A method for identifying adelphocoris ngritylus populations by utilizing molecular markers

Abstract

A method for identifying adelphocoris ngritylus populations by utilizing molecular markers is provided. The method is especially used for identifying a Northeast China population and a North China population. Specific primers are designed according to two gene segments which are adelphocoris ngritylus mitochondrial cytochrome oxidase I (COI) and mitochondrial NADH dehydrogenase subunit 4 (ND4) respectively. The primer sequences are shown as SEQ ID NO.1-2 and SEQ ID NO.3-4 respectively. A genome DNA of a variety to be identified is adopted as a template to perform PCR, and PCR amplification products are subjected to series connection and comparison, thus achieving identification of the adelphocoris ngritylus Northeast China population and the adelphocoris ngritylus North China population. The method is simple, reliable, high in sensitivity, high in specificity, short in consumed time, high in operationality, good in stability and high in repeatability and has a good application prospect.

Description

technical field

[0001] The invention relates to the technical field of molecular marker detection, in particular to a method for identifying Lygus black-lipped populations by using molecular markers, and a specific primer combination for identifying Lygus black-lipped populations. Background technique

[0002] The black-lipped alfalfa bug (Adelphocoris ngritylus) belongs to the genus Adelphocoris (Adelphocoris) of the family Miridae in the order Hemiptera. On other host plants, it has a wide distribution range, ranging from Heilongjiang in the north to Guizhou in the south. During the investigation, the applicant found that the population of this species is relatively large in Northeast China, such as Liaoning, Heilongjiang and other places; Henan, Hebei, Shandong, Beijing and other places in North China; although Ningxia, Guizhou and other places have this species distribution but relatively small populations. Although relevant studies have shown that the body color of th...

Examples

Embodiment 1

[0038] Example 1 Design and synthesis of PCR primer combinations for identification of Lygus black-lipped populations

[0039] Based on the mitochondrial cytochrome oxidase I (COI) gene and the mitochondrial NADH dehydrogenase subunit 4 (ND4) gene of Lygus alfalfa, the Primer Primer5 software was used to design specific primers for PCR amplification of the two genes. The primer sequences are as follows (SEQ ID NOs: 1-4):

[0040] ①Ngri-COI-F:

[0041] 5'-ATGAATAAATGATTATTTTCCACAAATCA-3' (SEQ ID NO. 1)

[0042] Ngri-COI-R:

[0043] 5'-CTGAATAATTAATATTTGTGCCATGAATG-3' (SEQ ID NO. 2)

[0044] ②Ngri-ND4-F:

[0045] 5'-AAAATACATTAAAGTAAACAAATCAATTT-3' (SEQ ID NO. 3)

[0046] Ngri-ND4-R:

[0047] 5'-ATATATTATTTATTTTTTGAGTGTAGTTTAATCCCT-3' (SEQ ID NO. 4)

[0048] The above primers were synthesized by Shanghai Bioengineering Technology Service Co., Ltd.

Embodiment 2

[0049] Embodiment 2 Utilize molecular markers to quickly identify the method for Northeast population and North China population of Lygus californica

[0050] 1. Collect the individuals of Lygus black-lipped alfalfa in Northeast China and North China, and extract genomic DNA

[0051] Representative populations were selected from Northeast China and North China, namely, Tieling City in Liaoning Province and Suihua City in Heilongjiang Province in Northeast China, Zhengzhou City in Henan Province, Hengshui City in Hebei Province and Dezhou City in Shandong Province in North China. . The field collection of Lygus black-lipped alfalfa adopts the net capture method. The adults are collected through a fluke tube, and then the adults are placed in analytically pure ethanol, and then brought back to the laboratory. The asana dissection microscope is used to refer to the relevant literature such as "Chinese Zoology" and so on. Further identification and confirmation of the external mo...

Embodiment 3

[0064] Example 3 Analysis of Specificity and Sensitivity of Detecting Lygus alfalfa by PCR Primer Ngri-COI-F / Ngri-COI-R

[0065] 1. Source of samples: In this example, Lygus pratensis (L.) was used as a control, and Lygus alfalfa came from Tieling, Liaoning in Northeast, Suihua in Heilongjiang and Zhengzhou in Henan, Hengshui in Hebei, and Dezhou in Shandong in North China; samples were used. It is preserved in the Plant Protection Laboratory, Cotton Research Institute, Chinese Academy of Agricultural Sciences.

