Method and device for identifying trachinotus anaginotus, wild population and cultured population

Abstract

The invention relates to a method for identifying trachinotus anaginotus, a wild population and a bred population, which comprises the following steps: collecting trachinotus anaginotus to be identified, and carrying out preliminary morphological identification, morphological measurement, DR imaging and DNA extraction on a sample; preliminarily determining the type of a to-be-identified sample based on morphological characteristics by using the retrieval table; amplifying the DNA to be identified by using the primer; comparing the sequence of the amplification product with a COI sequence downloaded from an NCBI database, constructing a phylogenetic tree, and determining the type of the to-be-identified sample from the molecular level; and finally determining the type of the to-be-identified sample by integrating morphological and molecular biological results. The method comprises the following steps: gradually discriminating and analyzing 22 standardized morphological measurement data of 108 trachinotus ovatus (50 natural populations and 58 cultured populations), and establishing a discriminating function equation; the trachinotus anaginotus, the wild population and the cultured population can be accurately and quickly identified in a large scale.

Description

Technical Field

[0001] The present invention relates to the technical field related to morphology and molecular marker identification, and in particular to a method and a device for identifying anaconda pomfret and its wild population and cultured population. Background Art

[0002] Golden pomfret belongs to the class Actinopterygii, order Perciformes, family Carangidae, and genus Trachinotus, and is one of the main marine farmed fish in Asia. The name "golden pomfret" is used to refer to different species, including T. ovatus, T. mookalee, T. anak, and T. blochii. The first three are collectively known as short-fin golden pomfret, while the latter is known as long-fin golden pomfret. The classification of these species has long been unclear, which is not conducive to the protection of golden pomfret germplasm resources and hinders the sustainable development of the aquaculture industry. In addition, it is difficult to distinguish between farmed and wild populations, resulting in the phenomenon of farmed fish posing as wild fish, which not only damages the legitimate rights and interests of consumers, but also disrupts the market competition order.

[0003] Currently, there is a lack of research on the classification of golden pomfret. Therefore, constructing a method that can accurately, simply and quickly identify the golden pomfret and determine the wild and farmed populations is of great significance for the protection of the germplasm resources of wild populations of the golden pomfret and the sustainable development of the aquaculture industry. Summary of the invention

[0004] The purpose of the present invention is to solve at least one of the deficiencies of the prior art and to provide a method for identifying the Ana pomfret and its wild population and cultured population.

[0005] In order to achieve the above object, the present invention adopts the following technical solutions: Specifically, a method for identifying the Ana pomfret and its wild and farmed populations is proposed, including the following: Obtaining golden pomfret samples to be identified; Performing morphological measurement on the golden pomfret sample to be identified to obtain its morphological measurement data, and performing DR imaging on the golden pomfret sample to be identified to obtain its DR imaging result; Based on the morphological measurement data and the DR imaging results, combined with the pre-established search table, it is determined whether the golden pomfret sample to be identified is the ana pomfret, and this determination result is recorded as the first determination result; Extracting DNA from the golden pomfret sample to be identified to obtain its DNA; Using the DNA as a template and primers to perform PCR amplification to obtain an amplified product, purifying the amplified product and sequencing it to obtain a sequencing result; Compare the sequencing results with the COI sequences downloaded from the NCBI database, construct a phylogenetic tree, and determine whether the identified golden pompano sample is Trachinotus anak, and record this judgment result as the second judgment result; If both the first judgment result and the second judgment result indicate that the identified golden pompano sample is Trachinotus anak, then determine the identified golden pompano sample as Trachinotus anak; Standardize the morphological measurement data and substitute it into a pre-established discriminant function equation, and determine the category of the golden pompano sample in Trachinotus anak according to the calculation result of the discriminant function equation, where the category includes a cultured population and a wild population.

[0006] Furthermore, specifically, the morphological measurement data includes fork length FL, distance from the tip of the snout to the origin of the first dorsal fin Sn-D1O, distance from the tip of the snout to the origin of the second dorsal fin Sn-D2O, distance from the tip of the snout to the origin of the pelvic fin Sn-P2O, distance from the tip of the snout to the origin of the anal fin Sn-A2O, distance from the origin of the first dorsal fin to the origin of the pelvic fin D1O-P2O, distance from the origin of the first dorsal fin to the origin of the anal fin D1O-A2O, distance from the origin of the second dorsal fin to the origin of the pelvic fin D2O-P2O, distance from the origin of the second dorsal fin to the origin of the anal fin D2O-A2O, body height MBD, half body height at the origin of the pectoral fin P1-D2I, length of the second dorsal fin HD2L, length of the anal fin HAL, length of the pectoral fin P1L, length of the pelvic fin P2L, base length of the second dorsal fin LDB, base length of the anal fin LAB, head length HL, postorbital head length PoHL, eye diameter ED, snout length SnL, length of the maxilla UjL, height of the maxilla MaD.

[0007] Furthermore, specifically, the pre-established retrieval table is as follows: 1(2) Body elliptical, body silvery white, with multiple elliptical dark spots on the body side, dorsal fin and anal fin white, tips black, caudal fin black, only the edge white Trachinotus ovatus Trachinotus ovatus (Eastern Atlantic); 2(1) Body rhomboid, body golden yellow, dorsal region grayish yellow, or body silvery white, dorsal region grayish green, no spots on the side, dorsal fin and anal fin yellow, no black tips, caudal fin yellow; 3(4) The first interspinous bone of the myeloid is elliptical and bead-shaped, the anterior fin rays of the second dorsal fin are quite extended, 1.5 - 2.0 times the head length, and for individuals of 100 - 400 mm, it is 35 - 60% of the fork length ····· Trachinotus blochii Trachinotus blochii (Indo-Pacific); 4(3) The first interspinous bone of the myeloid is inverted "L"-shaped, the anterior fin rays of the second dorsal fin and the anal fin are both short, and the length of the second dorsal fin is 24 - 30% of the fork length; 5(6) The ventral ribs did not increase in size, and the preorbital and nasal bones did not increase in bone density. The supraoccipital bones of individuals with a fork length of more than 200 mm became wider and shaped like an oblong column, and this was more obvious in individuals with a fork length of more than 300 mm. Trachinotus mookalee (Indo-Pacific); 6(5) The supracervical bone is thin and blade-shaped. The second or third ventral rib is enlarged, and the preorbital and nasal bones of individuals with a fork length of more than 300 mm show increased bone density. Ana Trachinotus anak (Western Pacific).

