Molecular markers related to carotenoid accumulation in scallops and their applications

Abstract

To develop a molecular marker related to carotenoid accumulation for the Patinopecten yessoensis variety "Haidu Jinbei" and to provide a method for maintaining population quality.SOLUTION: An InDel molecular marker related to carotenoid accumulation of Patinopecten yessoensis and an application thereof are disclosed. A base sequence of the InDel molecular marker in carotenoid accumulating Patinopecten yessoensis and a base sequence of the InDel molecular marker in common Patinopecten yessoensis are disclosed. Furthermore, an upstream primer sequence and a downstream primer sequence of a primer pair for amplifying the InDel molecular marker related to carotenoid accumulation of Patinopecten yessoensis are disclosed. By detecting the InDel molecular marker, homozygous Patinopecten yessoensis individuals capable of accumulating carotenoids can be conveniently and rapidly screened in a seedling stage and from parents, and effective technical support is provided for breeding of "Haidu Jinbei" and optimization of characters.SELECTED DRAWING: Figure 2

Description

[Technical Field]

[0001] The present invention belongs to the technical field of molecular breeding, and specifically relates to an InDel molecular marker involved in carotenoid accumulation in scallops and its application. [Background technology]

[0002] Carotenoids are a class of ubiquitous polyene compounds with yellow, red, and orange hues. They are important provitamin A and antioxidants. Dietary carotenoids can effectively suppress the development of cataracts and macular degeneration while reducing the incidence of cancer, cardiovascular disease, and obesity. Because animals can only obtain carotenoids from food and cannot synthesize them de novo, studying their absorption, metabolism, and bioavailability is crucial. At the same time, the economic value of carotenoids continues to rise, with the global carotenoid market expected to reach $1.84 billion annually in 2023, growing at a compound annual growth rate (CAGR) of 4.64% over the next five years (https: / / www.mordorintelligence.com).

[0003] The scallop (Patinopecten yessoensis) is a cold-water bivalve found naturally in northern Japan, the Russian Far East, and the northern coastal waters of the Korean Peninsula. It was introduced to China from Japan in 1982 and is one of the world's highest-quality cultivated species. In 2003, several mutant scallops with bright orange adductor muscles were discovered in wild scallop populations. The adductor muscles of normal wild scallops are white. Statistics show that orange adductor muscles are a rare mutation, accounting for approximately 0.2% of scallops. Previous breeding studies have demonstrated the following: (1) adductor muscle color is a stable, inherited quality trait; (2) the growth rate of scallops with orange adductor muscles is significantly higher than that of controls; and (3) stress tolerance experiments have shown that the color mutant scallops have higher stress tolerance than controls, demonstrating excellent stress tolerance. Through breeding, they obtained a new variety of scallop enriched in carotenoids, which they named "Haitajinbei (Sea Giant Gold Shell)."

[0004] In actual breeding processes, carotenoid enrichment in the adductor muscle is easily observed through phenotypes at the adult stage. However, the low levels of carotenoids accumulated in the adductor muscle at the seedling stage make identification through observation or content detection difficult, increasing the difficulty of identifying seedlings and directional breeding. Furthermore, reversion mutations have occurred in scallops with white adductor muscles in the Haitajinbei population, which is detrimental to maintaining the genetic stability of the Haitajinbei population. Developing molecular markers related to carotenoid accumulation in scallops allows for easy and rapid screening of scallop seedlings with homozygous carotenoid accumulation. Removing reversion mutations in white adductor muscles from the Haitajinbei population can be used to improve breeding efficiency and maintain the quality of the Haitajinbei population. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] China Patent Publication No. CN103740729A Summary of the Invention [Problem to be solved by the invention]

[0006] The present invention aims to provide an InDel molecular marker related to carotenoid accumulation in scallops, and also provides specific applications of the InDel molecular marker to address the deficiencies of the prior art. [Means for solving the problem]

[0007] To achieve the above object, the present invention adopts the following specific technical solutions. An InDel molecular marker for carotenoid accumulation in scallops, the molecular marker being located at base 23111924 of the Chr8 chromosome of the scallop, the base sequence of the InDel molecular marker in scallops with accumulated carotenoids being shown in SEQ ID NO: 1, and the base sequence of the InDel molecular marker in normal scallops being shown in SEQ ID NO: 2, the InDel molecular marker being located at base 99 of the base sequences of SEQ ID NO: 1 and SEQ ID NO: 2, and the base here is TACGT (ALT type, represented by 1) or T (REF type, represented by 0).

