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Complete set of molecular markers for detecting aegilops longissima chromosome arm, and application thereof

A technology of DNA molecules, sequences, applied in the field of complete sets of molecular markers

Inactive Publication Date: 2018-04-03
INST OF CROP SCI CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, so far, there are few reports on the research on I. talliana, especially the cloning of specific DNA fragments on specific chromosomes of I. talliana and the development of molecular markers.
Therefore, the research results obtained so far about the tall goat grass are still far from meeting the needs of translocation transfer of a large number of excellent genes in the tall goat grass to wheat.

Method used

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  • Complete set of molecular markers for detecting aegilops longissima chromosome arm, and application thereof
  • Complete set of molecular markers for detecting aegilops longissima chromosome arm, and application thereof
  • Complete set of molecular markers for detecting aegilops longissima chromosome arm, and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0187] Example 1. Design of a complete set of primers for detecting each chromosome arm of A. talliae

[0188] The total RNA was extracted from the leaves of A. talliana PI542196 seedlings. After transcriptome sequencing, sequence analysis and comparison, several primers for identifying the chromosome arms of A. talliana were obtained. By comparing the sensitivity, specificity and other properties, a set of primer combinations for detecting the chromosome arms of A. tallis was obtained. As follows:

[0189] For the detection of tall goat grass 1S 1 Primer pair 1 for the long arm of the chromosome (product size: 275bp):

[0190] 1SL-F (sequence 1 of the sequence listing): 5'-CCATACACCAGTACATCCATA-3';

[0191] 1SL-R (sequence 2 of the sequence listing): 5'-GTACATTACCATCTGACATAGG-3';

[0192] For the detection of tall goat grass 1S 1 Primer pair 2 for the short arm of the chromosome (product size: 752bp):

[0193] 1SS-F (sequence 3 of the sequence listing): 5'-TACCACATCCATC...

Embodiment 2

[0231] Embodiment 2, the establishment of the detection method of each chromosome arm of A. tallii

[0232] 1. Extract the total DNA of the plant to be tested.

[0233] 2. Using the total DNA obtained in step 1 as a template, perform PCR amplification using primer pair 1 to primer pair 14 in Example 1, respectively, to obtain PCR amplification products.

[0234] Reaction system for PCR amplification: template DNA 100ng, upstream primer 0.5μL, downstream primer 0.5μL, 2×TaqMasterMix (Mg 2+ , dNTP) 7.5μL, add ddH 2 0 to 15 μL.

[0235] Both the upstream primer and the downstream primer have been added to the reaction system in the form of a primer solution, and the concentration of each primer in the primer solution is 10 μmol L -1 .

[0236] The reaction program of PCR amplification: 95°C for 5min; 95°C for 30s, 55°C for 30s, 72°C for 1min, 35 cycles; 72°C for 8min, then store at 4°C.

[0237] 3. Carry out agarose gel electrophoresis detection to the PCR amplification prod...

Embodiment 3

[0252] Embodiment 3, detection method verification

[0253] Materials to be tested: Tall goatgrass PI542196, wheat variety China Spring, Chinese spring-tall goatgrass supplementary line DA1S#3, Chinese spring-tall goatgrass supplementary line DA2S#3, Chinese spring-tall goatgrass supplementary line DA3S#2, Chinese spring-tall ibex addition line DA4S#3, Chinese spring-tall ibex addition line DA5S#3, Chinese spring-tall ibex addition line DA6S#3 and Chinese spring-tall ibex addition line DA7S#3.

[0254] The total DNA of the seedling leaves of the material to be tested is extracted; the total DNA is used as a template, and the method in Example 2 is used for detection.

[0255] The result is as Figure 1-14 shown. Figure 1-14In the middle, lane M is Marker 2000+, lane 2 is the Chinese spring-tall goat grass addition line DA1S#3, lane 3 is the Chinese spring-tall goat grass addition line DA2S#3, and lane 3 is the Chinese spring-tall goat grass addition line DA3S#2, Lane 4 is ...

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Abstract

The invention discloses a complete set of molecular markers for detecting an aegilops longissima chromosome arm, and the application thereof. The protective specific primer combination consists of single-chain DNA molecules as shown in sequence 1 to sequence 28 of a sequence table. The primer combination can be applied to detection of the aegilops longissima chromosome arm. The inventor of the invention further proves and develops a sect of specific molecular markers for identifying the aegilops longissima chromosome arm and a chromosome segment by utilizing a group of common wheat-aegilops longissima additional system through an RNA-seq and bioinformatics analysis combined method. The molecular markers are very useful for transferring aegilops longissima chromosome segments required by wheat breeding.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a set of molecular markers for detecting the chromosome arms of A. tallii and its application. Background technique [0002] Tall goat grass (2n=2x=14, S l S l , Aegilops longissima L.) is a diploid species of the Triticea genus, which is mainly distributed in the coasts of Egypt and Palestine in the Middle East. In addition, there are also a small amount of distribution in Jordan and other places, which are annual plants. In terms of evolutionary relationship, the genome of A. tallis is relatively close to the B genome of hexaploid wheat. Goat grass has a wide range of resistance to biotic stress (such as rust, powdery mildew, cyst nematode, root-knot nematode, wheat fly and green aphid, etc.) and abiotic stress (such as drought, cold and high salinity, etc.), And it contains high-quality genes such as specific glutenin, so it is a high-quality gene source for wheat resistance br...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6895C12N15/11
CPCC12Q1/6895
Inventor 叶兴国王坤杨王轲林志珊杜丽璞李仕金王静石磊陈海强马晓兰
Owner INST OF CROP SCI CHINESE ACAD OF AGRI SCI