Method for predicting mature period of pear based on detection of NOR1 copy number variation
By detecting NOR1 copy number variations and establishing a mapping relationship using next-generation sequencing technology, the maturity period of pears can be predicted, solving the problem of low efficiency in existing pear breeding technologies and realizing efficient breeding of early-maturing varieties.
Patent Information
- Application Number
- CN202511857988.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-12-10
- Publication Date
- 2026-01-09
AI Technical Summary
The lack of effective molecular markers in existing technologies for predicting pear maturity leads to low efficiency in fruit tree breeding, especially in perennial fruit trees where it is difficult to identify the maturity of hybrid offspring.
By detecting NOR1 copy number variations, a mapping relationship between NOR1 and pear maturity was established. Next-generation sequencing technology was used to determine the NOR1 copy number, and the pear maturity was predicted by combining standardized sequencing depth, classifying them into early-maturing, mid-maturing, and late-maturing types.
It enables the prediction of the maturity period of hybrid offspring during the seedling stage, significantly improving the breeding efficiency of early-maturing varieties and simplifying the fruit tree breeding process.
Smart Images
Figure CN121294727A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of plant molecular marker technology, and in particular to a method for predicting the maturity period of pear based on detecting NOR1 copy number variations. Background Technology
[0002] Pears are one of my country's important fruit crops, with a wide cultivation area and large total output. Maturity is a crucial trait for fruit trees. Early-maturing varieties can capture a larger market share and thus generate higher economic benefits. Therefore, breeding high-quality, early-maturing pear varieties has become an important breeding goal for the pear industry.
[0003] Perennial fruit trees have long breeding cycles and require large land areas. Molecular marker-assisted selection, which uses molecular markers closely linked to traits for early screening of breeding parents or hybrid offspring, can greatly improve the efficiency of fruit tree breeding.
[0004] The genome contains a wealth of genetic variation, forming the basis for crop trait improvement and serving as ideal design targets for molecular markers. Copy number variation (CNV) refers to the variation in the number of repeats in a DNA segment, a type of structural variation. When a repeating segment contains a gene, it can alter gene expression levels, ultimately changing crop traits. Sequencing depth analysis is a commonly used method for detecting copy number variation. When copy number variation exists in a region, the sequencing depth of that region will be significantly higher than the average sequencing depth of the entire genome. Higher copy numbers generally correspond to higher sequencing depths. With the development of next-generation sequencing technology, the technology has become increasingly mature and cost-effective, leading to the further promotion of sequencing depth-based methods.
[0005] The tomato NOR gene belongs to the NAC transcription factor family and is expressed in the fruit. Numerous studies have shown a close relationship between NOR and its homologs and fruit ripening time. Genomic analysis reveals the presence of the tomato NOR gene homolog NOR1 on chromosome 3 of the pear genome. The region containing this gene is closely linked to ripening time. However, to date, there are no reports of NOR1 copy number variations affecting pear ripening time, nor are there molecular markers for detecting related copy number variations. Summary of the Invention
[0006] The purpose of this invention is to address the shortcomings of existing technologies by providing a method for predicting pear maturity based on the detection of NOR1 copy number variations.
[0007] The objective of this invention is achieved through the following technical solution: the application of NOR1 copy number variation in predicting pear maturity, and determining pear maturity based on NOR1 copy number.
[0008] Furthermore, the pear maturity period was determined based on the NOR1 copy number, including: Establish a mapping relationship between NOR1 copy number and pear maturity period; pear maturity period is divided into early maturity, mid maturity and late maturity.
[0009] Furthermore, when the NOR1 copy number is 2, the pear matures late; when the NOR1 copy number is 4, the pear matures mid-season; and when the NOR1 copy number is 6, the pear matures early.
[0010] Furthermore, a mapping relationship between standardized NOR1 sequencing depth and NOR1 copy number is established, thereby establishing a mapping relationship between standardized NOR1 sequencing depth and pear ripening period, and thus determining the pear ripening period based on standardized NOR1 sequencing depth.
