RPMs, short-read resequencing was performed on both RP and RPM samples. To rule out differences due to cell type and sequencing variations, two libraries were constructed for each sample, and sequencing was performed at different depths. Variation analysis identified a total of 13,924,894 variant sites, but only 528 differentially identified variant sites were found in both libraries, involving 840 variations (some sites had multiple genotypes). Among these, 562 were single-base variations and 278 were InDel sites.

[0032] To identify the presence of large-segment variations in RP and RPM, structural variations between RP and RPM were identified using HiFi reads from RP and RPM using four software programs: CuteSV, PBSV, Sniffles, and SVIM. Only one variation was detected by all four programs: a 91 bp deletion at chr10 1898733. This deletion, as noted, occurs 161 bp upstream of the MYB transcription factor Dlrp10A0146 initiator. This variation was also present in the variations detected by the aforementioned short-read analysis. Figure 3 a).

[0033] To verify whether the mutation in the DlMYB1 promoter region truly exists, PCR amplification was performed on this region in RP and RPM using the forward primer 5'-TCCATTAGCTCTAGAACAACT-3' (SEQ ID NO. 6) and the reverse primer 5'-ACACCAAGTAAATGCGACATG-3' (SEQ ID NO. 7), respectively. Cloning was performed using the pTOPO-Blunt Simple blunt-end cloning kit (Beijing Adley Biotechnology Co., Ltd., catalog number CV1702), and the cloned sequences were sequenced using the Sanger method. A tandem repeat sequence was found in the promoter region 177 bp upstream of the DlMYB1 start codon, consisting of two repeat units of 35 bp (TR1) and 18 bp (TR2). Two genotypes, P1 and P2, were found in the RP DlMYB1 promoter. The P1 promoter had three TR1 units and two TR2 units, while the P2 promoter had only one TR1 unit and one TR2 unit, lacking both TR1 and TR2 units. In contrast, only the P2 genotype was found in RPM. Figure 3 (b) This is consistent with the results of structural variation detection. Further testing of this variation was conducted in other red-skinned and non-red-skinned longan using the forward primer 5'-TTGATGATAGAGTAGAGGACAG-3' (SEQ ID NO.1) and the reverse primer 5'-AAGACTTCAGTAGTGGATACG-3' (SEQ ID NO.2). The results showed that in all tested longan germplasm, the longan pericarp color was red for all three genotypes in this region containing the P1 promoter (P1:P1 genotype and P1:P2 genotype), while the pericarp color was non-red for longan without the P1 promoter (P2:P2 genotype). Figure 3 c) indicates that the deletion of the tandem repeat sequence in the DlMYB1 promoter region is the key variation that leads to the inhibition of anthocyanin synthesis in RPM in bud mutation materials, while the P1 type promoter containing the tandem repeat sequence is positively correlated with the red peel trait of longan, and the presence or absence of this sequence can significantly distinguish between red-skinned longan and non-red-skinned longan.

[0034] Further LUC experiments revealed that the promoter allele containing the complete TR (P1 type) exhibited significantly higher transcriptional initiation activity, while the promoter allele lacking TR (P2 type) showed almost no transcriptional initiation activity. Figure 3 d) indicates that this area is a key functional area that affects the color of longan peel.

[0035] Example 2: Effect of functional marker region on DlMYB1 expression on anthocyanins in pericarp To determine how this functional marker region located in the DlMYB1 promoter affects DlMYB1 expression and anthocyanin biosynthesis, transcriptome sequencing was performed on the pericarps of RP (DlMYB1 promoter genotype P1:P2) and RPM (DlMYB1 promoter genotype P2:P2) fruits at three developmental stages: S1 (10 days post-flowering, DAF), S2 (45 days DAF), and S3 (90 days DAF). After removing low-quality and adapter sequences from the transcriptome sequences of each sample, the sequences were matched to the red-skinned longan genome using HISAT2 (v2.2.1), and the expression levels of each gene in the samples were calculated using Stringtie2 software (v2.2.3). Gene expression differences among the samples were compared using the R package DESeq2, and gene co-expression analysis was performed using the R package WGCNA.

[0036] Principal component analysis (PCA) showed that the repeated samples exhibited significant clustering. Figure 4 a), but the clustering of some RP and RPM samples indicates a high degree of similarity in their genetic backgrounds. Differential expression analysis (|log2FC|>1, Q value<0.01) showed that only 325, 276, and 213 differentially expressed genes were found between RP and RPM in S1, S2, and S3, respectively. Figure 4 b).

