[논문]Development of a Molecular Marker Linked to the A4 Locus and the Structure of HD Genes in Pleurotus eryngii

Lee, Song Hee; Ali, Asjad; Ha, Byeongsuk; Kim, Min-Keun; Kong, Won-Sik; Ryu, Jae-San

doi:10.1080/12298093.2019.1619989

Development of a Molecular Marker Linked to the A4 Locus and the Structure of HD Genes in Pleurotus eryngii 원문보기

Mycobiology, v.47 no.2, 2019년, pp.200 - 206

Lee, Song Hee (Department of Mushroom Science, Korea National College of Agriculture and Fisheries) , Ali, Asjad (EnvironmentFriendly Research Division, Gyeongsangnam-do Agricultural Research and Extension Services) , Ha, Byeongsuk (EnvironmentFriendly Research Division, Gyeongsangnam-do Agricultural Research and Extension Services) , Kim, Min-Keun (EnvironmentFriendly Research Division, Gyeongsangnam-do Agricultural Research and Extension Services) , Kong, Won-Sik (Mushroom Research Division, National Institute of Horticultural & Herbal Science, Rural Development Administration) , Ryu, Jae-San (Department of Mushroom Science, Korea National College of Agriculture and Fisheries)

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Allelic differences in A and B mating-type loci are a prerequisite for the progression of mating in the genus Pleurotus eryngii; thus, the crossing is hampered by this biological barrier in inbreeding. Molecular markers linked to mating types of P. eryngii KNR2312 were investigated with randomly amplified polymorphic DNA to enhance crossing efficiency. An A4-linked sequence was identified and used to find the adjacent genomic region with the entire motif of the A locus from a contig sequenced by PacBio. The sequence-characterized amplified region marker $7-2_{299}$ distinguished A4 mating-type monokaryons from KNR2312 and other strains. A BLAST search of flanked sequences revealed that the A4 locus had a general feature consisting of the putative HD1 and HD2 genes. Both putative HD transcription factors contain a homeodomain sequence and a nuclear localization sequence; however, valid dimerization motifs were found only in the HD1 protein. The ACAAT motif, which was reported to have relevance to sex determination, was found in the intergenic region. The SCAR marker could be applicable in the classification of mating types in the P. eryngii breeding program, and the A4 locus could be the basis for a multi-allele detection marker.

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gov/portal/). Alignment analysis was performed with DNAMAN (Lynnon Biosoft) with default parameters to determine the nucleotide frequency, including the transcription initiation site.

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marker. Because the A4 allele was shared in KNR2523 and KNR2522 but not in KNR2501 [8], 8 monokaryons, two from each mating type, were selected for the new marker. The 7-2₂₉₉marker amplified unique bands from KNR2523(2)-15, 34 (A4B3) and KNR2523(2)-24, 26(A4B12) but not from the remaining strains(KNR2523(2)-6 and -28 for A12B3 and KNR2523(2)-23 and -32 for A12B12).
The SCAR primer was manually designed using the 7-2 sequence and a flanked region (~1 kb) obtained from a contig of KNR2312P6 sequenced by PacBio. The developed primer sequences were F: 50-AATCACGGGAAGATCTGGTG-30 and loci-R: 50-GTGGTAGGGTTCCCGCCT-30. PCR conditions for the SCAR marker were optimized as follows: initial denaturation at 98°C for 30 sec, followed by 35 cycles of a 10 sec denaturation at 98°C, 15 sec annealing at 70°C, and 10 sec extension at 72°C, and a final extension of 10 min at 72°C.
A critical annealing temperature of 70°C was determined to best identify the mating type. Thus, to observe the strong linkage of 7-2₂₉₉ with the A4Bx locus, 17 monokaryons from KNR2312 were tested. Eight monokaryotic isolates showed a specific band for mating-type A4B3, A4B4, and P6 (A4B3), while no amplification was detected for A3B3, A3B4, and P5 (A3B4) (Figure 2).

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