[0011] Furthermore, the determination of standardized NOR1 sequencing depth includes the following steps: (1) Extract DNA from the variety to be predicted; (2) Use a sequencing platform to perform 150bp paired-end sequencing, the sequencing data volume is greater than 5G and the sequencing data quality is controlled to remove low-quality bases and sequencing adapter sequences. (3) Establish an index of the pear reference genome; (4) Align the paired-end data to the pear reference genome and sort them to obtain the BAM alignment file; (5) Mark the BAM alignment files with repetitive sequencing sequences; (6) Using the BAM alignment file after labeling the repetitive sequencing sequence as the input file, calculate the average sequencing depth of the whole genome and the average sequencing depth of the NOR1 gene region respectively; (7) The average sequencing depth of the NOR1 gene region is divided by the average sequencing depth of the whole genome to obtain the normalized NOR1 sequencing depth.
[0012] The present invention also provides a method for predicting the ripening period of pears based on detecting NOR1 copy number variation, wherein the ripening period of pears is determined according to the NOR1 copy number.
[0013] Furthermore, the pear maturity period was determined based on the NOR1 copy number, including: Establish a mapping relationship between NOR1 copy number and pear maturity period; pear maturity period is divided into early maturity, mid maturity and late maturity.
[0014] Furthermore, a mapping relationship between standardized NOR1 sequencing depth and NOR1 copy number is established, thereby establishing a mapping relationship between standardized NOR1 sequencing depth and pear ripening period, and thus determining the pear ripening period based on standardized NOR1 sequencing depth.
[0015] Furthermore, the determination of standardized NOR1 sequencing depth includes the following steps: (1) Extract DNA from the variety to be predicted; (2) Use a sequencing platform to perform 150bp paired-end sequencing, the sequencing data volume is greater than 5G and the sequencing data quality is controlled to remove low-quality bases and sequencing adapter sequences. (3) Establish an index of the pear reference genome; (4) Align the paired-end data to the pear reference genome and sort them to obtain the BAM alignment file; (5) Mark the BAM alignment files with repetitive sequencing sequences; (6) Using the BAM alignment file after labeling the repetitive sequencing sequence as the input file, calculate the average sequencing depth of the whole genome and the average sequencing depth of the NOR1 gene region respectively; (7) The average sequencing depth of the NOR1 gene region is divided by the average sequencing depth of the whole genome to obtain the normalized NOR1 sequencing depth.
[0016] Furthermore, when the NOR1 copy number is 2, the pear matures late; when the NOR1 copy number is 4, the pear matures mid-season; and when the NOR1 copy number is 6, the pear matures early.
[0017] Compared with existing technologies, the beneficial effects of this invention are as follows: Pear trees have a long juvenile period, and plants developed from seeds require many years of cultivation before bearing fruit, making it difficult to identify the maturity period of pear hybrid offspring. Currently, there are few molecular markers related to pear maturity. The molecular markers developed in this invention can predict the maturity period of hybrid offspring during the seedling stage, greatly increasing the breeding efficiency of early-maturing varieties. Attached Figure Description
[0018] To more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings used in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.
[0019] Figure 1 This diagram illustrates the copy number variation in the region containing NOR1. Detailed Implementation
[0020] The present invention will now be described in detail with reference to the accompanying drawings. Unless otherwise specified, the features of the following embodiments and implementations can be combined with each other.
[0021] Unless otherwise specified, the experimental methods used in the following examples are conventional methods; the materials and reagents used are commercially available unless otherwise specified.
