Development of a Molecular Marker for Fruiting Body Pattern in Auricularia auricula-judae

Figures & data

Table 1. Parent strains of isogenic lines and verified strains.

Number^a	Name	Fruiting body pattern^b	Number	Name	Fruiting body pattern
A14	Qihei No. 1	CL	B8	H10	CH
A18	Qihei No. 2	CH	B9	Yante 5	CL
A184	Wild strain	CH	B10	Au 8129	CH
A14-5	Monokaryon of A14	–	B11	Xinke	CH
A18-119	Monokaryon of A18	–	B12	Heimeur 1	CH
A184-57	Monokaryon of A184	–	B13	Heimuer 2	CL
B1	Heiwei 981	CL	B14	Heier 4	CH
B2	Hei 793	CH	B15	Heier 5	CH
B3	Hei A	CL	B16	Heier 6	CL
B4	Zhongnonghuang	CH	B17	Xueping 10	CH
B5	Tianjuhua	CH	B18	Fengshou 2	CL
B6	Zheer 1	CL	B19	Huer 3	CH
B7	Jiza 1	CH	B20	AU2	CL

^aA14, A18, A184 are parent strains of isogenic lines, and A14-5, A18-119, A184-57 are their respective monokaryotic strains. B1–B20 are verified strains.

^bCH: chrysanthemum-type fruiting body pattern; CL: cluster-type of fruiting body pattern.

Figure 1. Schematic representation of development of SCAR marker for fruiting body pattern in A. auricula-judae. The test strains were shown in the orange text boxes. CH-type: chrysanthemum-type fruiting body pattern; CL-type: cluster-type of fruiting body pattern.

Table 2. List of SRAP primer sequences.

Primer name	Primer sequence 5′–3′	Primer name	Primer sequence 5′–3′
Me1	TGAGTCCAAACCGGATA	Em5	GACTGCGTACGAATTAAC
Me2	TGAGTCCAAACCGGAGC	Em6	GACTGCGTACGAATTGCA
Me3	TGAGTCCAAACCGGAAT	Em7	GACTGCGTACGAATTATG
Me4	TGAGTCCAAACCGGACC	Em8	GACTGCGTACGAATTAGC
Me5	TGAGTCCAAACCGGAAG	Em9	GACTGCGTACGAATTACG
Me6	TGAGTCCAAACCGGTAG	Em10	GACTGCGTACGAATTTAG
Me7	TGAGTCCAAACCGGTTG	Em11	GACTGCGTACGAATTTCG
Me8	TGAGTCCAAACCGGTGT	Em12	GACTGCGTACGAATTGTC
Me9	TGAGTCCAAACCGGTCA	Em13	GACTGCGTACGAATTGGT
Em1	GACTGCGTACGAATTAAT	Em14	GACTGCGTACGAATTCAG
Em2	GACTGCGTACGAATTTGC	Em15	GACTGCGTACGAATTCTG
Em3	GACTGCGTACGAATTGAC	Em16	GACTGCGTACGAATTCGG
Em4	GACTGCGTACGAATTTGA	Em17	GACTGCGTACGAATTCCA

Em1–Em17: reverse primers; Me1–Me9: forward primers.

Table 3. List of SCAR primer sequences.

Number	Name of SCAR	Primer sequence (5′–3′)	Annealing temperature
1	SCH-16	F:TATTCTGGATCGGAGTCGACAG R:TTGCATACAATTTATCACCAGCA	58 °C
2	SCL-18	F:TCCAAACCGGATACCGCAG R:CTCTACCCTCCTTCCATCCGA	54 °C
3	SCH-34	F:GACTGCGTACGAATTTGAGGT R:CGTTCCTCCCGCAAATGAAA	58 °C
4	SCL-39	F:AGGTATGTTGTTCGGCGGT R:ATACACGTTCCTCCCGCAAA	58 °C
5	SCL-49	F:GAGTCCAAACCGGACCATCC R:CGAATAACACTCGTCACTCCAC	64 °C
6	SCL-412	F:AATATGTGGTGAGCTTGACGGG R:TGAGTCCAAACCGGACCC	58 °C
7	SCL-514	F:TGAGTCCAAACCGGAAGGAA R:CGTACGAATTCAGTAGGGACGA	60 °C
8	SCL-64	F:GTCCAAACCGGTAGAGCGAT R:GACTGCGTACGAATTGCAGA	62 °C
9	SCL-75	F:TTGGTCATCACACGCCAAAG R:CGGAAGCGTGGGAATATCGG	64 °C

F: forward primer; R: reverse primer.

