AU2021106292A4 - Rapid, accurate and noninvasive method for sex identification of dromaius novaehollandia nestlings - Google Patents
Rapid, accurate and noninvasive method for sex identification of dromaius novaehollandia nestlings Download PDFInfo
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Abstract
The present invention provides a rapid, accurate and noninvasive method for sex
identification of Dromaius novaehollandia nestlings and relates to the technical field of
nestling sex identification. In the present invention, by analyzing sequence differences in Z
chromosome and W chromosome in newly announced Dromaius novaehollandia genome, a
group of sex identification primers (Emu-SD1, Emu-SD2 and Emu-SD3) are designed. These
primers target a 15-bp insertion/deletion region on the W and Z chromosomes and produce
two amplicons of 166bp and 428bp for the W chromosome and a single amplicon of 409bp
for Z chromosome , respectively. The rapid, accurate and noninvasive method for sex
identification of Dromaius novaehollandia nestlings comprises the following Dromaius
novaehollandia sex identification steps of Dromaius novaehollandia feather sampling, DNA
extraction, primer design, PCR reaction and agarose electrophoresis. The sex of Dromaius
novaehollandia is identified with PCR and electrophoresis, and samples for identification are
a small quantity of feathers rather than blood and meat, so that the Dromaius
novaehollandia is not hurt, attacks to a human from the Dromaius novaehollandia are also
reduced in the catching and sampling process at the same time, and the rapid, accurate and
noninvasive method for sex identification of Dromaius novaehollandia nestlings is a
noninvasive identification technology. By optimizing a genome DNA extraction method and
a PCR annealing temperature, extraction of trace DNA and PCR amplification in the feathers
are achieved; and the specificity of the sex identification primers is good, an electrophoretic
band for sex identification is clear and bright, and the present invention is an accurate and
reliable identification technology.
Description
The present invention relates to the technical field of nestling sex identification and provides a rapid, accurate and noninvasive method for sex identification of Dromaius novaehollandia nestlings.
Emu, (Dromaius novaehollandiae), flightless bird of Australia that is the second largest living bird:the Dromaius novaehollandia is widely farmed in many countries due to important economic value. The Dromaius novaehollandia has very high nutritive value and contains proteins, vitamins, heme iron and creatine with relatively low contents of fat and cholesterol. The Dromaius novaehollandia contains a great quantity of polyunsaturated fatty acids and antioxidant and has the strong efficacy of inflammation resistance, cholesterol lowering, transdermal permeation promotion and
the like. The Dromaius novaehollandia attracts much attention due to the nutritive value and a medicinal value and is introduced into all the countries to be largely farmed.
The Dromaius novaehollandia is sexually homomorphic, and sex identification is difficultly conducted on the Dromaius novaehollandia aged with 16 months through features of appearance. Dromaius novaehollandia sex identification can help feeders provide more suitable management and feeding to the Dromaius novaehollandia based on different sex demands. The Dromaius novaehollandia is long in growth cycle and high in feeding cost, sex identification may shorten the feeding time for nontarget sex
individuals, and thus a great quantity of feeding cost is saved. With development of a genome sequencing and nucleic acid detection technology, molecular detection technologies, such as PCR, provide a tool to early rapid identification of the sex of the
Dromaius novaehollandia. The sex of the Dromaius novaehollandia is decided by combination of sex chromosomes. Male Dromaius novaehollandia contains two Z chromosomes, being of ZZ type; while female Dromaius novaehollandia contains one Z chromosome and one W chromosome, being of ZW type, and the W chromosome is unique to the female Dromaius novaehollandia, so that the sex of the Dromaius novaehollandia may be identified by detecting a sequence difference between the Z chromosome and the W chromosome.
Existing detection methods of determining the sex of the Dromaius novaehollandia by detecting the sequence difference between the Z chromosome and the W chromosome mostly conduct detection by detecting blood and meat of the Dromaius novaehollandia, may cause hurts on the Dromaius novaehollandia and easily causes attack to a human from the Dromaius novaehollandia when detection samples are obtained so as to easily cause a worker to be hurt.