[0008] Further, specifically, the primers used for PCR amplification include, FishF1 is shown in the sequence listing as SEQ ID 50: 5'-TCAACCAA CCACAAAGACATTGGCAC-3', FishR1 is shown in the sequence ID 51 of the sequence listing: 3'-TAGACTTCTGGGTGGCCAAAC AATCA-5', The amplification system was: DNA template 2 μL, TaqPCR Mix 12.5 μL, ddH2O 6.5 μL, MgCl2 2 μL, primer FishF1 1 μL, primer FishR1 1 μL.

[0009] The PCR amplification program is as follows: pre-denaturation at 95°C for 3 min, denaturation at 92°C for 45 s, annealing at 50°C for 45 s, extension at 72°C for 1 min, 35 cycles, and finally extension at 72°C for 10 min.

[0010] Further, specifically, the sequencing result is compared with the COI sequence downloaded from the NCBI database, a phylogenetic tree is constructed, and it is determined whether the golden pomfret sample to be identified is an ana pomfret, including: The amplified product sequence in the sequencing result is compared with the COI sequence downloaded from the NCBI database to construct a phylogenetic tree; wherein the COI sequence downloaded from the NCBI database is shown as SEQ ID No.1-45, SEQ ID No.1-43 is the COI sequence of the genus Pomfret, and SEQ ID No.44-45 is the outgroup sequence required for constructing the phylogenetic tree. If in the phylogenetic tree, the amplified product sequence and the COI sequence corresponding to the Ana Pomfret are clustered into one branch, then the molecular marker identification technology indicates that the golden pomfret sample to be identified is Ana Pomfret.

[0011] Further, specifically, the morphometric data is standardized as follows, The postorbital head length PoHL, eye diameter ED, snout length SnL, maxilla length UjL, and maxilla height MaD were divided by the head length, and other measurement data were divided by the fork length for standardization.

[0012] Furthermore, specifically, a discriminant function equation was constructed based on the stepwise discriminant method. The process included Constructing a statistical model for the standardized morphological measurement data by the Wilk-Lambda method to F use the chi-square distribution value as a benchmark. When F the chi-square value was greater than 3.84, the corresponding variable was introduced into the model. When F the chi-square value was less than 2.71, the corresponding variable was removed. Finally, five standardized morphological measurement data were selected: the distance from the snout tip to the origin of the pelvic fin / fork length denoted as X1, the semi-body height at the origin of the pectoral fin / fork length denoted as X2, the basal length of the anal fin / fork length denoted as X3, the postorbital head length / head length denoted as X4, and the eye diameter / head length denoted as X5. The established discriminant function equations included the discriminant equation for the wild population and the discriminant equation for the cultured population. Among them, the discriminant equation for the wild population was as follows: F1 = 3504.230*X1 - 650.078*X2 + 2317.691*X3 + 1635.079*X4 + 1324.686*X5 - 1467.609; The discriminant equation for the cultured population was as follows: F2 = 3428.068*X1 - 596.227*X2 + 2251.891*X3 + 1661.023*X4 + 1373.527*X5 - 1462.008.

[0013] Furthermore, specifically, the category of the golden pompano sample among Trachinotus anak was determined according to the calculation results of the discriminant function equation, including Calculating the values of F1 and F2 for the golden pompano sample respectively, and taking the category corresponding to the discriminant equation to which the relatively higher value of F1 and F2 belongs as the category of the golden pompano sample among Trachinotus anak.

[0014] The present invention also proposes a device for identifying Trachinotus anak and wild and cultured populations, including the following: A sample acquisition module for acquiring a golden pompano sample to be identified; A data acquisition module for performing morphological measurements on the golden pompano sample to be identified to obtain its morphological measurement data, and performing DR imaging on the golden pompano sample to be identified to obtain its DR imaging results; The first judgment module is used to judge whether the to-be-identified golden pompano sample is *Trachinotus anak* based on the morphometric data and the DR imaging results, in combination with a pre-established retrieval table, and record this judgment result as the first judgment result; The DNA extraction module is used to extract the DNA of the to-be-identified golden pompano sample; The PCR amplification module is used to use the DNA as a template, perform PCR amplification with primers to obtain an amplification product, purify and sequence the amplification product to obtain a sequencing result; The second judgment module is used to compare the sequencing result with the COI sequence downloaded from the NCBI database, construct a phylogenetic tree, judge whether the to-be-identified golden pompano sample is *Trachinotus anak*, and record this judgment result as the second judgment result; The species determination module is used to determine the to-be-identified golden pompano sample as *Trachinotus anak* when both the first judgment result and the second judgment result indicate that the to-be-identified golden pompano sample is *Trachinotus anak*; The category determination module is used to standardize the morphometric data and then substitute it into a pre-established discriminant function equation, and determine the category of the golden pompano sample in *Trachinotus anak* according to the calculation result of the discriminant function equation, where the category includes a cultured population and a wild population.

