An AS-PCR marker for identifying 01-7 pomelos and its application

Abstract

This invention provides an AS-PCR marker for identifying the '01-7' pomelo variety and its application, the sequence of which is shown in SEQ ID NO: 1-2. Using the main cultivated pomelo variety as the research object, this invention conducted genome resequencing and, through bioinformatics analysis, identified a homozygous SNP that differs between the '01-7' pomelo and other pomelo varieties. Sanger sequencing verified its authenticity. Based on this SNP, AS-PCR primers were designed. PCR amplification and Sanger sequencing proved the primers to be correct and effective, yielding amplified bands on the '01-7' pomelo but not on other pomelo varieties. Thus, an AS-PCR marker capable of distinguishing the '01-7' pomelo from other pomelo varieties was developed. This marker can be used for the molecular identification of the '01-7' pomelo variety, which is of great significance for ensuring the purity of seedlings of this variety and promoting the development of improved and high-quality pomelo varieties.

Description

Technical Field

[0001] This invention belongs to the field of molecular technology development and application, specifically involving an AS-PCR marker for identifying '01-7' pomelo and its application. Background Technology

[0002] The Changshan pomelo (Citrus changshanensis KSChen et CXFu), also known as the golden pomelo, is a local specialty citrus fruit produced through natural hybridization. After multiple selection processes, it has become a unique hybrid citrus species in my country. The Changshan pomelo has a long history of cultivation; the "ancestral tree" in Chengtan Village, Qingshi Town, Changshan County, is the oldest existing seedling Changshan pomelo tree, nearly 120 years old. As a Chinese national geographical indication product and a key specialty product developed and promoted by Zhejiang Province, the Changshan pomelo has brought significant economic benefits to the local area. The fruit's cold resistance, high yield, good storage properties, and unique flavor have secured it a place in both the fresh and processed citrus markets, making it a primary fruit ingredient in double-pomelo juice beverages. Furthermore, young Changshan pomelo fruits are used as a medicinal ingredient, known as Quzhou tangerine peel, which has medicinal value for regulating qi, relieving chest congestion, and alleviating phlegm and cough.

[0003] Because the pomelo originated from seedlings and underwent a long period of sporadic planting and seedling propagation during the selection and breeding process, new varieties such as 'Crispy Red', 'Red Flesh Pomelo', 'Summer Red Tangerine Pomelo', 'Pomelo No. 10', 'Pomelo No. 11', and '01-7' pomelo have differentiated from the common pomelo. Among them, '01-7' pomelo has become the main variety promoted in the pomelo industry due to its rich flavor, good storage properties, and strong resistance. However, different pomelo varieties are closely related and highly similar in morphology, making them difficult to distinguish, especially at the seedling stage. Therefore, there is an urgent need to develop molecular markers based on DNA to establish a variety identification system to ensure the accuracy and purity of seedlings and promote the industry's development.

[0004] Discovering SNPs through genome sequencing and then developing molecular markers based on sequence differences has become an important approach for molecular marker development. However, to date, there are no reports on SNP-based molecular markers that can distinguish different varieties of pomelo. Summary of the Invention

[0005] The purpose of this invention is to overcome the shortcomings of molecular identification technology for pomelo varieties and to provide an AS-PCR marker for identifying '01-7' pomelo, which is an AS-PCR molecular marker used to distinguish '01-7' pomelo from other pomelo varieties. The AS-PCR marker is a set of primer pairs, the sequences of which are shown in SEQ ID NO: 1 and SEQ ID NO: 2, specifically: 5'-GCAGAGAAATCGCCTTTGTCA-3' (SEQ ID NO: 1, AS-PCR-F) and 5'-CGTGTGGTCTCCAAAAGTCC-3' (SEQ ID NO: 2, AS-PCR-R).

[0006] The AS-PCR marker of the present invention is obtained by the following method:

[0007] (1) SNP discovery. Whole genome resequencing was performed on the '01-7' pomelo and the ancestral tree of pomelo (common pomelo). Based on the obtained data, bioinformatics analysis was carried out using pomelo as the reference genome to discover homozygous SNPs between the two materials;

[0008] (2) Verification of SNPs. Primers were designed on both sides of the SNP using Primer3web version 4.1.0 (https: / / primer3.ut.ee / ), and the correctness of the SNPs was verified by PCR, cloning, and Sanger sequencing.

