Molecular identification method for homologous, near-source and hybrid species of leiocassis longirostris

Abstract

The invention discloses a molecular identification method for homologous, near-source and hybrid species of leiocassis longirostris, and belongs to the technical field of fish genetic breeding. The method comprises the following steps: carrying out sequence comparison by virtue of 15 mitochondrial gene complete sequences of a plurality of existing species in a GenBank database, and calculating a similarity percentage index and a sorting index of a gene corresponding to a to-be-detected species in comparison of the plurality of species; finally, the method can accurately judge whether the to-be-detected fish belongs to a plurality of possible species such as homologous species, close species and related hybridized species of the leiocassis longirostris at one time, and a basic identification method is provided for actual production of genetic breeding and cultivation of the homologous species, the close species and the related hybridized species of the leiocassis longirostris.

Description

Technical Field

[0001] The invention belongs to the technical field of fish genetic breeding, and more specifically, relates to a species gene detection technology of a long-snout catfish in molecular biology. Background Art

[0002] The long-snouted catfish (Leiocassis longirostris) belongs to the Siluriformes, Bagridae, and Catfish. Because of its long snout, it is called the long-snouted catfish, commonly known as catfish, Jiangtuan, Feituo, Huaiwangyu, etc. The long-snouted catfish is a bottom-water fish that often lives in areas with slow currents, deep water, and many rocks. It is widely distributed in the Liaohe, Huaihe, Yangtze, Minjiang, and Pearl River systems in eastern China and western North Korea. The long-snouted catfish has no scales or intermuscular spines, and its meat is very delicious and rich in nutrition. These characteristics make it deeply loved by people. There is a saying among the people that "If you don't eat Jiangtuan, you don't know the taste of fish." The long-snouted catfish is a large economic fish with a fast growth rate. The body length of the long-snouted catfish can reach about 1 meter. The minimum age to reach sexual maturity is 3 years old, and it is generally 4-5 years old.

[0003] Yellow catfish (Tachysurusfulvidraco) belongs to the genus Pelteobagrus of the family Catfish. The body length of yellow catfish is 180-250mm. It is mainly distributed in China, North Korea, Laos, Vietnam and southeastern Siberia, Russia. The sexual maturity age of yellow catfish is generally 2 years old. It is 1-2 years old in South China, 2 years old in Central China, and 2-3 years old or more in the northern region. Yellow catfish (Tachysurus vachelli) is a fish of the genus Pelteobagrus of the family Catfish. It has an elongated body and males are generally larger than females. Yellow catfish is mainly distributed in the main and tributary rivers of the Yangtze River Basin in China and in the lakes connected to the Yangtze River. It is larger than yellow catfish, with a maximum length of 300 mm. Pseudobagrus ussuriensis belongs to the family Pseudobagrus and is widely distributed in the waters of Heilongjiang, Wusuli, Nenjiang, Songhua, and Pearl rivers, as well as Hongze and Taihu lakes. The body length of Pseudobagrus ussuriensis ranges from 60 to 450 mm, and the average individual weighs 0.5 to 2 kg, with the largest individual weighing more than 5 kg. It reaches sexual maturity at 3 years old.

[0004] Hybrid breeding is one of the most traditional methods in aquatic genetic breeding. About 1 / 3 of the new aquatic species approved use hybridization. A lot of research has been done on the hybridization of catfish: based on the characteristics of longnose catfish, such as fast growth and large male individuals, yellow catfish has become an important freshwater fish breeding object due to its high survival rate and strong adaptability. For example, longnose catfish has the characteristics of fast growth and high nutritional value compared to yellow catfish. In order to integrate the advantages of the two fish, the hybrid F1 generation obtained by yellow catfish (♀) and longnose catfish (♂) has the advantages of fast growth and strong resistance to adversity, and has become a new strain of yellow catfish. For example, the yellow catfish is closely related to the yellow catfish, but its individual size is much larger than that of the yellow catfish. Therefore, researchers selected the yellow catfish as the male parent and hybridized it with the yellow catfish to produce a new variety "Huangyou No. 1", which has morphological characteristics similar to those of the yellow catfish, and has a better growth rate and survival rate than the yellow catfish, and is favored by farmers. Compared with the Ussuri catfish and the yellow catfish, the longnose catfish is large in size and is an excellent male parent material for the improvement of the Ussuri catfish. The hybrid offspring obtained by hybridizing the Ussuri catfish as the female parent and the longnose catfish as the male parent showed good growth advantages and could reach the market size of the one-year-old Ussuri catfish in half a year.