[0015] The present invention proposes a method for identifying *Trachinotus anak* and differentiating between wild populations and cultured populations, provides the COI sequence for identifying *Trachinotus anak*, and establishes a discriminant equation for natural populations and cultured populations of *Trachinotus anak*. The method of the present invention is economical, simple, highly repeatable, can identify a large number of *Trachinotus anak* at one time, can judge the origin through morphological data without harming *Trachinotus anak*, and is beneficial to the protection of the germplasm resources of the wild population of *Trachinotus anak* and the sustainable development of the aquaculture industry. Description of the Drawings

[0016] By elaborating on the embodiments shown in combination with the drawings, the above and other features of the present disclosure will become more obvious. The same reference numerals in the drawings of the present disclosure represent the same or similar elements. Obviously, the drawings in the following description are only some embodiments of the present disclosure. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings. In the drawings: Figure 1 Shows a flowchart of the method for identifying *Trachinotus anak* and differentiating between wild populations and cultured populations of the present invention; Figure 2 Shows a schematic diagram of the morphometric measurement of golden pompano in the present invention; Figure 3 Shows a DR schematic diagram of *Trachinotus anak*, *Trachinotus mookalee*, and *Trachinotus blochii*; Figure 4 The figure shows a schematic phylogenetic tree of the genus Trachinotus. Detailed implementation mode

[0017] The following will clearly and completely describe the concept, specific structure and technical effects of the present invention in combination with embodiments and drawings, so as to fully understand the purpose, scheme and effects of the present invention. It should be noted that, without conflict, the embodiments in the present application and the features in the embodiments can be combined with each other. The same reference numerals used throughout the drawings indicate the same or similar parts.

[0018] The present invention collected golden pompano samples from cultured and natural populations of different sizes. Through skeletal analysis, it was revealed that the preorbital bone and nasal bone of fish with a fork length exceeding 300 mm showed an increase in bone density, and the second and third ventral ribs were enlarged and showed an increase in bone density. Compared with the type specimen, these samples were identified as Trachinotus anak. The phylogenetic tree constructed based on COI showed that all sequences clustered together with the sequences identified as Trachinotus anak. According to the above results, the commonly found golden pompano is Trachinotus anak, not Trachinotus ovatus. The true Trachinotus ovatus only exists in the eastern Atlantic. Traditionally, the presence of lingual teeth has been used to distinguish Trachinotus anak (toothless) and Trachinotus mookalee (toothed). However, the present invention found that most Trachinotus anak samples also have lingual teeth, indicating that this feature is not a reliable distinguishing feature.

[0019] Based on the above analysis, in Example 1, referring to Figure 1 , the present invention proposes a method for identifying Trachinotus anak and wild populations and cultured populations, including the following: Obtain a golden pompano sample to be identified; Perform morphological measurement on the golden pompano sample to be identified to obtain its morphological measurement data, and perform DR imaging on the golden pompano sample to be identified to obtain its DR imaging result (refer to Figure 3 ); Based on the morphological measurement data and the DR imaging result, in combination with a pre-established retrieval table (where the retrieval and comparison method can be manual comparison, or a relevant network model can be established to extract and classify the features of the retrieval table through the network model, automatically identify the input morphological measurement data and DR imaging result, and determine whether the golden pompano sample to be identified is Trachinotus anak according to the category of the retrieval table to which the identification result belongs), determine whether the golden pompano sample to be identified is Trachinotus anak, and record this judgment result as the first judgment result (where if the comparison is carried out manually, the comparison result input manually can be directly obtained, and if it is an intelligent identification through a relevant network model, the identification result can be directly obtained and the result can be checked and corrected manually); Extract DNA from the golden pompano sample to be identified to obtain its DNA; Using the said DNA as a template, PCR amplification is carried out with primers to obtain an amplification product, and the amplification product is purified and sequenced to obtain a sequencing result; The said sequencing result is compared with the COI sequences downloaded from the NCBI database, a phylogenetic tree is constructed, and it is judged whether the said golden pompano sample to be identified is Trachinotus anak, and this judgment result is recorded as the second judgment result; Among them, the COI sequences downloaded from the NCBI database are shown as SEQ ID No.1-45, SEQ ID No.1-43 are the COI sequences of Trachinotus, and SEQ ID No.44-45 are the outgroup sequences required for constructing the phylogenetic tree.

[0020] If both the first judgment result and the second judgment result indicate that the said golden pompano sample to be identified is Trachinotus anak, then the said golden pompano sample to be identified is determined to be Trachinotus anak; The said morphological measurement data are standardized and then substituted into a pre-established discriminant function equation, and the belonging category of the said golden pompano sample among Trachinotus anak is determined according to the calculation result of the discriminant function equation, where the belonging category includes a cultured population and a wild population.

[0021] Refer to Figure 2 , as a preferred embodiment of the present invention, specifically, the said morphological measurement data include, Fork length FL, distance from the tip of the snout to the origin of the first dorsal fin Sn-D1O, distance from the tip of the snout to the origin of the second dorsal fin Sn-D2O, distance from the tip of the snout to the origin of the pelvic fin Sn-P2O, distance from the tip of the snout to the origin of the anal fin Sn-A2O, distance from the origin of the first dorsal fin to the origin of the pelvic fin D1O-P2O, distance from the origin of the first dorsal fin to the origin of the anal fin D1O-A2O, distance from the origin of the second dorsal fin to the origin of the pelvic fin D2O-P2O, distance from the origin of the second dorsal fin to the origin of the anal fin D2O-A2O, body height MBD, half body height at the origin of the pectoral fin P1-D2I, length of the second dorsal fin HD2L, length of the anal fin HAL, length of the pectoral fin P1L, length of the pelvic fin P2L, basal length of the second dorsal fin LDB, basal length of the anal fin LAB, head length HL, postorbital head length PoHL, eye diameter ED, snout length SnL, length of the maxilla UjL, height of the maxilla MaD.

[0022] As a preferred embodiment of the present invention, specifically, the pre-established retrieval table is as follows: 1(2) Body oval, body silvery white, with multiple oval dark spots on the body side, dorsal fin and anal fin white, tips black, caudal fin black, only the edge white Trachinotus ovatus Trachinotus ovatus (East Atlantic); Among them, this key table adopts the continuous key table format. The number at the beginning of the sentence indicates the item number, which is used to identify the current feature description; the number in the bracket indicates the target item to jump to if the characteristics of the current item are not met. Here, "1" is the number of the item. If the morphological characteristics of item 1 are met, it indicates that the species is oval pomfret; if the morphological characteristics of item 1 are not met, it jumps to item 2.

[0023] 2(1) Body rhombus-shaped, golden yellow with grayish yellow dorsal area, or body silvery white with grayish green dorsal area, no spots on the sides, dorsal and anal fins yellow without black tip, caudal fin yellow; Among them, "2" is the number of the item. If it meets the morphological characteristics of item 2, it will jump to item 3 to continue checking other morphological characteristics; if it does not meet the morphological characteristics of item 2, it will jump to item 1.