[0009] (3) Design AS-PCR primers. AS-PCR primers were designed using BatchPrimer3 v1.0 provided on the BatchPrimer3 website (https: / / wheat.pw.usda.gov / demos / BatchPrimer3 / ). The upstream primer was an allele-specific primer, with the last base at its 3' end corresponding to the '01-7' sequence and the second-to-last base introducing a mismatch. The downstream primer was a common allele primer.

[0010] (4) Preparation of template DNA. Genomic DNA of pomelo was extracted using the CTAB (hexadecyltriethylammonium bromide) method;

[0011] (5) Run AS-PCR. The AS-PCR reaction system is as follows: 3 μL 2×Taq Master Mix, 0.8 μL 10 μmol·L⁻¹ -1 Upstream and downstream primers, 30 ng template DNA, and ddH2O added to a final volume of 10 μL. The PCR reaction program was 95℃ for 10 min; 95℃ for 10 s, 60℃ for 15 s, 72℃ for 5 s, for 30 cycles; finally, 72℃ for 10 min, and stored at 4℃.

[0012] (6) Perform agarose gel electrophoresis. PCR products were detected by agarose gel electrophoresis. The gel concentration was 1.5%, and the electrophoresis conditions were 110V constant voltage for 25 min. After GelRed staining, the gel was placed in a gel imaging system for imaging.

[0013] (7) Expanding Application and Validation. AS-PCR was applied to 12 pomelo materials, and the sequences of the SNP sites in the 12 materials were sequenced using the method described in (2) above. The consistency between the AS-PCR results and the SNP sequences was compared. These 12 pomelo materials were taken from the main production bases in the main production area, including 01-7 (01-7a, 01-7b, 01-7c, 01-7d) from 4 bases, common pomelo (PTa, PTb, PTc) from 3 bases, two other new varieties (HY10, HY11), 'Crispy Red' (CH), 'Red Flesh' (HR) and 'Summer Red' (XH).

[0014] Another object of the present invention is to provide the application of the aforementioned AS-PCR marker in distinguishing different varieties of pomelo.

[0015] This invention utilizes resequencing data from the '01-7' pomelo and its ancestral tree (common pomelo) to identify a homozygous SNP. Based on this SNP, an AS-PCR marker was developed and applied to 12 pomelo accessions. Results show that this marker can distinguish the '01-7' pomelo from other pomelo varieties. This marker can be used for the molecular identification of the '01-7' pomelo, which is of great significance for ensuring the purity of this variety's seedlings and promoting the development of improved and high-quality pomelo varieties.

[0016] This invention focuses on the main cultivated varieties of pomelo, conducting genome resequencing and then using bioinformatics analysis to identify a homozygous SNP that differs from other pomelo varieties in the '01-7' variety. Sanger sequencing verified its authenticity. Based on this SNP, allele-specific upstream primers and allele-shared downstream primers were designed. PCR amplification and Sanger sequencing confirmed the primers' correctness and effectiveness, yielding amplified bands on the '01-7' pomelo variety but not on other varieties. This led to the development of an AS-PCR marker that can distinguish the '01-7' pomelo from other varieties. This marker can be used for the molecular identification of the '01-7' pomelo variety, which is of great significance for ensuring the purity of seedlings and promoting the development of improved and high-quality pomelo varieties. Attached Figure Description

[0017] Figure 1 A schematic diagram of the design of molecular marker primers for AS-PCR.

[0018] Figure 2This is a comparison diagram of Sanger sequencing verification of the Chr1_7111834_G / A site in '01-7' pomelo and the ancestral tree of pomelo (common pomelo). A. Direct sequencing of PCR products from '01-7' pomelo; B. Direct sequencing of PCR products from the ancestral tree of pomelo; C. Sequencing of plasmids after cloning PCR products from '01-7' pomelo and the ancestral tree of pomelo. ZZ represents the ancestral tree of pomelo.

[0019] Figure 3 Electrophoresis images for using AS-PCR molecular markers to distinguish between '01-7' pomelo and the ancestral tree of pomelo (common pomelo) (ZZ represents the ancestral tree of pomelo).