Fig. 1 is a schematic view of three primers targeted region in W and Z chromsomes for sex identification of Dromaius novaehollandia nestlings of the present invention. Query for W chromosome region, Sbjct for Z chromosome region.
Fig. 2 is a schematic view of three primers in a rapid, accurate and noninvasive method for sex identification of Dromaius novaehollandia nestlings of the present invention.
Fig. 3 is a schematic view of an identification result of a rapid, accurate and noninvasive method for sex identification of Dromaius novaehollandia nestlings of the present invention at different annealing temperatures(42C and51°C).
Fig. 4 is a schematic view of a PCR result of a rapid, accurate and noninvasive method for sex identification of Dromaius novaehollandia nestlings of the present invention for 16 Dromaius novaehollandia samples.
In order to more clearly understand the above objective, characteristics and advantages of the present invention, the present invention is further described in combination with the drawings and the embodiments below. It should be noted that the embodiments of the present application and the characteristics in the embodiments can be combined with other another without conflict.
In the following description, numerous specific details are set forth to facilitate full understanding of the present invention. However, the present invention may also be implemented in ways other than those described here, and therefore, the present invention is not limited to the specific embodiments of the specifications disclosed below.
Embodiment: referring to Figs. 1-4, the present invention provides a rapid, accurate and noninvasive method for sex identification of Dromaius novaehollandia nestlings which comprises the following steps of:
Si: enabling a Dromaius novaehollandia nestling to wear a foot ring marker marked with a number in a Dromaius novaehollandia farm, then conducting sampling, picking 5 feathers from the back and the tail of each Dromaius novaehollandia nestling by a sampler wearing disposable gloves during sampling, overturning and wrapping the feathers with the gloves after feather picking each time, clamping the gloves with a clip, then putting the feathers in a low-temperature refrigerating box with the gloves together, and sending the low-temperature refrigerating box to a laboratory for detection;
S2: shearing roots of 0.5cm distant of two feathers, then cutting the sheared portions into pieces, putting the pieces in a 1.5ml centrifugal tube equipped with 180pl of tissue lysis solution TL, adding 20pl of protease k (20mg/ml), and immediately conducting vortex oscillation for full and uniform mixing to obtain a lysate;
S3: putting the lysate in a water bath of 55°C overnight until tissues are digested thoroughly, then adding 200pl of binding solution CB, immediately conducting vortex oscillation for full and uniform mixing, still standing a product for 10min at 70°C, adding 100pl of isopropanol after natural cooling, and immediately conducting vortex oscillation for full and uniform mixing to obtain a mixture;
S4: sucking the mixture with a Iml gun head, adding the mixture in an adsorption column AC, putting the adsorption column in a collection tube for centrifugation for s at 13000rpm, pouring out waste liquid in the collection tube, adding 200pl of inhibitor removing solution IR for centrifugation for 30s at 12000rpm, pouring out waste liquid, adding 700pl of rinsing solution WB for centrifugation for 30s at 12000rpm, pouring out waste liquid, adding 500pl of rinsing solution WB for centrifugation for 30s at 12000rpm, and pouring out waste liquid;
S5: putting the adsorption column AC back into the empty collection tube for centrifugation for 2min at 13000rpm, removing the rinsing solution, then taking out the adsorption column AC, putting the adsorption column AC in a clean centrifugal tube, adding 50pl of elution buffer EB at the middle part of an adsorption film, and putting the centrifugal tube at a room temperature for 3-5min, wherein before use of the elution buffer, the centrifugal tube may be put in water of 65-70°C for preheating;
S6: then conducting centrifugation for1min at 12000rpm, then discarding the adsorption column AC, keeping the collection tube containing a DNA solution, determining a concentration of DNA with a Nanodrop ND-2000 super trace nucleic acid-protein determinator from America to obtain extracted genome DNA, and storing the extracted genome DNA at -20°C serving as a template for subsequent PCR;
