SNP (Single Nucleotide Polymorphism) molecular marker and application thereof in identifying honeybee shin length character

Abstract

The invention relates to the technical field of bee breeding, in particular to an SNP (Single Nucleotide Polymorphism) molecular marker and application of the SNP molecular marker to identification of a honeybee shin length character. The SNP molecular marker is located at the 8751308th site of the 14 # chromosome of the bee, and the polymorphism is T / C. Based on a whole genome association analysis result of bee sample data, the SNP molecular marker which is very related to the character of the shin segment length of the bee is obtained through screening, and a polymorphism detection result of the SNP molecular marker can reflect the shin segment length of the bee to be detected. The SNP molecular marker provided by the invention can be applied to breeding bees with different tibia lengths, is beneficial to improving the pollen collection efficiency of bees, and has important value.

Description

Technical Field

[0001] The 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 include Chinese honey bees, Japanese honey bees, Indian honey bees and Philippine honey bees. Compared with Western honey bees, they are often more disease-resistant, but the amount of honey they produce is relatively small. Chinese honey bees show different traits and characteristics according to different geographical environments, and they also show different honey production abilities. Therefore, accurately and quickly judging 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 refer to the recognizable features of an organism in morphology, which are used to distinguish different species, subspecies or individuals. These markers can be subtle differences in structure, color, size or shape, and are usually used in taxonomy, genetics and evolutionary biology research. Traditional bee species identification and performance evaluation generally use morphological markers, but the morphology of Chinese bees is easily affected by external interference factors such as nutritional conditions during development. In addition, the detection of markers has strict requirements on the proficiency of measurement personnel, low operability, and human differences, which affect the accuracy of discrimination.

[0004] Tibia length is one of the traits that is closely related to the production performance of honey bees. It is also one of the important factors that affect the length of the hind legs of Chinese honey bees. For example, worker bees have pollen baskets (also called pollen baskets) on their hind legs to carry pollen. Longer tibiae help worker bees carry more pollen. In addition, tibia length affects the ability of bees to crawl and move on different surfaces. Long tibiae provide a wider range of motion, helping bees to complete cleaning tasks more efficiently. Developing molecular markers related to honey bee tibia length will help obtain high-quality honey bee 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 chromosome 14 of the honey bee and has a polymorphism of T / C; the genome 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, and the polymorphism is T / C.

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

[0009] The present invention further provides a primer pair for amplifying the SNP molecular marker: F: 5'-AATTTCGAATTGTTTGATGG-3',

[0010] R: 5'-TGGTCGTAGAGCATGGTGAG-3'.

[0011] In a second aspect, the present invention provides a kit, comprising the SNP molecular marker or the primer pair.

[0012] 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.

[0013] 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.

[0014] Furthermore, the bee is an oriental honey bee; preferably a cerana cerana cerana.

[0015] In a fourth aspect, the present invention provides a method for identifying the length of the tibia of a honey bee, comprising: 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 result.

[0016] Furthermore, the polymorphism of the SNP molecular marker is detected by sequencing.

[0017] Further, judging the tibia length of the bee to be tested according to the test result includes: In the test results, the SNP molecular marker identified that the tibia length of the T / C genotype Chinese honey bee was significantly shorter than that of the T / T genotype Chinese honey bee.

[0018] The present invention has the following beneficial effects: The present invention screened and obtained a SNP molecular marker related to the tibia length of Chinese honey bees, which can be used to identify the tibia length of Chinese honey bees. The polymorphism detection can reflect the relevant characteristics of the tibia length trait. The SNP molecular marker can be used to breed bees with specific tibia length traits (or used for molecular marker-assisted breeding related to tibia length), improve the efficiency of bee pollination, and obtain high-quality bee germplasm resources. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the technical solutions in the present invention or the prior art, the following briefly introduces 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 creative work.

[0020] Figure 1 This is a comparison chart of the tibia length of individuals with different genotypes at the Chr14_8751308 locus of the Chinese honey bee provided in Example 2 of the present invention. DETAILED DESCRIPTION

[0021] In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be described clearly and completely below. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0022] The experimental methods involved in the following examples, unless otherwise mentioned, are all conventional methods in the art, for example, reference may be made to experimental manuals in the art, or the conditions recommended in the manufacturer's instructions.

[0023] Unless otherwise specified, the experimental materials and reagents involved in the following examples can be obtained from commercial sources.

[0024] Example 1

[0025] The present invention provides a method for screening SNP molecular markers, which specifically includes the following process: 1. The present invention is based on 110 groups of Chinese honey bees. One worker bee is selected from each group to measure the length of its front tibia. For the above-mentioned dissected worker bees, the genomic DNA of the worker bee chest tissue is extracted, and then the library is constructed using the Truseq Nano DNA HT kit (Illumina, USA). The DNA is randomly broken into 350bp fragments, and the DNA library is obtained through end repair, ployA tailing, sequencing adapter, amplification, purification and other steps. The insert size of the library is quality inspected using Agilent 2100, and the effective concentration of the library is accurately quantified using the qPCR method. The DNA library construction is completed after the quality meets the standard.

