SNP (Single Nucleotide Polymorphism) molecular marker related to growth of macrobrachium rosenbergii under low-protein feed intake condition and application of SNP molecular marker

Abstract

The invention discloses an SNP molecular marker related to growth of macrobrachium rosenbergii under the condition of ingestion of low-protein feed and application of the SNP molecular marker, and relates to the field of genetic breeding of macrobrachium rosenbergii. A nucleotide sequence containing the SNP molecular marker is shown as SEQ ID NO.1, the nucleotide sequence is located at the 501st site from the 5'end on the SEQ ID NO.1 and corresponds to the 76447836th site on the 10th chromosome of the macrobrachium rosenbergii, and the polymorphic site of the SNP molecular marker is C or T. The SNP molecular marker is closely related to the body weight and body length of the macrobrachium rosenbergii under the condition of ingestion of the low-protein feed, so that the locus can provide reference for screening and cultivating new varieties of the macrobrachium rosenbergii resistant to the low-protein feed.

Description

Technical Field

[0001] The invention relates to the field of genetic breeding of Macrobrachium rosenbergii, and in particular to SNP molecular markers related to the growth of Macrobrachium rosenbergii under the condition of feeding on low-protein feed and applications thereof. Background Art

[0002] Protein is the main component of aquatic feed and the biggest factor affecting feed cost. However, my country's protein resources are relatively scarce. It is estimated that by 2030, my country's protein feed gap will reach 27.9 million tons ([1] Yang Zhenhai, Zhang Zhiqing. (2001) Research on the Development Strategy of China's Feed Industry in the Early 21st Century [J]. China Feed, 16.). In addition, with the continuous development of aquaculture and the decline of wild fishing resources, fish meal prices have soared, and some plant-based alternative protein sources such as soybean meal are heavily dependent on imports. The development of low-protein feed has gradually become a development trend ([2] Ashraf Y, Shalaby SM, Salama AN, et al. (2023) Effects of dietary β-mannanase (Hemicell®) and Lavandula angustifoliaon Oreochromis niloticus fed a low level of dietary protein: Growth, digestive enzymes, and hemato-biochemical indices. Aquaculture Reports , 30:101604.). Breeding new aquatic varieties (lines) that are tolerant to low protein can help to appropriately reduce the protein content of feed, save breeding costs, reduce nitrogen and phosphorus emissions, and reduce dependence on imported raw materials ([3] Liang H, Wu L, Chama MKH, et al. (2021) Culture salinity modulates Nrf2 antioxidant signaling pathway and immune response of juvenile Genetically Improved Farmed Tilapia (GIFT) ( Oreochromis niloticus ) under different dietary protein levels. Fish Shellfish Immunol, 117:220-227. [4] Miao S, Han B, Li J, et al. (2020) Effects of dietary protein level on the growth performance, feed utilization and immunity of red swamp crayfish Procambarus clarkia . Aquaculture Reports , 18:100540.), which is of great significance to the green and sustainable development of the economy.

[0003] Macrobrachium rosenbergii Macrobrachium rosenbergii ), also known as the "king of freshwater shrimp", is one of the main aquaculture varieties in my country and is deeply loved by consumers. According to the "2024 Fisheries Statistical Yearbook", my country's aquaculture output of Macrobrachium rosenbergii exceeded 190,000 tons in 2023, and the number of farmers and aquaculture output are still increasing. The protein content of Macrobrachium rosenbergii feed formula should generally not be less than 30% ([5] Li Aijie, Zheng Shuhe. (2005) Research on the nutritional requirements of Macrobrachium rosenbergii. Feed Industry, 26(4):3.). Genome-wide association analysis (GWAS) can identify high-density molecular markers in the positioning population and detect molecular markers associated with target traits.