Table 4. Fruiting body pattern of hybrids.

Name of monokaryotic strain	Name of hybrid	Fruiting body pattern	Name of monokaryotic strain	Name of hybrid	Fruiting body pattern
119 × 5-1	C1	CH	119 × 5-50	C50	CH
119 × 5-2	C2	CH	119 × 5-51	C51	CL
119 × 5-3	C3	CL	119 × 5-55	C55	CL
119 × 5-6	C6	CL	119 × 5-57	C57	CH
119 × 5-7	C7	CL	119 × 5-58	C58	CL
119 × 5-8	C8	CL	119 × 5-60	C60	CL
119 × 5-11	C11	CL	119 × 5-61	C61	CH
119 × 5-12	C12	CH	119 × 5-63	C63	CL
119 × 5-15	C15	CH	119 × 5-64	C64	CH
119 × 5-17	C17	CH	119 × 5-65	C65	CH
119 × 5-18	C18	CH	119 × 5-66	C66	CH
119 × 5-19	C19	CL	119 × 5-68	C68	CL
119 × 5-20	C20	CH	119 × 5-71	C71	CH

CH: chrysanthemum-type fruiting body pattern; CL: cluster-type fruiting body pattern.

Table 5. Sequencing information of target fragments.

Number	Name of target fragment	From primer combination	Gene pool of expanded specific band^a	Fragment size^b (bp)
1	1 × 6-CH-450	Me1 × Em6	CH	424
2	1 × 8-CL-510	Me1 × Em8	CL	522
3	3 × 4-CH-250	Me3 × Em4	CH	247
4	3 × 9-CL-300	Me3 × Em9	CL	405
5	4 × 9-CL-300	Me4 × Em9	CL	293
6	4 × 12-CL-240	Me4 × Em12	CL	234
7	5 × 14-CL-240	Me5 × Em14	CL	216
8	6 × 4-CL-200	Me6 × Em4	CL	189
9	6 × 6-CL-250	Me6 × Em6	CL	–
10	7 × 5-CL-300	Me7 × Em5	CL	295

^a CH: chrysanthemum-type fruiting body pattern; CL: cluster-type fruiting body pattern.

^b Fragment size was determined from sequencing results for each specific band. Several bands were produced using 6 × 6-CL-250, so the fragment was not sequenced.

Figure 2. Test results of positive clones of SRAP fragments. Lanes 1 and 2, plasmid target fragment of 7 × 5-CL-300; lanes 3 and 4, plasmid target fragment of 4 × 12-CL-240; lanes 5 and 6, plasmid target fragment of 1 × 8-CL-510; lanes 7 and 8, plasmid target fragment of 3 × 9-CL-300; lanes 9 and 10, plasmid target fragment of 4 × 9-CL-300; lanes 11 and 12, plasmid target fragment of 3 × 4-CH-250; lanes 13 and 14, plasmid target fragment of 5 × 14-CL-240; lanes 15 and 16, plasmid target fragment of 6 × 4-CL-200; lanes 17 and 18, plasmid target fragment of 6 × 6-CL-250; lanes 19 and 20, plasmid target fragment of 1 × 6-CH-450; M: 2,000-bp ladder marker.

Figure 3. Verification of SCAR marker SCL-18. Lanes 1–20 indicate verified strains (B1–B20) of nationally accredited varieties in China (Table 1). Single 522-bp band was amplified by SCL-18 primer in lane 1, 3, 6, 9, 13, 16, 18, and 20, corresponding to strains with cluster-type fruiting body pattern (Table 1). M: 1500-bp ladder marker.