S7: deciding the sex of Z Dromaius novaehollandia with a combination form of a Z sex chromosome and a W sex chromosome with the male Dromaius novaehollandia being of ZZ type and female Dromaius novaehollandia being of ZW type. By analyzing the Z chromosome sequence (CM027974.1) and a W chromosome sequence (CM027973.1) published by NCBI database in 2020, finding out a difference short sequence for the Z chromosome and the W chromosome, and designing 3 primers, i.e. Emu-SD1, Emu-SD2 and Emu-SD3, for distinguishing between male and female, (wherein the three primers and a difference short sequence region are as shown in Fig. 1, a region corresponding to the Emu-SD2 only exists on the W chromosome and is unique to female Dromaius novaehollandia) selecting a Z sequence identical region and a W sequence identical region from the upstream and the downstream of an Emu-SD2 region respectively, wherein sequences of the three primers are as shown in Fig. 2;
S8: then conducting PCR reaction, wherein a PCR reaction system (50pl) is as follows:
Dromaius novaehollandia genome DNA 1pl
1OpM/L Emu-SD1, Emu-SD2 and Emu-SD3 1pl of each
2x EasyTaq PCR SuperMix 25pl
H20 21pl
and the PCR reaction conditions are as follows:
0predegeneration: 94°C 5min
@degeneration: 94°C 30s
@annealing: 51°C 30s
@ elongation: 72°C 40s
@final elongation: 72°C 10min
and @-@ cycle for 35 times; and
selection of annealing temperatures of the primers is as follows: based on base sequences of the primers, Tm values of the 3 primers are 58.5°C, 53.8°C and 47.0°C respectively, two annealing temperatures of 42°C and 51°C are tried with genomes of two adult male and female Dromaius novaehollandia with known sexes as templates, from which discovered that an amplification result at 51°C is good, and furthermore, PCR amplification is conducted on genomes of 16 Dromaius novaehollandia with unknown sexes; and
S9: separating a PCR amplification product with 2% agarose gel electrophoresis, conducting electrophoresis with 0.5XTBE electrophoretic buffer solution for 40min at 150V with a DNA dye of NA-Red, photographing a product under ultraviolet light, and then comparing the electrophoretic band with DL2000 DNA ladder with the following situations: ( PCR product electrophoretic results at different annealing temperatures are as follows: with genomes of two Dromaius novaehollandia with known sexes as templates, amplification is conducted with two annealing temperatures of 51°C and 42 0C, and when the annealing temperature is 51°C, the electrophoretic result shows that an amplification product of a female sample F51 is 2 bands (of 166bp and 428bp), an amplification product of a male sample is 1 band (of 409bp), the female has one more band than the male, the positions and the amount of the bands accord with those in theory, the bands are all clear, and 51°C may be used as the annealing temperature for sex identification; and when the annealing temperature is 42°C, the female sample only has the band of 166bp and lacks of the band of 428bp, while the band of the male sample is very light, as shown in Fig. 3, and thus 42°C is not a suitable annealing temperature for sex identification.
@ Genome DNA is extracted from feathers of 16 Dromaius novaehollandia nestlings with unknown sexes, PCR amplification and electrophoresis are conducted on the genome DNA with the three primers, the Dromaius novaehollandia with a female specific band of 166bp is the female Dromaius novaehollandia, and thus samples 1, 3, 4, 5, 9, 12, 13 and 15 are female Dromaius novaehollandia samples; while samples 2, 6, 7, 8, 10, 11, 14 and 16 lack of the female specific band of 166bp and contains one amplification band of about 409bp and thus being male Dromaius novaehollandia samples, as shown in Fig. 4.
Meanwhile, when the 16 Dromaius novaehollandia, subjected to PCR electrophoresis identification, grow to 18-month old, the sexes of the Dromaius novaehollandia are identified with an anus turning method, results of the anus turning method are compared with results of PCR detection one by one, and a result shows that the results of sex identification with PCR are thoroughly consistent to those obtained by the anus turning method with the accuracy of 100%.