[0037] Among these differentially expressed genes, DlMYB1 was expressed only in RP samples at fruit ripening stage (S3), and not at any stage of RPM. Figure 4 c). To further explore the regulatory network associated with DlMYB1, a weighted gene co-expression network analysis was performed. A total of 18 co-expression modules were detected ( Figure 4 (d) The cyan module showed the most significant enrichment of anthocyanin biosynthesis structural genes. This module contains 163 annotated genes, 11 of which are related to anthocyanin biosynthesis and transport. The module also contains six transcription factors, including MYB1, MYB102, bHLH41, bHLH79, WD91, Dp-1, and TFIIIB. The characteristic gene (MEcyan) of this module was specifically highly expressed in the S3 stage of the RP pericarp, but lowly expressed in the S1 and S2 stages of the RP and all stages of the RPM. Figure 4 e). This co-expression pattern indicates a significant correlation between DlMYB1 and anthocyanin biosynthesis.

[0038] Co-expression network analysis further showed that DlMYB1 is closely associated with genes such as UFGT, GST, DFR, and bHLH. Figure 4f). Based on the expression levels of these genes, a regulatory model of anthocyanin synthesis in the pericarp of red longan was constructed. In this model, DlMYB1 interacts with the proteins bHLH41, bHLH79, and WD40, regulating the expression of anthocyanin synthesis genes (CHS-1, CHS-2, CHI-1, CHI-2, F3H-1, F3H-2, DFR-1, DFR-2, ANS-1, ANS-2), modification genes (UFGT), and transporter protein genes (GST). Figure 4 g), thereby promoting the accumulation of anthocyanins in RP pericarp.

[0039] Example 3: Application of functional markers in screening the peel color of longan hybrid progeny InDel molecular markers were developed based on the differences in the DlMYB1 promoter region. The forward primer for marker detection was 5'-TTGATGATAGAGTAGAGGACAG-3' (SEQ ID NO.1), and the reverse primer was 5'-AAGACTTCAGTAGTGGATACG-3' (SEQ ID NO.2). Genomic DNA was extracted from the leaves of 120 self-pollinated populations of red-skinned longan seedlings using a plant genomic DNA extraction kit (Beijing Adley Biotechnology Co., Ltd., catalog number DN1402). Then, the DNA from each sample was amplified by PCR using KOD FX high-fidelity enzyme (TOYOBO, catalog number KOD-201) according to the manufacturer's instructions, and the PCR products were detected by 1.5% agarose gel electrophoresis. The results showed that the homozygous genotype samples lacking the DlMYB1 promoter tandem repeat sequence (P2:P2) all had green leaves and no anthocyanin synthesis, while the genotypes containing the DlMYB1 promoter tandem repeat sequence (P1:P1 and P1:P2) had red leaves and could synthesize anthocyanins. This indicates that this molecular marker can predict different peel color traits in longan materials at an early stage by identifying whether they can synthesize anthocyanins. Figure 5 ).

[0040] It should be noted that, in this document, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Unless otherwise specified, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes that element.

[0041] The sequence numbers of the above embodiments of the present invention are for descriptive purposes only and do not represent the superiority or inferiority of the embodiments.

[0042] The above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. An InDel molecular marker for identifying the color of longan peel, characterized in that, The molecular marker is located upstream of the ATG start codon of the DlMYB1 gene on chromosome 10 of longan. It contains or omits a nucleotide fragment of 50–120 bp in length; The nucleotide fragment is a tandem repeat sequence region; The molecular markers can affect the color of longan peel.

2. The InDel molecular marker according to claim 1, characterized in that, The molecular marker contains or omits a nucleotide fragment of 91 bp in length, the nucleotide sequence of which is shown in SEQ ID NO.

3.

3. The application of the InDel molecular marker as described in claim 1 or 2 in identifying the color trait of longan peel.

4. The application of the InDel molecular marker as described in claim 1 or 2 in molecular breeding of longan.

5. A primer for identifying the InDel molecular marker as described in claim 1 or 2, characterized in that, It includes a forward primer and a reverse primer, the nucleotide sequence of which is shown in SEQ ID NO.1 and the nucleotide sequence of which is shown in SEQ ID NO.

2.

6. A kit for identifying the InDel molecular marker as described in claim 1 or 2, characterized in that, It contains the primers as described in claim 5.

7. The application of the primers of claim 5 or the kit of claim 6 in the identification of longan peel color traits or in longan molecular breeding.

8. The application according to claim 7, characterized in that, If the longan genome lacks the nucleotide fragment shown in SEQ ID NO.3, it is a yellow-skinned longan; if it contains the nucleotide fragment shown in SEQ ID NO.3, it is a red-skinned longan.

9. A method for identifying the color of longan peel, characterized in that, It includes the following steps: (1) Extract DNA from the longan sample to be tested; (2) Using the longan DNA extracted in step (1) as a template, and the primers described in claim 5 as amplification primers, perform PCR amplification, and perform electrophoresis detection and / or sequencing on the PCR amplification products. (3) Based on the results of step (2), determine the genotype and the color of the longan peel.

10. The method for identifying the color of longan peel according to claim 9, characterized in that, Longan samples include seedlings, leaves, pericarps, flowers, or embryonic tissues.

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