Ryu, Jae-San
(Eco-friendliness Research Department, Gyeongsangnam-do Agricultural Research and Extension Services)
,
Kim, Min Keun
(Eco-friendliness Research Department, Gyeongsangnam-do Agricultural Research and Extension Services)
,
Ro, Hyeon-Su
(Department of Microbiology and Research Institute for Life Science, Gyeongsang National Univesity)
,
Kang, Young Min
(Eco-friendliness Research Department, Gyeongsangnam-do Agricultural Research and Extension Services)
,
Kwon, Jin-Hyeuk
(Eco-friendliness Research Department, Gyeongsangnam-do Agricultural Research and Extension Services)
,
Kong, Won-Sik
(Mushroom Research Division, National Institute of Horticultural and Herbal Science, Rural Development Administration)
,
Lee, Hyun-Sook
(Department of Microbiology and Research Institute for Life Science, Gyeongsang National Univesity)
In order to estimate how diverse the mating types in Pleurotus eryngii from different regions are, pairings between monokaryons derived from inter- and intra-groups were done. Sixteen and 15 alleles were identified at loci A and B from the 12 strains. In the P. eryngii KNR2312, widely used for comme...
In order to estimate how diverse the mating types in Pleurotus eryngii from different regions are, pairings between monokaryons derived from inter- and intra-groups were done. Sixteen and 15 alleles were identified at loci A and B from the 12 strains. In the P. eryngii KNR2312, widely used for commercial production, four mating loci, A3, A4, B3, and B4, were determined. Those loci, except A3, were found in 4 strains out of 12 strains. To improve breeding efficiency, especially in mating type determination, RAPD and BSA were performed to screen for a mating type specific marker. The SCAR marker 13-$2_{2100}$ was developed based on the RAPD-derived sequence typing B3 locus. The sequence analysis of 13-$2_{2100}$ revealed that it contained a conserved domain, the STE3 super-family, and consensus sequences like the TATA box and GC box. It seems likely that the SCAR marker region is a part of the pheromone receptor gene.
In order to estimate how diverse the mating types in Pleurotus eryngii from different regions are, pairings between monokaryons derived from inter- and intra-groups were done. Sixteen and 15 alleles were identified at loci A and B from the 12 strains. In the P. eryngii KNR2312, widely used for commercial production, four mating loci, A3, A4, B3, and B4, were determined. Those loci, except A3, were found in 4 strains out of 12 strains. To improve breeding efficiency, especially in mating type determination, RAPD and BSA were performed to screen for a mating type specific marker. The SCAR marker 13-$2_{2100}$ was developed based on the RAPD-derived sequence typing B3 locus. The sequence analysis of 13-$2_{2100}$ revealed that it contained a conserved domain, the STE3 super-family, and consensus sequences like the TATA box and GC box. It seems likely that the SCAR marker region is a part of the pheromone receptor gene.
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제안 방법
Amplification was done using SCAR (13-22100 F and R) primers with the monokaryons derived from KNR2523, KNR2525, KNR2501, and KNR2522. Two monokaryons were selected from each mating type.
To verify why RAPD primer (OPS-13) occasionally did not work or amplified smear bands upon strains containing AxB3 and was likely caused by a mismatch on the sequences of the genome and the primer, the technique of genome walking was performed. Based on the sequence of the 13-2 fragment, upstream primers (RGSP1, GGTCAGTTCA ACCAATCCAG TATCTT for primary PCR; and RGSP2, GTGAACGCTA CAATCTGGTC TGATAAT for nested PCR) and downstream primers (LGSP1, GTCGCCAACC CTGTCGCCCC TTGGATCAGC for primary PCR; and LGSP2, ATCCCTCTCT CCATCTTTAT CATCTG for nested PCR) were designed to extend the gene sequence using a Universal GenomeWalker kit (CloneTech, Mountain View, CA, USA).
Developed primer sequences for the B3 locus were 13-22100-F: 5'- CAAAGGTCCCCCCGATAGTGAT-3'; and 13-22100-R: 5'-CAGAGG TCCCAGCTGGAAGTGT-3' (SCAR marker primers were the registered Korean patent, #100993814). In order to optimize PCR conditions for the SCAR marker, annealing temperature was tested at a range of 37 to 67oC and a number of reaction cycles. To verify the marker’s reproducibility and reliability upon different concentrations of template DNA and Taq DNA polymerases, PCR reaction was performed with 10, 30, 100, and 300 ng/µl of genomic DNA as a template and several Taq DNA polymerases including Go-Taq (Promega, USA), Accupower PCR Premix (Bioneer, Korea), and Super-therm (Southern Cross Biotech, South Africa).
The results of sequencing were used to design more reliable primers for identifying mating type in the monokaryotic offspring derived from KNR2312 and other strains. The SCAR primer was manually designed using the sequence of 13-2 and flanked region.
To determine and assign mating types of the monokaryotic sibling from the strains, 20 monokaryons were selected randomly and all possible combinations crossed, sorted, and grouped based on the morphological characters including true clamp connection, pseudo-clamp connection, and flat form. The 3-4 isolates identified were selected from each mating type group in all strains crossed inter-specifically.
To verify the marker’s reproducibility and reliability upon different concentrations of template DNA and Taq DNA polymerases, PCR reaction was performed with 10, 30, 100, and 300 ng/µl of genomic DNA as a template and several Taq DNA polymerases including Go-Taq (Promega, USA), Accupower PCR Premix (Bioneer, Korea), and Super-therm (Southern Cross Biotech, South Africa).
대상 데이터
3. DNA fragments obtained in a set of monokaryons derived from P. eryngii KNR2312 using the SCAR (13-22100 F and R) as primers.
For evaluation of the SCAR marker’s applicability to other strains, monokaryons derived from KNR2501, KNR2514, KNR2519, KNR2522, KNR2523, and KNR2525 were tested.
The SCAR marker saved time by 13 days over the conventional method (taking 15 days) to identify the mating types. Over 150 monokaryotic isolates from various P. eryngii strains including KNR2312, KNR2514, KNR2522, and KNR2523 were tested with 13-22100 primers, and every monokaryon containing B3 showed the specific band. No recombination was observed between the marker and the phenotype (mating type).
eryngii KNR2312. Specific PCR products ranged from 450-2,500 bp, linked to all cases of mating type, A3B3, A4B3, A3B4, and A4B4 were screened (data not shown). All of them were cloned, sequenced, and analyzed.
Recombinant plasmid was conducted into host JM109 cells by transformation according to the supplier’s protocol. The clones possessing mating type specific region were sequenced by an ABI 377 sequencer with the M13 universal primer (Genotech, Korea). In case the length of cloned DNA was over 1,500 bp, an additional primer for sequencing was designed based on the previously sequenced region by the Bioneer primer software (Bioneer, Korea).
성능/효과
The 8 monokaryons including 4 mating types evenly from the above strains were used for amplification with the 13-22100 primers. As the result, the unique 13-22100 marker was amplified only in isolates bearing B3; thus, the SCAR marker was effective to detect the B3 locus in other P. eryngii strains (Fig. 4).
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