[0020] Figure 4 Electrophoresis images of 12 pomelo materials were used to identify them using AS-PCR molecular markers. Materials 1 to 12 are: 01-7a, 01-7b, 01-7c, 01-7d, PTa, PTb, PTc, HY10, HY11, 'Crispy Red', 'Red Flesh Pomelo', and 'Summer Red'. PT represents common pomelo. HY10 and HY11 are other new varieties.

[0021] Figure 5 This is a sequence alignment diagram of 12 pomelo samples for sequencing verification, where information from samples 1 to 12 is compared with... Figure 4 Similarly, the base in the red box is the base where the SNP is located. Detailed Implementation

[0022] The present invention will be further described below with reference to specific accompanying drawings and embodiments.

[0023] Example 1: Whole genome resequencing analysis of pomelo

[0024] Fresh, healthy, and tender leaves from the '01-7' pomelo and its ancestral tree (common pomelo) were used for whole-genome resequencing. A DNBSEQ-T7 sequencer was used, employing a PCR-free library construction method, with a sequencing read length of PE150. The raw data was filtered using FastQC, and adapters and low-quality data were removed using Trimmomatics v0.39. An index was built using the 'Late White Pomelo' (http: / / citrus.hzau.edu.cn / orange / download / index.php) genome as a reference. Sequencing fragments were aligned to the reference genome using BWA-MEM v0.7.17, and fragments with a mapping quality value less than 30 were removed using a shell script. PICARD (http: / / picard.sourceforge.net) was used to remove duplicate and redundant fragments. As shown in Table 1, after data contamination removal, adapter removal, and low-quality data removal from the raw data, a total of 120.21M high-quality fragments and 36.06Gb of high-quality bases were obtained. The average scores for Q20 and Q30 were 96.55 and 89.97, respectively, indicating high data quality. Alignment of the sequenced fragments with the reference genome revealed an average base alignment rate of 98.54% and a 1X genome coverage exceeding 95%, indicating that 'Late White Pomelo' can be used as a reference genome for pomelo analysis and achieves near-whole genome coverage. The average sequencing depth reached 46.75X, suitable for resequencing analysis of closely related samples. To further improve the accuracy of variant detection, SNP-calling was performed using both BCFtools v1.9 and GATK-HaplotypeCaller v3.6 modules. The intersection of the two software loci yielded 6,280,517 differentially expressed loci compared to the reference genome.

[0025] Table 1. Statistics on the quality assessment of whole-genome resequencing data of *Pomelo*

[0026]

[0027]

[0028] Example 2: SNP mining of grapefruit

[0029] SNPs were extracted from the detected whole-genome variations and filtered. For ease of subsequent application, only homozygous SNPs between the '01-7' pomelo and the ancestral tree of the pomelo (common pomelo) were mined. The SNP variation sets of the two materials were filtered using the following methods and parameters: (1) Initial filtering was performed using vcftools software to filter out sites with the smallest gene quality value less than 30 and sequencing depth greater than 60 or less than 10, and biselary sites were selected; (2) Secondary filtering was performed using the VariantFiltration module of GATK software, with the filtering conditions being "QUAL<30.0||QD<2.0||MQ<40.0||FS>60.0||SOR>3.0||MQRankSum<-12.5||ReadPosRankSum<-8.0". After two steps of quality filtering, high-confidence SNPs with quality values ​​greater than 30 and sequencing depths between 10 and 60 were selected. Finally, the IGV genome browser was used to examine the base distribution of the SNP site sequencing fragments in the bam file, and nine SNPs with an allele frequency greater than 90% were screened out. One of the SNPs located at the 7111834th base on chromosome 1 was selected for sequence verification and subsequent molecular marker development.

[0030] Example 3: SNP Verification

[0031] SNP validation was performed using gene cloning and Sanger sequencing. Based on the location information in the vcf file, 600bp sequences upstream and downstream of the SNP were extracted from the reference genome. Primers Chr1_7111834_G / AF and Chr1_7111834_G / AR were designed flanking the SNP using Primer3web version 4.1.0 (https: / / primer3.ut.ee / ). The expected PCR amplification length was 498bp (see attached image). Figure 1 Using genomic DNA from the '01-7' pomelo and its ancestral tree (common pomelo) as templates, PCR amplification, cloning, and Sanger sequencing were performed. The results showed that the genotypes of the '01-7' pomelo and its ancestral tree (common pomelo) were A / A and G / G, respectively. (See attached image) Figure 2 This indicates that the homozygous SNP information mined through resequencing combined with bioinformatics is true.