[0005] In addition, the hybridization method also involves which species each parent chooses as the paternal parent, which results in the morphological characteristics of the hybrid offspring being different from or similar to those of the parents. How to identify these various hybrid species, homologous and closely related species? Identification by morphological methods alone will result in low accuracy, affecting breeding production management. Molecular identification has always been favored for its high accuracy. There have been some reports on molecular identification patents for hybrid varieties related to long-nosed catfish. Representative patents are as follows:

[0006] [1] Luo Hui, Ye Hua, Zhou Jian, et al. A molecular marker for identifying the genetic sex of long-nosed catfish and its application [P]. Chongqing: CN202210030085.2, 2023-08-01.

[0007] [2] Ye Huan, Ruan Rui, Li Chuangju, et al. A male sex-specific molecular marker for long-nosed catfish, its amplification primers and genetic sex identification method [P]. Hubei Province: CN202111232187.4, 2022-06-17.

[0008] [3] Mei Jie, Liao Qian, Gong Gaorui, et al. Primers, methods and kits for identifying yellow catfish, longnose catfish and hybrids [P]. Hubei Province: CN202211438645.4, 2023-04-11.

[0009] [4] Wang Tao, Wang Lingling, Peng Ye, et al. A method for identifying yellow catfish, longnose catfish and their hybrid F1 generation and their specific primers [P]. Jiangsu Province: CN202211448943.1, 2023-03-28.

[0010] However, the above patents are often only for the molecular identification of two fish parents and their hybrid F1 species, and cannot be used for more other homologous or closely related species, especially for different geographical populations, and the differences between hybrids of different male and female parents cannot be distinguished. In view of this, we specifically focus on the more complex situation of long-snouted catfish and develop a method based on the comparison of existing nucleic acid data resources of long-snouted catfish-related species. This method is more detailed and accurate in classification and identification. Summary of the invention

[0011] 1. Technical problem to be solved by the invention

[0012] Through the application of this method, various types of species closely related to the long-snouted catfish can be identified, including homologous species, closely related species and various possible hybrids.

[0013] 2. Technical solution

[0014] In order to solve the above technical problems, the specific technical solution steps provided by the present invention are as follows:

[0015] (1) For the long-snout catfish-related species to be tested, if they are alive, total DNA can be extracted by sampling blood from the tail with minimal damage. For non-living species, total DNA can be directly extracted from the muscle or liver tissue of the fish. The mitochondrial genome of the species to be tested can be obtained directly by shotgun first-generation sequencing or second-generation sequencing. The complete sequences of 15 mitochondrial genes including 12S rRNA, 16S rRNA, ND1, ND2, ND3, ND4, ND4L, ND5, ND6, COI, COII, COIII, ATP6, ATP8 and Cytb genes can be further located and isolated.

[0016] (2) Select the longnose catfish related species available in GenBank, including longnose catfish, yellow catfish, yellow catfish (female) x longnose catfish (male) hybrid, longnose catfish (female) x yellow catfish (male) hybrid, yellow catfish (female) x longnose catfish (male), yellow catfish (female) x yellow catfish (male) and Pseudomonas ussuri x yellow catfish, etc., in the Yangtze River or Huaihe River basin, with a total of no less than 10 species. Obtain their mitochondrial genome sequence, and then directly extract the complete sequence of the 15 genes mentioned above.

[0017] (3) Construct the fasta file of the full nucleic acid sequence of each gene of the species to be tested and the species that can be identified, use Clustal X1.8 software to perform local sequence alignment, obtain the alignment results of each gene of the species to be tested and the known species, and extract their similarity percentage index and ranking index.

[0018] Another solution for this step is to use online BLAST direct comparison to implement it, put the 15 gene nucleic acid sequences of the species to be tested into the Enter Query Sequence sequence area of ​​the BLASTn interface of GenBank, select more than 10 species related to the long-snouted catfish in the Organism option of Choose SearchSet, and obtain the similarity percentage index and ranking index of the online comparison from the Blast results that are run subsequently.

[0019] (4) Combined analysis of the two indicators: the similarity percentage indicator should have 90% of the gene sequences with a maximum similarity greater than 99%, and a known species sequence position with a frequency of occurrence greater than 90% in the first sequence. We believe that this known species may be the same population, the same species, or the same hybrid as the species to be identified.

[0020] The following two points need to be emphasized:

[0021] (1) In most cases, the identification result of this method should be the same population or species as the species to be tested. The actual identification result of the species to be tested is a homologous, closely related or hybrid species of the long-snouted catfish, which is determined only by whether the known species to which it is compared is a homologous, closely related or hybrid species of the long-snouted catfish.