[0004] However, the application of GWAS in the field of molecular breeding of aquatic animals is still in its infancy. Although there have been reports on SNPs related to the growth of Macrobrachium rosenbergii in recent years, there has been no detailed study on SNP loci that are simultaneously associated with the weight and length of Macrobrachium rosenbergii under the condition of long-term low-protein diet. Therefore, identifying and verifying these key SNP loci will provide new possibilities and directions for the breeding of Macrobrachium rosenbergii and the sustainable development of the industry. Summary of the invention

[0005] The purpose of the present invention is to provide a SNP molecular marker related to the growth of Macrobrachium rosenbergii juveniles under the condition of feeding low-protein feed, so as to provide a basis for breeding Macrobrachium rosenbergii varieties (lines) with better tolerance to low-protein feed.

[0006] Through whole genome association analysis, this application screened out SNP sites that were significantly correlated with the weight and body length of Macrobrachium rosenbergii under the condition of long-term intake of low-protein feed (29.79% protein level). The results will help in molecular marker-assisted breeding of low-protein-tolerant Macrobrachium rosenbergii and save protein resources.

[0007] To achieve the above-mentioned purpose, the technical solution of the present invention is as follows: The present invention provides a SNP molecular marker associated with the growth of Macrobrachium rosenbergii under the condition of feeding low-protein feed. The nucleotide sequence containing the SNP molecular marker is shown in SEQ ID NO.1, which is located at the 501st position (black bold font) from the 5' end of SEQ ID NO.1, corresponding to the 76447836th position on chromosome 10 of Macrobrachium rosenbergii, and the polymorphic site of the SNP molecular marker is C or T.

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

[0009] Preferably, when the genotype of the SNP molecular marker on the two chromosomes of Macrobrachium rosenbergii is TT, CT or CC, the body weight and body length of Macrobrachium rosenbergii fed with low-protein feed decrease successively.

[0010] The present invention also provides the use of the SNP molecular marker in screening Macrobrachium rosenbergii varieties or strains tolerant to low-protein feeds.

[0011] Preferably, the low protein content is ≤30%.

[0012] The present invention also provides a method for screening a variety or strain of Macrobrachium rosenbergii that is tolerant to low-protein feed, the method comprising detecting the genotype of the SNP molecular marker in the genome of Macrobrachium rosenbergii and selecting Macrobrachium rosenbergii with a specific genotype for breeding. The specific genotype is TT or CT type.

[0013] The present invention also provides a method for molecular marker-assisted selection breeding based on Macrobrachium rosenbergii tolerance to low-protein feed, the method comprising: a) extracting DNA from Macrobrachium rosenbergii to be bred; b) performing genotyping on the extracted DNA to determine the genotype of the SNP molecular marker; c) Based on the identification results, select Macrobrachium rosenbergii with the characteristics of tolerance to low-protein feed for breeding.

[0014] Preferably, the Macrobrachium rosenbergii with low-protein feed tolerance refers to Macrobrachium rosenbergii with TT or CT type SNP molecular marker genotype.

[0015] Preferably, the low protein content is ≤30%.

