Goldfish molecular marker assisted breeding method
By using molecular marker-assisted breeding and deep learning image recognition technology, the problems of germplasm degradation and imperfect strains caused by inbreeding in goldfish breeding have been solved. A new strain of Wangtianzhu, with visible pupils and a rounded body when viewed from above, has been obtained, realizing germplasm management and intellectual property protection.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- BEIJING ACADEMY OF AGRICULTURE & FORESTRY SCIENCES
- Filing Date
- 2026-04-24
- Publication Date
- 2026-06-09
AI Technical Summary
There is a problem of genetic degradation caused by inbreeding in existing goldfish breeding, and there is a lack of ball pearl goldfish strains on the market that combine visible pupils when viewed from above with a round and beautiful body shape. Furthermore, there is insufficient genetic management and intellectual property protection.
Using molecular marker-assisted breeding, genotype screening was conducted using specific mutation sites of the LRP2 gene. Combined with deep learning image recognition technology, the characteristics of Wangtianyan and Zhenzhulin were constructed. Independent families were constructed through reciprocal crosses and phenotypic verification was performed. Finally, a new strain of Wangtianzhu was obtained, and germplasm was tracked through molecular markers.
It enables early and precise identification of homozygotes, shortens the breeding cycle, reduces costs, obtains healthy Wangtianzhu strains, meets market aesthetic demands, and achieves germplasm protection through molecular markers.
Smart Images
Figure CN122162734A_ABST
Abstract
Claims
1. A marker-assisted breeding method for goldfish, characterized in that, include: S1: Goldfish with the "Sky-eye" trait and homozygous for both the first and second molecular markers were selected as the first parent, and goldfish with the "Pearl Scale" trait were selected as the second parent. The first molecular marker was the termination mutation site chr9: 28,575,379 C>T located in exon 38 of the LRP2 gene on chromosome 9, and the second molecular marker was the missense mutation site chr9: 28575713 G>A located in exon 38 of the LRP2 gene on chromosome 9. S2: Perform orthogonal and reciprocal crosses between the first parent and the second parent to construct two hybrid families and obtain the F1 generation; S3: The F1 generation of the two hybrid families is interbred with siblings within the same family to obtain the F2 generation; S4: Extract fin DNA from each individual in the F2 generation of the two hybrid families, detect the genotype of each individual at the first molecular marker, and screen out individuals that are homozygous for the first molecular marker as the first candidate individuals; S5: Detect the genotype of the first candidate individual at the second molecular marker, and screen out individuals that are homozygous for the second molecular marker as second candidate individuals; S6: The second candidate individuals were raised to 10 months of age, and individuals that simultaneously possessed the traits of "Sky Eye" and "Pearl Scale" were selected as candidate Sky Pearl strains. S7: Crossbreed the candidate Wangtianzhu strains selected from two families through S6 to obtain the F3 generation, which is the target new goldfish strain.
2. The goldfish molecular marker-assisted breeding method according to claim 1, characterized in that, The method for detecting the genotype of each individual at the first molecular marker in S4 is as follows: PCR amplification is performed using the first primer pair, the sequence of which is shown in SEQ ID NO: 1-2. The first amplified fragment is obtained, and the first amplified fragment is digested with the restriction endonuclease DdeI. If the digestion product consists of two bands of 139 bp and 222 bp, the individual is determined to be homozygous for the first molecular marker.
3. The goldfish molecular marker-assisted breeding method according to claim 1, characterized in that, The method for detecting the genotype of the first candidate individual at the second molecular marker in S5 is as follows: PCR amplification is performed using the second primer pair, the sequence of which is shown in SEQ ID NO: 3-4, to obtain the second amplified fragment. The second amplified fragment is then digested with the restriction endonuclease BsrFI. If the digestion product is a 278 bp band, the individual is determined to be homozygous for the second molecular marker.
4. The goldfish molecular marker-assisted breeding method according to claim 1, characterized in that, In S1, the standard Sky Eye morphology and standard Pearl Scale morphology are determined using the following image recognition method: Collect side and top views of the goldfish to be identified; The acquired side view and top view images are input into a pre-trained first neural network model, which includes at least an eye feature extraction branch and a scale feature extraction branch. The eye feature extraction branch outputs the angle value of the goldfish's eyes turning upwards. When the angle value reaches or exceeds the predetermined angle threshold, it is judged as the standard sky-gazing eye characteristic. The scale feature extraction branch outputs a calcification deposition feature map of the goldfish's body surface scales. When the scales present a regular arrangement of hemispherical protrusions and the area covering the body surface reaches or exceeds a predetermined area ratio threshold, it is judged as a standard pearl scale trait.
