Efficient prawn DNA mixed pool construction method and breeding application

Abstract

The invention belongs to the technical field of biology, and particularly relates to an efficient prawn DNA mixed pool construction method and breeding application. The method specifically comprises the following steps: taking prawn tails of prawns as tissues of equivalent mixed single individuals, taking one prawn tails of each to-be-treated individual, merging and mixing the taken prawn tails, cutting the same length to realize uniform mixing of the equivalent tissues of the individuals, and then extracting DNA of the mixed tissues, namely constructing a DNA mixed pool. The technical scheme provided by the invention effectively solves the problems of difficulty in accurate quantification, complex operation and high cost in construction of a large number of DNA mixed pool tissues in traditional BSA analysis, simplifies the identification process of the character-related markers, is suitable for genetic analysis of large-scale prawn samples and identification of the character-related markers, and has a wide application prospect in the field of prawn breeding.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology, and particularly relates to a high-efficiency method for constructing a DNA pool of prawns and breeding application. BACKGROUND

[0002] The development of high-throughput genotyping technology promotes the application of genome-wide association study (GWAS) and genomic selection (GS). However, traditional genotyping methods are usually targeted at a single individual. The number of offspring of aquatic species such as prawns is large, and the population size is large. Large-scale individual genotyping operation is complex and costly, which limits its wide application in molecular breeding of aquatic animals.

[0003] Pooled genotyping method significantly reduces the number and cost of DNA genotyping by mixing individuals with the same or similar phenotype for DNA genotyping. Previous studies have confirmed that the genotyping results of whole-genome resequencing of DNA pool are highly correlated with the genotyping results of single sample (Fracassetti, M., Griffin, P.C., Willi, Y., 2015. Validation of Pooled Whole-Genome Re-Sequencing in Arabidopsis lyrata. PLoS One 10(10), e0140462). Therefore, the BSA-seq (Pooled genotyping-based bulked segregant analysis) technology based on the pooled genotyping technology is widely used in crops, livestock and aquatic products, and promotes the identification of markers of important economic traits and breeding application.

[0004] The main steps of BSA-seq include DNA pool construction, DNA pool sequencing and trait-associated marker identification. The DNA pool construction is a key step and the most complex step. There are two commonly used methods for pool construction. The first method is to extract DNA from individual samples and then mix the DNA equally. The DNA pool prepared by this method has small differences in the amount of DNA from different individuals, and the typing results of the mixed samples are stable. However, this method requires a large number of individual DNA extraction, which is costly and not suitable for large-scale breeding applications. The second method is to mix the tissues of individual individuals equally to form a mixed sample, and then extract the DNA from the mixed sample. This method reduces the number of DNA extraction times, but requires ensuring that the amount of tissue from different individuals in each mixed sample is equal, and that the tissue is completely digested during DNA extraction to reduce the error of individual DNA extraction. In the second method, the most important thing is to mix equal amounts of tissue, and the existing method is to mix equal amounts of muscle or appendages. This method has a large sampling volume, high weighing precision requirements, a complicated operation process, and a large workload. To realize the large-scale application of this method, a simple and efficient DNA pool construction method is urgently needed. SUMMARY

[0005] The present application aims to provide an efficient DNA pool construction method for shrimp and breeding applications.

[0006] To achieve the above-mentioned purpose, the technical scheme adopted by the present application is as follows: An efficient DNA pool construction method for shrimp, which uses shrimp antennae as equal amounts of mixed tissues of individual individuals, takes one shrimp antenna from each individual to be processed, mixes the taken shrimp antennae, cuts them to the same length, realizes the uniform mixing of equal amounts of individual tissues, and then extracts the DNA from the mixed tissues to construct a DNA pool.

[0007] One shrimp antenna is taken from each individual shrimp to be processed in anhydrous ethanol, the selected shrimp antennae of each individual are mixed and aligned towards the head and carapace end, and the same length is cut as the equal mixing of each sample tissue for DNA extraction.

[0008] The shrimp is Litopenaeus vannamei, Chinese white shrimp, Marsupenaeus japonicus, Japanese shrimp, Exopalaemon modestus, Procambarus clarkii, and Procambarus viridis.

[0009] An application of the method, the method is applied in shrimp breeding.

