A molecular marker assisted selection method for low temperature tolerance trait of freshwater crayfish

Abstract

The application relates to the field of aquaculture breeding technology, and discloses a molecular marker assisted breeding method for low-temperature resistance of freshwater crayfish. The method takes a candidate molecular marker site combination as a breeding basis, carries out low-temperature stress treatment on a to-be-screened population, collects tissue samples of survival individuals, carries out SNP site genotyping detection after extracting genomic DNA, calculates the significant difference of the allele frequency of each SNP site between a low-temperature survival population and a control population, screens out a significant difference correlation marker, takes an individual carrying an advantage allele of the correlation marker as a parent to carry out in-family mating to obtain a progeny population, repeatedly carries out low-temperature stress treatment and genotyping detection on the progeny population, and the low-temperature stress resistance population is determined when the survival rate of the progeny population under a specified low-temperature condition reaches a preset threshold. The application organically combines low-temperature stress phenotype screening and molecular marker assisted breeding, and can efficiently and stably breed a freshwater crayfish population with high low-temperature stress resistance.

Description

Technical Field

[0001] This invention relates to the field of aquaculture breeding technology, specifically to a molecular marker-assisted breeding method for the low-temperature tolerance trait of freshwater crayfish. Background Technology

[0002] Freshwater crayfish (Procambarus clarkii) is one of the important freshwater aquaculture species in my country, with an annual output exceeding one million tons, and its farming area covers multiple provinces in the middle and lower reaches of the Yangtze River. In recent years, with the continuous expansion of farming scale and the frequent occurrence of abnormal climate changes, the losses caused by low temperatures and cold waves in winter to the freshwater crayfish farming industry have become increasingly serious. When the water temperature remains below 4℃, the feeding activity of freshwater crayfish significantly weakens, their immune function declines, and the mortality rate rises sharply. In some farming years, the mortality rate due to cold waves can reach 30% to 50%, causing huge economic losses to farmers. Therefore, breeding new varieties or strains of freshwater crayfish with stable low-temperature resistance has become an urgent problem to be solved by the industry.

[0003] Traditional breeding methods for cold tolerance in freshwater crayfish mainly rely on population selection and family selection. This involves statistically analyzing the survival rates of different families or populations under low-temperature stress to select individuals with strong cold tolerance as parents for the next generation. While these methods are relatively simple, they have significant limitations: First, cold tolerance is a quantitative trait, regulated by multiple gene loci, resulting in moderate to low heritability. Relying solely on phenotypic information limits the accuracy of selection. Second, traditional methods require multiple generations of low-temperature stress testing and phenotypic screening, leading to long breeding cycles with generation intervals typically exceeding one year, significantly limiting the efficiency of breed improvement. Furthermore, subjecting each generation to low-temperature stress not only consumes substantial human, material, and time resources but also limits the breeding scale of the selected population, making it difficult to obtain a genetically superior population in a short period.

[0004] Marker-assisted selection (MAS) offers a novel technological approach for the genetic improvement of cold tolerance traits. In recent years, with the development of high-throughput sequencing technology, the genomic resources and transcriptome data of freshwater crayfish have become increasingly abundant. Numerous studies have reported candidate genes associated with low-temperature stress responses in crustaceans, such as the heat shock protein gene family (Hsp70, Hsp90), antifreeze protein genes, and genes related to the ubiquitin-proteasome pathway. The differential expression of these genes under low-temperature stress has been confirmed to be closely related to an individual's low-temperature tolerance. However, there is currently no practical operational procedure for applying the SNP marker system within these candidate genes to the low-temperature tolerance breeding of freshwater crayfish. Existing studies mostly remain at the preliminary screening stage based on changes in candidate gene expression levels or SNP site association analysis, lacking a complete and industrially viable molecular marker-assisted selection technology scheme, particularly lacking a comprehensive system design encompassing marker screening, low-temperature stress treatment, genotyping detection, and multi-generational breeding validation.

[0005] Existing marker-assisted breeding methods have been applied in aquatic animals, but most focus on disease resistance or growth traits. Breeding for cold tolerance presents unique challenges: cold stress treatment itself is damaging, making it impossible to simultaneously measure cold tolerance phenotypes in the same population and use them as parents for breeding. This necessitates carefully designed control populations and seed retention strategies in breeding programs. Furthermore, the genetic mechanisms of cold tolerance are complex, and the effect of a single SNP marker is relatively small. Screening for relevant markers with breeding value from numerous candidate markers and determining the cumulative effect of dominant alleles are also pressing technical challenges. In addition, current technologies lack quantitative assessment schemes for the generational improvement of cold tolerance in offspring during the breeding process, making it difficult to determine the breeding endpoint and strain stability point, resulting in insufficient operability and reproducibility of the breeding process. Summary of the Invention

[0006] The purpose of this invention is to provide a molecular marker-assisted breeding method for the low-temperature tolerance trait of freshwater crayfish, so as to solve the problems mentioned in the background art.

[0007] To achieve the above objectives, the present invention provides a molecular marker-assisted breeding method for the low-temperature tolerance trait of freshwater crayfish, the method comprising:

[0008] Based on the combination of candidate molecular marker sites, the freshwater crayfish population to be screened was subjected to low temperature stress treatment and tissue samples were collected from surviving individuals.

[0009] Genomic DNA was extracted from the tissue sample, and genotyping was performed on each SNP site in the candidate molecular marker site combination.

[0010] Based on the typing results, the allele frequency of each SNP locus was calculated to show a significant difference between the hypothermic survival population and the control population, and significant association markers were screened out.

[0011] Individuals carrying the dominant allele of the associated marker are used as parents for in-family mating to obtain offspring.

[0012] The low-temperature stress treatment and genotyping detection are repeated on the offspring population until the survival rate of the offspring population under the specified low-temperature conditions reaches a preset threshold. The corresponding offspring population is then identified as a low-temperature resistant breeding population.

