Unlock instant, AI-driven research and patent intelligence for your innovation.

Fluorescent molecular marker for molecular assisted breeding of corn haploid inducible line and primer of fluorescent molecular marker

A technology of haploid induction system and fluorescent molecules, which is applied in the determination/inspection of microorganisms, biochemical equipment and methods, DNA/RNA fragments, etc., can solve the problems of inaccurate selection, heavy workload, and low efficiency, and achieve The test process is simple, efficient and cost-effective, and the effect of accurate identification

Pending Publication Date: 2020-11-06
JILIN ACAD OF AGRI SCI
View PDF4 Cites 5 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] The current method of breeding high-frequency haploid induction lines generally relies on generation-by-generation induction cross-pollination, and determines the induction rate of the male parent through the identification of the seed color and oil content of the progeny. The workload is large, and the experimental data is easily affected by the maternal The material genotype, corn growth environment and human factors lead to inaccurate selection and low efficiency

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Fluorescent molecular marker for molecular assisted breeding of corn haploid inducible line and primer of fluorescent molecular marker
  • Fluorescent molecular marker for molecular assisted breeding of corn haploid inducible line and primer of fluorescent molecular marker
  • Fluorescent molecular marker for molecular assisted breeding of corn haploid inducible line and primer of fluorescent molecular marker

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] The primers were designed for the variant sites of the ZmPLA1 gene (GRMZM2G471240) and the ZmDMP gene (GRMZM2G465053) by the five-primer amplified-arrested mutation system (PARMS) technique, and the design results are shown in Table 1 and Table 2.

[0034] Table 1 ZmPLA1 gene PARMS primer sequence

[0035]

[0036] 2 primers designed according to the target genotype of ZmPLA1 gene (CGAG insertion and no insertion) and common primers were added to the PCR reaction system at the same time for amplification. The CGAG non-insertion genotype of ZmPLAd can be compared with the haplotype primer PLA-2Fg Match and amplify to obtain the FAM fluorescence signal value; CGAG insertion genotype can match with the haplotype primer PLA-2Fc according to the SNP difference and amplify to obtain the FAM fluorescence signal value; PLA-2R is a common primer; the heterozygous site has both FAM and HEX signals.

[0037] Schematic diagram of amplification for identification of CGAG inserti...

Embodiment 2

[0043] The method for using the above-mentioned fluorescent molecular marker and its primers to assist in the breeding of maize haploid induction lines, the specific steps are as follows:

[0044] (1) Quickly and low-cost extraction of genomic DNA from maize leaves by alkaline boiling method, the specific steps are:

[0045] ①Take 1cm 2 Add 100 μl of 0.3M sodium hydroxide to grind the leaves; ②Centrifuge at 3000rpm for 1min, then add 200μl of 0.2M Tris-HCl (pH7.0) to neutralize and lower the pH; The supernatant was diluted 10-30 times and used for PARMS reagent detection.

[0046] (2) ZmPLA gene haplotype primers PLA-2Fg, PLA-2Fc and common primer PLA-2R, or ZmDMP gene haplotype primer DMP-A, DMP-B and common primer DMP-C participate in PCR reaction simultaneously, reaction The system is:

[0047] Element Amount added (10μl) Final concentration 2x PARMS master mix 5μl 1x PLA-2Fg / DMP-A 0.15μl 150nM PLA-2Fc / DMP-B 0.15μl 150nM PLA-2R / ...

Embodiment 3

[0051] Embodiment 3 accuracy verification

[0052] The homozygous inducible line and the basic material DNA for inducible line breeding were extracted, and two sets of PARMS primers of the present invention were used for PCR reaction to analyze the genotype. The results are shown in Table 3.

[0053] table 3

[0054]

[0055]

[0056] Note: Jiyu 101, CAU5, W7-4, WY7 are homozygous inducible lines; JNX6 and JNX22 are parents of waxy corn variety Jinongnuo 7.

[0057] The results in Table 3 show that Jiyu 101 and other high-frequency induction lines have CGAG insertion of ZmPLA1 gene and C genotype of ZmDMP gene, and the intermediate breeding materials have C or T or C / T heterozygous genotype of ZmDMP gene and ZmPLA1 gene respectively. CGAG insertion and non-insertion genotypes, the test results meet expectations, which proves the accuracy and breeding practicability of the present invention.

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

Abstract

The invention discloses a fluorescent molecular marker for molecular assisted breeding of a corn haploid inducible line and a primer of the fluorescent molecular marker, and belongs to the technical field of molecular biological breeding. According to the fluorescent molecular marker for molecular assisted breeding of the corn haploid inducible line and the primer of the fluorescent molecular marker, the detection test process of the molecular marker is simple and convenient, fluorescent data are obtained accurately and environmentally friendly, and compared with results obtained through similar methods, the fluorescent molecular marker is accurate and efficient in target genotype recognition with lower in cost, and in the practice of breeding the induction line, the fluorescent molecularmarker is more intuitive and accurate, the test task amount is smaller, and the breeding efficiency is higher.

Description

technical field [0001] The invention relates to the technical field of molecular biological breeding, in particular to a fluorescent molecular marker and a primer for molecular-assisted breeding of maize haploid induction lines. Background technique [0002] Maize Doubled Haploid Breeding (DH, Doubled Haploid Breeding) technology is a revolution of traditional breeding technology, and has constituted a new system of modern maize breeding technology together with molecular breeding technology and transgenic technology. In recent years, the application of maize haploid breeding technology in conventional maize has become more mature, and the induction mechanism of haploid induction lines has been analyzed. In 2017, researchers at home and abroad (kelliher, et al., 2017; Liu, et al., 2017) successively reported the relevant content of the induced gene GRMZM2G471240, Liu et al. named it PLA1, and 4 bases were inserted into the gene The resulting loss of gene function is the key...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6895C12Q1/686C12N15/11
CPCC12Q1/6895C12Q1/686C12Q2600/156C12Q2600/13C12Q2563/107
Inventor 王薪淇卢实路明李穆何欢张梓心周旭东刘文国孟令聪郑淑波岳尧海张建新梅楠王敏董亚琳周小辉
Owner JILIN ACAD OF AGRI SCI