CN115521984A - Method for identifying nondestructive sexes in middle stage of chicken embryo by utilizing tibia heredity and transmission spectrum - Google Patents
Method for identifying nondestructive sexes in middle stage of chicken embryo by utilizing tibia heredity and transmission spectrum Download PDFInfo
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Abstract
The invention discloses a method for carrying out nondestructive male and female identification on chicken embryos by utilizing shank color inheritance and transmission spectra, wherein 1 insertion/deletion fragment which is completely linked with shank color inheritance is screened on a Z chromosome of a chicken genome for the first time, cocks inserted with homozygotes and hens of the deletion fragment are utilized for breeding, the shank color of the offspring hen embryos becomes dark at the age of 11-13 days, melanin is deposited, and the melanin-free deposition of the cock embryos is expressed as transparent light yellow, the change enables the transmission intensity of the eggs at the wavelengths of 716 +/-5 nm and 809 +/-5 nm to generate a distinguishable significant difference, and the male and female identification can be carried out at the embryo stage by utilizing the difference.
Description
Technical Field
The invention belongs to the technical field of livestock molecular markers and breeding, and particularly relates to a molecular marker linked with shank color, wherein the marker is 1 143bp insertion/deletion fragment located on a chicken Z chromosome, and can be used for nondestructive male and female identification in the middle stage of chicken embryo at a specific wavelength after being used for hybridization of chicken.
Background
The shank color of the chicken is an important quality character of the chicken, has a certain economic value and is mainly regulated and controlled by an inhibitor of dermatan (Id), and the site is discovered by Knox in 1935 for 100 years, but cannot be accurately positioned yet. Id is positioned on a Z chromosome of the chicken, and a self-distinguished male-female matched line with shank color can be theoretically generated by using the Id for inter-species hybridization, wherein the offspring cocks are all light-color shanks, and the hens are all dark-color shanks. However, the study found that the color of the shank cannot be used to judge the gender of the cock at birth, because some hens had light-colored shanks at birth and gradually changed into dark-colored shanks with the age of the day, which interfered with sex determination. This phenomenon is independent of feed nutrition, disease and growth environment and is called shank transformation. The Id locus of the shank color of the chicken is positioned and controlled, the origin of the shank color conversion phenomenon is explored, the shank color self-distinguishing male and female population can be promoted to be established, the application of molecular breeding in the domestic chicken industry is promoted, and genetic resources are enriched.
The shank color of chicken is derived from skin color, and the variety of chicken shank color is various, and the chicken shank color can be roughly divided into dark shank and light shank, which are determined by the existence of lutein in the epidermis layer and melanin in the dermis layer: the deposition of melanin in the dermis appears as a dark shin, whereas it appears as a light shin. Studies by Knox on chicken shank in 1935 predicted comprehensively the loci that affect shank. He believes that there are mainly 6 loci affecting the shank phenotype, 3 of which are located on autosomes, namely extended black E (extended black), yellow legs W (yellow legs) and dominant white I (dominant white), and 3 are located on sex chromosome Z, namely dermal melanin inhibitor Id (epidermal mean), streak B (seat-lined gene) and recessive yellow (seat-lined yellow skin). The Id, W and E loci play a determining role in shank, the rest play a regulating role. The Id locus controls the formation and phenotype of a chicken dark shin, when a chicken sex chromosome Z carries an Id gene, the deposition of the melanin of the shin is inhibited, the phenotype of the chicken shin is a yellow shin or a white shin or other light shin, when the Id gene is pure, the deposition function of the melanin is normal, and the phenotype of the chicken shin is a dark shin; the W/W locus mainly controls the deposition of the pigment of the control epidermis, no lutein deposits and shows white when W exists, and lutein deposits and shows yellow when W is recessive homozygous; the B locus is characterized in that the feather color has transverse stripes, while the B without the transverse stripes has no inhibition or promotion effect on the deposition of the black pigment of the shank color, namely the chicken shank color without the transverse stripes is not influenced by the reed flower gene; the recessive yellow Y locus has a certain dilution effect on epidermal melanin when being recessive homozygous, and the corresponding allele Y dominant does not influence the shank phenotype; the dominant white I locus inhibits the deposition of the melanin of the foot shin and is the feather presenting white feather; chicken feathers appear black in the presence of extended black E, and shins may still appear black when Id is expressed. Therefore, the shank color is an extremely complex quality character regulated by multiple genes.
