Genetic Diversity of Amylomyces rouxii from Ragi tapai in Java Island Based on Ribosomal Regions ITS1/ITS2 and D1/D2

Figures & data

Figure 1. Location of Ragi tapai sample collection sites in Java Island.1. Bandung, 2. Sumedang, 3. Muntilan, 4. Yogyakarta, 5. Blora, 6. Bondowoso. Map from Google Maps.

Table 1. Macroscopic and microscopic characteristics of Ragi tapai isolates^a.

Characters	BDG	SMD	MTL	YOG	BLR	BDS
Macroscopic
1. Colony color	White	White	White	White	Black with white margin	Greyish white with white margin
2. Reverse color	Light cream	Light cream	Light cream	Light cream	Dull cream	Light cream
3. Diameter	8 cm	8 cm	8 cm	8 cm	8 cm	8 cm
4. Texture	Floccose	Floccose	Floccose	Floccose	Fibrous	Floccose
Microscopic
1. Abortive sporangia	+	+	+	+	+	+
2. Terminal chlamydospore	+	+	+	+	+	+
3. Intercalary chlamydospore	–	+	+	+	+	+
4. Septa	−	−	−	−	−	−

^a +: present; −: absent.

Figure 2. Abortive sporangia of Ragi tapai isolates (400x). (A) BDG, (B) SMD, (C) MTL, (D) BLR, (E) YOG, (F) BDS.

Figure 3. Microscopic features of Ragi tapai isolates. (A) oval abortive sporangia with enlarged apophyses SMD (400x); (B) Coenocytic hypha YOG (400x); (C) Zygospore YOG (400x); (D) Chlamydospores BLR (100x); (E) Intercalary chlamydospores BDS (400x); (F) Y-shaped spore-like bodies BLR (400x).

Table 2. NCBI BLAST results of ITS1/ITS2 region of ribosomal DNA.

Isolate	Homolog species	Query cover	Similarity	Accession number	Reference
BDG	Amylomyces rouxii	98%	98.06%	AY238888	[23]
	Amylomyces rouxii SDM29	95%	99.49%	AY228094	[23]
MTL	Amylomyces rouxii	99%	98.06%	AY238888	[23]
	Amylomyces rouxii	95%	99.32%	AB181331	[24]
SMD	Amylomyces rouxii	96%	99.83%	AY238888	[23]
	Amylomyces rouxii	94%	100.00%	JX393890	[25]
BLR	Rhizomucor sp. Rm-33	99%	99.52%	EF583634	[26]
	Mucor indicus F5-02	99%	99.52%	JN561265	[27]
BDS	Rhizomucor sp. Rm-33	99%	99.50%	EF583634	[26]
	Mucor indicus AOM7	99%	99.33%	MK396498	[28]
YOG	Mucor indicus F5-02	99%	100.00%	JN561265	[27]
	Rhizomucor sp. Rm-33	99%	100.00%	EF583634	[26]

Table 3. NCBI BLAST results of D1/D2 region of ribosomal DNA.

Isolate	Homologue species	Query cover	Similarity	Accession number	Reference
BDG	Amylomyces rouxii CBS 438.76	92%	99.54%	AB250194	[29]
	Amylomyces rouxii	92%	99.24%	AF157174	[30]
MTL	Amylomyces rouxii CBS 438.76	93%	99.54%	AB250194	[29]
	Amylomyces rouxii	92%	99.24%	AF157174	[30]
SMD	Amylomyces rouxii CBS 438.76	93%	100.00%	AB250194	[29]
	Amylomyces rouxii	92%	99.69%	AF157174	[30]
BLR	Mucor indicus CBS 423.71	98%	99.03%	MH871967	[22]
	Mucor indicus Q1088	98%	98.89%	KT359360	[31]
BDS	Mucor indicus CBS 423.71	98%	99.31%	MH871967	[22]
	Mucor indicus CBS 226.29	96%	99.58%	NG057878	[32]
YOG	Mucor indicus Q1088	99%	98.91%	KT359360	[31]
	Mucor indicus CBS 424.71	99%	98.50%	MH871968	[22]

Figure 4. Phylogenetic tree of ITS1/ITS2 region using the Neighbor-Joining (NJ) method and Kimura-2-parameter (K2P) with 1000 bootstrap value.

Figure 5. Phylogenetic tree of D1/D2 region using the Neighbor-Joining (NJ) method and Kimura-2-parameter (K2P) with 1000 bootstrap value.

Table 4. Genetic variation between clades of ITS1/ITS2 and D1/D2 regions of ribosomal DNA.

Parameters	ITS1/ITS2		D1/D2
	Clade A	Clade B	Clade A	Clade B
Fragment length	565 bp	565 bp	564 bp	564 bp
Monomorphic sites	558 bp	556 bp	560 bp	560 bp
Polymorphic sites	7 bp	9 bp	4 bp	4 bp
Parsimony sites	0 bp	1 bp	2 bp	1 bp
Haplotypes	3	3	3	4
Haplotype diversity	0.600	0.600	0.800	0.810
Nucleotide diversity	0.00413	0.00566	0.00355	0.00253

Figure 6. Distribution pattern of ragi isolates using Principal Coordinates Analysis (PCoA) based on ribosomal genes ITS1/ITS2.

Figure 7. Distribution pattern of ragi isolates using Principal Coordinates Analysis (PCoA) based on ribosomal genes D1/D2.

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