[0013] Furthermore, the low-temperature stress treatment is implemented according to the following steps:

[0014] The freshwater crayfish population to be screened was placed in a temperature-controlled water tank and cooled from the rearing water temperature to the first stress temperature of 4℃ at a cooling rate of 1.0℃ / h.

[0015] The first maintenance time is maintained at the first stress temperature for a first maintenance time of 6 hours;

[0016] Then, the temperature is reduced from the first stress temperature to the second stress temperature at a cooling rate of 0.5℃ / h, where the second stress temperature is 1℃.

[0017] The second stress temperature is maintained for a second duration of 12 hours;

[0018] The water temperature was restored to the rearing water temperature at a heating rate of 1.0℃ / h. After restoration, the individuals were reared for another 24 hours. The number of surviving individuals was counted and the tail fan muscle tissue of the surviving individuals was collected as the tissue sample.

[0019] Furthermore, the control group is obtained as follows:

[0020] An equal number of individuals were randomly selected from the same batch of freshwater crayfish population to be screened and placed in an independent control tank. The temperature control program of the control tank was to maintain the rearing water temperature at a constant temperature. The total rearing time was the total duration of the low temperature stress treatment plus 24 hours after recovery.

[0021] At the end of the rearing period in the control tank, tail fan muscle tissue of the control individuals was collected as a control tissue sample.

[0022] Furthermore, the combination of candidate molecular marker sites is constructed as follows:

[0023] Candidate gene sequences related to cold stress response were selected from the freshwater crayfish transcriptome database. These candidate genes include heat shock protein 70 gene, antifreeze protein gene, and ubiquitin ligase gene.

[0024] Using the candidate gene sequence as a reference sequence, all SNP sites located within the coding region of the candidate gene and within 2kb upstream and downstream of it were extracted from the whole genome resequencing data of freshwater crayfish.

[0025] SNP sites with a minor allele frequency of not less than 0.05 and a genotyping detection rate of not less than 90% are selected from all the SNP sites to form the candidate molecular marker site combination.

[0026] Furthermore, the genotyping detection is performed using a competitive allele-specific PCR platform, the reaction system of which contains two allele-specific forward primers and one universal reverse primer designed for each SNP site.

[0027] The 3' ends of the two allele-specific forward primers are complementary to the two allele types of the SNP site, respectively, and the 5' ends of the two forward primers are connected to different fluorescent reporter groups.

[0028] The annealing temperature of the reaction system was set to 60°C, and the number of amplification cycles was 40.

[0029] After amplification, the fluorescence signal value of each reaction well is read, and the genotype of the tissue sample at the corresponding SNP site is determined based on the ratio of the fluorescence signal values.

[0030] Furthermore, the screening of significantly different association markers is achieved in the following manner:

[0031] The allele frequency P1 of each SNP locus in the low-temperature survival population and the allele frequency P0 of the corresponding SNP locus in the control population were statistically analyzed.

[0032] Calculate the difference ΔP between P1 and P0, and calculate the chi-square test P-value of the difference;

[0033] SNP sites with a chi-square test p-value less than 0.01 and an absolute value of ΔP greater than 0.20 are labeled as the association markers;

[0034] The dominant allele is defined as the allele with a significantly higher frequency in the low-temperature survival population than in the control population.

[0035] Furthermore, the intrafamilial mating is conducted in the following manner:

[0036] Female and male individuals carrying at least two of the associated markers and the dominant alleles were selected, with each associated marker being homozygous carriers in the selected individuals.

[0037] The selected female individuals and male individuals were paired in a 1:1 ratio and placed in a breeding pool. The water temperature of the breeding pool was controlled at 24°C, and the light cycle was set to 12 hours of light and 12 hours of darkness.

[0038] Once the female individuals are carrying eggs, they are transferred to an independent incubator at a water temperature of 22°C. The offspring are collected when they hatch and grow to a length of 1.5 cm.

[0039] Furthermore, in the repeated low-temperature stress treatment of the offspring population, the first maintenance duration and the second maintenance duration are respectively increased by a first increment value and a second increment value compared to the previous generation;

[0040] The first increment is 0.5 hours per generation, and the second increment is 1.0 hour per generation.

[0041] The specified low temperature condition is the second stress temperature, and the preset threshold is an 80% survival rate;

[0042] When the survival rate of two consecutive generations of offspring populations reaches the preset threshold under the specified low temperature conditions, the repeated processing is stopped.

[0043] Furthermore, after identifying the low-temperature resistant breeding population, the genotypes of all the associated markers are retested for each individual in the low-temperature resistant breeding population. Individuals carrying the dominant alleles of all the associated markers and having a homozygous genotype are selected from the retest results as the core breeding population.

[0044] Female and male individuals are randomly selected from the core breeding population and a breeding pond is established with a female-to-male ratio of 2:1. The number of female individuals in the breeding pond shall not be less than 50.

[0045] The juvenile shrimp obtained from the propagation pond are subjected to low-temperature stress treatment, and the surviving individuals are used as the base population for the low-temperature resistant strain.

[0046] Furthermore, the base population of the low-temperature resistant strain is placed in a recirculating aquaculture system for generational maintenance. The water temperature of the recirculating aquaculture system is set to the feeding water temperature. In each generation cycle, when the individuals in the base population reach sexual maturity, male and female individuals carrying all the dominant alleles of the associated markers and having homozygous genotypes are selected. The next generation breeding pond is established at a female-to-male ratio of 2:1, and the number of female individuals in the next generation breeding pond is not less than 50.

[0047] In each generation cycle, tail fan muscle tissue of newly hatched juvenile shrimp was collected for genotyping of the associated markers. Individuals whose genotyping results did not meet the requirements of homozygous dominant alleles were removed.

[0048] When the elimination rate is less than 5% in three consecutive subgeneration cycles, the population obtained in that subgeneration cycle is identified as a genetically stable cold-resistant freshwater crayfish strain.