[0032] Example 4: AS-PCR Primer Design

[0033] AS-PCR primers were designed using BatchPrimer3v1.0 from the BatchPrimer3 website (https: / / wheat.pw.usda.gov / demos / BatchPrimer3 / ). The resulting upstream primer AS-PCR-F is an allele-specific primer, with its 3' terminal base identical to that of the '01-7' pomelo variety but different from the ancestral tree of the pomelo (common pomelo). The downstream primer AS-PCR-R is an allele-shared primer. Simultaneously, the second-to-last base at the 3' end of the upstream primer was designed as a mismatched base (see attached). Figure 1 The specific sequences of the two primers are: 5'-GCAGAGAAATCGCCTTTGTCA-3' (SEQ ID NO: 1, AS-PCR-F) and 5'-CGTGTGGTCTCCAAAAGTCC-3' (SEQ ID NO: 2, AS-PCR-R). Using these primers, the expected PCR amplification results are: using '01-7' pomelo DNA as a template, a 205bp fragment can be amplified; however, no PCR product is formed when using DNA from the ancestral pomelo tree (common pomelo) as a template.

[0034] Example 5: AS-PCR reaction

[0035] Because AS-PCR is highly sensitive to PCR reaction systems and procedures, the PCR reaction system and procedure were optimized based on gradient PCR experiments to establish a suitable AS-PCR reaction system. The AS-PCR reaction used a Taq enzyme (Vazyme 2×Taq Master Mix) without 3'-5' exonuclease activity to prevent the correction of introduced mismatched bases. The optimal reaction system obtained from the experiments was 3 μL 2×Taq Master Mix and 0.8 μL 10 μmol·L⁻¹. -1 Upstream and downstream primers, 30 ng template DNA, and ddH2O were added to a final volume of 10 μL. The PCR reaction program was 95℃ for 10 min; 95℃ for 10 s, 60℃ for 15 s, 72℃ for 5 s, for 30 cycles; finally, 72℃ for 10 min, and stored at 4℃. Electrophoresis conditions were as follows: 1.5% agarose gel, constant voltage at 110V for 25 min, GelRed staining, and imaging using a gel imaging system.

[0036] From the appendix Figure 3 It can be seen that a band can be amplified from the '01-7' pomelo, with a length consistent with the expected 205bp, while a band cannot be amplified from the ancestral pomelo tree (common pomelo), indicating that this AS-PCR marker is correct and effective.

[0037] Example 6: Application and Validation of AS-PCR

[0038] AS-PCR markers were further applied to 12 pomelo samples, covering major production areas and main varieties. Specifically, these included 01-7 (01-7a, 01-7b, 01-7c, 01-7d) from four production bases, common pomelo (PTa, PTb, PTc) from three production bases, two new varieties (HY10, HY11), 'Crispy Red' (CH), 'Red Flesh' (HR), and 'Summer Red' (XH). AS-PCR results are attached. Figure 4 As shown, all four '01-7' pomelo materials amplified bands of the expected length, while the other pomelo materials did not amplify.

[0039] To verify the accuracy of AS-PCR amplification, the sequence of this SNP site in all pomelo materials was analyzed based on gene cloning and Sanger sequencing. The results showed that only the '01-7' pomelo had an A / A genotype at this SNP site, while the genotype of other pomelo materials was G / G (see appendix). Figure 5 AS-PCR results (attached) Figure 4 ) and the actual SNP sequence (attached) Figure 5 The match confirms the correctness and effectiveness of the AS-PCR marker.

[0040] In summary, the AS-PCR marker developed in this invention can effectively distinguish '01-7' pomelo from other pomelos, and can be applied to the identification of genuine and counterfeit seedlings of this variety, which is conducive to ensuring the purity of seedlings and thus helps to promote the planting of '01-7' pomelo.

Claims

1. An AS-PCR marker for identifying '01-7' shatang pomelo, characterized in that, The AS-PCR marker is a pair of primers, and the sequences are shown as SEQ ID NO: 1 and SEQ ID NO:

2.

2. Application of the AS-PCR marker in claim 1 in distinguishing '01-7' from other pomelo varieties.

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