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

doi:10.11983/CBB18229

Abstract

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

Fan Li,Jiwei Ruan. High-throughput Identification of Meiotic Anti-CO Mutants by Fluorescent Reporters[J]. Chinese Bulletin of Botany, 2019, 54(4): 522-530.

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URL: https://www.chinbullbotany.com/EN/10.11983/CBB18229

https://www.chinbullbotany.com/EN/Y2019/V54/I4/522

Figures/Tables 6

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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