[0016] Beneficial effects of the present invention: The SNP molecular marker of the present invention is closely related to the body weight and body length of Macrobrachium rosenbergii under the condition of feeding low-protein feed, so that the site can provide a reference for screening and breeding new varieties of Macrobrachium rosenbergii tolerant to low-protein feed. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 The growth and feed coefficient of Macrobrachium rosenbergii after feeding on low-protein and normal-protein feeds for 8 weeks in Example 1; A is the average final weight of Macrobrachium rosenbergii; B is the survival rate of Macrobrachium rosenbergii; C is the weight gain rate of Macrobrachium rosenbergii; D is the specific growth rate of Macrobrachium rosenbergii; E is the feed coefficient. "*" indicates significant difference ( P <0.05), where “**” indicates extremely significant difference ( P <0.01); Figure 2The weight distribution of 200 Macrobrachium rosenbergii individuals with large growth differences after feeding on low-protein feed in Example 1; wherein A is the weight of 200 Macrobrachium rosenbergii individuals; B is the phenotypic distribution of the weight of 200 Macrobrachium rosenbergii individuals; Figure 3 The distribution of body lengths of 200 Macrobrachium rosenbergii individuals with large growth differences after feeding on low-protein feed in Example 1; wherein A is the body lengths of 200 Macrobrachium rosenbergii individuals; and B is the phenotypic distribution of body lengths of 200 Macrobrachium rosenbergii individuals; Figure 4 The distribution diagram of the SNP detected on each chromosome in Example 2; Figure 5 It is the Manhattan plot of the GWAS of the body weight trait of Macrobrachium rosenbergii in Example 2; there are 2 SNPs above the threshold line, among which 2, 0 and 2 SNP sites are screened out by GLM, MLM and FarmCPU models respectively; the dotted line is the threshold line; Figure 6 This is the Manhattan plot of the GWAS of the body length trait of Macrobrachium rosenbergii in Example 2. There are 5 SNPs above the threshold line, among which 5, 0 and 5 SNP sites were screened out by GLM, MLM and FarmCPU models respectively; the dotted line is the threshold line; Figure 7 This is a violin plot of the body weight of different mutant populations at the 76447836 site on chromosome 10 of Macrobrachium rosenbergii in Example 3; where "*" indicates significant difference ( P <0.05), “ns” means no significant difference ( P >0.05); Figure 8 This is a violin plot of the body length of different mutant populations at the 76447836 site on chromosome 10 of Macrobrachium rosenbergii in Example 3; "*" indicates significant difference ( P <0.05), where “**” indicates extremely significant difference ( P <0.01), “ns” means no significant difference ( P >0.05). DETAILED DESCRIPTION

[0018] Example 1

[0019] Establish a low-protein model and screen individuals with significant growth differences under low-protein feeding conditions.

[0020] 1. Feed preparation With fish meal, casein, gelatin, and soybean meal as the main protein sources (as shown in Table 1), and a mixture of fish oil and soybean oil (mass ratio of 1:1) as the main fat source, two groups of feeds with different protein levels were prepared (the main protein sources were increased in equal proportions). All feed raw materials were fully crushed and passed through a 60-mesh sieve, accurately weighed according to the formula, and the feed raw materials were fully mixed by the step-by-step expansion method. Finally, a twin-screw extruder (model: SYSLG30-Ⅳ) was used to make a 2 mm particle size pellet feed. The prepared feed was air-dried at room temperature in a ventilated place until the moisture content was below 10%, then stored in a sealed bag, and finally stored in a -20° freezer.

[0021] Table 1 Feed formula (%, mass ratio)

[0022] 2. Experimental animals and breeding management The experiment was conducted at Hengyuan Aquatic Shrimp Farm (Wuxing District, Huzhou City, Zhejiang Province). Juvenile Macrobrachium rosenbergii were sourced from local farmers. A total of 640 shrimps (0.228 ± 0.008 g, mean ± SD) were evenly distributed to 8 randomly distributed 0.5 m 3 In a rectangular plastic barrel (length × width × height = 1.35 × 0.83 × 0.625 meters), two treatment groups (4 parallel barrels for each treatment, 80 shrimps in each barrel) were fed with two groups of experimental feeds respectively. The daily feeding amount was 8% of the shrimp body weight, and the sewage was sucked at 8 am every day, and 1 / 2 of the water volume was replaced with clean aerated filtered water every day. During the breeding period, the water quality conditions were monitored every day. The experimental water temperature was 23-27℃, the pH value was 7.4-8.3, the dissolved oxygen was not less than 7 mg / L, and the ammonia nitrogen was less than 0.05 mg / L.

[0023] 3. Phenotyping and tissue sampling After 8 weeks of feeding, the number and total weight of shrimp in each bucket were counted. Figure 1 Compared with the normal protein diet 2, the average final weight of the low protein diet 1 ( Figure 1 A in), the weight gain rate was significantly reduced ( Figure 1 C in P <0.01), the specific growth rate was significantly reduced ( Figure 1 D in P <0.05), the feed coefficient increased significantly ( Figure 1 The E in P <0.01). This indicates that the protein level of feed 1 cannot meet the normal growth needs. In this experiment, a low-protein feeding model was successfully established by feeding low-protein feed 1.