Moreover, through genome amplification on the female Dromaius novaehollandia samples and the male Dromaius novaehollandia samples by using the three primers, each band after electrophoresis is subjected to gel cutting recovery and then is sent for sequencing, and sequencing results are as follows:
O female fragment WI: ( AGCTGCTTTGCTACTGCTTTATCCT) CCTCTCCACTGCAATCAGGTTTATCTCATTTTTTCATGTGACCATGAGGACT CTGGGACGCTCTTACAGACCTGTGTCCCATCAGTGCACGGATGTCCAACTG CTGAAGCCAGATGCAAAGGAGTACTGAGACACTCATTACAGCAGAAACAA AAAGCCAATATTAAAGGCAAAACTAGAGAATGCTCAATGAAAATAAACAG ATCCGTTAAGGCACATAGAAGCATATACAGTATACCTAAGCCAGCTTTTTAC
® female fragment W2: ( AGCTGCTTTGCTACTGCTTTATCCT) CCTCTCCACTGCAATCAGGTTTATCTCATTTTTTCATGTGACCATGAGGACT CTGGGACGCTCTTACAGACCTGTGTCCCATCAGTGCACGGATGTCCAACTG CTGAAGCCAGATG (GTTTTACCTGAGTAATTAATTCCTA)
@ male fragment Z1: ( AGCTGCTTTGCTACTGCTTTATCCT
which in parentheses are primer regions.
By analyzing the sequence difference in the Z chromosome and the W chromosome in the newly announced Dromaius novaehollandia genomes, a group of sex identification primers are designed. A sex of the Dromaius novaehollandia is identified with PCR and electrophoresis, and samples for identification are a small quantity of the feathers rather than the blood and the meat, so that the Dromaius novaehollandia is not hurt, attacks to the human from the Dromaius novaehollandia are also reduced in the catching and sampling process at the same time, and the rapid, accurate and noninvasive method for sex identification of Dromaius novaehollandia nestlings is the noninvasive identification technology. By optimizing the genome DNA extraction method and the PCR annealing temperature, extraction of the trace DNA and PCR amplification in the feathers are achieved; and the specificity of the sex identification primers is good, an electrophoretic band for sex identification is clear and bright, and the present invention is the accurate and reliable identification technology.
The above description is only the preferred embodiments of the present invention, and is not intended to limit the present invention in other forms. Any skilled in the art may apply equivalent embodiments with changes or variations of the technical contents disclosed above as equivalent changes to other fields. However, any simple modifications, equivalent changes and variations made to the above embodiments according to the technical substance of the present invention without departing from the contents of the technical solution of the present invention still belong to the protection scope of the technical solution of the present invention.
Claims (5)
1. A rapid, accurate and noninvasive method for sex identification of Dromaius novaehollandia nestlings, wherein, comprising the following steps of:
Si: enabling a Dromaius novaehollandia nestling to wear a foot ring marker marked with a number in a Dromaius novaehollandia farm, then sampling 5 feathers from the back and the tail of the nestling, putting the feathers in a low-temperature refrigerating box, and sending the low-temperature refrigerating box to a laboratory for detection;
S2: shearing roots of 0.5cm distant of two feathers, then cutting the sheared portions into pieces, putting the pieces in a 1.5ml centrifugal tube equipped with 180pl of tissue lysis solution TL, adding 20pl of protease k (20mg/ml), and immediately conducting vortex oscillation for full and uniform mixing to obtain a lysate;
S3: putting the lysate in a water bath of 55°C overnight until tissues are digested thoroughly, then adding 200pl of binding solution CB, immediately conducting vortex oscillation for full and uniform mixing, still standing a product for 10min at 70°C, adding 100pl of isopropanol after natural cooling, and immediately conducting vortex oscillation for full and uniform mixing to obtain a mixture;