[0008] A primer pair for amplifying an InDel molecular marker associated with carotenoid accumulation by scallops, the upstream primer sequence being shown in SEQ ID NO:3 and the downstream primer sequence being shown in SEQ ID NO:4. The application of a primer pair for amplifying an InDel molecular marker related to carotenoid accumulation in scallops or an InDel molecular marker associated with carotenoid accumulation in scallops in the breeding of scallops that accumulate carotenoids, for example, in the breeding of ``Hai Ta Jin Bei''.

[0009] A method for breeding scallops that accumulate carotenoids, comprising: (1) Sampling target scallops, including sampling the entire soft tissue for identification of the population of seedlings at the small-scale seedling stage, non-destructive sampling of gill filaments for directional breeding of seedlings at the large-scale seedling stage, and non-destructive sampling of gill filaments at the adult stage for parent selection, and then placing the sample in alcohol for later use; (2) Extracting DNA from the individual to be detected using the phenol chloroform method, detecting the concentration and purity of the extracted DNA sample using Nanodrop and 1% agarose gel, and storing it in a refrigerator at -20°C for later use; (3) performing PCR amplification using DNA of a sample to be detected as a template and the sequences shown in SEQ ID NOs: 3 and 4 as primers to obtain a PCR product; (4) Sanger sequencing the PCR product to determine the genotype of the InDel site in the individual to be detected; (5) If the result of the genotyping is expressed as 1 / 1 type, the individual is a carotenoid-enriched scallop individual, and if the result of the genotyping is expressed as 0 / 0 type, the individual is a normal scallop individual that is not significantly enriched in carotenoids.

[0010] Since the genotype frequency of the InDel molecular marker in the carotenoid-enriched "Hitajinbei" population is significantly different from that in normal scallops, it is possible to effectively distinguish between scallops with accumulated carotenoids and those without, thereby realizing the breeding of "Hitajinbei." [Effects of the Invention]

[0011] Compared with the prior art, the present invention has the following advantages and beneficial effects: The present invention provides a combination of InDel molecular markers related to scallop carotenoids and their corresponding primers. By detecting the InDel molecular markers, scallop individuals with homozygous carotenoid accumulation can be easily and quickly screened from seedlings and parents, providing effective technical support for the breeding and optimization of the characteristics of "Haitajinbei." [Brief explanation of the drawings]

[0012] [Figure 1] FIG. 1 shows the results of a comparison of the total carotenoid content of normal scallops with white adductor muscles and "Hai Ta Jin Bei" scallops with orange adductor muscles. [Figure 2] This shows the results of breeding the carotenoid-enriched "Hai Ta Jin Bei." DETAILED DESCRIPTION OF THE INVENTION

[0013] DETAILED DESCRIPTION OF THE INVENTION Reference will now be made in detail to the embodiments of the present invention, examples of which are illustrated in the accompanying drawings. The following examples are intended to further illustrate the present invention, but are not intended to limit the present invention.

[0014] Example 1: The method for identifying InDel molecular markers associated with carotenoid accumulation by scallops comprises the following steps. (1) The scallops used in the experiment were sampled from Zhangzi Dao Yangzhichang (the Chinese character for "scallop" is composed of the dog radical and the chapter radical aligned on the left and right) farm in Dalian, Liaoning Province, China. A total of 63 scallops were sampled, including 42 regular scallops with white adductor muscles and 21 Haita Jinbei scallops with orange adductor muscles. The results showed that the total carotenoid content of the Haita Jinbei scallops was significantly higher than that of regular scallops (see Figure 1). (2) Using the phenol-chloroform method, extract the whole genomic DNA from the adductor muscle tissue of the individuals obtained in step (1), construct a whole genome resequencing library, and send it to Tianjin Novogene Company for next-generation sequencing. (3) Quality control analysis was performed on the off-machine resequencing data to obtain high-quality data for subsequent analysis. The high-quality data was compared with the scallop reference genome using the MEM algorithm in BWA software, and the data was sorted, repetitive sequences were removed, and mutation sites were detected and typed using Samtools, picard software, and the Haplotyper command in GATK software, respectively, to obtain genotype data for each individual of the normal scallop and the "Hai Ta Jin Bei" scallop. (5) The difference in genotype frequency of the mutation site between the normal scallop and the "Hai Ta Jin Bei" populations was calculated. As shown in Table 1, an InDel site was identified at base 23111924 of the Chr8 chromosome of the scallop. The genotype frequency of this site was significantly different (P = 2.45e-13) between the "Hai Ta Jin Bei" and normal scallop populations, and it can be used as a molecular marker related to carotenoid accumulation by scallops.

[0015] [Table 1]

[0016] P geno : p-value of the difference in genotype frequency between the two types of scallops.