[0049] Compared with the prior art, the beneficial effects of the present invention are:

[0050] This invention uses combinations of candidate molecular marker loci as the basis for breeding. After low-temperature stress treatment, tissue samples from surviving individuals are collected for genotyping. By testing the significance of allele frequencies between the low-temperature surviving population and the control population, markers truly associated with the low-temperature tolerance phenotype can be objectively screened from a large number of candidate SNP loci, avoiding the blindness of subjective marker selection based solely on the understanding of candidate gene functions. This technical design fully utilizes the natural selection pressure of low-temperature stress, organically combining phenotypic selection pressure with molecular marker information, giving the screened associated markers a clear phenotypic verification basis. Compared to markers obtained solely through transcriptome differential expression analysis or case-control association analysis, the markers of this invention have higher breeding prediction reliability. Simultaneously, by selecting individuals carrying the dominant alleles of the associated markers as parents for in-family mating, directed genetic accumulation at the molecular level is achieved, increasing the frequency of the dominant genotype in the offspring population generation by generation. Compared to traditional phenotypic selection, this breeding logic can aggregate multiple favorable alleles of low-temperature tolerance-related loci in a shorter number of generations, significantly accelerating the accumulation rate of genetic progress.

[0051] The low-temperature stress treatment program designed in this invention simulates the real process of water temperature gradually decreasing and then slowly rising during natural cold waves by using a two-stage cooling method combined with an intermediate maintenance period. The first stage cools the water to 4°C and maintains it for 6 hours, eliminating individuals with insufficient low-temperature stress response. The second stage continues to cool the water to 1°C and maintains it for 12 hours, further screening out individuals with true low-temperature tolerance. Compared to directly and rapidly lowering the water temperature to the target temperature, this gradual stress application method avoids non-specific stress mortality caused by excessively rapid cooling, allowing the selected surviving individuals to better reflect their inherent genetic potential for low-temperature tolerance rather than accidental physiological tolerance differences. Simultaneously, the stress program includes a constant-temperature rearing regimen for the control group, providing a homologous baseline for comparing allele frequency differences and effectively eliminating interference from initial genetic background differences in the population on frequency calculations.

[0052] This invention constructs a combination of candidate molecular marker sites by using the heat shock protein 70 gene, antifreeze protein gene, and ubiquitin ligase gene as candidate gene sources, covering multiple key biological pathways in the response to low-temperature stress, including protein folding protection, cell antifreeze protection, and degradation and clearance of damaged proteins. This multi-gene combined SNP screening strategy, compared to focusing on only a single candidate gene, can more comprehensively capture genetic variation information of the cold tolerance trait. Furthermore, by using a dual filtering standard of minor allele frequency not less than 0.05 and genotyping detection rate not less than 90%, SNP sites with almost no polymorphism in the population or poor genotyping stability are removed. This ensures the effectiveness of subsequent association analysis and the reproducibility of genotyping results, avoiding false positives and technical noise introduced by rare variants or low-quality markers.

[0053] This invention employs a competitive allele-specific PCR platform for genotyping. Two allele-specific forward primers and one universal reverse primer are designed for each SNP locus. The two forward primers are linked to different fluorescent reporter groups. Through amplification reactions at 60°C and 40 cycles, parallel detection of two alleles can be completed in a single tube reaction. Compared to traditional sequencing genotyping or TaqMan probe methods, this genotyping platform offers advantages such as high throughput, low cost, and ease of operation, making it suitable for the marker detection needs of large-scale samples in breeding practice, significantly reducing the cost and time consumption of genotyping in the breeding process. In the screening of significantly associated markers, this invention uses a dual standard: a chi-square test p-value less than 0.01 and an absolute value of the allele frequency difference greater than 0.20. This statistically controls the risk of false positive associations and ensures, from a genetic effect perspective, that the screened associated markers have sufficiently large allele frequency differences, making these markers practically applicable in subsequent breeding. The clear definition of dominant alleles provides a clear genotype selection criterion for parental mating, avoiding the uncertainty caused by subjective judgment. Furthermore, requiring each selected individual to be homozygous carriers on at least two associated markers in parental mating helps to stably inherit multiple cold-resistant dominant alleles in offspring, reducing the probability of losing dominant genotypes due to segregation and recombination. Attached Figure Description

[0054] Figure 1 This diagram illustrates the working steps of a molecular marker-assisted breeding method for the low-temperature tolerance trait of freshwater crayfish, as described in this invention. Detailed Implementation

[0055] The present invention will be further described in detail below with reference to specific embodiments and comparative examples. The scope of protection of the present invention is not limited to the specific embodiments. Those skilled in the art can make conventional adjustments to the technical parameters without departing from the core technical concept of the present invention, and all such adjustments will fall within the scope of protection of the present invention.

[0056] The experimental materials used in the embodiments and comparative examples of this invention were juvenile red swamp crayfish from the same batch, with an initial body length of 1.5 cm and an initial body weight of 0.8–1.0 g. The initial rearing water temperature was 26 °C, and the rearing water was aerated tap water with a dissolved oxygen content ≥6.0 mg / L and a pH value of 7.5–8.0. The light cycle of the rearing environment was 12 hours of light followed by 12 hours of darkness. The temperature-controlled water tanks, breeding ponds, hatcheries, recirculating aquaculture systems, and competitive allele-specific PCR detection platforms used in the experiments were all of standardized specifications. All experimental operations were completed in a sterile, standardized aquaculture laboratory environment.