[0010] The DNA pool is constructed by the method, the DNA pool is genotyped by using the DNA targeted sequencing typing technology, then the Ridit method is used to analyze the correlation between the phenotype and genotype data of different DNA pools, the molecular markers related to the target phenotype traits are identified, and the molecular markers are used for breeding applications.

[0011] The DNA targeted sequencing and typing technique described herein utilizes targeted sequencing technology based on probe capture or multiplex PCR amplification to perform high-depth targeted sequencing on DNA pools.

[0012] The method for identifying candidate markers for the target trait uses the Ridit (Relative to an identified distribution in tegral transformation) algorithm to select the sites with the most significant differences as trait-related markers.

[0013] Advantages of this invention: (1) The method for constructing DNA pools using shrimp whisker tissue of equal length established in this invention simplifies the operation steps and greatly reduces costs while ensuring accurate and stable genotyping results compared with traditional pool DNA construction methods.

[0014] (2) This invention makes full use of the high depth of targeted sequencing to perform mixed-pool sequencing and typing. A depth greater than 100× can obtain accurate typing results, which improves the accuracy of mixed-pool DNA typing and also improves the efficiency of trait-related marker identification. Attached Figure Description

[0015] Figure 1 The results of the mixed-pond typing identification of growth trait-related markers for population No. 124 provided in this embodiment of the invention are presented.

[0016] Figure 2 The population in pond No. 127 provided in this embodiment of the invention was identified using mixed-pond typing and growth trait-related markers. Detailed Implementation

[0017] The following examples further illustrate specific embodiments of the present invention. It should be noted that the specific embodiments described herein are merely for illustration and explanation and are not intended to limit the scope of the present invention.

[0018] This invention proposes an efficient method for constructing mixed-sample shrimp DNA using shrimp whisker tissue, followed by the use of this mixed-sample construction method for trait-related marker identification and breeding applications. For mixed-sample samples, a method based on targeted sequencing for mixed-sample typing is proposed, achieving efficient and low-cost mixed-sample typing in shrimp.

[0019] The following section will use Litopenaeus vannamei as an example to explain the technical solution, implementation process, and principles of this invention by conducting mixed population genotyping and trait-related marker identification on genetic materials for growth traits. Samples from other species can also refer to this implementation scheme for DNA mixed-pond construction and target trait-related marker identification.

[0020] Example 1: Construction of mixed-pond DNA from Litopenaeus vannamei individuals and its application in the identification of trait-related markers. 1) Construction of mixed-pond samples of Litopenaeus vannamei The shrimp genetic material used in this study consisted of two hybrid families from Bohai Aquatic Breeding (Hainan) Co., Ltd. (cultured in ponds 124 and 127, respectively). The individuals were closely related and their body weights followed a normal distribution. Based on the weight of each individual, the individuals in each pond were divided into three groups: large, medium, and small, with 150 individuals in each group. The average body weight of individuals in each group within each pond is shown in Table 1. Due to the large number of individuals, each group was further divided into three parallel groups (1, 2, and 3). Therefore, the constructed hybrid populations are denoted as 124L1, 124L2, 124L3, 124M1, 124M2, 124M3, 124S1, 124S2, 124S3, 127L1, 127L2, 127L3, 127M1, 127M2, 127M3, 127S1, 127S2, and 127S3. Shrimp whisker tissue from individuals in each mixed population was aligned near the cephalothorax and preserved together in anhydrous ethanol for DNA extraction.

[0021] Table 1. Average weight of individuals in each group in the culture pond.

[0022]

[0023] 2) Construction of DNA Mixture Pools Shrimp whiskers were removed from the mixed population from anhydrous ethanol and cut into approximately 5mm lengths using scissors for extraction. During lysis, the whiskers were transferred to 1.5ml centrifuge tubes, and DNA lysis buffer (Tiangen, Beijing) and proteinase K were added. The mixture was then thoroughly ground using a tissue homogenizer and digested in a 65℃ water bath for 48 hours. After complete digestion, the supernatant was aspirated. DNA extraction was then performed according to the Tiangen DNA extraction kit, yielding a DNA pool. Furthermore, the extracted DNA was tested for quality and concentration using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA) and 1% agarose gel electrophoresis. Samples meeting the quality standards were then used for further analysis.