In order to understand the shank character more comprehensively and deeply, people carry out genetic localization and functional research on a plurality of shank regulation gene loci. In 1988, bitgood reported that the sites B, id and y were linked on the Z chromosome, with the linear relationship being B-Id-y, where B was located on the long arm of the Z chromosome away from the centromere and Id was 13.7cM from the B site. The Liuwei utilizes a physical map to position a melanin inhibiting gene at the tail end of a Z chromosome, and is 20cM away from ACO 1. Dorshorst uses the F2 segregating population to localize the chicken dermal melanin inhibitor within 5.2Mb of 67.1-72.3Mb of the Z chromosome using whole genome association analysis techniques. Tianming found linkage with the 71,945,632-73,291,823bp region in the Id family. Li et al found 3 SNPs significantly associated with the corium pigment inhibitor in the interval 78.5-79.2Mb (galGal 4) in 2014 using the Tibetan chicken 600K Affymetrix Axiom HD chip data, but none on a known gene, consistent with previous studies. In 2016, jin et al found a site on chromosome 24 that could quantify the shank trait in Korean native chickens. In 2017, the 79.02-79.44Mb interval on the Z chromosome was found to be significantly associated with the corilagin inhibitory gene in the Gushi chicken, and a candidate gene with GRAMD3 as Id was identified, but no variation associated with the shank color was found in the gene. No more elaborate reports are reported since then, and it can be seen from these studies that the Id locus is significantly associated with 72Mb to 79Mb of the Z chromosome in different varieties. The W site controls the deposition of cortical lutein, located in the autosome. It was found that point mutations in the BCDO2 gene inhibit the decomposition of carotenoids by BCDO2 enzyme, resulting in the production of yellow skin. The Barring trait reported by Dorshorst is controlled by CDKN2A/2B, which is the first report that CDKN2A is related to pigmentation function. In D1911, bateson and Punnett demonstrated that shank is a sex-linked inheritance using hybridization experiments. In 1932, john W.Macarthur also reported that the characters of dark and light shank, fast and slow feathers, and honeysuckle feathers in poultry are linked with gender. Before 1935, two views were held on the inheritance of shank: one considers that yellow shin is sexually linked, its genetic rule conforms to simple Mendel's Law, and yellow shin is dominant to cyan shin; the other is that the yellow shank is located in autosome and is recessive character for white shank. However, knox in 1935 proposed another hypothesis that yellow-shin individuals had dominant linked dark-shin inhibitory factors, whereas dark-shin individuals lacked expression of this gene. In 1979, mcGibbon reported that the green shin is sexually linked, lacking the expression of melanin inhibiting factor (Id). Zhang Xie, sunzui Rong and Yi Hua Gui, etc. also demonstrated that dark shank is a sex-linked inheritance in different chicken breeds. The above studies show that: shank color is a concomitant hereditary trait controlled by the melanin-inhibiting gene of chicken dermis.
The invention discovers that a 143bp insertion/deletion sequence on a Z chromosome is completely linked with an ID locus, is suspected to be causative mutation of the locus, and simultaneously eliminates the interference of a shank color transformation genotype, so that the identification of male and female by using the ID locus becomes practical, and the nondestructive identification of male and female can be carried out by using the transmission intensity of a transmission spectrum at different wavelengths when a chicken embryo grows to the age of 13-day old embryo, wherein the transmission intensity of the transmission spectrum is different between a dark shank color embryo (hen) and a bright yellowish shank color embryo (cock).
