SNP Molecular Markers Related to the Growth of Macrobrachium rosenbergii Fed with Low-Protein Diet and Their Applications

Abstract

The present invention discloses SNP molecular markers related to the growth of Macrobrachium rosenbergii under the condition of feeding on low-protein feed and their applications, which relates to the field of genetic breeding of Macrobrachium rosenbergii. The nucleotide sequence containing the SNP molecular marker of the present invention is as shown in SEQ ID NO.1, located at the 501st site from the 5' end on SEQ ID NO.1, corresponding to the 76447836th site on chromosome 10 of Macrobrachium rosenbergii, and the polymorphic site of the SNP molecular marker is C or T. 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 on low-protein feed, so that this site can provide a reference for screening and cultivating new varieties of Macrobrachium rosenbergii resistant to low-protein feed.

Description

Technical Field

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

[0002] Protein is the main component of aquatic feed and also the biggest factor affecting feed cost. However, protein resources in China are relatively scarce, and it is estimated that the protein feed gap in China will reach 27.9 million tons in 2030 ([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.). And with the continuous development of aquaculture and the decline of wild fishing resources, the price of fish meal has soared, and some plant-based alternative protein sources such as soybean meal are highly dependent on imports. The development of low-protein feed has gradually become a trend ([2] Ashraf Y, Shalaby SM, Salama AN, et al. (2023) Effects of dietary β-mannanase (Hemicell®) and Lavandula angustifolia on Oreochromis niloticus fed a low level of dietary protein: Growth, digestive enzymes, and hemato-biochemical indices. Aquaculture Reports , 30:101604.). Breeding new varieties (lines) of aquatic products that are tolerant to low protein helps to appropriately reduce the protein content of feed, save breeding costs, reduce nitrogen and phosphorus emissions, and reduce the dependence on imports of 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 economic, green and sustainable development.

[0003] Macrobrachium rosenbergii ( Macrobrachium rosenbergii ), also known as the "king of freshwater shrimps", is one of the main aquaculture varieties in China and is deeply loved by consumers. According to the "Fishery Statistical Yearbook 2024", the aquaculture output of Macrobrachium rosenbergii in China exceeded 190,000 tons in 2023, and currently both the number of farmers and the aquaculture output are still increasing continuously. The protein content in the feed formula of Macrobrachium rosenbergii generally cannot be lower than 30% ([5] Li Aijie, Zheng Shuhe. (2005) Study on the nutritional requirements of Macrobrachium rosenbergii. Feed Industry, 26(4):3.). Genome-wide association study (GWAS) can identify and locate high-density molecular markers in a population and detect molecular markers associated with target traits.

[0004] However, in the field of molecular breeding of aquatic animals, the application of GWAS is still in its infancy. Although there have been research reports on SNPs related to the growth of Macrobrachium rosenbergii in recent years, there is no detailed study on SNP loci simultaneously related to the body weight and body length of Macrobrachium rosenbergii under the condition of long-term feeding with low-protein feed. Therefore, identifying and validating 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 an SNP molecular marker related to the growth of juvenile Macrobrachium rosenbergii under the condition of feeding with low-protein feed, so as to provide a basis for breeding Macrobrachium rosenbergii varieties (lines) with better tolerance to low-protein feed.

[0006] Through genome-wide association analysis, the present application screened SNP loci that were simultaneously significantly related to the body weight and body length of Macrobrachium rosenbergii under the condition of long-term feeding with low-protein feed (protein level of 29.79%). The results are helpful for the molecular marker-assisted breeding of low-protein-tolerant Macrobrachium rosenbergii and the conservation of protein resources.

[0007] To achieve the above purpose, the technical solution of the present invention is as follows:

[0008] The present invention provides an SNP molecular marker related to the growth of Macrobrachium rosenbergii under the condition of feeding on low-protein feed. The nucleotide sequence containing the SNP molecular marker is as shown in SEQ ID NO.1, located at the 501st site (in bold and black font) from the 5'-end on SEQ ID NO.1, corresponding to the 76447836th site on chromosome 10 of Macrobrachium rosenbergii. The polymorphic site of the SNP molecular marker is C or T.

[0009] The sequence shown in SEQ ID NO.1 is as follows:

[0010]

[0011] Preferably, when the genotypes of the SNP molecular markers on two chromosomes of Macrobrachium rosenbergii are TT, CT or CC, the body weight and body length of Macrobrachium rosenbergii fed with low-protein feed decrease in turn.

[0012] The present invention also provides the application of the SNP molecular marker in screening Macrobrachium rosenbergii varieties or strains resistant to low-protein feed.

[0013] Preferably, the content of the low protein ≤ 30%.

[0014] The present invention also provides a method for screening Macrobrachium rosenbergii varieties or strains resistant 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.