5. The goldfish molecular marker-assisted breeding method according to claim 4, characterized in that, Perform eye region detection on the input top-view image and extract the regions of interest for the left and right eyes; Heatmap regression networks are used to predict the coordinates of key points on the eyeball for the regions of interest of the left and right eyes, respectively. The key points include at least the center point of the eyeball, the vertex of the upper edge of the eyeball, and the outermost point of the eyeball in the horizontal direction. Calculate the upward rotation angle θ of the left eyeball based on the coordinates of the key points of the left eye. L Calculate the upward rotation angle θ of the right eyeball based on the coordinates of the key points of the right eye. R ; When θ L and θ R All reached or exceeded the predetermined angle threshold, and θ L With θ R When the absolute difference between them does not exceed the predetermined symmetry deviation threshold, it is judged as the standard Sky Eye characteristic.
6. The goldfish molecular marker-assisted breeding method according to claim 4, characterized in that, Perform scale instance segmentation on the input side view and top view images, and output the contour mask and class confidence of each scale protrusion; For each scale protrusion, its morphological quality parameters are calculated, including: contour roundness, the ratio of protrusion height to base diameter, and surface curvature uniformity. When the outline roundness of the scale protrusion is greater than or equal to a first predetermined threshold, the ratio of the protrusion height to the base diameter is within a predetermined range, and the surface curvature uniformity is greater than or equal to a second predetermined threshold, the scale protrusion is determined to be an effective hemispherical protrusion. Calculate the ratio of the total area of effective hemispherical protrusions to the total surface area of the goldfish. When this ratio reaches or exceeds a predetermined area ratio threshold, further calculate the spatial arrangement regularity score of the effective hemispherical protrusions. The spatial arrangement regularity score is calculated based on the variance of the spacing between adjacent protrusions and the grid arrangement deviation of the protrusion centers. When the spatial arrangement regularity score reaches or exceeds the predetermined regularity threshold, it is judged as a standard pearl scale morphology.
7. The goldfish molecular marker-assisted breeding method according to claim 1, characterized in that, In S2, both orthogonal and reciprocal crosses are conducted during the goldfish breeding season, and artificial wet insemination is used. The eggs and sperm are simultaneously released into a hatching basin containing tap water that has been aerated for 24 hours by squeezing the abdomen of the male and female fish. After fertilization, the eggs are left to stand for 30 minutes, rinsed, and then fresh tap water that has been aerated for 24 hours is added. The hatching basin is then suspended in the hatching pond to maintain a natural incubation temperature range of 17-22℃. Both orthogonal and reciprocal cross families have at least two repeat breeding groups, and in each repeat group, the male and female parents are paired in a one-male-one-female pairing.
8. The goldfish molecular marker-assisted breeding method according to claim 6, characterized in that, In S6, after the second candidate individuals are raised to 10 months of age, an image acquisition pipeline is set up, with a first acquisition station and a second acquisition station set up in sequence. The first acquisition station is equipped with a top camera, and the second acquisition station is equipped with a side camera. A temporary storage pool is set up between the two stations. Goldfish are allowed to enter the first collection station one by one in turn. Top-view images are collected, and the angles of the left and right eyeballs turning upwards are calculated. If the angles of the left and right eyes both reach or exceed the predetermined angle thresholds and the absolute difference between the two does not exceed the predetermined symmetry deviation threshold, the individual is judged to be qualified for the "sky-gazing eye" trait and is released to the temporary storage pool and enters the second collection station; otherwise, it is directly diverted and rejected. When goldfish enter the second collection station and are viewed from the side, the proportion of the total area of effective hemispherical protrusions to the surface area of the goldfish's body is first calculated. If this proportion does not reach the predetermined area proportion threshold, the goldfish is directly eliminated. If it does reach the threshold, the spatial arrangement regularity score of the effective hemispherical protrusions is calculated. When the score reaches or exceeds the predetermined regularity threshold, the goldfish is judged to be qualified for the pearl scale trait and the individual is output as a candidate for the Wangtianzhu strain. If the regularity score does not reach the threshold, the goldfish is eliminated.