Gene sequence for identifying sex of tapisicia sinensis, and applications of gene sequence

A technology of gene sequence and gall pepper tree is applied in the field of molecular biology DNA molecular marker detection, and achieves the effect of wide application range and saving time and cost

Inactive Publication Date: 2017-05-31
NORTHWEST UNIV(CN)
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] In view of the problems existing in the prior art, the purpose of the present invention is to provide a method for quickly carrying out the sex identification of the pepper tree by using SCAR molecular markers, so as to solve the problem that the early sex identification of the pepper tree cannot be carried out accurately and quickly in the prior art

Method used

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  • Gene sequence for identifying sex of tapisicia sinensis, and applications of gene sequence
  • Gene sequence for identifying sex of tapisicia sinensis, and applications of gene sequence
  • Gene sequence for identifying sex of tapisicia sinensis, and applications of gene sequence

Examples

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Embodiment 1

[0031] The present embodiment provides a kind of gene sequence that is used to identify the sex of the pepper tree, and the method for obtaining the gene sequence is:

[0032] 1. Use TIANGEN's Plant Genomic DNA Kit to extract genomic DNA from the male and hermaphrodite plants of P. japonica collected from seven regions (as shown in Table 1).

[0033] The specific steps are:

[0034] (1) Take about 30 mg of plant leaves dried and preserved in silica gel, and add liquid nitrogen to fully grind;

[0035] (2) Quickly transfer the ground powder to a centrifuge tube pre-filled with 700 μl 65°C preheated buffer GP1 (add mercaptoethanol to the preheated GP1 before the experiment to make the final concentration 0.1%), and quickly invert After mixing, place the centrifuge tube in a 65°C water bath for 20 minutes. During the water bath, invert the centrifuge tube to mix the sample several times;

[0036] (3) Add 700 μl of chloroform, mix thoroughly, and centrifuge at 12,000 rpm for 5 m...

Embodiment 2

[0073] This embodiment provides a SCAR marker primer, the specific steps are:

[0074] 1. Use Primer PREMIER 5.0 to design specific primers based on the sequencing results of Example 1, so as to convert the obtained male-specific SRAP molecular markers into SCAR markers.

[0075] Specific primers are as follows:

[0076] TS-SCAR-F: 5'-ATCTTCATAGCCGCATAGCAAA-3'

[0077] TS-SCAR-R: 5'-CCAAATCCTTCAACAATATACCTG-3'

[0078] 2. SCAR-PCR amplification system and amplification procedure. The PCR product was detected by 1% agarose gel and recovered by gel cutting, connected to the PMD19-T (TAKARA) vector, transformed into DH5α competent cells, and sent to Shanghai Sangong for sequencing to confirm that the designed SCAR-specific primers could amplify Add the gene sequence that only exists in male plants obtained in step 7 in Example 1. Sequencing results showed that the designed SCAR-specific primers could successfully amplify the gene sequence obtained in step 7 of Example 1 that ...

Embodiment 3

[0085] In this example, the correctness of gender identification is verified for the obtained gene sequence and the TS-SCAR primers designed according to the gene sequence. The specific verification method is as follows:

[0086] 1 Use plants of known sex to verify the correctness of the primers, the DNA extraction method is the same as in Example 1, and the SCAR-PCR amplification system and amplification procedure are the same as in Example 2.

[0087] First, 12 plants of known sex in XY (campus) artificial cultivation population are used for verification, such as figure 2 As shown, TS-SCAR primers accurately distinguished 12 plants of known sex in the campus. Then use two male plants and two hermaphroditic plants from different populations in the field to verify the accuracy. like image 3 As shown, TS-SCAR primers accurately discriminated plants of known sex from different field populations. figure 2 ,The capital letters in the three are capital letters of the Chinese ...

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Abstract

The invention provides a gene sequence for identifying the sex of tapisicia sinensis. The gene sequence is used for identifying the male and amphiprotic sex of tapisicia sinensis, and through the SCAR molecular marker primer acquired by utilizing the gene sequence, the male and amphiprotic sex of tapisicia sinensis can be rapidly and simply judged. The SCAR marker primer for identifying the sex of tapisicia sinensis comprises a forward primer TS-SCAR-F and a reverse primer TS-SCAR-R, and the specific sequences are TS-SCAR-F:5'-ATCTTCATAGCCGCATAGCAAA-3', and TS-SCAR-F:5'-CCAAATCCTTCAACAATATACCTG-3'. The acquired male-specific SRAP molecular marker is successfully converted into a stable, accurate and simple SCAR marker which can be used for completely identifying different sexes of tapisicia sinensis. The SCAR primer combination can be used for accurately identifying sexes of manually cultured tapisicia sinensis and tapisicia sinensis at different regions of wild populations, and the application range is wide.

Description

technical field [0001] The invention belongs to the technical field of DNA molecular marker detection in molecular biology, and in particular relates to a method for quickly identifying the sex of the pepper tree by using SCAR. Background technique [0002] Gall pepper tree (Tapiscia sinensis Oliver.), also known as silver magpie tree, is an androdioecy breeding system, androdioecy breeding system is the rarest type in angiosperms, so it is also an important research object for angiosperms. Important materials on the evolution of breeding systems and the evolutionary life history of plants. Determining the early sex of P. japonica will help to study the genetic mechanism of sex determination in androdioecious plants, and help to understand the evolution and maintenance of plant breeding systems. In addition, different genders of the pepper tree have different economic values, so in practical application, the appropriate sex ratio can be selected for cultivation according to...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
Inventor 任小龙刘文哲彭春喜王玲丽辛桂亮杜晓敏
Owner NORTHWEST UNIV(CN)
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