[0066] 2. DNA extraction: as described in Example 2.

[0067] 3. PCR amplification: The PCR reaction system and PCR amplification procedure are the same as those described in Example 2.

[0068] 4. Result analysis: Take 5 μL of PCR amplification product, use 2% agarose gel for electrophoresis, the voltage is 120V, and test the result under ultraviolet light after 25 to 30 minutes. If there is a characteristic band of about 1100bp in size, it proves that The tested s...

technical field

[0001] The invention relates to the technical field of molecular marker detection, in particular to a method for identifying Lygus black-lipped populations by using molecular markers, and a specific primer combination for identifying Lygus black-lipped populations. Background technique

[0002] The black-lipped alfalfa bug (Adelphocoris ngritylus) belongs to the genus Adelphocoris (Adelphocoris) of the family Miridae in the order Hemiptera. On other host plants, it has a wide distribution range, ranging from Heilongjiang in the north to Guizhou in the south. During the investigation, the applicant found that the population of this species is relatively large in Northeast China, such as Liaoning, Heilongjiang and other places; Henan, Hebei, Shandong, Beijing and other places in North China; although Ningxia, Guizhou and other places have this species distribution but relatively small populations. Although relevant studies have shown that the body color of th...

Examples

Embodiment 1

[0038] Example 1 Design and synthesis of PCR primer combinations for identification of Lygus black-lipped populations

[0039] Based on the mitochondrial cytochrome oxidase I (COI) gene and the mitochondrial NADH dehydrogenase subunit 4 (ND4) gene of Lygus alfalfa, the Primer Primer5 software was used to design specific primers for PCR amplification of the two genes. The primer sequences are as follows (SEQ ID NOs: 1-4):

[0040] ①Ngri-COI-F:

[0041] 5'-ATGAATAAATGATTATTTTCCACAAATCA-3' (SEQ ID NO. 1)

[0042] Ngri-COI-R:

[0043] 5'-CTGAATAATTAATATTTGTGCCATGAATG-3' (SEQ ID NO. 2)

[0044] ②Ngri-ND4-F:

[0045] 5'-AAAATACATTAAAGTAAACAAATCAATTT-3' (SEQ ID NO. 3)

[0046] Ngri-ND4-R:

[0047] 5'-ATATATTATTTATTTTTTGAGTGTAGTTTAATCCCT-3' (SEQ ID NO. 4)

[0048] The above primers were synthesized by Shanghai Bioengineering Technology Service Co., Ltd.

Embodiment 2

[0049] Embodiment 2 Utilize molecular markers to quickly identify the method for Northeast population and North China population of Lygus californica

[0050] 1. Collect the individuals of Lygus black-lipped alfalfa in Northeast China and North China, and extract genomic DNA

[0051] Representative populations were selected from Northeast China and North China, namely, Tieling City in Liaoning Province and Suihua City in Heilongjiang Province in Northeast China, Zhengzhou City in Henan Province, Hengshui City in Hebei Province and Dezhou City in Shandong Province in North China. . The field collection of Lygus black-lipped alfalfa adopts the net capture method. The adults are collected through a fluke tube, and then the adults are placed in analytically pure ethanol, and then brought back to the laboratory. The asana dissection microscope is used to refer to the relevant literature such as "Chinese Zoology" and so on. Further identification and confirmation of the external mo...

Embodiment 3

[0064] Example 3 Analysis of Specificity and Sensitivity of Detecting Lygus alfalfa by PCR Primer Ngri-COI-F / Ngri-COI-R

[0065] 1. Source of samples: In this example, Lygus pratensis (L.) was used as a control, and Lygus alfalfa came from Tieling, Liaoning in Northeast, Suihua in Heilongjiang and Zhengzhou in Henan, Hengshui in Hebei, and Dezhou in Shandong in North China; samples were used. It is preserved in the Plant Protection Laboratory, Cotton Research Institute, Chinese Academy of Agricultural Sciences.

[0066] 2. DNA extraction: as described in Example 2.

[0067] 3. PCR amplification: The PCR reaction system and PCR amplification procedure are the same as those described in Example 2.

[0068] 4. Result analysis: Take 5 μL of PCR amplification product, use 2% agarose gel for electrophoresis, the voltage is 120V, and test the result under ultraviolet light after 25 to 30 minutes. If there is a characteristic band of about 1100bp in size, it proves that The tested s...

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