De novo transcriptome analysis of a medicinal fungi Phellinus linteus and identification of SSR markers

Figures & data

Figure 1. Morphological characteristics of colony and mycelia from P. linteus strains.

Note: Fruit of P. linteus from M. alba (A), solid colony (B) and liquid mycelia (C).

Table 1. Parameters used for development of SSR.

No.	SSR type	Set of repeating bases	Repetition number for the set
1.	Mononucleotide	Repeats 1	≥10 bases
2.	Dinucleotide	Repeats 2	≥6 bases
3.	Trinucleotide	Repeats 3	≥5 bases
4.	Tetranucleotide	Repeats 4	≥5 bases
5.	Pentanucleotide	Repeats 5	≥5 bases
6.	Hexanucleotide	Repeats 6	≥5 bases

Note: SSR detection was done with software MicroSAtellite (MISA) using unigenes as reference.

Table 2. Statistics of Illumina sequencing.

Total raw reads	17,467,362
Total clean reads	16,383,818
Total clean nucleotides (Nt)	1,474,543,620
Q20 percentage	99.00%
N percentage	0.01%
GC percentage	51.49%

Note: Total raw reads and total clean reads are actually total number of raw reads and total number of clean reads. Total clean nucleotides is the total number of clean nucleotides. Q20 percentage is the proportion of nucleotides with quality value larger than 20 in reads; N percentage is the proportion of unknown nucleotides in clean reads. GC percentage is the proportion of guanidine and cytosine nucleotides among the total nucleotides.

Table 3. Statistics of assembly quality.

	Contig	Unigene
Total number	35,532	25,811
Total length (nt)	17,841,209	25,229,528
Mean length (nt)	502	977
N50	1262	1658
Total consensus sequences	−	25,811
Distinct clusters	−	9395
Distinct singletons	−	16,416

Note: Transcriptome de novo assembly is carried out by short reads assembling program -- Trinity. The statistical parameters of the assembly quality in cotig and unigene included total number, total length (nt) and mean length (nt); measure of average contig or unigene length (N50 length), total consensus sequences, distinct clusters and distinct singletons.

Table 4. Summary statistics of functional annotation of Phellinus linteus unigenes in public databases.

Public protein database	Number of unigene hits	Percentage (%)
Nr	19,266	74.64
NT	4562	17.67
Swiss-Prot	10,978	42.53
KEGG	11,088	42.96
COG	7831	30.34
GO	6845	26.52
ALL	19,350	74.97

Note: Unigene annotation provides information of the expression and function of unigene. Information of functional annotation gives protein functional annotation, COG functional annotation and gene ontology (GO) functional annotation of Unigenes. Unigene sequences are firstly aligned by BLASTX to protein databases like Nr, Swiss-Prot, KEGG and COG (E-value < 1.0 E⁻⁵) and aligned by BLASTN to nucleotide databases NT (E-value < 1.0 E⁻⁵), retrieving proteins with the highest sequence similarity with the given Unigenes along with their protein functional annotations, the results about this are included in the folder annotation.

Figure 2. Characteristics of sequence homology of P. linteus blasted against NCBI non-redundant (Nr) database.

Note: E-value distribution of BLAST hits for matched unigene sequences, using an E-value cutoff of 1.0 E⁻⁴⁵ (A), similarity distribution of top BLAST hits for each unigene (B) and species distribution of the top BLAST hits (C).

Figure 3. Histogram presentation of clusters of orthologous groups (COG) classification (unigene).

Note: All unigenes were aligned to the COG database to predict and classify possible functions. Out of 19,266 NR hits, 7831unigenes were assigned to 25 COG classifications.

Figure 4. Gene ontology classification of assembled unigenes.

Note: The results are summarized in three main categories: biological process, cellular component and molecular function. The right y-axis indicates the number of genes in a category. The left y-axis indicates the percentage of a specific category of genes.

Table 5. Unigenes related to sterol biosynthesis in Phellinus linteus.

Pathway	Enzyme name	Symbol/gene name	Number of annotated sequences transcripts
Terpenoid backbone biosynthesis	Acetyl-CoA C-acetyltransferase	E.9, atoB	2
	Hydroxymethylglutaryl-CoA synthase	E.10	1
	Hydroxymethylglutaryl-CoA reductase (NADPH)	HMGCR	2
	Phosphomevalonate kinase	mvaK2	2
	Diphosphomevalonate decarboxylase	MVD, mvaD	1
	Isopentenyl-diphosphate delta-isomerase	IDI	1
	Farnesyl diphosphate synthase	FDPS	4
Ergosterol biosynthesis	Lanosterol synthase	LSS, ERG7	2
	Sterol 14-demethylase	CYP51	7
	Delta14-sterol reductase	TM7SF2, ERG24	3
	Methylsterol monooxygenase	SC4MOL, ERG25	2
	Sterol-4alpha-carboxylate 3-dehydrogenase (decarboxylating)	NSDHL, ERG26	2
	3-keto steroid reductase	ERG27	2
	Sterol 24-C-methyltransferase	SMT1, ERG6	2
	C-8 sterol isomerase	ERG2	1
	Lathosterol oxidase	ERG3	1
	C-22 sterol desaturase	ERG5	6
	Delta24(24(1))-sterol reductase	ERG4	1
Phytosterol biosynthesis	Sterol 24-C-methyltransferase	SMT1, ERG6	2
	Sterol 14-demethylase	CYP51	7
	Delta14-sterol reductase	TM7SF2, ERG24	3
	Cholestenol delta-isomerase	EBP	1
	Lathosterol oxidase	SC, ERG3	1

Table 6. Statistics of SSRs identified from Phellinus linteus transcriptome.

1. Total number of examined sequences	25,811
2. Total size of examined sequences (bp)	25,229,528
3. Total number of identified SSRs	1129
4. Number of sequences containing one SSR	873
5. Number of sequences containing more than one SSR	177
6. Number of SSR present in compound formation	94

Figure 5. Simple sequence repeats: distribution of SSRs into di-, tri-, tetra-, penta- and hexarepeat types.