[0022] (2) If the identification results show that the known species and the species to be tested do not meet the conditions of being the same species (the similarity percentage index should have 90% of the gene sequences with a maximum similarity greater than 99%, and there is a known species ranked first with a ranking frequency greater than 90%), then the species most likely to be identified is a homologous, closely related, or hybrid species of the known species ranked first. This means that the relationship between the species to be tested and any of the more than 10 species provided is not the same species.

[0023] 3. Beneficial effects

[0024] Compared with the prior art, the technical method provided by the present invention has the following significant effects:

[0025] (1) Improvement in the number of species identified. The present invention conducts comparative analysis of species to be tested based on 15 mitochondrial genes of more than 10 species, which can improve the existing molecular identification technology of long-snouted catfish-related species from 2-3 species to more than 10 species.

[0026] (2) Improvement in identification accuracy and breadth. The present invention is based on more than 10 identifiable species known in the GenBank database. These species may involve different geographical populations or populations of the same species (homologous species), or may involve closely related species or hybrids between closely related species. Among them, the existing closely related species of long-snouted catfish include at least three species such as yellow catfish, yellow catfish and Ussuri catfish. Therefore, compared with the prior art that can only identify a pair of clear closely related species and their hybrids, the present invention has a higher accuracy in species identification: it can involve different populations of homologous species, and can identify the exchange of parental species of two closely related species and their hybrids; it has a wider breadth: the identified species are not necessarily known and clear different closely related species, but can also be other species related to or close to the long-snouted catfish. Specific implementation methods

[0028] In order to further understand the content of the present invention, the present invention is described in detail in conjunction with implementation cases.

[0029] Example 1

[0030] In this implementation case, the species related to the long-snouted catfish to be tested is a live fish weighing about 100g, which looks very similar to the long-snouted catfish. It needs to be accurately identified to determine whether it is a hybrid of the long-snouted catfish. If it is a long-snouted catfish, it is also necessary to identify whether it belongs to the Yangtze River species or the Huaihe River Basin. The specific identification steps are as follows:

[0031] (1) For living species related to long-snouted catfish, total DNA was extracted by blood sampling from the tail and sequenced using next-generation sequencing to obtain the mitochondrial genome. The complete sequences of 15 mitochondrial genes, including 12S rRNA, 16S rRNA, ND1, ND2, ND3, ND4, ND4L, ND5, ND6, COI, COII, COIII, ATP6, ATP8 and Cytb genes, were located and isolated.

[0032] (2) Ten existing longnose catfish-related species in GenBank were selected, including longnose catfish, yellow catfish (female) x longnose catfish (male) hybrid, longnose catfish (female) x yellow catfish (male) hybrid, yellow catfish (female) x longnose catfish (male), yellow catfish (female) x yellow catfish (male) and other species (as shown in Table 1 below), and the complete sequences of 15 genes corresponding to their mitochondrial genomes were obtained.

[0033] Table 1 Information on 10 homologous, closely related or hybrid species of Longnose Catfish used to identify the species to be tested in Example 1

[0034] Serial number Species Information Accession Number Possible origin 1 Longnose catfish MK458524 Huaihe River 2 Longnose catfish NC_014586 Yangtze 3 Yellow catfish (female) x longnose catfish (male) MF583740 Nanjing, self-hybridization 4 Yellow catfish (female) x longnose catfish (male) NC_036665 Probably also Hubei Jingzhou 5 Longnose catfish (female) x Yellow catfish (male) NC_030628 Jingzhou City, Hubei Province 6 Yellow catfish (female) x longnose catfish (male) MW841469 Location unknown 7 Yellow catfish (female) x Yellow catfish (male) NC_026544 Nanjing Fisheries Research Institute 8 Pelteobagrus vachelli (female) x Pelteobagrus fulvidraco (male) MN365880 Anhui Academy of Agricultural Sciences 9 Ussuri catfish x yellow catfish MZ343379 Anhui Academy of Agricultural Sciences 10 Yellow catfish NC_015888 Jingzhou, Hubei, is located in the middle reaches of the Yangtze River

[0035] (3) The fasta files of the complete nucleic acid sequences of each gene of the 11 species were constructed, and the local sequence alignment was performed using Clustal X 1.8 software to obtain the alignment results of each gene of the tested species with the known species, and to extract their similarity percentage index and ranking index.

[0036] (4) Through calculation, it was found that 90% of the gene sequences of the tested species and the 10 known species had a maximum similarity percentage greater than 99%. There were two species, namely, Longnose Catfish (MK458524) and Longnose Catfish (NC_014586), and the known species Longnose Catfish (MK458524) ranked first in the ranking index with a ranking frequency of 100%. Therefore, it was determined that the tested species was most likely the Longnose Catfish species produced in the Huaihe River Basin.