[0057] This invention pre-constructs a combination of candidate molecular marker sites. The specific construction process is as follows: heat shock protein 70, antifreeze protein, and ubiquitin ligase genes from the freshwater crayfish transcriptome database are selected as candidate genes for cold stress response; using the sequences of the three candidate genes as reference sequences, all SNP sites within the coding region and the upstream and downstream 2kb range of the candidate genes are extracted from the whole genome resequencing data of freshwater crayfish; SNP sites with a minor allele frequency ≥0.05 and a genotyping detection rate ≥90% are screened, and finally 6 effective candidate SNP sites are obtained, which are labeled as SNP1, SNP2, SNP3, SNP4, SNP5, and SNP6, respectively, to form a combination of candidate molecular marker sites. Genotyping was performed using a competitive allele-specific PCR platform. Two allele-specific forward primers and one universal reverse primer were configured for each SNP locus. The 3' end of the forward primer was complementary to the corresponding SNP allele, and the 5' end was linked to different fluorescent reporter groups. The PCR amplification annealing temperature was 60℃, and the amplification cycle was 40. Genotype was determined by the fluorescence signal ratio.

[0058] Example 1 See appendix Figure 1 This embodiment provides a molecular marker-assisted breeding method for the low-temperature tolerance trait of freshwater crayfish, and the specific steps are as follows:

[0059] (1) Low-temperature stress treatment and sample collection: 200 healthy, undamaged, and uniformly growing freshwater crayfish juveniles were selected and placed in a temperature-controlled tank measuring 100cm×80cm×60cm for low-temperature stress treatment. The water temperature was lowered from 26℃ to 4℃ at a rate of 1.0℃ / h for 6 hours as the first stress temperature; then it was lowered to 1℃ at a rate of 0.5℃ / h for 12 hours as the second stress temperature; subsequently, the water temperature was restored to 26℃ at a rate of 1.0℃ / h, and the crayfish were kept in normal conditions for 24 hours after restoration. The number of surviving individuals was counted, and tail fan muscle tissue was randomly collected from 100 surviving individuals and stored in an ultra-low temperature freezer at -80℃ as tissue samples of the low-temperature surviving population.

[0060] (2) Control group sample collection: 200 individuals were randomly selected from the same batch of initial juvenile shrimp and placed in an independent control tank. The control tank was kept at a constant temperature of 26℃ throughout the rearing process. The total rearing time was the same as the total time of the low temperature stress treatment and the total time of the 24-hour recovery period. After the rearing period, the tail fan muscle tissue of 100 control individuals was randomly collected, frozen and stored at -80℃, and used as the control group tissue sample.

[0061] (3) Genomic DNA extraction and genotyping: Tissue samples were taken from the cryogenic survival population and the control population, and genomic DNA was extracted from the samples using a marine animal genomic DNA extraction kit. The purity and concentration of the DNA were tested to ensure that the OD260 / OD280 value was between 1.8 and 2.0 and the DNA concentration was ≥50 ng / μL. Genotyping was performed on the six pre-selected candidate SNP loci (SNP1~SNP6) using a competitive allele-specific PCR platform, and the genotype data of each sample were recorded.

[0062] (4) Screening of association markers: The allele frequencies P1 of each SNP locus in the hypothermia-surviving population and the corresponding allele frequencies P0 in the control population were statistically analyzed. The frequency difference ΔP and the chi-square test P value were calculated. SNP loci with chi-square test P < 0.01 and |ΔP| > 0.20 were screened as hypothermia-resistant association markers. The alleles expressed frequently in the hypothermia-surviving population were defined as dominant alleles. In this embodiment, four significantly different association markers were obtained, namely SNP1, SNP2, SNP3, and SNP5.

[0063] (5) Construction of the first generation of offspring: Individuals were selected from the low-temperature surviving population, requiring that they simultaneously carry the dominant alleles of 4 associated markers, and that all associated markers be homozygous. Thirty healthy females and thirty healthy males were selected and paired at a 1:1 ratio and placed in a breeding tank at a water temperature of 24℃ and a light / dark cycle of 12h for inbreeding. After the females carried eggs, they were transferred to an independent incubator at a water temperature of 22℃ for incubation until the juveniles reached a body length of 1.5cm. All juveniles were then collected to obtain the first generation of offspring, totaling 1860 individuals.

[0064] (6) Multigenerational low-temperature stress acclimatization and screening: The first generation of offspring was subjected to repeated low-temperature stress treatment. In each generation of acclimatization, the first maintenance time was increased by 0.5 h compared to the previous generation, and the second maintenance time was increased by 1.0 h compared to the previous generation. The second stress temperature of 1℃ was used as the detection condition, and the preset survival rate threshold was 80%. The survival rate of the first generation of offspring after stress was 72.3%, which did not reach the threshold. The second generation of offspring was acclimatized and the stress time was increased. The survival rate of the second generation of offspring was 81.5%. The third generation of offspring was acclimatized with the same parameters. The survival rate was 83.2%. The survival rate of two consecutive generations met the standard. The repeated stress treatment was stopped, and the offspring population was determined to be the preliminary low-temperature resistant breeding population.

[0065] (7) Screening and propagation of the core breeding population: All individuals in the initial low-temperature tolerant breeding population underwent marker genotyping, and 426 individuals carrying all four dominant alleles and homozygous were selected as the core breeding population. Sixty females and 30 males from the core population were selected and propagated in a 2:1 female-to-male ratio in a propagation pond at 26℃. Under normal aquaculture management, offspring shrimp were obtained. The offspring shrimp were subjected to standard low-temperature stress treatment, and the surviving individuals constituted the basic population of the low-temperature tolerant strain.

[0066] (8) Subgeneration Purification and Maintenance: The basic population was cultured in a recirculating aquaculture system at a constant temperature of 26℃ for three consecutive generations of subgeneration selection. After each generation reached sexual maturity, male and female individuals with homozygous dominant alleles were selected and propagated in a 2:1 female-to-male ratio, with no fewer than 50 female individuals. Genotyping was performed on each newborn juvenile shrimp, and individuals that did not meet the criteria were removed, and the removal rate was calculated. The removal rates for the three generations were 4.2%, 3.1%, and 2.8%, respectively, all below 5%, ultimately yielding a genetically stable cold-resistant freshwater crayfish strain.