[0015] The present invention also provides a method for molecular marker-assisted selection breeding based on Macrobrachium rosenbergii resistant to low-protein feed, the method comprising:

[0016] a) Extracting the DNA of the Macrobrachium rosenbergii to be bred;

[0017] b) Conducting genotype identification on the extracted DNA to determine the genotype of the SNP molecular marker;

[0018] c) Selecting Macrobrachium rosenbergii with the characteristics of being resistant to low-protein feed for breeding according to the identification result.

[0019] Preferably, the Macrobrachium rosenbergii with the characteristics of being resistant to low-protein feed refers to Macrobrachium rosenbergii with the SNP molecular marker genotype of TT or CT type.

[0020] Preferably, the content of the low protein ≤ 30%.

[0021] Beneficial effects of the present invention:

[0022] 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 with low-protein feed, so that this locus can provide a reference for screening and cultivating new varieties of Macrobrachium rosenbergii resistant to low-protein feed. Description of the drawings

[0023] Figure 1 For the growth and feed coefficient of Macrobrachium rosenbergii after 8 weeks of feeding with low-protein and normal-protein feed in Example 1; wherein, 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 a significant difference ( P < 0.05), wherein "**" indicates a highly significant difference ( P<0.01);

[0024] Figure 2 It is the weight distribution of 200 Macrobrachium rosenbergii individuals with large growth differences when feeding on low - protein feed in Example 1; among them, A is the weight of 200 Macrobrachium rosenbergii individuals; B is the phenotypic distribution of the weights of 200 Macrobrachium rosenbergii individuals.

[0025] Figure 3 It is the body - length distribution of 200 Macrobrachium rosenbergii individuals with large growth differences when feeding on low - protein feed in Example 1; among them, A is the body length of 200 Macrobrachium rosenbergii individuals; B is the phenotypic distribution of the body lengths of 200 Macrobrachium rosenbergii individuals.

[0026] Figure 4 It is the distribution map of SNPs on each chromosome in Example 2.

[0027] Figure 5 It is the GWAS Manhattan plot of the body - weight trait of Macrobrachium rosenbergii in Example 2; there are a total of 2 SNPs above the threshold line. Among them, 2, 0, and 2 SNP loci are screened out using the GLM, MLM, and FarmCPU models respectively; the dotted line among them is the threshold line.

[0028] Figure 6 It is the GWAS Manhattan plot of the body - length trait of Macrobrachium rosenbergii in Example 2. There are a total of 5 SNPs above the threshold line. Among them, 5, 0, and 5 SNP loci are screened out using the GLM, MLM, and FarmCPU models respectively; the dotted line among them is the threshold line.

[0029] Figure 7 It is the violin plot of the weights of different mutant populations at the 76447836 locus on chromosome 10 of Macrobrachium rosenbergii in Example 3; among them, "*" indicates significant difference ( P <0.05), "ns" indicates no significant difference ( P >0.05);

[0030] Figure 8 It is the violin plot of the body lengths of different mutant populations at the 76447836 locus on chromosome 10 of Macrobrachium rosenbergii in Example 3; "*" indicates significant difference ( P <0.05), among which "**" indicates extremely significant difference ( P <0.01), "ns" indicates no significant difference ( P >0.05). Specific implementation manners

[0031] Example 1

[0032] Establish a low - protein model and screen individuals with significant growth differences under low - protein feeding conditions.

[0033] 1. Feed preparation

[0034] Two diets with different protein levels were prepared, using fish meal, casein, gelatin, and soybean meal as the primary protein sources (Table 1) and a mixture of fish oil and soybean oil (1:1 by mass) as the primary fat source. All feed ingredients were thoroughly ground and passed through a 60-mesh sieve. They were accurately weighed according to the recipe and thoroughly mixed using a step-by-step method. Finally, the feed was extruded using a twin-screw extruder (model: SYSLG30-IV) to produce 2 mm pellets. The prepared feeds were air-dried in a well-ventilated area at room temperature until the moisture content was below 10%. The feeds were then stored in resealable bags and finally in a -20°C freezer.

[0035] Table 1 Feed formula (%, mass ratio)

[0036]

[0037] 2. Experimental animals and breeding management

[0038] The experiment was conducted at Hengyuan Aquatic Shrimp Farm (Wuxing District, Huzhou City, Zhejiang Province). Juvenile Macrobrachium rosenbergii were obtained from local farmers. 640 shrimp (0.228 ± 0.008 g, mean ± standard deviation) were evenly distributed to 8 randomly distributed 0.5 m 3 Two experimental feeds were fed to shrimp in rectangular plastic barrels (length × width × height = 1.35 × 0.83 × 0.625 m) in each treatment group (four replicate barrels per treatment, 80 shrimp per barrel). The daily feed intake was 8% of shrimp body weight. Sewage was aspirated daily at 8:00 AM, and half of the water volume was replaced daily with clean, aerated, filtered water. Water quality was monitored daily during the culture period. The experimental water temperature was 23-27°C, the pH was 7.4-8.3, the dissolved oxygen level was no less than 7 mg / L, and the ammonia nitrogen level was less than 0.05 mg / L.