[0017] Example 2: The method for validating the InDel molecular markers associated with carotenoid accumulation by scallops of the present invention in a scallop population and supporting the breeding of "Hai Ta Jin Bei" comprises the following steps: (1) For the identified InDel sites (see SEQ ID NOs: 1 and 2), a corresponding primer pair (see SEQ ID NOs: 3 and 4) was developed based on Table 2 to verify the reliability of the InDel molecular marker in the scallop population. The primer information is shown in Table 3.

[0018] [Table 2]

[0019] [Table 3]

[0020] (2) 92 scallops are randomly selected and genotyped for the above InDel sites by Sanger sequencing. This method includes the following steps: 1) Extract genomic DNA from the target scallop. 2) Using the genomic DNA obtained in step 1) as a template, PCR amplification is carried out using the base sequences shown in SEQ ID NOs: 3 and 4 as primers to obtain PCR amplification products for each individual scallop. 3) Sanger sequencing is performed on the PCR amplification products obtained in step 2) to determine the genotype of each individual scallop to be detected at that site. 4) Statistical analysis of the genotype frequency of the InDel site in normal scallop and "Hitajinbei" populations. 5) By combining the sequencing results with the phenotypic characteristics, if the genotype determination result at the InDel site is 0 / 0 type, the individual is a normal scallop individual, and if the genotype determination result at the InDel site is 1 / 1 type, the individual is a "Haitajinbei" individual.

[0021] (3) Methods for supporting the breeding and germplasm maintenance of "Haitajinbei" 1) Randomly select 50 to 100 scallops to be detected, and sample the entire soft tissue at the small-scale seedling stage, non-destructively sample the gill filaments at the large-scale seedling stage, and similarly non-destructively sample the gill filaments at the adult stage. 2) Sample DNA is extracted using the phenol-chloroform extraction method as a template, and PCR amplification is performed using the base sequences shown in SEQ ID NOs: 3 and 4 as primers to obtain PCR amplification products. Sanger sequencing is performed on the PCR amplification products to determine the genotype of each individual scallop. 3) Scallop individuals whose genotype results are homozygous 0 / 0 and heterozygous 0 / 1 are removed, and scallop individuals whose genotype results are 1 / 1 are retained.

[0022] By identifying and screening scallops using the above method, it is possible to screen for "Haitajinbei" individuals enriched in carotenoids. The parent "Haitajinbei" that was screened is shown in Figure 2. This example demonstrates the effectiveness of utilizing the InDel site obtained by the screening of the present invention to breed scallops with accumulated carotenoids.

[0023] Of course, the above examples are merely examples provided for ease of explanation and are not intended to limit the embodiments. Those skilled in the art can make other different modifications or alterations based on the above description. It is not necessary and impossible to cover all embodiments here. Therefore, any obvious modifications or alterations derived therefrom still fall within the scope of protection of the present invention.

Claims

1. A method for breeding scallops that accumulate carotenoids, comprising: The method uses InDel molecular markers to determine the genotype, The InDel molecular marker is located at base 23111924 of scallop Chr8 chromosome; The base sequence of the InDel molecular marker in normal scallops is shown in SEQ ID NO: 2, The base sequence of the InDel molecular marker in carotenoid-accumulating scallops is shown in SEQ ID NO:

1. The InDel molecular marker is It is located at the 99th position of the base sequence of SEQ ID NO: 2, the base here is T, the genotype is determined as REF type, and is represented by 0, or It is located at positions 99 to 103 of the base sequence of SEQ ID NO: 1, the bases here are TACGT, the genotype is ALT type, and is represented by 1, The method for breeding scallops with accumulated carotenoids comprises: (1) Sampling target scallops, including sampling the entire soft tissue of small-scale seedlings for identification of small-scale seedlings, non-destructively sampling gill filaments of large-scale seedlings for directional breeding, and non-destructively sampling gill filaments of adult scallops for parent selection, after sampling, placing the sample in alcohol for later use; (2) extracting DNA from the individual to be detected, detecting the concentration and purity of the extracted DNA sample, and storing it for later use; (3) performing PCR amplification using DNA of a sample to be detected as a template and the sequences shown in SEQ ID NOs: 3 and 4 as primers to obtain a PCR product; (4) performing Sanger sequencing on the PCR product to determine the genotype of the InDel site in the individual to be detected; (5) determining that if the genotype determination result is expressed as 1 / 1 type, the individual is a carotenoid-enriched scallop individual, and if the genotype determination result is expressed as 0 / 0 type, the individual is a normal scallop individual that is not significantly enriched in carotenoids; (6) A method for breeding scallops with accumulated carotenoids, comprising the steps of selecting and continuously culturing scallop individuals whose genotype results are expressed as 1 / 1 type.

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