[0024] 3(4) The first interspinal bone is oval-shaped. The anterior ray of the second dorsal fin is quite elongated, 1.5 to 2.0 times the length of the head. For individuals of 100 to 400 mm, it is 35 to 60% of the length of the fork. Trachinotus blochii (Indo-Pacific); Among them, "3" is the number of the entry. If the morphological characteristics of entry 3 are met, it indicates that the species is Brandt's pomfret; if the morphological characteristics of entry 3 are not met, jump to entry 4.

[0025] 4(3) The first interspinal bone is inverted "L" shape, the anterior fin rays of the second dorsal fin and anal fin are both short, and the length of the second dorsal fin is 24-30% of the length of the fork; Among them, "4" is the number of the item. If it meets the morphological characteristics of item 4, jump to item 5 to continue checking other morphological characteristics; if it does not meet the morphological characteristics of item 4, jump to item 3.

[0026] 5(6) The ventral ribs did not increase in size, and the preorbital and nasal bones did not increase in bone density. The supraoccipital bones of individuals with a fork length of more than 200 mm became wider and shaped like an oblong column, and this was more obvious in individuals with a fork length of more than 300 mm. Trachinotus mookalee (Indo-Pacific); Among them, "5" is the number of the entry. If the morphological characteristics of entry 5 are met, it indicates that the species is Mukh Pomfret; if the morphological characteristics of entry 5 are not met, jump to entry 6.

[0027] 6(5) The supracervical bone is thin and blade-shaped. The second or third ventral rib is enlarged, and the preorbital and nasal bones of individuals with a fork length of more than 300 mm show increased bone density. Ana Trachinotus anak (Western Pacific); Among them, "6" is the number of this entry. If the morphological characteristics of entry 6 are met, it indicates that the species is *Trachinotus anak*. If the morphological characteristics of entry 6 are not met, jump to entry 5.

[0028] As a preferred embodiment of the present invention, specifically, the primers used for PCR amplification include, FishF1 is shown in SEQ ID NO: 50 of the sequence listing: 5’-TCAACCAACCACAAAGACATTGGCAC-3’; FishR1 is shown in SEQ ID NO: 51 of the sequence listing: 3’-TAGACTTCTGGGTGGCCAAACAATCA-5’; The amplification system is: 2 μL of DNA template, 12.5 μL of TaqPCR Mix, 6.5 μL of ddH2O, 2 μL of MgCl2, 1 μL of primer FishF1, and 1 μL of primer FishR1.

[0029] The PCR amplification program is: pre-denaturation at 95 °C for 3 min, denaturation at 92 °C for 45 s, annealing at 50 °C for 45 s, extension at 72 °C for 1 min, 35 cycles, and finally extension at 72 °C for 10 min.

[0030] As a preferred embodiment of the present invention, specifically, comparing the sequencing result with the COI sequence downloaded from the NCBI database to determine whether the *Trachinotus* sample to be identified is *Trachinotus anak* includes, Comparing the amplified product sequence in the sequencing result with the COI sequence downloaded from the NCBI database to construct a phylogenetic tree (refer to Figure 4 ); if in the phylogenetic tree, the amplified product sequence and the COI sequence corresponding to *Trachinotus anak* cluster into one branch, the molecular marker identification technology indicates that the *Trachinotus* sample to be identified is *Trachinotus anak*.

[0031] The COI sequence downloaded from the NCBI database is shown as SEQ ID No. 5-49. SEQ ID No. 1-43 is the COI sequence of the genus *Trachinotus*, and SEQ ID No. 44-45 is the outgroup sequence required for constructing the phylogenetic tree.

[0032] As a preferred embodiment of the present invention, specifically, standardize the morphological measurement data as follows, Standardize the postorbital head length (PoHL), eye diameter (ED), snout length (SnL), length of maxilla (UjL), and height of maxilla (MaD) by dividing them by the head length, and standardize other measurement data by dividing them by the fork length.

[0033] As a preferred embodiment of the present invention, specifically, constructing a discriminant function equation based on the stepwise discriminant method, the process includes, Construct a statistical model for the standardized morphological measurement data by the Wilk-Lambda method, with F the chi-square distribution value as the benchmark. When F the value is greater than 3.84, introduce the corresponding variable into the model. When F the value is less than 2.71, remove the corresponding variable. By gradually introducing variables, 22 morphological measurement data are gradually introduced after 5 operation steps. According to the contribution rate of the variables to the model, gradually eliminate the variables with small contribution rates. Finally, select 5 standardized morphological measurement data: the distance from the snout to the origin of the pelvic fin / fork length denoted as X1, the semi-body height at the origin of the pectoral fin / fork length denoted as X2, the length of the base of the anal fin / fork length denoted as X3, the length of the head behind the eye / head length denoted as X4, and the eye diameter / head length denoted as X5; The established discriminant function equations include the discriminant equation for the wild population and the discriminant equation for the cultured population. Among them, the discriminant equation for the wild population is as follows: F1 = 3504.230 * X1 - 650.078 * X2 + 2317.691 * X3 + 1635.079 * X4 + 1324.686 * X5 - 1467.609; The discriminant equation for the cultured population is as follows: F2 = 3428.068 * X1 - 596.227 * X2 + 2251.891 * X3 + 1661.023 * X4 + 1373.527 * X5 - 1462.008.

[0034] As a preferred embodiment of the present invention, specifically, determine the category of the golden pompano sample in Trachinotus anak based on the calculation results of the discriminant function equation, including calculate the values of F1 and F2 of the golden pompano sample respectively, and take the category corresponding to the discriminant equation to which the relatively higher value of F1 and F2 belongs as the category of the golden pompano sample in Trachinotus anak.

[0035] In this preferred embodiment set, the above discriminant function equation is proposed, and a significance test is carried out on the discriminant function. The result shows that P < 0.01, indicating that the discriminant function reaches a significant level and can effectively distinguish the natural population and the cultured population.

