A SNP molecular marker and its application in identifying the honeybee leg length trait

Abstract

The application relates to the technical field of bee breeding, and particularly relates to a SNP (Single Nucleotide Polymorphism) molecular marker and application thereof in identifying a honeybee tibia length character. The SNP molecular marker is located at the 8751308th position of a 14th chromosome of the honeybee, and the polymorphism is T / C. Based on the whole genome correlation analysis result of the honeybee sample data, the application screens a SNP molecular marker which is very relevant to the honeybee tibia length character. The polymorphism detection result of the SNP molecular marker can reflect the tibia length of the honeybee to be detected. The SNP molecular marker provided by the application can be applied to breeding honeybee species with different tibia lengths, is favorable to improving the pollen collection efficiency of the honeybee, and has important value.

Description

Technical Field

[0001] The present invention relates to the technical field of bee breeding, and in particular to a SNP molecular marker and an application thereof in identifying the tibia length trait of a bee. Background Art

[0002] Oriental honey bees, including the Chinese honey bee, Japanese honey bee, Indian honey bee, and Philippine honey bee, are often more disease-resistant than Western honey bees, but produce relatively less honey. Chinese honey bees exhibit different traits and characteristics depending on their geographical environment, and their honey production capacity also varies. Therefore, accurate and rapid evaluation of the morphological traits of Chinese honey bees is an important means of protecting, identifying, and utilizing bee species resources, and can effectively evaluate the performance of bee species.

[0003] Morphological markers are recognizable features of an organism's morphology, used to distinguish species, subspecies, or individuals. These markers can be subtle differences in structure, color, size, or form, and are commonly used in taxonomy, genetics, and evolutionary biology research. Traditionally, morphological markers are used for bee species identification and performance evaluation. However, the morphology of Chinese honeybees is easily affected by external factors such as nutritional conditions during development. Furthermore, marker detection requires strict proficiency, is difficult to perform, and is subject to human variability, affecting accuracy.

[0004] Tibia length is a trait closely linked to honeybee performance and a key factor influencing the length of the hind legs of Chinese honeybees. For example, worker bees carry pollen baskets on their hind legs, which carry pollen. Longer tibias help them carry more pollen. Furthermore, tibia length influences a bee's ability to crawl and move across surfaces. Longer tibias provide a wider range of motion, helping bees to more efficiently complete cleaning tasks. Developing molecular markers associated with tibia length in honeybees will contribute to the acquisition of high-quality honeybee germplasm resources. Summary of the Invention

[0005] In order to solve the problems existing in the prior art, the present invention provides a SNP molecular marker and its application in identifying the tibia length trait of honey bees.

[0006] In a first aspect, the present invention provides a SNP molecular marker, which is located at position 8751308 of honey bee chromosome 14 and has a polymorphism of T / C; the genomic version number of the SNP molecular marker is PRJNA738447.

[0007] Furthermore, the SNP molecular marker includes a nucleotide sequence as shown in SEQ ID NO. 1, and has a polymorphism at position 301, which is T / C.

[0008] The nucleotide sequence shown in SEQ ID NO.1 is as follows:

[0009] .

[0010] The present invention further provides a primer pair for amplifying the SNP molecular marker:

[0011] F: 5'-AATTTCGAATTGTTTGATGG-3',

[0012] R: 5'-TGGTCGTAGAGCATGGTGAG-3'.

[0013] In a second aspect, the present invention provides a kit comprising the aforementioned SNP molecular marker or the aforementioned primer pair.

[0014] In a third aspect, the present invention provides the use of the SNP molecular marker, or the primer pair, or the kit in detecting the tibia length trait of honey bees.

[0015] The present invention further provides the use of the SNP molecular marker, the primer pair, or the kit in breeding bees with different tibia lengths.

[0016] Furthermore, the bee is an oriental honey bee; preferably a cerana cerana cerana cerana.

[0017] In a fourth aspect, the present invention provides a method for identifying the length of the hind tibia of a honeybee, comprising:

[0018] The polymorphism of the SNP molecular marker is detected for the bees to be tested, and the tibia length of the bees to be tested is determined according to the detection results.

[0019] Furthermore, the polymorphism of the SNP molecular marker is detected by sequencing.

[0020] Furthermore, determining the tibia length of the bee to be tested according to the test result includes:

[0021] In the test results, the SNP molecular marker identified that the tibia length of the T / C genotype Chinese honeybee was significantly shorter than that of the T / T genotype Chinese honeybee.

[0022] The present invention has the following beneficial effects:

[0023] This study screened a single-nucleotide polymorphism (SNP) molecular marker associated with tibia length in Chinese honeybees. This marker can be used to identify tibia length in Chinese honeybees. Polymorphic detection can reveal relevant characteristics of tibia length. This SNP molecular marker can be used to breed bees with specific tibia length traits (or for marker-assisted breeding related to tibia length), thereby improving bee pollination efficiency and obtaining high-quality bee germplasm resources. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] In order to more clearly illustrate the technical solutions in the present invention or the prior art, a brief introduction is given below to the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0025] Figure 1 This is a comparison chart of the tibia length of individuals with different genotypes at the Chr14_8751308 locus of the Chinese honeybee provided in Example 2 of the present invention. DETAILED DESCRIPTION

[0026] To make the objectives, technical solutions, and advantages of the present invention more clear, the technical solutions of the present invention are described clearly and completely below. Obviously, the embodiments described are only some of the embodiments of the present invention, not all of them. All other embodiments derived by persons of ordinary skill in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention.

[0027] Unless otherwise specified, the experimental methods involved in the following examples are all conventional methods in the art. For example, reference can be made to experimental manuals in the art, or the conditions recommended by the manufacturer's instructions.

