Study on the Monokaryons Mating Types Identification Based on the Primers Design According to the Resequencing Results of the Conserved Region in Lentinula edodes Mating Type Locus

doi:10.7668/hbnxb.20194804

Abstract

Abstract:

The hybridization of Lentinula edodes mononuclear mycelium is influenced by A and B two mating type factors.Based on whole genome sequencing,mating type identification of Lentinula edodes mononuclear hyphae can be carried out,but there are limitations in terms of technical complexity and high cost.In order to achieve rapid identification of unknown mating type genes in Lentinula edodes mononuclear mycelium,we used H31 and BJ4 strains as materials,and determined PCR amplification primers that can be used for identifying unknown mating type genes through specific amplification and resequencing of mating type site regions.The specific method was to use the hyphae hybridization results to determine the two mononuclear hyphae that could be hybridized,and record their mating types as A1B1 and A2B2,respectively.Based on the alignment analysis of the A and B mating type site gene sequences obtained from NCBI,primers were designed in the conserved region of their mating type sites to amplify the different mating type mononuclear hyphae A1B1 and A2B2.By comparing the resequencing information of the amplified products,we obtained nonconserved regions in gene sequences of different mating types A1 and A2,B1 and B2.Then,four mating type gene specific amplification primers to identify mating type genes in H31 and BJ4 Lentinula edodes mycelium were designed in this region.Based on the molecular level mating type identification results,hybridization validation was conducted on H31 and BJ4 Lentinula edodes mononuclear mycelium,and the results showed that the molecular identification results of mating type genes were consistent with the hybridization results of mononuclear mycelium.The mating type gene identification method established in this study no longer relies on whole genome sequencing.This method uses Lentinula edodes as experimental material,but it is also applicable to the identification of mating type genes in other edible mushrooms.Therefore,the results of this study have broad application value for genetic breeding of edible mushrooms.

Key words: Lentinula edodes, Cross breeding, Mating type identification, Mononuclear mycelium, Primer design

JIAN Wencheng, XING Xin, WANG Qi, QUAN Jianyu, WANG Li'an, GE Rongchao. Study on the Monokaryons Mating Types Identification Based on the Primers Design According to the Resequencing Results of the Conserved Region in Lentinula edodes Mating Type Locus[J]. Acta Agriculturae Boreali-Sinica, 2024, 39(4): 87-93. doi: 10.7668/hbnxb.20194804.

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Figures/Tables 11

Fig.1 Design of mating type A locus specific primer sequences L808.Chromosome 1 of L808 strain;135P32.135P32 mating type A locus;Underlined.Primer sequence.

Fig.2 Design of mating type B locus specific primer sequences L808.Chromosome 2 of L808 strain;L26P2.L26P2 mating type B locus;Underlined.Primer sequence.

Fig.3 H31 and BJ4 mononuclear mycelium mating type amplification results 1.Primers were AF and AR;2.Primers were BF and BR;M.Standard DNA molecular weight.

Fig.4 A1 and A2 sequence align and primer design of H31 mating type A locus Underline.H31-4 A1 primer sequence; Wave line.H31-7 A2 primer sequence.

Fig.5 B1 and B2 sequence align and primer design of H31 mating type B-locus Underline.H31-4 B1 primer sequence; Wave line.H31-7 B2 primer sequence.

Fig.6 PCR identification of mating types of each H31 strains 1.Primers were 4A1F and 4A1R;2.Primers were 7A2F and 7A2R; 3.Primers were 4B1F and 4B1R;4.Primers were 7B2F and 7B2R;M.Standard DNA molecular weight.

Fig.7 Antagonistic performance of H31 monokaryons hybridization combination

Fig.8 A1 and A2 sequence align and primer design of BJ4 mating type A-locus Wave line.BJ4-6 A1 primer sequence;Underlined.BJ4-2 A2 primer sequence.

Fig.9 B1 and B2 sequence align and primer design of BJ4 mating type B-locus Wave line.BJ4-6 B1 primer sequence; Underlined.BJ4-2 B2 primer sequence.

Fig.10 PCR identification of mating types of each BJ4 strains 1.Primers were 6A1F and 6A1R;2.Primers were 2A2F and 2A2R;3.Primers were 6B1F and 6B1R;4.Primers were 2B2F and 2B2R;M.Standard DNA molecular weight.

Fig.11 Antagonistic performance of BJ4 monokaryons hybridization combination

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