Disclosure of Invention
One of the purposes of the invention is to provide a molecular marker linked with the shank, the marker is located on 1 143bp insertion/deletion fragment on a Z chromosome of a chicken, a nucleotide sequence is shown as SEQ ID NO.1, the 143bp insertion/deletion is utilized to control the shank to be different, the insertion of the chicken is the dark shank, the deletion of the chicken is the light shank, the insertion homozygote of the cock is the dark shank, and the insertion/deletion heterozygote and the deletion homozygote are both expressed as the light shank.
The invention also aims to provide a method for carrying out nondestructive male and female identification on chicken embryos by utilizing shank color inheritance and transmission spectrum, which comprises the following steps:
(1) Utilizing PCR amplification, selecting 143bp inserted homozygous black-shin cock as male parent and 143bp deleted yellow-shin hen as female parent, performing artificial insemination, collecting hatching eggs, and hatching, wherein the nucleotide sequence of the inserted or deleted fragment is shown as SEQ ID NO. 1;
(2) Hatching the chick embryos to 13 days old, and collecting the spectral intensity of the chick embryos under the wavelength of 716 +/-5 nm or 809 +/-5 nm; at the wavelength of 716 +/-5 nm, the spectral intensity of a male individual is less than 5900, and the transmission intensity of a female individual is more than 6700; at a wavelength of 809 ± 5nm, the transmission intensity of male individuals is less than 5900, while the transmission intensity of female individuals is greater than 6300.
Further, the sequences of the PCR amplification primers of the insertion or deletion fragment in step (1) above are: f: ggtagagtgtgttgtgaga, R: ccaggattcccagttatgagt.
Compared with the prior art, the invention has the following advantages and beneficial effects:
testis in a male reproductive system of the chicken contracts into an abdominal cavity, penis is degenerated, the appearance characteristics of male and female parts are not obvious in the process of chicks, and male and female parts are identified by a second sex characteristic identification method, a 1-day-old identification method and in-embryo identification. The second sex characteristics identification method is an original identification method used in the poultry industry as the economic period of a backyard, and male and female sex characteristics can be identified as the hormone levels of male and female individuals are different and the second sex characteristics appear about one month as the day age of chicks increases; the 1-day-old identification method comprises the steps of performing anus turning identification by using morphological and histological subtle differences of reproductive protrusions and reproductive bulges within 12 hours of hatching of chicks, and performing identification by using feather color, feather speed, feather spot and the like of accompanying characters; the 1-day-old sex identification promotes the industrial development of poultry (male and female removal in laying hen breeding), brings animal welfare problems, and causes the waste of social resources because more than half of chicks (males) are killed after being out of shells. For sex determination of embryonated eggs, there are three methods: the Raman spectroscopy method, the hormone detection method and the hyperspectral measurement method have the advantages that the probability of large-scale industrial application is low due to the problems of high cost and low accuracy of opening eggshells, the hyperspectral measurement is researched by AAT and In Ovo HQ companies, the male and female are identified by using the difference of the plume color of the gold and silver plumes with the accompanying characters at the high spectral transmission intensity after 13 days of age, the program can be completely and automatically carried out, the accuracy is about 95 percent, the eggshells are not opened, the risks of pollution and embryo damage are avoided, any chemical substance is not needed, and the environment is protected. Because the highlight spectrum can only distinguish brown (the golden feather is the hen) amount, but can not distinguish the pattern, its precision can only reach about 95%, this is useless to large-scale production, 5% is the cock, the price of chick will reduce, make whole profit reduce more than 50%, for million laying hens, fifty thousand cocks mix wherein, the work load of choosing is big, and the cock is difficult to be utilized when can distinguishing, is difficult to handle.