[0067] Example 2 This embodiment provides a molecular marker-assisted breeding method for the low-temperature tolerance trait of freshwater crayfish, and the specific steps are as follows:

[0068] (1) Low temperature stress treatment and sample collection: 200 freshwater crayfish juveniles of the same batch and size as in Example 1 were selected and placed in a standard temperature-controlled water tank. A gradient cooling mode was adopted, with the temperature decreasing from 26℃ to 4℃ at a rate of 1.0℃ / h and maintained at a constant temperature for 6h; decreasing to 1℃ at a rate of 0.5℃ / h and maintained at a constant temperature for 12h; and increasing the temperature back to 26℃ at a rate of 1.0℃ / h. After 24h of recovery, the number of surviving individuals was counted, and the tail fan muscle tissue of 100 surviving individuals was randomly collected and frozen at -80℃ as the low temperature survival population sample.

[0069] (2) Collection of control group samples: 200 juvenile shrimp from the same batch were selected simultaneously and a constant temperature of 26℃ was set up as a control group. The entire culture time was exactly the same as that of the experimental group. After the culture was completed, 100 control individuals were randomly collected for tissue samples and frozen for later use.

[0070] (3) DNA extraction and genotyping: The DNA extraction method and PCR detection system were exactly the same as in Example 1. Genotyping was performed on the 6 candidate SNP sites. The purity and concentration of the DNA samples were strictly controlled to ensure that the genotyping data were accurate and effective.

[0071] (4) Screening of associated markers: Through allele frequency statistics, difference calculation and chi-square test, four low-temperature resistance associated markers (SNP1, SNP2, SNP3 and SNP5) consistent with those in Example 1 were screened and the dominant alleles corresponding to each marker were identified.

[0072] (5) Construction of the first generation of offspring: Individuals with homozygous dominant alleles for all four associated markers were selected from the low-temperature surviving population. 35 female shrimp and 35 male shrimp were selected and paired 1:1 and placed in a standardized breeding pond. The culture environment parameters were the same as in Example 1. The egg-bearing females were transferred to a 22℃ incubator for incubation. When the juvenile shrimp grew to 1.5cm, a total of 2120 first generation offspring were collected.

[0073] (6) Multi-generation low-temperature stress acclimatization: The acclimatization rule of first maintenance time + 0.5h and second maintenance time + 1.0h for each generation was strictly followed, with a low temperature of 1℃ and a survival rate of 80% as the screening threshold. The survival rate of the first generation offspring was 74.6%, which did not meet the standard; the survival rate of the second generation offspring was 82.1%, which met the standard; the survival rate of the third generation offspring was 84.5%, which met the standard for two consecutive generations, and the acclimatization was terminated, thus obtaining a preliminary low-temperature resistant breeding population.

[0074] (7) Core population expansion: The selected population underwent full genotypic retesting, and 482 homozygous dominant individuals were selected as the core breeding population. 70 female shrimp and 35 male shrimp were selected to form a propagation pond. After the offspring were screened under low temperature stress, a low temperature resistant strain basic population was obtained.

[0075] (8) Successive generation stabilization and purification: Three generations of continuous recirculating aquaculture were carried out for successive generation selection. In each generation, homozygous dominant genotype parents were strictly screened, and all newborn juvenile shrimp underwent genotyping and unqualified individuals were removed. The removal rates for the three generations were 3.8%, 2.9%, and 2.5%, respectively, all below 5%, and a genetically stable cold-resistant freshwater crayfish strain was successfully cultivated.

[0076] Example 3 This embodiment provides a molecular marker-assisted breeding method for the low-temperature tolerance trait of freshwater crayfish, and the specific steps are as follows:

[0077] (1) Low temperature stress treatment and sample collection: 200 standardized freshwater crayfish juveniles from the same batch were selected and subjected to a uniform gradient low temperature stress program: 26℃ was reduced to 4℃ at 1.0℃ / h and maintained for 6h; reduced to 1℃ at 0.5℃ / h and maintained for 12h; and the temperature was increased back to 26℃ at 1.0℃ / h and cultured for 24h. Surviving individuals were counted, and tail fan muscle tissue of 100 surviving individuals was randomly collected and cryopreserved.

[0078] (2) Collection of control group samples: A parallel control group was set up, and 200 juvenile shrimp of the same age were selected and cultured synchronously at a constant temperature of 26℃. After the cycle was completed, 100 control tissue samples were collected.

[0079] (3) Molecular detection and marker screening: Standardized DNA extraction and competitive allele-specific PCR typing techniques were used to complete the typing of 6 candidate SNP sites. Statistical analysis determined that SNP1, SNP2, SNP3 and SNP5 were effective low-temperature resistance associated markers.

[0080] (4) Family construction and offspring breeding: Individuals with homozygous dominant alleles at all loci were selected, and 32 female shrimp and 32 male shrimp were selected and bred in a 1:1 ratio. After standardized hatching and breeding, the first generation of offspring population was 1980.

[0081] (5) Gradual acclimatization and screening: The duration of low temperature stress was increased generation by generation according to the predetermined parameters. The survival rate of the first generation offspring was 73.5%, which did not reach the 80% threshold. The survival rate of the second generation offspring was 81.8%, and the survival rate of the third generation offspring was 83.9%. The two generations met the standard and the preliminary low temperature resistant breeding population was determined.

[0082] (6) Propagation and subgeneration purification: 451 core breeding individuals were screened by genotyping and established in a propagation pond with a female-to-male ratio of 2:1 to obtain the basic population. After three consecutive generations of subgeneration selection, the genotyping elimination rates of juvenile shrimp were 4.0%, 3.3%, and 2.6%, respectively, all below 5%, and a genetically stable low-temperature resistant crayfish strain was finally obtained.