[0022] Example 1: Development of molecular markers for pear ripening By analyzing the genomes of pear varieties at different maturity stages, this invention discovered copy number variations in the DNA fragment containing NOR1 (Ya pear reference genome Chr03:24680452-24682505). In one haplotype, the NOR1 fragment has only one copy. In another haplotype, the NOR1 segment has two tandem repeats, resulting in a copy number of 3 (e.g., ...). Figure 1 (As shown). Based on the analysis of genome assembly and third-generation sequencing data, this invention found that the copy number of NOR1 in the early-maturing pear variety 'Cuiguan' (development period within 120 days) is 6 (3+3); the copy number of NOR1 in the mid-maturing pear variety 'Qinghuali' (development period 120-150 days) is 4 (3+1); and the copy number of NOR1 in the late-maturing pear variety 'Dangshan Suli' (development period more than 150 days) is 2 (1+1), indicating that the copy number of NOR1 is positively correlated with the maturity period.
[0023] To fill the aforementioned technological gap, this invention designs a method for detecting NOR1 copy number variations based on second-generation sequencing data, which can be used to predict the fruit ripening period of pear varieties. The specific method is as follows: 1. Extract DNA from the variety to be predicted using common methods such as the CTAB method.
[0024] 2. Perform 150bp paired-end sequencing using common second-generation sequencing platforms such as Illumina. The sequencing data volume is greater than 5G. Use FastP software to perform quality control on the sequencing data, removing low-quality bases and sequencing adapter sequences.
[0025] 3. Use BWA software to create an index of the pear reference genome (containing 2 NOR1 copies). The software execution command is "bwa index -p reference_index reference.fa".
[0026] 4. Use BWA software to align the paired-end data to the pear reference genome and sort using SAMtools to obtain alignment files in BAM format. The software execution command is "bwa mem -R "@RG\tID:sample_WGS\tPL:Illumina\tSM:sample" reference_index reads_1.fq.gz reads_2.fq.gz | samtools sort -oaln.bam -". After completion, create an index for the BAM file using the software execution command "samtools index aln.bam".
[0027] 5. Use the GATK4 software package to label the BAM file with repetitive sequencing sequences. The command is "gatkMarkDuplicates -I aln.bam -O markdup.bam -M markdup.metric". After completion, create an index for the newly generated BAM file using the command "samtools index markdup.bam".
[0028] 6. Using the BAM file containing the labeled repetitive sequencing sequences as input, calculate the average sequencing depth of the whole genome and the average sequencing depth of the NOR1 gene region using PanDepth software. The software commands are "pandepth -i markdup.bam -o genome_depth" and "pandepth -imarkdup.bam -o NOR1_depth -b NOR1.bed" respectively.
[0029] 7. The normalized NOR1 depth is obtained by dividing the NOR1 depth by the genome depth. Table 1 lists varieties with known NOR1 copy numbers, normalized NOR1 sequencing depths, and maturity dates. Considering errors such as alignment bias in sequencing data, the normalized NOR1 sequencing depth for these varieties, plus or minus 0.25, is used as a reference range for estimating pear maturity dates. Maturity dates are predicted for different pear germplasm resources after obtaining normalized NOR1 sequencing depths, referring to Table 1.
[0030] Table 1: Reference for Standardized NOR1 Sequencing Depth of Pear Varieties at Different Maturity Stages Example 2: Pear Maturity Prediction 1. Data collection for pear second-generation sequencing DNA was extracted from 'New Century' plant tissue using the CTAB method. Second-generation (NGA) sequencing of 150 bp paired ends was performed using the Illumina sequencing platform, yielding a sequencing data volume greater than 5 GB. NGA sequencing data for 14 varieties, including 'Yakumo', were obtained from public databases. FastP software was used for quality control of the sequencing data, removing low-quality bases and sequencing adapter sequences.
[0031] 2. Calculation of Standardized NOR1 Sequencing Depth Calculate according to the method in Example 1.
[0032] 3. Prediction of pear ripening period Based on the standardized NOR1 sequencing depth calculated for each variety, and referring to Table 1, the maturity date was predicted. The results are shown in Table 2, with a concordance rate of 93%.