[0024] Further, 100 individuals with good growth and 100 individuals with poor growth were selected from the low-protein diet group, and their weight and length were measured, and the phenotypic data were recorded for subsequent analysis. The average weight of the 100 individuals with good growth was ensured to be higher than the average weight of the Macrobrachium rosenbergii fed 36% protein diet (2.23 g) during the screening process. Figure 2 and Figure 3 The distribution of weight and body length of 200 shrimps. The mean weight of 200 shrimps is 2.02 g, the standard deviation is 0.98, and the coefficient of variation is 48.33%; the mean body length is 4.67 cm, the standard deviation is 0.67, and the coefficient of variation is 14.3%. Among them, the mean weight of 100 shrimps with good growth is 2.79 g, and the mean body length is 5.22 cm; among them, the mean weight of 100 Macrobrachium rosenbergii with poor growth is 1.25 g, and the mean body length is 4.12 cm.

[0025] Example 2: Genotyping and genome-wide association analysis based on 100KcGPS liquid phase chip

[0026] 1. Genotype identification The genotype of Macrobrachium rosenbergii was genotyped using the 100KcGPS (precise positioning sequencing typing technology based on liquid phase capture of target sequences) liquid phase chip. First, the muscle tissue of the above 200 shrimps (preserved in 98% ethanol) was collected for DNA extraction. The sample DNA was extracted by magnetic beads, the concentration of the DNA sample was detected by Qubit fluorescence quantification instrument, and the integrity of the DNA sample was detected by 1% agarose gel electrophoresis. The samples that passed the quality control were used for cGPS library construction and preparation. After the library construction was completed, the library concentration was determined using Qubit fluorescence quantification instrument. After the library quality inspection was qualified, the sequencing chip was loaded and sequenced on the machine.

[0027] The raw sequencing sequences (Raw Reads) obtained by sequencing were filtered to obtain high-quality Clean Reads, which were used for subsequent analysis. The filtered Clean Reads were aligned with the reference genome using the alignment software BWA (Burrows-Wheeler transform), the Clean Reads were located on the reference genome, and the alignment results were statistically analyzed. Based on the alignment results of the Clean Reads reference genome sequence, the genotype of the targeted site was detected using the mutation analysis software GATK (GenomeAnalysis Toolkit) variation detection tool HaplotypeCaller. In order to clearly display the distribution of chip sites on each chromosome, a heat map of the SNP density distribution on the chromosome was drawn; the distribution of the target sites on the genome can be seen in Figure 4. The site detection rate of all samples was between 94.94% and 98.96%, with an average detection rate of 97.69%; the heterozygosity rate was between 21.37% and 33.11%, with an average heterozygosity rate of 29.31%; the genotype consistency rate of repeated samples was between 98.53% and 98.53%, with an average consistency rate of 98.53%. ANNOVAR software was used to perform functional annotations on the detected gene variants, and the regions of the genome where the variant sites occurred (intron regions, intergenic regions, coding regions, 5-terminal UTR regions, 3-terminal UTR regions, etc.) and the effects of the variants (synonymous and non-synonymous mutations, etc.) were obtained.

[0028] Genome-wide association study (GWAS) According to the test results, SNP sites were screened by VCFtools according to the filtering criteria of missing rate ≤ 0.1, MAF ≥ 0.05 and dimorphic SNPs, and 91,657 SNP sites were obtained. GWAS analysis was performed using rMVP combined with the 200 shrimp weight and body length traits measured previously. Manhattan plots were drawn using the R package CMplot, and sites were annotated using SnpEff. Three models were used in the analysis: general linear model (GLM), mixed linear model (MLM), and FarmCPU model.

[0029] The GWAS results based on body weight traits are shown in Figure 5 , three models were used to locate significant loci, of which the GLM model had 2 loci, the MLM model had 0 loci, and the FarmCPU model had 2 loci. The GWAS results based on body length traits are shown in Figure 6 , significant loci were located by three models, of which the GLM model had 5 loci, the MLM model had 0 loci, and the FarmCPU model had 5 loci. As shown in Table 2, through integration, a SNP locus significantly associated with both body weight and body length was finally obtained, located at position 76447836 of chromosome 10, in the intergenic region, and the polymorphic locus was C or T.