[0083] Comparative Example 1 This comparative example uses traditional phenotypic selection methods to cultivate cold-resistant freshwater crayfish strains without SNP molecular marker-assisted screening. All other culture, stress, and domestication parameters are completely consistent with those in Example 1. The specific steps are as follows:

[0084] (1) Initial selection under low temperature stress: 200 standardized freshwater crayfish juveniles from the same batch were selected and treated with the same gradient low temperature stress program as in Example 1. After 24 hours of warming and recovery, healthy individuals were selected as the initial parent stock without molecular genotyping.

[0085] (2) Parental pairing and breeding: 30 healthy female shrimp and 30 male shrimp were randomly selected from the surviving individuals and paired 1:1 and placed in a breeding pond with the same parameters. After carrying eggs, they were transferred to a standardized incubator and the first generation of offspring population was obtained by breeding 1790 shrimp.

[0086] (3) Multigenerational low-temperature acclimatization: The same rule of increasing stress duration per generation as in Example 1 was adopted. Acclimatization screening was carried out with 1℃ and 80% survival rate as thresholds. Individuals were screened based solely on phenotypic survival, without molecular marker screening.

[0087] (4) Propagation and subgeneration breeding: After domestication to obtain a qualified population, healthy individuals were randomly selected for propagation at a male-to-female ratio of 2:1. There was no genotype retesting or removal of unqualified individuals. Three generations of subgeneration breeding were carried out continuously, and the survival rate and genotype purification of each generation were statistically analyzed.

[0088] Comparative Example 2 This comparative example uses molecular marker screening but does not undergo successive generations of gradually increasing low-temperature stress acclimatization. The specific steps are as follows:

[0089] (1) Initial stress and marker screening: Using the same low temperature stress procedure, sample collection and molecular marker screening method as in Example 1, four low temperature resistance associated markers were obtained. Individuals with homozygous dominant alleles at all loci were selected as parents and paired to breed to obtain the first generation of offspring.

[0090] (2) Domestication treatment of offspring: The offspring population was subjected to initial standard low temperature stress treatment only once. The domestication was not carried out according to the rule of increasing stress duration with each generation. The stress parameters were fixed at 4℃ for 6 hours and 1℃ for 12 hours. The breeding was repeated for three generations without increasing the stress intensity.

[0091] (3) Propagation and succession: After the offspring screening is completed, propagation and three generations of succession breeding are carried out. There is no gradient stress enhancement throughout the process, only routine breeding management. The survival rate and genetic stability of each generation are statistically analyzed.

[0092] Comparative Example 3 After screening and associating markers in this comparative example, only two generations of subculture purification were performed, and three generations of stable subculture selection were not completed. The specific steps are as follows:

[0093] (1) The entire process of low temperature stress, molecular marker screening, family breeding, and gradient domestication as described in Example 1 was adopted to obtain a qualified low temperature resistant breeding population and a core breeding population.

[0094] (2) After the breeding and propagation are carried out, only two generations of genotyping and removal of unqualified individuals are carried out to terminate the breeding process and to statistically analyze the genetic stability and low-temperature survival performance of the population.

[0095] Table 1: Comparison of cryogenic survival rates of populations in each embodiment and comparative example (unit: %) Example 1 72.3 81.5 83.2 yes Example 2 74.6 82.1 84.5 yes Example 3 73.5 81.8 83.9 yes Comparative Example 1 65.2 68.7 70.1 no Comparative Example 2 71.8 75.3 76.9 no Comparative Example 3 72.1 81.2 82.7 yes

[0096] As shown in Table 1, the low-temperature survival rate of the offspring in the three sets of embodiments of the present invention showed a stable upward trend with each generation. The survival rate of the first generation offspring was concentrated in the range of 72.3% to 74.6%, and none of them reached the preset threshold of 80%. The survival rate of the second generation offspring all exceeded 80%, reaching 81.5% to 82.1%. The survival rate of the third generation offspring further improved to 83.2% to 84.5%, meeting the survival rate threshold requirement for two consecutive generations, and the breeding effect was stable and excellent. Comparative Example 1 adopted the traditional phenotypic breeding mode without molecular marker-assisted screening. The highest survival rate of the three generations of offspring was only 70.1%, which could not reach the preset screening threshold. This shows that traditional phenotypic breeding relies only on the appearance of survival status for screening, which cannot accurately screen individuals carrying the low-temperature resistance gene. The breeding efficiency is extremely low, and it is difficult to cultivate high-performance low-temperature resistant strains. Although Comparative Example 2 used molecular markers to screen parents, it did not undergo progressively increasing low-temperature stress acclimatization across generations. The improvement in offspring survival rate was minimal, with the highest survival rate after three generations reaching only 76.9%, failing to meet the target. This indicates that molecular marker screening can only optimize parental genotypes; combining it with progressively increasing stress acclimatization is necessary for continuous screening and solidification of low-temperature tolerance. Comparative Example 3 showed a survival rate trend in the first two generations that was largely consistent with the previous examples, achieving the target for two consecutive generations. However, subsequent stable succession was not achieved, raising concerns about trait stability. The data from the three sets of examples showed minimal differences and good reproducibility, demonstrating that the molecular marker-assisted breeding combined with progressive acclimatization technique of this invention can stably improve the low-temperature survival performance of freshwater crayfish, with significantly better breeding results than traditional breeding and single molecular screening methods.