[0036] Combining all the preferred embodiments proposed in the present invention, Example 2 includes the following steps 1) Collect the golden pompano samples to be identified.

[0037] 2) Conduct preliminary morphological identification, morphological measurement, DR imaging, and DNA extraction on the samples; weigh the body weight with an electronic balance, take a photo of the left side of the fish body using a camera, and then measure the fish body morphology with Mingmei to obtain the following data: fork length FL, distance from the tip of the snout to the origin of the first dorsal fin Sn-D1O, distance from the tip of the snout to the origin of the second dorsal fin Sn-D2O, distance from the tip of the snout to the origin of the pelvic fin Sn-P2O, distance from the tip of the snout to the origin of the anal fin Sn-A2O, distance from the origin of the first dorsal fin to the origin of the pelvic fin D1O-P2O, distance from the origin of the first dorsal fin to the origin of the anal fin D1O-A2O, distance from the origin of the second dorsal fin to the origin of the pelvic fin D2O-P2O, distance from the origin of the second dorsal fin to the origin of the anal fin D2O-A2O, body height MBD, half body height at the origin of the pectoral fin P1-D2I, length of the second dorsal fin HD2L, length of the anal fin HAL, length of the pectoral fin P1L, length of the pelvic fin P2L, base length of the second dorsal fin LDB, base length of the anal fin LAB, head length HL, length of the postorbital part of the head PoHL, eye diameter ED, snout length SnL, length of the maxilla UjL, height of the maxilla MaD.

[0038] The Mingmei mentioned above is a powerful 2D image processing and analysis software with rich measurement functions.

[0039] There is no special limitation on the method for extracting the DNA of the sample to be identified. Any conventional method for extracting animal genomic DNA in the art can be used, and it is preferably extracted using a commercial kit.

[0040] 3) Based on the morphological measurement data (length of the second dorsal fin / fork length) obtained in step 2) and the DR imaging results, compare with the descriptions in the retrieval table to determine whether the sample to be identified is Trachinotus anak from a morphological perspective.

[0041] The DR imaging results mainly observe the shapes of the first interspinous bones of the vertebra and the supraoccipital bone, whether there is an increase in bone density in the preorbital bone and nasal bone, whether the ventral ribs are enlarged, and whether there is an increase in bone density.

[0042] The content of the retrieval table is as follows: 1(2) Body oval. Body silvery white, with multiple oval dark spots on the body side. Dorsal fin and anal fin white, tips black. Caudal fin black, only the edge white - Trachinotus ovatus Trachinotus ovatus (Eastern Atlantic); 2(1) Body rhomboid. Body golden yellow, the dorsal region gray - yellow, or body silvery white, the dorsal region gray - green, without spots on the side. Dorsal fin and anal fin yellow, without black tips, caudal fin yellow; 3(4) The first interspinous bones of the vertebra are oval - bead - shaped. The anterior fin rays of the second dorsal fin are quite elongated, 1.5 - 2.0 times the head length, or for individuals of 100 - 400 mm, 35 - 60% of the fork length ···· Trachinotus blochii Trachinotus blochii (Indo - Pacific); 4(3) The first interspinal bone is inverted "L" shape, the anterior fin rays of the second dorsal fin and anal fin are both short, and the length of the second dorsal fin is 24-30% of the length of the fork; 5(6) The abdominal ribs did not increase in size. The preorbital bones and nasal bones did not increase in bone density. The supracervical bones of individuals with fork lengths exceeding 200 mm became wider and elliptical in shape, and this was more obvious in individuals with fork lengths exceeding 300 mm. Trachinotus mookalee (Indo-Pacific); 6(5) The supracervical bone is thin and blade-shaped. The second or third ventral rib is enlarged, and the preorbital and nasal bones of individuals with a fork length of more than 300 mm show increased bone density. Ana Trachinotus anak (Western Pacific); 4) Using the DNA obtained in step 2) as a template, performing PCR amplification with primers to obtain an amplified product, purifying and sequencing the amplified product, and comparing the sequencing results.

[0043] The template DNA is located on mitochondrial DNA (mtDNA) COI.

[0044] The primers for amplifying DNA include FishF1 and FishR1; the nucleotide sequence of the FishF1 is as follows: FishF1: 5'-TCAACCAA CCACAAAGACATTGGCAC-3'; the nucleotide sequence of the FishR1 is as follows, specifically as follows: FishR1: 3'-TAGACTTCTGGGTGGCCAAAC AATCA-5'.

[0045] The amplification system of the amplification is calculated in 25 μL and includes the following components: 2 μL of DNA template, 12.5 μL of TaqPCR Mix, 6.5 μL of ddH2O, 2 μL of MgCl2, 1 μL of primer FishF1, and 1 μL of primer FishR1.

[0046] The PCR amplification program was as follows: pre-denaturation at 95°C for 3 min, denaturation at 92°C for 45 s, annealing at 50°C for 45 s, extension at 72°C for 1 min, 35 cycles, and finally extension at 72°C for 10 min, and storage at 4°C.

[0047] After the amplification is completed, the amplification product is preferably purified and sequenced. The present invention has no particular limitation on the purification and sequencing methods, and the purification and sequencing are preferably performed by entrusting a biological sequencing company.

[0048] 5) Compare the amplified product sequence with the COI sequence downloaded from the NCBI database to construct a phylogenetic tree; if in the phylogenetic tree, the amplified product sequence and the Ana pomfret are clustered into a branch, then the molecular marker identification technology indicates that the sample to be identified is Ana pomfret.

[0049] The COI sequences used for constructing the phylogenetic tree are shown as SEQ ID No.1-49, where SEQ ID No.1-43 are the COI sequences of other fishes in the genus Trachinotus, SEQ ID No.44-45 are the outgroup sequences required for constructing the phylogenetic tree, and SEQ ID No.46-49 are the COI sequences of the measured Trachinotus anak.

[0050] 6) If the results of steps 3) and 5) are both Trachinotus anak, the sample to be identified is determined to be Trachinotus anak.

[0051] 7) Standardize the postorbital head length (PoHL), eye diameter (ED), snout length (SnL), maxilla length (UjL), and maxilla height (MaD) by dividing them by the head length, and divide other measurement data by the fork length using the morphological measurement data obtained in step 2).