[0039] 3. Phenotyping and Tissue Sampling

[0040] 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 was 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.

[0041] Further, 100 individuals of Macrobrachium rosenbergii with better growth performance and 100 individuals with poorer growth performance were selected from the group fed with low-protein feed. Their body weights and body lengths were measured, and the phenotypic data were recorded for subsequent analysis. During the screening process, the average weight of the 100 individuals with better growth performance was ensured to be higher than the average weight of Macrobrachium rosenbergii fed with 36% protein feed (2.23 g). Figure 2 and Figure 3 are the distribution of the body weights and body lengths of 200 shrimps, respectively. The average body weight of 200 shrimp individuals was 2.02 g, the standard deviation was 0.98, and the coefficient of variation was 48.33%; the average body length was 4.67 cm, the standard deviation was 0.67, and the coefficient of variation was 14.3%. Among them, the average body weight of 100 shrimps with better growth performance was 2.79 g, and the average body length was 5.22 cm; among the 100 individuals of Macrobrachium rosenbergii with poorer growth performance, the average body weight was 1.25 g, and the average body length was 4.12 cm.

[0042] Example 2: Genotype identification and genome-wide association analysis based on 100KcGPS liquid-phase chip

[0043] 1. Genotype identification

[0044] The 100KcGPS (Precision Positioning Sequencing Typing Technology Based on Target Sequence Liquid Capture) liquid-phase chip of Macrobrachium rosenbergii was used for genotype typing. First, the muscle tissues of the above 200 shrimps (preserved in 98% ethanol) were collected for DNA extraction. The magnetic bead method was used for sample DNA extraction. The concentration of the DNA sample was detected by a Qubit fluorescence quantifier, and the integrity of the DNA sample was detected by 1% agarose gel electrophoresis. Samples with qualified quality control were used for the construction and preparation of the cGPS library. After the library construction was completed, the library concentration was measured by a Qubit fluorescence quantifier. After the library quality inspection was qualified, the sequencing chip was loaded and sequenced on the machine.

[0045] The original sequencing sequences (Raw Reads) obtained by sequencing were filtered to obtain high-quality Clean Reads, and the Clean Reads were used for subsequent analysis. The filtered Clean Reads were aligned with the reference genome using the alignment software BWA (Burrows-Wheeler transform) to locate the positions of the Clean Reads on the reference genome and count the alignment results. Based on the alignment results of the Clean Reads and the reference genome sequence, the genotype of the target site was detected using the variant detection tool HaplotypeCaller of the mutation analysis software GATK (GenomeAnalysis Toolkit). To clearly show the distribution of chip sites on each chromosome, a SNP density distribution heat map on the chromosome was drawn; the distribution of the target sites on the genome is shown in Figure 4For all samples in this study, the locus detection rate ranged from 94.94% to 98.96%, with an average detection rate of 97.69%; the heterozygosity rate ranged from 21.37% to 33.11%, with an average heterozygosity rate of 29.31%; the genotype consistency rate of repeated samples ranged from 98.53% to 98.53%, with an average consistency rate of 98.53%. Using the ANNOVAR software, functional annotation was performed on the detected gene mutations to obtain the regions in the genome where the mutation sites occurred (intron region, intergenic region, coding region, 5'UTR region, 3'UTR region, etc.), as well as the effects caused by the mutations (synonymous, non-synonymous mutations, etc.).

[0046] Genome-wide association study (GWAS)

[0047] According to the detection results, 91,657 SNP loci were screened by VCFtools for SNP loci according to the filtering criteria of missing rate ≤ 0.1, MAF ≥ 0.05, and bi-allelic SNPs. The rMVP was used to perform GWAS analysis in combination with the previously measured body weight and body length traits of 200 shrimps, and the Manhattan plot was drawn using the R package CMplot, and locus annotation was performed by SnpEff. Three models were used in the analysis: general linear model (GLM), mixed linear model (MLM), and FarmCPU model.

[0048] The GWAS results based on body weight traits are shown in Figure 5 , and significant loci were mapped by three models. Among them, there were 2 loci in the GLM model, 0 loci in the MLM model, and 2 loci in the FarmCPU model. The GWAS results based on body length traits are shown in Figure 6 , and significant loci were mapped by three models. Among them, there were 5 loci in the GLM model, 0 loci in the MLM model, and 5 loci in the FarmCPU model. As shown in Table 2, by integration, a total of 1 SNP locus significantly related to both body weight and body length was obtained, located at position 76,447,836 on chromosome 10, in the intergenic region, and the polymorphic locus was C or T.