[0026] 2. Genome sequencing, alignment and SNPs identification: After the sample library was successfully constructed, the sample library was sequenced based on the Illumina HiseqPE150 platform (Illumina, USA). During the sequencing process, low-quality reads were deleted to ensure the quality of the results [quality control standards: delete reads containing more than 10% unknown nucleotides, delete reads containing adapter sequences, delete reads with low quality (phred quality <5) base content exceeding 50% of the length in the sequencing], and finally a single bee sample generated more than 4.5G high-quality paired-end clean reads, with Q20 and Q30 above 90% and 85%, respectively.

[0027] 3. The obtained high-quality paired-end clean reads were aligned to the reference genome Apis cerana (Genbank accession number: PRJNA738447) using BWA 0.7.8 software. The alignment results were deduplicated using SAMTOOLS 1.15 software, and the average alignment rate of the population samples was guaranteed to be above 95%, and the average sequencing depth of the genome was above 20X.

[0028] 4. The Bayesian model in SAMTOOLS 1.15 software was used to detect group SNPs, and high-quality SNPs were screened according to the quality control standards [delete SNPs with sequencing error rate >1% (Q20 quality control), delete SNPs with base spacing <5 between adjacent SNP sites, delete SNPs with coverage depth exceeding 1 / 3~5 times the average depth]. ANNOVAR 20130520 software was used to annotate the detected SNPs, identify exon regions, intron regions, variable splicing sites, upstream and downstream regions of genes, and intergenic regions, and distinguish between synonymous mutation SNPs and non-synonymous mutation SNPs.

[0029] 5. Genome-wide association analysis: Genome-wide association studies (GWAS) were conducted based on mrMLM 1.3 software to clarify the association between tibia length and SNPs. The SNPs quality control standard was MAF>5%, and the multi-locus random mixed linear model was selected as the model.

[0030] 6. Finally, the present invention obtains multiple molecular markers related to the tibia length of honey bees, one of which has a significant difference, namely the SNP molecular marker Chr14_8751308, which is located at position 8751308 of chromosome 14 of the honey bee, and the polymorphism is T / C. The polymorphism of the SNP molecular marker in the tested honey bees can be detected.

[0031] Example 2

[0032] The present invention selects 101 Chinese honey bee samples to carry out verification work, and verifies the application of SNP molecular markers in detecting the tibia length of Chinese honey bees in Example 1. Specifically, the 101 Chinese honey bees are sequenced, and the tibia length of the 101 honey bees is measured using a microscopic measurement system to obtain the tibia length data and SNP data of the 101 honey bees; The mice were grouped according to the genotype types at the SNP loci, and the tibia length data of different groups were analyzed for significant differences using SPSS 16.0 software to compare whether there were differences in tibia length among different genotypes.

[0033] Finally, 31 Chinese bees showed T / C genotype, and 70 Chinese bees showed T / T genotype. Through T-test data analysis, the T / T genotype and T / C genotype showed significant differences ( P <0.05), such as Figure 1 As shown, the tibia length of the T / C genotype Chinese honey bee is significantly shorter than that of the T / T genotype Chinese honey bee.

[0034] Table 1 Comparison of tibia length among different genotypes of Chr14_8751308 locus in A. cerana

[0035] It can be seen 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.

[0036] 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 embodiments of the present invention.

Claims

1. A SNP molecular marker, characterized in that: The SNP molecular marker is located at position 8751308 of chromosome 14 of the honey bee, and the polymorphism is T / C; the genome version number of the SNP molecular marker is PRJNA738447.

2. The SNP molecular marker according to claim 1, characterized in that: The SNP molecular marker includes a nucleotide sequence as shown in SEQ ID NO.1, and has a polymorphism at position 301, and the polymorphism is T / C.

3. A kit, characterized in that: Comprising the SNP molecular marker described in claim 1 or 2.

4. Use of the SNP molecular marker according to claim 1 or 2, or the kit according to claim 3, in detecting the tibia length trait of honey bees.

5. Use of the SNP molecular marker according to claim 1 or 2, or the kit according to claim 3 in breeding bees with different tibia lengths.

6. The use according to claim 4 or 5, characterized in that: The honey bee is an oriental honey bee; preferably a cerana ....

7. A method for identifying the length of the hind tibia of a honey bee, characterized in that: include: The polymorphism of the SNP molecular marker as claimed in claim 1 or 2 is detected for the bees to be tested, and the tibia length of the bees to be tested is determined according to the detection result.

8. The method according to claim 7, characterized in that The polymorphism of the SNP molecular marker as claimed in claim 1 or 2 is detected by sequencing.

9. The method according to claim 7 or 8, characterized in that: Determining the tibia length of the bee to be tested according to the test result comprises: In the test results, the SNP molecular markers as described in claim 1 or 2 identified that the tibia length of the T / C genotype Chinese honey bee was significantly shorter than that of the T / T genotype Chinese honey bee.

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