[0052] 8) Use the Wilk-Lambda method to construct a statistical model for the data in step 7), with the F-distribution value as the benchmark. When F the value is greater than 3.84, introduce the corresponding variable into the model. When F the value is less than 2.71, remove the corresponding variable. By gradually introducing variables, 22 morphological measurement data are gradually introduced through 5 operation steps. According to the contribution rate of the variables to the model, gradually eliminate the variables with small contribution rates, and screen out 5 variables: the distance from the snout tip to the origin of the pelvic fin / fork length, the semi-body height at the origin of the pectoral fin / fork length, the base length of the anal fin / fork length, the postorbital head length / head length, and the eye diameter / head length. Perform a collinearity analysis on the 5 variables, and the results show that the collinearity among the 5 variables is very low, and there is no collinearity problem. Let X1, X2, X3, X4, and X5 represent the distance from the snout tip to the origin of the pelvic fin / fork length, the semi-body height at the origin of the pectoral fin / fork length, the base length of the anal fin / fork length, the postorbital head length / head length, and the eye diameter / head length respectively, and establish the discriminant equation as follows: The discriminant equation for the natural population is as follows: F1 = 3504.230 * X1 - 650.078 * X2 + 2317.691 * X3 + 1635.079 * X4 + 1324.686 * X5 - 1467.609; The discriminant equation for the cultured population is as follows: F2 = 3428.068 * X1 - 596.227 * X2 + 2251.891 * X3 + 1661.023 * X4 + 1373.527 * X5 - 1462.008.

[0053] Conduct a significance test on the discriminant function, and the results show that P < 0.01, indicating that the discriminant function reaches a significant level and can effectively distinguish between the natural population and the cultured population.

[0054] 9) Substitute the individual morphological measurement data of each Trachinotus anak into the discriminant function equation, and the category corresponding to the discriminant function with a larger function value is the category to which it belongs.

[0055] Specifically, when the present invention is applied, it includes the following steps. 1. Collect a total of 108 samples of Trachinotus ovatus, including 58 samples from the cultured population and 50 samples from the natural population. Weigh the body weight with an electronic balance, take pictures of the left side of the fish body using a camera, and collect an appropriate amount of tissue for preservation at -20°C. Measure the fish body morphology using Mingmei to obtain the following data: Fork length FL, distance from the tip of the snout to the origin of the first dorsal fin Sn-D1O, distance from the tip of the snout to the origin of the second dorsal fin Sn-D2O, distance from the tip of the snout to the origin of the pelvic fin Sn-P2O, distance from the tip of the snout to the origin of the anal fin Sn-A2O, distance from the origin of the first dorsal fin to the origin of the pelvic fin D1O-P2O, distance from the origin of the first dorsal fin to the origin of the anal fin D1O-A2O, distance from the origin of the second dorsal fin to the origin of the pelvic fin D2O-P2O, distance from the origin of the second dorsal fin to the origin of the anal fin D2O-A2O, body height MBD, half body height at the origin of the pectoral fin P1-D2I, length of the second dorsal fin HD2L, length of the anal fin HAL, length of the pectoral fin P1L, length of the pelvic fin P2L, base length of the second dorsal fin LDB, base length of the anal fin LAB, head length HL, length of the postorbital head PoHL, eye diameter ED, snout length SnL, length of the maxilla UjL, height of the maxilla MaD.

[0056] 2. The obtained morphological measurement data shows that the ratio of the length of the second dorsal fin to the fork length is less than 30%; the DR imaging result shows that the first interspinous bone is in an inverted "L" shape, the occipital bone is blade-shaped, the second and third ventral ribs are enlarged, and there is an increase in bone density in the preorbital bone and nasal bone of individuals with a fork length exceeding 300 mm.

[0057] Comparing with the description in the retrieval table, the sample to be identified is Trachinotus anak.

[0058] 3. Extract the DNA of the sample to be identified, use the extracted DNA as a template for PCR amplification, purify and sequence the amplification result, and compare the sequencing result. The primer sequences are as follows: FishF1 is as shown in SEQ ID NO: 50 in the sequence listing: 5’-TCAACCAACCACAAAGACATTGGCAC-3’; FishR1 is as shown in SEQ ID NO: 51 in the sequence listing: 3’-TAGACTTCTGGGTGGCCAAACAATCA-5’.

[0059] Add the following reagents successively into a 0.2 ml centrifuge tube: 12.5 μL of TaqPCR Mix, 6.5 μL of ddH2O, 2 μL of MgCl2, 2 μL of DNA template, and 1 μL each of primers FishF1 and FishR1. The PCR amplification program is as follows: pre-denaturation at 95 °C for 3 min, denaturation at 92 °C for 45 s, annealing at 50 °C for 45 s, extension at 72 °C for 1 min, for 35 cycles, and finally extension at 72 °C for 10 min. Send the amplification product to Sangon Biotech (Shanghai) Co., Ltd. for sequencing.

[0060] 4. After the sequencing is completed, compare the sequencing results and construct a phylogenetic tree based on the sequencing results.

[0061] The results of the phylogenetic tree show that ( Figure 4 ), the sample to be identified clusters into one branch with Trachinotus anak.

[0062] 5. Combining the morphological analysis (Step 2) and the sequencing results (Step 5) indicates that the sample to be identified is Trachinotus anak.

[0063] 6. Substitute the 5 morphological measurement data of each Trachinotus anak sample to be identified into the discriminant function equation respectively, and the function with the larger calculated result is the category to which it belongs. The discrimination results show (see Table 1 below) that the discrimination accuracy rate of the natural population of Trachinotus anak is 80.0%, the discrimination accuracy rate of the cultured population is 86.2%, and the comprehensive discrimination accuracy rate is 83.3%.