S4: sucking the mixture with a 1ml gun head, adding the mixture in an adsorption column AC, putting the adsorption column in a collection tube for centrifugation for s at 13000rpm, pouring out waste liquid in the collection tube, adding 200pl of inhibitor removing solution IR for centrifugation for 30s at 12000rpm, pouring out waste liquid, adding 700pl of rinsing solution WB for centrifugation for 30s at 12000rpm, pouring out waste liquid, adding 500pl of rinsing solution WB for centrifugation for 30s at 12000rpm, and pouring out waste liquid;
S5: putting the adsorption column AC back into the empty collection tube for centrifugation for 2min at 13000rpm, removing the rinsing solution, then taking out the adsorption column AC, putting the adsorption column AC in a clean centrifugal tube, adding 50pl of elution buffer EB at the middle part of an adsorption film, and putting the centrifugal tube at a room temperature for 3-5min;
S6: then conducting centrifugation for1min at 12000rpm, then discarding the adsorption column AC, keeping the collection tube containing a DNA solution, determining a concentration of DNA with a nucleic acid-protein determinator to obtain extracted genome DNA, and packaging and storing the extracted genome DNA;
S7: designing 3 primers Emu-SD1, Emu-SD2 and Emu-SD3 to distinguish between male and female based on difference short sequence between the Z chromosome and the W chromosome of the Dromaius novaehollandia, then selecting a Z sequence identical region and a W sequence identical region from the upstream and the downstream of an Emu-SD2 region respectively, wherein a region corresponding to the Emu-SD2 only exists on the W chromosome and is unique to female Dromaius novaehollandia, a sequence of the designed primer Emu-SD1 is AGCTGCTTTGCTACTGCTTTATCCT, a sequence of the designed primer Emu-SD2 is AATGAGTGTCTCAGTACTCCTTTG, and a sequence of the designed primer Emu SD3 is TAGGAATTAATTACTCAGGTAAAAC;
S8: then conducting PCR reaction, wherein a PCR reaction system (50pl) is as follows:
Dromaius novaehollandia genome DNA 1pl
1OpM/L Emu-SD1, Emu-SD2 and Emu-SD3 1pl of each
2x EasyTaq PCR SuperMix 25pl
H20 21pl
the PCR reaction conditions are as follows:
0predegeneration: 94°C 5min
@degeneration: 94°C 30s
@annealing: 51°C 30s
@ elongation: 72°C 40s
@final elongation: 72°C 10min
and @-@ cycle for 35 times;
S9: separating a PCR amplification product with 2% agarose gel electrophoresis, conducting electrophoresis with 0.5XTBE electrophoretic buffer solution for 40min at
150V with a DNA dye of NA-Red, photographing a product under ultraviolet light, then comparing the electrophoretic band with DL2000 DNA ladder, determining the Dromaius novaehollandia with bands of 166bp and 428bp to be female Dromaius novaehollandia, and determining the Dromaius novaehollandia lacking the female specific band of 166bp and containing 1 band of ~ 409bp to be male Dromaius novaehollandia.
2. The rapid, accurate and noninvasive method for sex identification of Dromaius novaehollandia nestlings of claim 1, wherein in the step Si, during sampling, a sampler needs to wear disposable gloves for picking 5 feathers from the back and the tail of each Dromaius novaehollandia nestling; and after feather picking each time, the feathers need to be overturned and wrapped with the gloves, the gloves are clamped with a clip, and then the feathers are put in a low-temperature refrigerating box with the gloves together for storage.
3. The rapid, accurate and noninvasive method for sex identification of Dromaius novaehollandia nestlings of claim 1, wherein in the step S5, before use of the elution buffer, the centrifugal tube may be put in water of 65-70°C for preheating.
4. The rapid, accurate and noninvasive method for sex identification of Dromaius novaehollandia nestlings of claim 1, wherein in the step S6, the used nucleic acid protein determinator is Nanodrop ND-2000 super trace nucleic acid-protein determinator from America.
5. The rapid, accurate and noninvasive method for sex identification of Dromaius novaehollandia nestlings of claim 1, wherein in the step S6, the extracted genome DNA needs to be stored at -20°C.
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Fig. 2 Fig. 1
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Fig. 4 Fig. 3
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