[0030] Table 2 SNP loci associated with body weight and length traits of Macrobrachium rosenbergii under low-protein diet identified based on GWAS

[0031] Note: BW is body weight trait, BL is body length trait.

[0032] Example 3: Verification of the effect of SNP chr10:76447836 on the body weight and length of Macrobrachium rosenbergii fed a low-protein diet

[0033] In this example, a SNP molecular marker was identified that was simultaneously associated with the body weight and body length of Macrobrachium rosenbergii under low-protein feeding conditions. The SNP molecular marker was located at the 501st position from the 5' end of SEQ ID NO.1, corresponding to the 76447836th position of chromosome 10 of Macrobrachium rosenbergii. The reference genome of this position was C genotype and the mutant type was T.

[0034] The genotype of the SNP molecular marker site located at SEQ ID NO.1 was correlated with the weight and length traits of Macrobrachium rosenbergii under low-protein diet conditions. The statistical results are as follows: Figure 7 and Figure 8 . Under low-protein feeding conditions, the TT-type Macrobrachium rosenbergii has the largest weight and body length, the CC-type Macrobrachium rosenbergii has the smallest weight and body length, and the CT-type has a weight and body length between the TT-type and CC-type. Moreover, there is a significant difference in weight between the TT-type and CT-type Macrobrachium rosenbergii and the CC-type Macrobrachium rosenbergii; there is a significant difference in body length between the TT-type Macrobrachium rosenbergii and the CC-type Macrobrachium rosenbergii, and there is a very significant difference in body length between the CT-type Macrobrachium rosenbergii and the CC-type Macrobrachium rosenbergii. In summary, the molecular markers provided by the present invention can provide a basis for molecular marker-assisted selection breeding of Macrobrachium rosenbergii for tolerance to low-protein feeds, and have great scientific and commercial value.

Claims

1. A SNP molecular marker associated with the growth of Macrobrachium rosenbergii under low-protein feed conditions, characterized in that: The nucleotide sequence containing the SNP molecular marker is shown in SEQ ID NO.1, located at the 501st position from the 5' end of SEQ ID NO.1, corresponding to the 76447836th position on chromosome 10 of Macrobrachium rosenbergii, and the polymorphic site of the SNP molecular marker is C or T.

2. The SNP molecular marker according to claim 1, characterized in that: When the genotypes of the SNP molecular markers on the two chromosomes of Macrobrachium rosenbergii are TT, CT or CC, the body weight and body length of the Macrobrachium rosenbergii fed with low-protein feed decrease in turn.

3. Use of the SNP molecular marker according to claim 1 or 2 in screening Macrobrachium rosenbergii varieties or strains tolerant to low-protein feeds.

4. The use according to claim 3, characterized in that: The content of the low protein is ≤30%.

5. A method for screening Macrobrachium rosenbergii varieties or strains tolerant to low-protein feeds, characterized in that: The method comprises detecting the genotype of the SNP molecular marker according to claim 1 or 2 in the genome of Macrobrachium rosenbergii, and selecting Macrobrachium rosenbergii with a specific genotype for breeding.

6. The method according to claim 5, characterized in that The specific genotype is TT or CT type.

7. A method for molecular marker-assisted selection breeding based on Macrobrachium rosenbergii tolerance to low-protein feed, characterized in that: The method includes: a) extracting DNA from Macrobrachium rosenbergii to be bred; b) performing genotyping on the extracted DNA to determine the genotype of the SNP molecular marker according to claim 1 or 2; c) Based on the identification results, select Macrobrachium rosenbergii with the characteristics of tolerance to low-protein feed for breeding.

8. The method according to claim 7, characterized in that The Macrobrachium rosenbergii with the characteristic of being resistant to low-protein feed is a Macrobrachium rosenbergii with a TT or CT type SNP molecular marker genotype.

9. The method according to claim 7, characterized in that: The content of the low protein is ≤30%.

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