Chin Bull Bot ›› 2019, Vol. 54 ›› Issue (4): 522-530.doi: 10.11983/CBB18229

• TECHNIQUES AND METHODS • Previous Articles     Next Articles

High-throughput Identification of Meiotic Anti-CO Mutants by Fluorescent Reporters

Li Fan,Ruan Jiwei()   

  1. Yunnan Key Laboratory of Flower Breeding, National Engineering Research Center for Ornamental Horticulture, Flower Research Institute, Yunnan Academy of Agricultural Sciences, Kunming 650200, China
  • Received:2018-10-30 Accepted:2019-02-11 Online:2020-01-08 Published:2019-07-01
  • Contact: Ruan Jiwei E-mail:snruanjiwei@126.com

Abstract:

The forward genetic approach has become a widespread methodology to reveal genetic factors involved in meiosis, such as the crossover negative regulators, which limit the class II crossover formation. Here we developed a forward genetics mutant screen to identify anti-CO mutants under the Col wild-type background of Arabidopsis thaliana. We isolated 18 mutant lines showing more than three-fold increase in male meiotic recombination frequency as compared with the wild type, including dominant and recessive mutants. Thus, the EMS screen based on fluorescent reporters allows for high-throughput identification of meiotic anti-CO mutants and provides a novel approach and genetic materials to study the molecular mechanism of meiotic recombination regulation.

Key words: meiosis, anti-CO mutants, fluorescent marker, high-throughput visual assay

Figure 1

A schematic illustration of Arabidopsis fluorescent tagged line FTL-I2b (A) Fluorescence micrograph of a tetrad pollen of FTL-I2b, which contains a DsRed and an eYFP fluorescent marker and expresses red fluorescence and green fluorescence under different fluorescence excitations respectively (Bars=50 μm); (B) The genomic location of FTL-I2b fluorescent markers on the chromosome (Bar=1 Mb), the DsRed and eYFP fluorescent markers are indicated by filled circles colored by red (down) and green (up), respectively, which constructing a I2b interval (1.45 Mb). BF: Bright field"

Figure 2

The Arabidopsis hemizygous fluorescent markers (I2b/++) of FTL-I2b (A) A schematic illustration of FTL-I2b cross with Col, the hybrid F1 contains a hemizygous fluorescent markers after hybridization, the recombined and unrecombined gametes are formed through meiosis; (B) Fluorescence micrograph of pollen in hybrid F1 generation, in which yellow pollen (GR) is unrecombined gamete, and monochromatic red (R) and green (G) pollen is recombined gametes (Bars=50 μm); (C) The recombination frequency (RF) of I2b interval can be calculated by counting the fluorescent marker pattern in the gametes via flow cytometry, the formula calculating recombination frequency=(G+R)/(2GR+G+R)×100."

Figure 3

The germination rate of M1 Arabidopsis seeds by different treat time using 75 mmol∙L-1 EMS The average germination rate was derived from three repeats, different lowercase letters indicate significant differences (P<0.05)."

Figure 4

A schematic illustration of Arabidopsis meiotic anti-CO mutants forward genetics screen The forward genetics mutants screen was used the FTL-I2b containing linked hemizygous fluorescent reporters (I2b/++) on the genetic background of Arabidopsis wild type (WT). High-throughput detecting the recombination frequency by flow cytometry could identify the mutants with increased recombination rate. The DsRed and eYFP fluorescent reporters of FTL-I2b are indicated by R and G, respectively. The m refers to a dominant or recessive mutation. RF: Recombination frequency"

Figure 5

The pollen micrograph of Arabidopsis wild type and big pollen mutants (A) The pollen size of Arabidopsis wild type (WT) is (21.82±0.98) µm (n=10); (B) The large pollen rate of big pollen 1 mutant is 100%, pollen size is (38.36±1.37) µm (n=10); (C) The large pollen rate of big pollen 2 mutant is 50%, pollen size is (36.00±2.70) µm (n=10). Bars=50 µm"

Figure 6

Arabidopsis wild type and recombination suppressor mutants (A) The plant phenotype of Arabidopsis wild type (WT), dominant recombination suppressor mutant (drs) and recessive recombination suppressor mutant (rrs1) (Bars=1 cm); (B) The fluorescent pollen micrograph of Arabidopsis wild type (WT), dominant recombination suppressor mutant (drs) and recessive recombination suppressor mutant (rrs1) (Bars=50 μm)"

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