[0097] Table 2: Comparison of Genotype Elimination Rates in Three Generations for Each Group (Unit: %) Example 1 4.2 3.1 2.8 3.37 yes Example 2 3.8 2.9 2.5 3.07 yes Example 3 4.0 3.3 2.6 3.30 yes Comparative Example 1 12.6 10.8 9.5 10.97 no Comparative Example 2 8.9 7.6 6.8 7.77 no Comparative Example 3 4.1 3.2 / 3.65 no

[0098] Genetic stability is a core indicator for determining the success of breeding new aquatic varieties. This invention uses a genotype knockout rate of <5% across three consecutive generations as the stability standard. As shown in Table 2, the knockout rates in the three examples all exhibit a decreasing trend across generations. The knockout rates for Example 1 were 4.2%, 3.1%, and 2.8% for three generations; for Example 2, they were 3.8%, 2.9%, and 2.5%; and for Example 3, they were 4.0%, 3.3%, and 2.6%. All examples showed a knockout rate below 5% across each generation, with an average knockout rate of only 3.07% to 3.37%. This demonstrates that after three generations of molecular marker retesting, purification, and subculturing, the proportion of homozygous genotypes exhibiting low-temperature tolerance in the population is extremely high, while the number of heterozygous individuals decreases with each generation. The population's genetic traits tend to stabilize, and the superior trait of low-temperature tolerance can be stably inherited. Comparative Example 1, lacking molecular marker screening and genotype purification, relied solely on phenotypic selection. The knockout rate exceeded 9% across three generations, averaging 10.97%. The population exhibited chaotic genotypes, a high proportion of heterozygous individuals, and extremely poor genetic stability, making it impossible to establish a stable strain. Comparative Example 2 only performed initial molecular marker screening without gradient stress training to enhance traits. Genotype purification was slow, with knockout rates exceeding 6.5% across three generations, failing to meet stability standards. This demonstrates that trait training and genotype purification must be synchronized and indispensable. Comparative Example 3 only completed two generations of purification. Although the knockout rates in the first two generations met the standard, the lack of third-generation stability verification prevented determination of strain genetic stability. The data from the three examples showed strong consistency and no significant deviations, fully validating the stepwise purification, gradient training, and multi-generation breeding system of this invention, which effectively improves the genetic stability of the low-temperature tolerance trait in freshwater crayfish populations.

[0099] Table 3: Comparison of Low Temperature Tolerance Performance Indicators of Final Strains in Each Group Example 1 No mass deaths in 24 hours 16.8 96.7 Stablize Example 2 No mass deaths in 24 hours 15.5 97.2 Stablize Example 3 No mass deaths in 24 hours 16.1 96.9 Stablize Comparative Example 1 50% mortality rate within 12 hours 29.9 78.3 Unstable Comparative Example 2 40% mortality rate after 16 hours 23.1 85.6 Unstable Comparative Example 3 25% mortality rate after 20 hours 17.3 92.1 Basically stable (not up to standard)

[0100] As shown in Table 3, the performance data of the three sets of examples of this invention exhibit highly consistent performance. No large-scale mortality was observed under continuous stress at 1°C for 24 hours, with a low-temperature stress mortality rate of only 15.5%–16.8%. The homozygosity rate of the dominant genotype reached 96.7%–97.2%, demonstrating excellent genetic stability, superior low-temperature tolerance, and extremely high phenotypic homozygosity. Comparative Example 1, a traditionally bred strain, showed the worst low-temperature tolerance, with 50% of individuals dying after only 12 hours of 1°C low-temperature stress, a mortality rate as high as 29.9%. The homozygosity rate of the dominant genotype was only 78.3%, indicating severe heterozygosity and weak low-temperature resistance. Comparative Example 2, lacking gradient acclimatization and intensive training, experienced 40% mortality after 16 hours of low-temperature stress, a mortality rate of 23.1%, with a genotypic homozygosity rate of 85.6%, significantly lower than the strains of this invention. Although Comparative Example 3 showed short-term low-temperature survival performance similar to the Example, its genotypic homozygosity was only 92.1%, and it had not completed three generations of stable breeding, indicating a risk of phenotypic degradation, thus it could not be considered a stable strain. In summary, this invention, through precise screening of individuals with superior low-temperature genotypes using candidate SNP molecular markers, combined with progressively increasing low-temperature stress acclimatization, multi-generational family purification, and subsequent breeding, can efficiently solidify the excellent low-temperature tolerance trait in freshwater crayfish, significantly improving population low-temperature survival rate, genotypic homozygosity, and genetic stability. This addresses the industry pain points of traditional phenotypic breeding, such as low accuracy, long breeding cycles, unstable traits, and poor low-temperature resistance.

[0101] The three parallel test results showed good repeatability and small data deviation, confirming that the molecular marker-assisted breeding method provided by this invention can accurately locate individuals with dominant genotypes for low-temperature tolerance in freshwater crayfish. Through homozygous mating within families, gradient low-temperature stress acclimatization, and multi-generational genotype purification, it is possible to rapidly cultivate new low-temperature tolerant freshwater crayfish strains with high low-temperature survival rates, stable genetic traits, and high homozygosity of dominant genotypes. Compared with traditional phenotypic breeding, single molecular marker screening, and incomplete generational breeding control schemes, the technical solution of this invention improves breeding efficiency by more than 40%, significantly enhances strain genetic stability, and effectively solves the breeding problems of frostbite and large-scale mortality in freshwater crayfish during winter, making it suitable for large-scale breeding and promotion.

[0102] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A molecular marker-assisted breeding method for the low-temperature tolerance trait of freshwater crayfish, characterized in that, Includes the following steps: Based on the combination of candidate molecular marker sites, the freshwater crayfish population to be screened was subjected to low temperature stress treatment and tissue samples were collected from surviving individuals. Genomic DNA was extracted from the tissue sample, and genotyping was performed on each SNP site in the candidate molecular marker site combination. The candidate molecular marker site combination was constructed as follows: Candidate gene sequences related to cold stress response were selected from the freshwater crayfish transcriptome database. These candidate genes include heat shock protein 70 gene, antifreeze protein gene, and ubiquitin ligase gene. Using the candidate gene sequence as a reference sequence, all SNP sites located within the coding region of the candidate gene and within 2kb upstream and downstream of it were extracted from the whole genome resequencing data of freshwater crayfish. SNP sites with a minor allele frequency of not less than 0.05 and a genotyping detection rate of not less than 90% are selected from all the SNP sites to form the candidate molecular marker site combination; The genotyping detection was performed using a competitive allele-specific PCR platform. The reaction system of the platform contained two allele-specific forward primers and one universal reverse primer designed for each SNP site. The 3' ends of the two allele-specific forward primers are complementary to the two allele types of the SNP site, respectively, and the 5' ends of the two forward primers are connected to different fluorescent reporter groups. The annealing temperature of the reaction system was set to 60°C, and the number of amplification cycles was 40. After amplification, the fluorescence signal value of each reaction well is read, and the genotype of the tissue sample at the corresponding SNP site is determined based on the ratio of the fluorescence signal values. Based on the typing results, the allele frequency of each SNP locus was calculated to show a significant difference between the hypothermic survival population and the control population, and significant association markers were screened out. Individuals carrying the dominant allele of the associated marker are used as parents for in-family mating to obtain offspring. The low-temperature stress treatment and genotyping detection are repeated on the offspring population until the survival rate of the offspring population under the specified low-temperature conditions reaches a preset threshold. The corresponding offspring population is then identified as a low-temperature resistant breeding population.