The invention discovers that a 143bp insertion/deletion sequence on a Z chromosome is completely linked with an ID locus, is suspected to be causative mutation of the locus, finds a completely linked genotype of chick shank color transformation and non-transformation, eliminates interference of the shank color transformation genotype, and realizes self sex discrimination by using the ID locus. Meanwhile, according to offspring of mating with a preset genotype, when chick embryos grow to 11-13 days old, the black pigment of the shank of a hen is deposited, the shank of a cock is transparent and yellowish, and can be obviously distinguished by naked eyes, the nondestructive male and female identification can be carried out by utilizing the transmission intensity of a transmission spectrum at different wavelengths when the dark shank embryo (hen) is different from the transparent and yellowish shank embryo (cock), and the test result shows that the transmission intensity of the male and female embryoid eggs at a specific wavelength is very obvious difference in the period of 13 days old, for example, the transparent intensity of the male embryo is lower than 5900, the transmission intensity of the female embryo is higher than 6700 and the middle difference value is very large when the wavelength is 716 +/-5 nm. Compared with the method that the feather color of the gold and silver feathers and the hyperspectral measurement of the gold and silver feathers with the accompanying characters are combined after 13 days, the method is simpler and more convenient, only different wavelengths are used, and the method is more accurate, the accuracy of embryo and egg identification actually depends on the differentiation degree of genetic male and female embryo characteristics, the feather color of the gold and silver feathers is self-identified by female and male embryos mainly according to the feather color yellowness, and part of the feather color is limited by patterns, so that only the highlight spectrum of the yellowness can cause 5% of misjudgment, but according to the shank color, only transparent light yellow and the grey-black shank color of pigment deposition are used at 13 days, the middle difference of the transmission intensity is very large, and the expected accuracy can reach 99.99%. The utilization values of the embryo eggs after male and female identification are different, the embryo eggs of 12-13 days old can be used as raw materials of special food 'live beads', and the embryo eggs of 13 days old later lose the utilization value.
The invention is completed under the funding of national science fund (project number: 31772585), major animal and plant variety breeding and biological product research and development (project number: 2020ABA 016) of major science and technology in Hubei province.
Drawings
Figure 1 shows the GWAS analysis results.
Fig. 2 is a SNP manhattan plot associated with a black shank, with strongly correlated signals on chromosomes 2, 13, and 32, with the highest degree of signal correlation on chromosome 32.
FIG. 3 is a gel electrophoresis of the deletion fragment, wherein lane 1 is a black shank, 3-6 are yellow shank, and 2 is Marker.
Fig. 4 is a graph showing the difference between gray black shin (left) of female embryonated eggs and yellow shin (right) of male embryonated eggs.
FIG. 5 is a spectral intensity spectrum of 13 day old chick embryos.
Detailed Description
Example 1 development of molecular markers linked to shank color
(1) Whole genome association analysis and screening of black shank association sites
In order to obtain sites significantly associated with black shanks, 384 chicken individuals were genotyped on a 600K high density chip, and after quality control, the black shanks (76) were cast and the other shanks (286) were control, and genome wide association analysis was performed using FarmCPU, with the SNP significance threshold being set at 1.1e-07 (0.05/436875). The GWAS results show that there is a SNP site with a very high association to black shin at 78732319bp (galgal5.0) on chromosome 32, Z (P value = 9.54E-29), which is the T/C mutation (fig. 1, fig. 2). This site (rs 312851916) is also located on the first exon of LncRNA (LOC 107052436).
(2) Mining of associated sequence variations for suspected causal mutations
In order to discover possible causative mutations, candidate intervals (78.71-78.74 Mb) were selected near the SNP site for PCR amplification and re-sequencing, and the total amplification length was about 25000bp. By comparing the amplified genome sequences of the black shin and the yellow shin, a large sequence difference is found in the yellow shin and the black shin, and the results of verification of 64 black shin individuals and 18 yellow shin individuals randomly selected in the resource population (fig. 3) show that a 143bp fragment deletion exists in the yellow shin individuals, but no deletion exists in the black shin individuals, and the nucleotide sequence of the insertion/deletion fragment is shown as SEQ ID No. 1.