[0064] Table 1 Discrimination accuracy rates of the natural population and cultured population of Trachinotus anak

[0065] The present invention also proposes a device for identifying Trachinotus anak and its wild population and cultured population, including the following: A sample acquisition module for acquiring the Trachinotus ovatus samples to be identified; A data acquisition module for performing morphological measurements on the Trachinotus ovatus samples to be identified to obtain their morphological measurement data, and performing DR imaging on the Trachinotus ovatus samples to be identified to obtain their DR imaging results; A first judgment module for judging whether the Trachinotus ovatus samples to be identified are Trachinotus anak based on the morphological measurement data and the DR imaging results, in combination with a pre-established retrieval table, and recording this judgment result as the first judgment result; A DNA extraction module for extracting DNA from the Trachinotus ovatus samples to be identified; A PCR amplification module for using the DNA as a template, performing PCR amplification with primers to obtain an amplification product, purifying and sequencing the amplification product to obtain a sequencing result; A second judgment module, configured to compare the sequencing result with the COI sequence downloaded from the NCBI database, construct a phylogenetic tree, judge whether the identified golden pompano sample is Trachinotus anak, and record this judgment result as the second judgment result; A species determination module, configured to determine the identified golden pompano sample as Trachinotus anak when both the first judgment result and the second judgment result indicate that the identified golden pompano sample is Trachinotus anak; A category determination module, configured to standardize the morphological measurement data and then substitute it into a pre-established discriminant function equation, and determine the category of the golden pompano sample in Trachinotus anak according to the calculation result of the discriminant function equation, where the category includes a cultured population and a wild population.

[0066] Although the description of the present invention has been quite detailed and particularly describes several of the described embodiments, it is not intended to be limited to any of these details or embodiments or any particular embodiment, but should be regarded as providing a broad interpretation of these claims in light of the prior art by reference to the appended claims, so as to effectively cover the intended scope of the present invention. In addition, the present invention is described above with embodiments foreseeable by the inventor for the purpose of providing a useful description, and non-substantive modifications to the present invention that are not currently foreseeable may still represent equivalent modifications of the present invention.

[0067] As described above, it is only a preferred embodiment of the present invention. The present invention is not limited to the above-mentioned embodiments. As long as it achieves the technical effects of the present invention by the same means, it should fall within the protection scope of the present invention. Within the protection scope of the present invention, various different modifications and changes can be made to its technical solutions and / or embodiments.

Claims

1. A method for identifying wild and cultured populations of Ana pomfret, characterized in that: Includes the following: Obtaining golden pomfret samples to be identified; Performing morphological measurement on the golden pomfret sample to be identified to obtain its morphological measurement data, and performing DR imaging on the golden pomfret sample to be identified to obtain its DR imaging result; Based on the morphological measurement data and the DR imaging results, combined with the pre-established retrieval table, it is determined whether the golden pomfret sample to be identified is the ana pomfret, and this determination result is recorded as the first determination result; Extracting DNA from the golden pomfret sample to be identified to obtain its DNA; Using the DNA as a template and primers to perform PCR amplification to obtain an amplified product, purifying the amplified product and sequencing it to obtain a sequencing result; Compare the sequencing result with the COI sequence downloaded from the NCBI database, construct a phylogenetic tree, determine whether the golden pomfret sample to be identified is an ana pomfret, and record this determination result as the second determination result; If the first judgment result and the second judgment result both indicate that the golden pomfret sample to be identified is Ana pomfret, the golden pomfret sample to be identified is determined to be Ana pomfret; The morphological measurement data is standardized and then input into a pre-established discriminant function equation, and the category of the golden pomfret sample in the anaconda is determined according to the calculation result of the discriminant function equation, wherein the categories include farmed populations and wild populations.

2. The method for identifying the Ana pomfret and its wild population and cultured population according to claim 1, characterized in that: Specifically, the morphological measurement data includes: Fork length FL, distance from snout tip to starting point of first dorsal fin Sn-D1O, distance from snout tip to starting point of second dorsal fin Sn-D2O, distance from snout tip to starting point of pelvic fin Sn-P2O, distance from snout tip to starting point of anal fin Sn-A2O, distance from starting point of first dorsal fin to starting point of pelvic fin D1O-P2O, distance from starting point of first dorsal fin to starting point of anal fin D1O-A2O, distance from starting point of second dorsal fin to starting point of pelvic fin D2O-P2O, distance from starting point of second dorsal fin to starting point of anal fin D2O-A2O, body height MBD, half body height at starting point of pectoral fin P1-D2I, length of second dorsal fin HD2L, anal fin length HAL, pectoral fin length P1L, pelvic fin length P2L, length of base of second dorsal fin LDB, length of base of anal fin LAB, head length HL, length of head behind eye PoHL, eye diameter ED, snout length SnL, maxillary length UjL, maxillary height MaD.

3. The method for identifying the Ana pomfret and its wild population and cultured population according to claim 2, characterized in that: Specifically, the pre-established search table is as follows: 1(2) The body is oval, silvery white, with multiple oval dark spots on the sides, the dorsal and anal fins are white with black tips, and the caudal fin is black with only white edges; Among them, this key table adopts a continuous key table format. The number at the beginning of the sentence indicates the item number, which is used to identify the current feature description; the number in the bracket indicates the target item to jump to if the characteristics of the current item are not met. Here, 1 is the number of the item. If the morphological characteristics of item 1 are met, it indicates that the species is oval pomfret; if the morphological characteristics of item 1 are not met, jump to item 2; 2(1) Body rhombus-shaped, golden yellow with grayish yellow dorsal area, or silvery white with grayish green dorsal area, no spots on the sides, dorsal and anal fins yellow without black tip, caudal fin yellow; Wherein, 2 is the number of the item. If the morphological characteristics of item 2 are met, then jump to item 3 to continue checking other morphological characteristics; if the morphological characteristics of item 2 are not met, then jump to item 1; 3(4) The first interspinous bone is oval-shaped, and the anterior ray of the second dorsal fin is quite elongated, 1.5 to 2.0 times the length of the head, and 35 to 60% of the fork length for individuals 100 to 400 mm long; Among them, 3 is the number of the entry. If the morphological characteristics of entry 3 are met, it indicates that the species is the Brandt's pomfret; if the morphological characteristics of entry 3 are not met, jump to entry 4; 4(3) The first interspinal bone is inverted L-shaped, the anterior rays of the second dorsal and anal fins are short, and the length of the second dorsal fin is 24-30% of the length of the fork; Wherein, 4 is the number of the item. If the morphological characteristics of item 4 are met, then jump to item 5 to continue checking other morphological characteristics; if the morphological characteristics of item 4 are not met, then jump to item 3; 5(6) The ventral ribs did not increase in size, and the preorbital and nasal bones did not increase in bone density. The supraoccipital bones of individuals with a fork length of more than 200 mm became wider and shaped like an elongated ellipse, which was more obvious in individuals with a fork length of more than 300 mm. Among them, 5 is the number of the entry. If the morphological characteristics of entry 5 are met, it indicates that the species is Muke pomfret; if the morphological characteristics of entry 5 are not met, jump to entry 6; 6(5) The supraoccipital bone is thin and blade-shaped, the second or third ventral rib is enlarged, and individuals with a fork length of more than 300 mm have increased bone density in the preorbital and nasal bones; Among them, 6 is the number of the entry. If the morphological characteristics of entry 6 are met, it indicates that the species is the Ana pomfret; if the morphological characteristics of entry 6 are not met, jump to entry 5.