[0024] 3) Targeted sequencing genotyping of mixed samples The mixed sample obtained in the previous step was targeted sequenced using the "Zhongke Xin 2" 10K SNP chip for Litopenaeus vannamei (patent application number 202310120257.X), with a sequencing depth >100×. The sequencing data were used for SNP genotyping using GATK (v 4.3.0.0), identifying a total of 75,285 SNPs. The genotyping results were further filtered using VCFtools (v 0.1.13) with parameters "--max-missing 0.9 --maf 0.05", retaining 42,419 SNPs. The filtered SNPs showed a relatively uniform distribution in the Litopenaeus vannamei genome.

[0025] 4) Calculation of allele frequencies in different group groups In subsequent analyses, the average allele frequency from each mixed population within the same pool and level is used as the allele frequency for that pool and level of DNA pool. In this example, taking pool 124 as an example, the average allele frequency of the mixed populations 124L1, 124L2, and 124L3 is used as the allele frequency for the highest weight class in pool 124. The calculation method for allele frequencies in other levels of DNA pools is the same.

[0026] 5) Identification of growth trait-related markers and genes Ridit analysis was used to determine the significant differences in allele frequencies at each SNP locus between different levels of DNA pools within the same pool. The analysis results for pools 124 and 127 are presented as Manhattan plots, as shown below. Figure 1 and Figure 2 As shown in the figure. In this example, p<0.01 was used as the selection threshold to screen for significantly different SNP sites. 3448 candidate markers were identified in pool 124 and 2927 candidate markers were identified in pool 127.

[0027] To identify universally relevant growth-related candidate genes, 285 candidate markers shared by the two families were further analyzed. For SNPs in exons and introns, the gene at that location was considered a candidate gene. For upstream or downstream SNPs, the nearest gene was considered a candidate gene. For SNPs in intergenic regions, genes within a 20 kb range upstream and downstream were considered candidate genes. Functional enrichment results of the candidate genes showed that they were enriched in growth-related signaling pathways such as the Rap1 signaling pathway, indole alkaloid biosynthesis, and the Notch signaling pathway, and can be used for subsequent gene function analysis.

[0028] This invention provides a highly efficient and low-cost method for genotyping mixed populations of Litopenaeus vannamei. DNA extraction and genotyping were performed on a total of 18 mixed populations from two ponds. Using a single-sample genotyping method would require DNA extraction and genotyping of all 900 individuals involved in the analysis, representing a workload 50 times greater than the method used in this invention. Furthermore, the genotyping method employed in this example is a low-cost DNA-targeted sequencing genotyping technique, significantly reducing the sequencing costs required to obtain genotyping data.

Claims

1. A highly efficient method for constructing mixed-pond shrimp DNA, characterized in that: Using shrimp whiskers as an equal amount of tissue for mixing individual individuals, one whisker was taken from each individual to be treated, and the whiskers were combined and mixed, and cut to the same length to achieve uniform mixing of equal amounts of tissue from individuals. Then, the DNA of the mixed tissue was extracted, thus constructing a DNA mixing pool.

2. The efficient shrimp DNA mixed-pond construction method according to claim 1, characterized in that: Take one shrimp antennae from each shrimp individual to be treated and place it in anhydrous ethanol. Mix the selected shrimp antennae and cut them into equal lengths, aligning them with the end closest to the cephalothorax. Mix these equal amounts as the sample tissues for DNA extraction.

3. The efficient method for constructing mixed-pond shrimp DNA according to claim 1 or 2, characterized in that: The prawns mentioned include Litopenaeus vannamei, Litopenaeus sinensis, Penaeus monodon, Penaeus japonicus, and Litopenaeus vannamei, as well as freshwater prawns such as Macrobrachium rosenbergii, Macrobrachium japonicum, Procambarus clarkii, and Redclaw crayfish.

4. An application of the method according to claim 1, characterized in that, Application of the method in shrimp breeding.

5. The application according to claim 4, characterized in that, DNA pools were constructed using the method described above, and genotyping of the DNA pools was performed using DNA targeted sequencing genotyping technology. Subsequently, the association between phenotypic and genotypic data from different DNA pools was analyzed using the Ridit method to identify molecular markers associated with the target phenotypic traits for application in breeding.

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