[0037] Example 2

[0038] In this implementation case, the long-snout catfish-related species to be tested is a piece of fish tissue, which needs to be identified. The specific identification steps are as follows:

[0039] (1) Total DNA was directly extracted from fish tissues and sequenced using the shotgun method to obtain the mitochondrial genome of the tissue to be tested, and the corresponding 15 mitochondrial gene sequences were located and isolated.

[0040] (2) 10 existing long-snout catfish related species in GenBank were selected, including long-snout catfish, yellow catfish, yellow catfish (female) x long-snout catfish (male) hybrid, etc. The details are shown in Table 2 below for the next step of online analysis.

[0041] Table 2 Information on 10 homologous, closely related or hybrid species of Longnose Catfish used to identify the species to be tested in Example 2

[0042] Serial number Species Information Accession Number Possible origin 1 Longnose catfish MK458524 Huaihe River 2 Yellow catfish (female) x longnose catfish (male) MF583740 Nanjing, self-hybridization 3 Yellow catfish (female) x longnose catfish (male) NC_036665 Probably also Hubei Jingzhou 4 Longnose catfish (female) x Yellow catfish (male) NC_030628 Jingzhou City, Hubei Province 5 Yellow catfish (female) x longnose catfish (male) MW841469 Location unknown 6 Yellow catfish (female) x Yellow catfish (male) NC_026544 Nanjing Fisheries Research Institute 7 Pelteobagrus vachelli (female) x Pelteobagrus fulvidraco (male) MN365880 Anhui Academy of Agricultural Sciences 8 Ussuri catfish x yellow catfish MZ343379 Anhui Academy of Agricultural Sciences 9 Yellow catfish NC_015888 Jingzhou, Hubei, is located in the middle reaches of the Yangtze River 10 Yellow catfish MH192350 Yancheng City, Jiangsu Province, lower reaches of the Yangtze River

[0043] (3) Online BLAST was used to directly perform the comparison. The nucleic acid sequences of the 15 genes of the species to be tested were placed in the Enter Query Sequence region of the BLASTn interface of GenBank. The 10 species related to the long-snouted catfish listed in Table 2 were selected in the Organism option of Choose Search Set. The similarity percentage index and ranking index of the online comparison were obtained from the Blast results of the subsequent run.

[0044] (4) The gene sequences of the tested species with 90% similarity percentage index with the known 10 species were obtained by calculation. The maximum similarity percentage index was 100%, and only Pelteobagrus fulvidraco (female) x Longnose Catfish (male) (NC_036665), and the known species Longnose Catfish (MK458524) ranked first in the ranking index and the ranking frequency was also 100%. Therefore, it was judged that the species corresponding to the tested tissue was most likely a hybrid species of the female Pelteobagrus fulvidraco and the male Longnose Catfish.

[0045] Example 3

[0046] In this implementation case, we will select reference sequences in GenBank that are likely to be known identifiable species, and construct a similarity comparison matrix for them, attempting to analyze their similarity percentage indicators and ranking indicators.

[0047] (1) The 11 known species mentioned in Examples 1 and 2 were selected to analyze the 15 corresponding mitochondrial genes.

[0048] (2) Taking the long-snouted catfish (MK458524) as the object to be analyzed, the similarity percentage index and ranking index were calculated, and the results obtained are shown in Table 3 (due to the large amount of data, only three gene examples are given here).

[0049] Table 3 Calculation results of two indicators for three genes of 10 species in Example 3

[0050]

[0051] (3) Directly analyze the similarity percentage index between the 10 known identified species and determine whether the similarity percentage is specific between species and genes. For species specificity, the results of pairwise comparison of the 10 species were 45, as shown in Table 4. Among them, it was found that the number of genes obtained by comparison of 25, 31, 33, 36, 37, and 40 was the largest, with the highest being more than 65%. Therefore, when the identification results involve the species corresponding to the above accession numbers, the threshold of the similarity index needs to be increased to ensure the credibility of the results.

[0052] Table 4 shows the percentage statistics of 100% similarity between 15 genes of 10 species in Example 3

[0053]

[0054]

[0055] From the gene level, the frequency of genes with a similarity percentage index value of 100% in the 45 pairs of comparisons between the 10 species was calculated. The results are shown in Table 5. It was found that the lowest frequencies were 16S rRNA and ND4 genes. Therefore, when identifying related species, these two genes are more likely to be used as an important basis for indicating that the species are the same species.

[0056] Table 5 shows the frequency of genes in Example 3 showing that 15 genes in 10 species have 100% similarity between each other.

