KR-102964658-B1 - Primer set for identification of Haplotropis brunneriana and species identification method using the same

Abstract

The present invention relates to a primer set for identifying Haplotropis brunneriana and a method for identifying Haplotropis brunneriana using the same. The primer set of the present invention relates to a primer set that can identify the species of Haplotropis brunneriana, which is difficult to find in its habitat, using the molted shell debris without damaging the Haplotropis brunneriana, and a non-damaging method for species identification.

Inventors

• 김만년
• 이혜린
• 차덕재

Assignees

• 국립생태원

Dates

Publication Date

20260513

Application Date

20240222

Claims (11)

1. A primer set for identifying *Haplotropis brunneriana*, comprising: a forward primer consisting of the nucleotide sequence of SEQ ID NO. 1; and a reverse primer consisting of the nucleotide sequence of SEQ ID NO. 2.
2. In paragraph 1, The above primer set is a primer set for identifying the fat wrinkled grasshopper, using the molted shell of the fat wrinkled grasshopper as a sample.
3. In paragraph 2, The above molted shell is a primer set for identifying the fat wrinkled grasshopper, comprising epithelial cells isolated in vitro from the body of the fat wrinkled grasshopper.
4. A kit for identifying the fat wrinkled grasshopper, comprising a primer set according to any one of claims 1 to 3.
5. In paragraph 4, The above kit is characterized by further including DNA polymerase for amplification reaction, dNTPs, and reaction buffer, for the identification of the fat wrinkled grasshopper.
6. i) A step of isolating gDNA from a sample of the fat wrinkled grasshopper; ii) a step of performing an amplification reaction of a target sequence using a primer set comprising a forward primer consisting of the nucleotide sequence of SEQ ID NO. 1 and a reverse primer consisting of the nucleotide sequence of SEQ ID NO. 2, using the gDNA isolated in step i) as a template; and iii) A method for identifying a fat wrinkled grasshopper comprising the step of detecting the amplified product of step ii).
7. In paragraph 6, In step i) above, the sample of the fat wrinkled grasshopper is molting imprinted, fat wrinkled grasshopper identification method.
8. In paragraph 6, A method for identifying a fat-wrinkled grasshopper, wherein the target sequence in step ii) above is fat-wrinkled grasshopper cytochrome b.
9. In paragraph 6, A method for identifying a fat wrinkled grasshopper, further comprising, after step iii) above, step iv) confirming species identification between the fat wrinkled grasshopper and other grasshoppers through comparison of PCR amplification products.
10. In Paragraph 9, A method for identifying the fat wrinkled grasshopper, wherein the above other grasshoppers are one or more species selected from the group consisting of the katydid, the bean cricket, and the red bean cricket.
11. In paragraph 6, A method for identifying the fat-faced grasshopper, wherein the detection in step iii) above confirms whether a DNA band is formed through electrophoresis or whether fluorescence is emitted using a luminescent substance.

Description

Primer set for identification of Haplotropis brunneriana and species identification method using the same The present invention relates to a species-specific primer for the fat wrinkled grasshopper and a non-damaging species identification method using the same. Specifically, the present invention relates to a species identification method that can distinguish the fat wrinkled grasshopper and closely related species such as the katydid, katydid, and katydid using genetic methods without damaging the target species, the fat wrinkled grasshopper. Haplotropis brunneriana is a Class II endangered wild animal designated by the Ministry of Environment. While it previously inhabited grasslands nationwide, it currently resides in grasslands in only three specific regions: Yeongwol-gun in Gangwon-do, Jecheon-si in Chungcheongbuk-do, and Uiseong-gun in Gyeongsangbuk-do. It is primarily found in grasslands with dry soil and feeds on herbaceous plants. Unlike other grasshoppers, it becomes active starting in early March, and a distinguishing feature is that it cannot fly even after reaching adulthood. Furthermore, it presents challenges in determining its presence within its habitat due to morphological characteristics that provide camouflage similar to the surrounding environment, as well as a short life cycle of approximately four months. To secure genetic sequences for comparative population genetic studies of the target species based on habitat, it is unavoidable to collect tissue samples from the species. However, according to Ministry of Environment Regulation No. 621, the ‘Regulations on the Artificial Propagation of Endangered Wildlife,’ the capture of species designated as endangered wildlife is limited to a maximum of 20 individuals per instance, and re-capture in the same area is prohibited for three years. Therefore, genetic research methods and species identification techniques using gene sequence amplification, which are typically performed after collecting tissue samples from the target species, are not suitable for endangered wildlife. Accordingly, the present invention has developed a method to amplify a specific genetic sequence using molted exoskeletons that are discarded as the target species, the fat wrinkled grasshopper, grows, without harming the target species. Figure 1 is a photograph of the habitat of the fat wrinkled grasshopper, the subject of the present invention, showing that it is difficult to search for as it is difficult to distinguish it from fallen leaves within the habitat, and depicts the mating of the fat wrinkled grasshopper (male above, female below; difficult to find within the habitat due to body coloration similar to fallen leaves). Figure 2 illustrates a method for extracting genetic material from the molted shell of the fat wrinkled grasshopper, which is the subject of the present invention. Figure 3 shows the sequence of the primers for cytochrome b gene sequence amplification and species identification within the molted shell of the chubby-legged grasshopper according to the present invention. Figure 4 shows the results of confirming the target sequence amplified by PCR of the gDNA of the obese wrinkled grasshopper using the electrophoresis of the present invention. (M: 100 base pair DNA marker, L.mig: Grasshopper tissue gDNA, G.mar: Common katydid tissue gDNA, O.inf: Red-billed katydid tissue gDNA, H.bru-t: Obese wrinkled grasshopper tissue gDNA, H.bru-e: Obese wrinkled grasshopper molted shell gDNA, Black triangle: PCR amplification product of approximately 560 base pairs in size) Figure 5 shows the results of amplifying and comparing target sequences via the PCR method after extracting gDNA from the molted exoskeletons of the chubby-winged grasshopper and the katydid grasshopper using the electrophoresis of the present invention. (M: 100 base pair DNA marker, L.mig-e Repeat 1-3: katydid grasshopper molted exoskeleton gDNA repeat 1-3, H.bru-e Repeat 1-3: chubby-winged grasshopper molted exoskeleton gDNA repeat 1-3, H.bru-t: chubby-winged grasshopper tissue gDNA, black triangle: PCR amplification product of approximately 560 base pairs) Figure 6 shows the results of confirming how long residual gDNA can be detected in molted exoskeletons of *Scutellaria baicalensis* by exposing them to an artificial natural environment (26 ± 2℃, relative humidity 50-60%) at 10-day intervals for up to 40 days using the electrophoresis of the present invention. (M: 100 base pair DNA marker, Day 10 Repeat 1-3: Molted exoskeleton repeat 1-3 after 10 days of environmental exposure, Day 20 Repeat 1-3: Molted exoskeleton repeat 1-3 after 20 days of environmental exposure, Day 30 Repeat 1-3: Molted exoskeleton repeat 1-3 after 30 days of environmental exposure, Day 40 Repeat 1-3: Molted exoskeleton repeat 1-3 after 40 days of environmental exposure, Black triangle: PCR amplification product of approximately 560 base pairs) FIG. 7 is a schematic diagram of genetic sequence comparison showing vari