4. The method for identifying the Ana pomfret and its wild population and cultured population according to claim 1, characterized in that: Specifically, the primers used for PCR amplification include: FishF1 is shown in the sequence listing as SEQ ID 50: 5'-TCAACCAA CCACAAAGACATTGGCAC-3', FishR1 is shown in the sequence ID 51: 3'-TAGACTTCTGGGTGGCCAAAC AATCA-5', The amplification system was as follows: DNA template 2 μL, TaqPCR Mix 12.5 μL, ddH2O 6.5 μL, MgCl2 2 μL, primer FishF1 1 μL, primer FishR1 1 μL; The PCR amplification program is as follows: pre-denaturation at 95°C for 3 min, denaturation at 92°C for 45 s, annealing at 50°C for 45 s, extension at 72°C for 1 min, 35 cycles, and finally extension at 72°C for 10 min.

5. The method for identifying the Ana pomfret and its wild population and cultured population according to claim 1, characterized in that: Specifically, the sequencing result is compared with the COI sequence downloaded from the NCBI database, a phylogenetic tree is constructed, and it is determined whether the golden pomfret sample to be identified is an ana pomfret, including: Compare the amplified product sequence in the sequencing result with the COI sequence downloaded from the NCBI database to construct a phylogenetic tree; If in the phylogenetic tree, the amplified product sequence and the COI sequence corresponding to the ana pomfret are clustered into one branch, then the molecular marker identification technology indicates that the golden pomfret sample to be identified is the ana pomfret.

6. The method for identifying the Ana pomfret and its wild population and cultured population according to claim 2, characterized in that: Specifically, the morphometric data are standardized as follows: The back-of-eye head length PoHL, eye diameter ED, snout length SnL, maxillary length UjL, and maxillary height MaD were divided by the head length, and the other measurements were divided by the fork length for standardization.

7. The method for identifying the Ana pomfret and its wild population and cultured population according to claim 6, characterized in that: Specifically, the discriminant function equation is constructed based on the stepwise discriminant method, and the process includes: The standardized morphometric data were used to construct a statistical model using the Wilk-Lambda method. F The type distribution value is used as a benchmark. F When the value is greater than 3.84, the corresponding variable is introduced into the model. F When the value was less than 2.71, the corresponding variables were removed, and finally five standardized morphological measurement data were selected: the distance from the snout to the starting point of the pelvic fin / fork length was recorded as X1, the half-body height at the starting point of the pectoral fin / fork length was recorded as X2, the base length of the anal fin / fork length was recorded as X3, the length of the head behind the eye / head length was recorded as X4, and the eye diameter / head length was recorded as X5; The discriminant function equations established include the discriminant equations of wild populations and the discriminant equations of farmed populations. The discriminant equation of the wild population is as follows: F1=3504.230*X1-650.078*X2+2317.691*X3+1635.079*X4+1324.686*X5-1467.609; The discriminant equation of the breeding population is as follows: F2=3428.068*X1-596.227*X2+2251.891*X3+1661.023*X4+1373.527*X5-1462.

008.

8. The method for identifying the Ana pomfret and its wild population and cultured population according to claim 7, characterized in that: Specifically, determining the category of the golden pomfret sample in the anaconda according to the calculation result of the discriminant function equation includes: The F1 and F2 values ​​of the golden pomfret sample are calculated respectively, and the category corresponding to the discriminant equation to which the relatively higher value of the F1 and F2 values ​​belongs is taken as the category of the golden pomfret sample in the Ana pomfret.

9. A device for distinguishing wild and cultured populations of Ana pomfret, characterized in that: Includes the following: A sample acquisition module, used to obtain the golden pomfret sample to be identified; A data acquisition module, used for performing morphological measurement on the golden pomfret sample to be identified to obtain its morphological measurement data, and performing DR imaging on the golden pomfret sample to be identified to obtain its DR imaging result; A first judgment module is used to judge whether the golden pomfret sample to be identified is an ana pomfret based on the morphological measurement data and the DR imaging result in combination with a pre-established search table, and record the judgment result as a first judgment result; A DNA extraction module, used to extract DNA from the golden pomfret sample to be identified to obtain its DNA; A PCR amplification module is used to use the DNA as a template and primers to perform PCR amplification to obtain an amplified product, and to purify and sequence the amplified product to obtain a sequencing result; The second judgment module is used to compare the sequencing result with the COI sequence downloaded from the NCBI database, construct a phylogenetic tree, determine whether the golden pomfret sample to be identified is an ana pomfret, and record this judgment result as the second judgment result; A species determination module, for determining the golden pomfret sample to be identified as Ana pomfret when both the first judgment result and the second judgment result indicate that the golden pomfret sample to be identified is Ana pomfret; The category determination module is used to standardize the morphological measurement data and then input them into a pre-established discriminant function equation, and determine the category of the golden pomfret sample in the anaconda according to the calculation result of the discriminant function equation, wherein the category includes a cultured population and a wild population.

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