[0057]

Claims

1. A molecular identification method for homologous, closely related and hybrid species of long-snouted catfish, characterized in that By using the full sequences of 15 mitochondrial genes of multiple known identifiable species in the GenBank database, and comparing them with the mitochondrial genes of the species to be tested, the similarity percentage index and ranking index of the corresponding genes of the species to be tested in the comparison of multiple known species are calculated. Finally, it is possible to accurately determine at one time whether the fish to be tested belongs to the homologous, closely related, or related hybrid species of the long-snouted catfish.

2. The method for molecular identification of homologous, closely related and hybrid species of long-snouted catfish according to claim 1, characterized in that The proposed molecular identification method involves identifiable species including the homologous species Huaihe population of longnose catfish and the Yangtze population of longnose catfish, closely related species including yellow catfish, yellow catfish walleye, and pseudocatfish of ussuri, hybrid species including yellow catfish (female) x longnose catfish (male) hybrid, longnose catfish (female) x yellow catfish (male) hybrid, yellow catfish walleye (female) x longnose catfish (male), yellow catfish (female) x yellow catfish walleye (male) and pseudocatfish of ussuri x yellow catfish, etc.

3. The method for molecular identification of homologous, closely related and hybrid species of long-snouted catfish according to claim 1, characterized in that The proposed molecular identification method involves 15 mitochondrial genes including 12S rRNA, 16S rRNA, ND1, ND2, ND3, ND4, ND4L, ND5, ND6, COI, COII, COIII, ATP6, ATP8 and Cytb, and only nucleic acid sequences are selected for sequence analysis.

4. The method for molecular identification of homologous, closely related and hybrid species of long-snouted catfish according to claim 1, characterized in that It is recommended to use the offline Clustal software or the online BLAST software for sequence alignment.

5. The method for molecular identification of homologous, closely related and hybrid species of long-snouted catfish according to claim 1, characterized in that The similarity percentage index between the tested species and known species refers to the similarity percentage obtained by comparing each gene of the tested species with the corresponding genes of all known species. The number of known species sequences is no less than 10, and the number and specific sequences must be determined before the comparison analysis and should not be changed.

6. The method for molecular identification of homologous, closely related and hybrid species of long-snouted catfish according to claim 1, characterized in that The comparison and ranking index between the species to be tested and the known species refers to the ranking of each gene of the species to be tested with the corresponding genes of all known species from high to low similarity. Each gene will obtain the ranking position of the known species, and the ranking positions with the same similarity have the same number.

7. The method for molecular identification of homologous, closely related and hybrid species of long-snouted catfish according to claim 3, characterized in that When the percentage of 16S rRNA and ND4 gene similarity between the tested species and the known species was determined to be 100%, except for the top known species involving Pelteobagrus fulvidraco (MH192350), Pelteobagrus fulvidraco (NC_015888), Pelteobagrus fulvidraco (female) x Longnose catfish (male) (NC_036665), Pelteobagrus fulvidraco (female) x Longnose catfish (male) (MF583740), and Pelteobagrus fulvidraco (female) x Pelteobagrus vachelli (male) (NC_026544), they are more likely to be the same species.

8. The method for molecular identification of homologous, closely related and hybrid species of long-snouted catfish according to claim 5, characterized in that In most cases, the maximum value of the similarity percentage index to determine that the species to be tested is the same species as the known species should be 90% of the gene sequence similarity greater than 99%; the known species with the highest ranking are Pelteobagrus fulvidraco (female) x Longnose catfish (male) (MF583740), Pelteobagrus fulvidraco (MH192350), Pelteobagrus fulvidraco (NC_015888), Pelteobagrus vachelli (female) x Pelteobagrus fulvidraco (male) (MN3 65880), Pelteobagrus vachelli (female) x Longnose Catfish (male) (MW841469), Pelteobagrus vachelli (female) x Longnose Catfish (male) (NC_036665), and Pelteobagrus vachelli (female) x Pelteobagrus vachelli (male) (NC_026544), the maximum value of the similarity percentage index for determining that the tested species and the known species are the same species should be increased to 90%, and the gene sequence similarity is preferably equal to 100%.

9. The method for molecular identification of homologous, closely related and hybrid species of long-snouted catfish according to claim 6, characterized in that The ranking index for determining that the species to be tested is the same species as the known species should have more than 90% of the ranking positions be the first.

10. A molecular identification method for homologous, closely related and hybrid species of long-snouted catfish according to claim 8 and claim 9, characterized in that The similarity percentage index and ranking index to determine whether the species to be tested is the same species as the known species must be met at the same time.

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