Example 2 method for identifying chicken embryos without damage to males and females in middle stage by utilizing tibia heredity and transmission spectrum
(1) Mating cock and hen
Performing artificial insemination by using PCR amplification (F: GGTAGAGGTGTGTTGTGAGA, R: CCAGGATTCCAGTTATGAGT) by selecting a homozygous cock with 143bp insertion as a male parent and a yellow-shin hen with 143bp deletion as a female parent, and collecting hatching eggs for hatching;
(2) Transmission spectrum intensity measurement of embryo ages of different days
Hatching the chick embryos to the ages of 11 days, 12 days, 13 days and 15 days, and collecting the full spectrum intensity;
(3) Identification of male and female individuals and fitting of spectral intensities
After the chicken embryo is hatched, sex identification of molecular biology and anatomy are carried out, then ovary and testis are observed for sex identification, the sex and the male sex are subjected to spectrum fitting with the spectral intensity, and the wavelength and the transmission intensity which can be used for identifying the sex of the embryo are selected. .
(4) Shank color distinction of 13-day-old male and female embryonated eggs
The reproductive system is observed after dissecting 13-day-old embryonated eggs to distinguish male and female, male individual grows symmetrically and is a pair of testicles, female individual develops in the left ovary and regresses in the right ovary. The shins of the female individuals used had pigment deposition, the shins became darker, while the shins of the male individuals used had a clear yellowish coloration, as shown in fig. 4. At a wavelength of 716 ± 5nm, the transmission intensity of a male individual is less than 5900, and the transmission intensity of a female individual is greater than 6700; at a wavelength of 809 ± 5nm, the transmission intensity of the male individuals is below 5900, while the transmission intensity of the female individuals is above 6300. Therefore, when the chicken embryos bred in a matched way are 13 days old, the transmission intensity of the wavelengths of 716 +/-5 nm and 809 +/-5 nm can be used for carrying out nondestructive male and female identification on the embryos.
Sequence listing
<110> university of agriculture in Huazhong
<120> method for identifying chicken embryo middle-stage lossless male and female by utilizing shank color heredity and transmission spectrum
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 143
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
gtcgtaaata gaatttttta agccatggaa gtgggaagaa tacaagcaga aagtaatatt 60
ttccatacct tttcaggatg ttttcttaga atagcattca taacataact taagacatta 120
actgtcacca aaccccaggg agt 143
Claims (3)
1. The molecular marker linked with the shank color is characterized in that the molecular marker is positioned on a Z chromosome, and the nucleotide sequence is shown as SEQ ID NO. 1.
2. The method for carrying out nondestructive male and female identification on chicken embryos by utilizing shank color inheritance and transmission spectrum is characterized by comprising the following steps:
(1) Utilizing PCR amplification, selecting 143bp inserted homozygous black-shin cock as male parent and 143bp deleted yellow-shin hen as female parent, performing artificial insemination, collecting hatching eggs, and hatching, wherein the nucleotide sequence of the inserted or deleted fragment is shown as SEQ ID NO. 1;
(2) Hatching the chick embryos to 13 days old, and collecting the spectral intensity of the chick embryos under the wavelength of 716 +/-5 nm or 809 +/-5 nm; at the wavelength of 716 +/-5 nm, the spectral intensity of a male individual is less than 5900, and the transmission intensity of a female individual is more than 6700; at a wavelength of 809 ± 5nm, the transmission intensity of the male individual is less than 5900, while the transmission intensity of the female individual is greater than 6300.
3. The method according to claim 2, wherein the sequences of the PCR amplification primers inserted or deleted in the fragment in step (1) are: f: ggtagagtgtgttgtgaga, R: CCAGGATTCCAGTTATGAGT.
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