[0028] Unless otherwise specified, the experimental materials and reagents involved in the following examples can be obtained from commercial sources.

[0029] Example 1

[0030] The present invention provides a method for screening SNP molecular markers, which specifically includes the following process:

[0031] This method is based on a sample of 110 swarms of Apis cerana cerana. One worker bee from each swarm was selected and its anterior tibia length measured. Genomic DNA was extracted from the thorax tissue of the dissected worker bees, and a library was constructed using the Truseq Nano DNA HT Kit (Illumina, USA). The DNA was randomly fragmented into 350 bp fragments. The DNA library was obtained through end-repair, PloyA tailing, sequencing adapter addition, amplification, and purification. The insert size of the library was quality-checked using an Agilent 2100, and the effective concentration of the library was accurately quantified using qPCR. Once the quality met the standards, the DNA library was constructed.

[0032] 2. Genome Sequencing, Alignment, and SNP Identification: After successful sample library construction, genome sequencing was performed on the Illumina HiseqPE150 platform (Illumina, USA). Low-quality reads were removed during sequencing to ensure quality [quality control criteria: reads containing more than 10% unknown nucleotides, reads containing adapter sequences, and reads with low-quality (phred quality < 5) bases exceeding 50% of the sequence length]. Finally, high-quality paired-end clean reads of at least 4.5G were generated for each honey bee sample, with Q20 and Q30 values ​​exceeding 90% and 85%, respectively.

[0033] 3. High-quality paired-end clean reads were aligned to the reference genome, Apis cerana (Genbank accession number: PRJNA738447), using BWA 0.7.8 software. Alignment results were deduplicated using SAMTOOLS 1.15 software. The average alignment rate for the population samples was maintained at over 95%, and the average sequencing depth across the genome was over 20X.

[0034] 4. A Bayesian model in SAMTOOLS 1.15 software was used to detect cluster SNPs. High-quality SNPs were screened based on quality control criteria (removing SNPs with a sequencing error rate >1% (Q20 quality control), removing SNPs with a base spacing of <5 between adjacent SNPs, and removing SNPs with a coverage depth exceeding 1 / 3 to 5 times the average). Detected SNPs were annotated using ANNOVAR 20130520 software to identify exons, introns, alternative splice sites, upstream and downstream regions, and intergenic regions, and to distinguish between synonymous and nonsynonymous SNPs.

[0035] 5. Genome-wide association analysis: Genome-wide association studies (GWAS) were performed using mrMLM 1.3 software to clarify the association between tibia length and SNPs. The SNP quality control standard was MAF>5%, and the multilocus random mixed linear model was selected as the model.

[0036] 6. Finally, the present invention obtained multiple molecular markers related to the tibia length of honey bees, one of which had a significant difference, namely the SNP molecular marker Chr14_8751308. This SNP molecular marker is located at position 8751308 of chromosome 14 of honey bees, and the polymorphism is T / C. The polymorphism of this SNP molecular marker in the tested honey bees can be detected.

[0037] Example 2

[0038] The present invention selected 101 Chinese honeybee samples to carry out verification work, verifying the application of SNP molecular markers in detecting the tibia length of Chinese honeybees in Example 1. Specifically, sequencing was performed on these 101 Chinese honeybees, and the tibia length of the 101 honeybees was measured using a microscopic measurement system to obtain tibia length data and SNP data for the 101 honeybees;

[0039] The genotypes at the SNP loci were grouped, and the tibia length data of different groups were analyzed for significance of differences using SPSS 16.0 software to compare whether there were differences in tibia length among different genotypes.

[0040] Finally, 31 Chinese bees showed T / C genotype, and 70 Chinese bees showed T / T genotype. Through T-test data analysis, T / T genotype and T / C genotype showed significant difference ( P <0.05), such as Figure 1 As shown in the figure, the tibia length of the T / C genotype Chinese honey bee is significantly shorter than that of the T / T genotype Chinese honey bee.

[0041] Table 1 Comparison of tibia length among different genotypes of Chr14_8751308 locus in Apis cerana

[0042]

[0043] It can be seen from this that the SNP molecular marker Chr14_8751308 can be used to identify the tibia length of honey bees. The polymorphism of this site reflects the tibia length trait of honey bees. In the actual research process, sequencing can be used to identify the polymorphism of this SNP molecular marker, or KASP primers can be designed to determine the polymorphism of this SNP molecular marker through primer amplification results.

[0044] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the various embodiments of the present invention.

Claims

1. A SNP molecular marker for identifying the tibia length trait of the Chinese honey bee, characterized in that: The nucleotide sequence of the SNP molecular marker is shown in SEQ ID NO. 1, and has a polymorphism at position 301, which is T / C.

2. Use of the SNP molecular marker according to claim 1 in detecting the tibia length trait of the Chinese honeybee.

3. Use of the SNP molecular marker according to claim 1 in breeding Chinese honeybees with different tibia lengths.

4. A method for identifying the length of the hind tibia of the Chinese honey bee, characterized in that: include: The polymorphism of the SNP molecular marker according to claim 1 is detected for the tested honeybee, and the tibia length of the tested honeybee is determined according to the detection result.

5. The method according to claim 4, characterized in that The polymorphism of the SNP molecular marker as claimed in claim 1 is detected by sequencing.

6. The method according to claim 4 or 5, characterized in that Determining the tibia length of the tested honeybee according to the test results includes: In the test results, the SNP molecular markers as described in claim 1 identified that the tibia length of the T / C genotype Chinese honeybee was significantly shorter than that of the T / T genotype Chinese honeybee.

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