[0049] Table 2 SNP loci related to the body weight and body length traits of Macrobrachium rosenbergii fed with low-protein diet identified by GWAS

[0050]

[0051] Note: BW represents body weight trait, and BL represents body length trait.

[0052] Example 3: Verification of the effects of SNP chr10:76447836 on the body weight and body length of Macrobrachium rosenbergii fed with low-protein diet

[0053] One SNP molecular marker identified in this example is simultaneously related to the body weight and body length of Macrobrachium rosenbergii under the condition of feeding on a low-protein diet. The SNP molecular marker is located at the 501st site from the 5' end on SEQ ID NO.1, corresponding to the 76447836th site on chromosome 10 of Macrobrachium rosenbergii. The reference genome of this site is genotype C, and the mutant type is T.

[0054] The genotype of the SNP molecular marker locus located on SEQ ID NO.1 was analyzed for its correlation with the body weight and body length traits of Macrobrachium rosenbergii under the condition of feeding on a low-protein diet. The statistical results are as Figure 7 and Figure 8 . Under the condition of feeding on a low-protein diet, the Macrobrachium rosenbergii with the largest body weight and body length is of the TT type, the Macrobrachium rosenbergii with the smallest body weight and body length is of the CC type, and the body weight and body length of the CT type are between those of the TT type and the CC type. Moreover, there are significant differences in body weight between the TT and CT types of Macrobrachium rosenbergii and the CC type; there are significant differences in body length between the TT type and the CC type of Macrobrachium rosenbergii, and there are extremely significant differences in body length between the CT type and the CC type of Macrobrachium rosenbergii. In summary, the molecular marker provided by the present invention can provide a basis for molecular marker-assisted selection breeding of Macrobrachium rosenbergii resistant to low-protein feed, and has great scientific and commercial value.

Claims

1. Application of SNP molecular markers related to the growth of Macrobrachium rosenbergii under the condition of feeding on low-protein feed in screening varieties or strains of Macrobrachium rosenbergii resistant to low-protein feed; The SNP molecular marker is located at the 501st site from the 5' end of SEQ ID NO.1, corresponding to the 76447836th site on chromosome 10 of Macrobrachium rosenbergii, and the polymorphism of the SNP molecular marker is C or T; The protein content of the low-protein feed ≤ 30%.

2. The application according to claim 1, wherein When the genotype of the SNP molecular marker on the chromosome of Macrobrachium rosenbergii is TT, CT or CC, the body weight and body length of Macrobrachium rosenbergii fed with low-protein feed decrease in turn.

3. A method for screening Macrobrachium rosenbergii varieties or strains resistant to low-protein feed, characterized in that, The method includes detecting the genotype of the SNP molecular marker related to the growth of Macrobrachium rosenbergii under the condition of feeding on low-protein feed in the genome of Macrobrachium rosenbergii, and selecting Macrobrachium rosenbergii with a specific genotype for breeding; The SNP molecular marker is located at the 501st site from the 5' end of SEQ ID NO.1, corresponding to the 76447836th site on chromosome 10 of Macrobrachium rosenbergii, and the polymorphism of the SNP molecular marker is C or T; When the genotype of the SNP molecular marker on the chromosome of Macrobrachium rosenbergii is TT, CT or CC, the body weight and body length of Macrobrachium rosenbergii fed with low-protein feed decrease in turn; The protein content of the low-protein feed ≤ 30%; The specific genotype is TT or CT type.

4. A method for molecular marker-assisted selection breeding of Macrobrachium rosenbergii resistant to low-protein feed, characterized in that, The method includes: a) Extracting the DNA of Macrobrachium rosenbergii to be bred; b) Conducting genotype identification on the extracted DNA to determine the genotype of the SNP molecular marker related to the growth of Macrobrachium rosenbergii under the condition of feeding on low-protein feed; The SNP molecular marker is located at the 501st site from the 5' end of SEQ ID NO.1, corresponding to the 76447836th site on chromosome 10 of Macrobrachium rosenbergii, and the polymorphism of the SNP molecular marker is C or T; When the genotype of the SNP molecular marker on the chromosome of Macrobrachium rosenbergii is TT, CT or CC, the body weight and body length of Macrobrachium rosenbergii fed with low-protein feed decrease in turn; c) According to the identification result, selecting Macrobrachium rosenbergii with the characteristic of being resistant to low-protein feed for breeding; The Macrobrachium rosenbergii with the characteristic of being resistant to low-protein feed is the Macrobrachium rosenbergii with the TT or CT type SNP molecular marker genotype; The protein content of the low-protein feed ≤ 30%.

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