2. The molecular marker-assisted breeding method for the low-temperature tolerance trait of freshwater crayfish according to claim 1, characterized in that, The low-temperature stress treatment is implemented according to the following steps: The freshwater crayfish population to be screened was placed in a temperature-controlled water tank and cooled from the rearing water temperature to the first stress temperature of 4℃ at a cooling rate of 1.0℃ / h. The first maintenance time is maintained at the first stress temperature for a first maintenance time of 6 hours; Then, the temperature is reduced from the first stress temperature to the second stress temperature at a cooling rate of 0.5℃ / h, where the second stress temperature is 1℃. The second stress temperature is maintained for a second duration of 12 hours; The water temperature was restored to the rearing water temperature at a heating rate of 1.0℃ / h. After restoration, the individuals were reared for another 24 hours. The number of surviving individuals was counted and the tail fan muscle tissue of the surviving individuals was collected as the tissue sample.

3. The molecular marker-assisted breeding method for the low-temperature tolerance trait of freshwater crayfish according to claim 2, characterized in that, The control group was obtained as follows: An equal number of individuals were randomly selected from the same batch of freshwater crayfish population to be screened and placed in an independent control tank. The temperature control program of the control tank was to maintain the rearing water temperature at a constant temperature. The total rearing time was the total duration of the low temperature stress treatment plus 24 hours after recovery. At the end of the rearing period in the control tank, tail fan muscle tissue of the control individuals was collected as a control tissue sample.

4. The molecular marker-assisted breeding method for the low-temperature tolerance trait of freshwater crayfish according to claim 3, characterized in that, The filtering of significantly different association markers is achieved in the following way: The allele frequency P1 of each SNP locus in the low-temperature survival population and the allele frequency P0 of the corresponding SNP locus in the control population were statistically analyzed. Calculate the difference ΔP between P1 and P0, and calculate the chi-square test P-value of the difference; SNP sites with a chi-square test p-value less than 0.01 and an absolute value of ΔP greater than 0.20 are labeled as the association markers; The dominant allele is defined as the allele with a significantly higher frequency in the low-temperature survival population than in the control population.

5. The molecular marker-assisted breeding method for the low-temperature tolerance trait of freshwater crayfish according to claim 4, characterized in that, Intrafamilial mating is conducted in the following manner: Female and male individuals carrying at least two of the associated markers and the dominant alleles were selected, with each associated marker being homozygous carriers in the selected individuals. The selected female individuals and male individuals were paired in a 1:1 ratio and placed in a breeding pool. The water temperature of the breeding pool was controlled at 24°C, and the light cycle was set to 12 hours of light and 12 hours of darkness. Once the female individuals are carrying eggs, they are transferred to an independent incubator at a water temperature of 22°C. The offspring are collected when they hatch and reach a body length of 1.5 cm.

6. The molecular marker-assisted breeding method for the low-temperature tolerance trait of freshwater crayfish according to claim 5, characterized in that, In the repeated low-temperature stress treatment of the offspring population, the first maintenance duration and the second maintenance duration are respectively increased by a first increment value and a second increment value compared with the previous generation; The first increment is 0.5 hours per generation, and the second increment is 1.0 hour per generation. The specified low temperature condition is the second stress temperature, and the preset threshold is an 80% survival rate; When the survival rate of two consecutive generations of offspring populations reaches the preset threshold under the specified low temperature conditions, the repeated processing is stopped.

7. The molecular marker-assisted breeding method for the low-temperature tolerance trait of freshwater crayfish according to claim 6, characterized in that, After identifying the low-temperature resistant breeding population, the genotypes of all the associated markers of each individual in the low-temperature resistant breeding population are re-examined. Individuals carrying the dominant alleles of all the associated markers and having a homozygous genotype are selected from the re-examination results as the core breeding population. Female and male individuals are randomly selected from the core breeding population and a breeding pond is established with a female-to-male ratio of 2:

1. The number of female individuals in the breeding pond shall not be less than 50. The juvenile shrimp obtained from the propagation pond are subjected to low-temperature stress treatment, and the surviving individuals are used as the base population for the low-temperature resistant strain.

8. The molecular marker-assisted breeding method for the low-temperature tolerance trait of freshwater crayfish according to claim 7, characterized in that, The base population of the low-temperature resistant strain is placed in a recirculating aquaculture system for generational maintenance. The water temperature of the recirculating aquaculture system is set to the feeding water temperature. In each generation cycle, when the individuals in the base population reach sexual maturity, male and female individuals carrying all the dominant alleles of the associated markers and having homozygous genotypes are selected. The next generation breeding pond is established at a female-to-male ratio of 2:1, and the number of female individuals in the next generation breeding pond is not less than 50. In each generation cycle, tail fan muscle tissue of newly hatched juvenile shrimp was collected for genotyping of the associated markers. Individuals whose genotyping results did not meet the requirements of homozygous dominant alleles were removed. When the elimination rate is less than 5% in three consecutive subgeneration cycles, the population obtained in that subgeneration cycle is identified as a genetically stable cold-resistant freshwater crayfish strain.

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