[0033] Table 2: Pear Maturity Prediction Based on Standardized NOR1 Sequencing Depth The above embodiments are only used to illustrate the design concept and features of the present invention, and their purpose is to enable those skilled in the art to understand the content of the present invention and implement it accordingly. The protection scope of the present invention is not limited to the above embodiments. Therefore, all equivalent changes or modifications made based on the principles and design ideas disclosed in the present invention are within the protection scope of the present invention.
Claims
1. The application of NOR1 copy number variation in predicting pear maturity, characterized in that, The pear ripening period was determined based on the NOR1 copy number.
2. The application as described in claim 1, characterized in that, The pear maturity period was determined based on the NOR1 copy number, including: Establish a mapping relationship between NOR1 copy number and pear maturity period; pear maturity period is divided into early maturity, mid maturity and late maturity.
3. The application as described in claim 1 or 2, characterized in that, When the NOR1 copy number is 2, the pear matures late; when the NOR1 copy number is 4, the pear matures mid-season; when the NOR1 copy number is 6, the pear matures early.
4. The application as described in claim 1, characterized in that, A mapping relationship between standardized NOR1 sequencing depth and NOR1 copy number was established, thereby establishing a mapping relationship between standardized NOR1 sequencing depth and pear maturity, and thus determining the pear maturity based on standardized NOR1 sequencing depth.
5. The application as described in claim 1, characterized in that, Determining the standardized NOR1 sequencing depth includes the following steps: (1) Extract DNA from the variety to be predicted; (2) Use a sequencing platform to perform 150bp paired-end sequencing, the sequencing data volume is greater than 5G and the sequencing data quality is controlled to remove low-quality bases and sequencing adapter sequences. (3) Establish an index of the pear reference genome; (4) Align the paired-end data to the pear reference genome and sort them to obtain the BAM alignment file; (5) Mark the BAM alignment files with repetitive sequencing sequences; (6) Using the BAM alignment file after labeling the repetitive sequencing sequence as the input file, calculate the average sequencing depth of the whole genome and the average sequencing depth of the NOR1 gene region respectively; (7) The average sequencing depth of the NOR1 gene region is divided by the average sequencing depth of the whole genome to obtain the normalized NOR1 sequencing depth.
6. A method for predicting pear maturity based on detecting NOR1 copy number variation, characterized in that, The pear ripening period was determined based on the NOR1 copy number.
7. The method as described in claim 6, characterized in that, The pear maturity period was determined based on the NOR1 copy number, including: Establish a mapping relationship between NOR1 copy number and pear maturity period; pear maturity period is divided into early maturity, mid maturity and late maturity.
8. The method as described in claim 7, characterized in that, A mapping relationship between standardized NOR1 sequencing depth and NOR1 copy number was established, thereby establishing a mapping relationship between standardized NOR1 sequencing depth and pear maturity, and thus determining the pear maturity based on standardized NOR1 sequencing depth.
9. The method as described in claim 8, characterized in that, Determining the standardized NOR1 sequencing depth includes the following steps: (1) Extract DNA from the variety to be predicted; (2) Use a sequencing platform to perform 150bp paired-end sequencing, the sequencing data volume is greater than 5G and the sequencing data quality is controlled to remove low-quality bases and sequencing adapter sequences. (3) Establish an index of the pear reference genome; (4) Align the paired-end data to the pear reference genome and sort them to obtain the BAM alignment file; (5) Mark the BAM alignment files with repetitive sequencing sequences; (6) Using the BAM alignment file after labeling the repetitive sequencing sequence as the input file, calculate the average sequencing depth of the whole genome and the average sequencing depth of the NOR1 gene region respectively; (7) The average sequencing depth of the NOR1 gene region is divided by the average sequencing depth of the whole genome to obtain the normalized NOR1 sequencing depth.
10. The method as described in claim 6, characterized in that, When the NOR1 copy number is 2, the pear matures late; when the NOR1 copy number is 4, the pear matures mid-season; when the NOR1 copy number is 6, the pear matures early.