Validation of Internal Control Genes for Quantitative Real-Time PCR Gene Expression Analysis in Morchella

et al. 2020

PLoS ONE

The development of fungal fruiting bodies from a hyphal thallus is inducible under low temperature (cold stress). The molecular mechanism has been subject to surprisingly few studies. Analysis of gene expression level has become an important means to study gene function and its regulation mechanism. But identification of reference genes (RGs) stability under cold stress have not been reported in famous medicinal mushroom-forming fungi Cordyceps militaris. Herein, 12 candidate RGs had been systematically validated under cold stress in C. militaris. Three different algorithms, geNorm, NormFinder and BestKeeper were applied to evaluate the expression stability of the RGs. Our results showed that UBC and UBQ were the most stable RGs for cold treatments in short and long periods, respectively. 2 RGs (UBC and PP2A) and 3 RGs (UBQ, TUB and CYP) were the suitable RGs for cold treatments in short and long periods, respectively. Moreover, target genes, two-componentsystem histidine kinase genes, were selected to validate the most and least stable RGs under cold treatment, which indicated that use of unstable expressed genes as RGs leads to biased results. Our results provide a good starting point for accurate reverse transcriptase quantitative polymerase chain reaction normalization by using UBC and UBQ in C. militaris under cold stress and better support for understanding the mechanism of response to cold stress and fruiting body formation in C. militaris and other mushroom-forming fungi in future research.

Section: Discussionsupporting

confidence: 90%

Analysis of reference genes stability and histidine kinase expression under cold stress in Cordyceps militaris

et al. 2020

PLoS ONE

“…Compared with microarray and RNA sequencing, RT-qPCR technique has the advantages of high accuracy, high sensitivity, good repeatability and low cost for quantifying gene expression [16,17]. [29], which was similar to our findings. An interesting result is that, in…”

Section: Discussionsupporting

confidence: 89%

Analysis of Reference Genes stability and Histidine Kinase Expression under Cold Stress in Cordyceps militaris

et al. 2020

Preprint

Background: The development of fungal fruiting bodies from a hyphal thallus represents a transition from simple to complex multicellularity that is inducible under low temperature (cold stress). The molecular mechanism has been subject to surprisingly few studies. Analysis of gene expression level has become an important means to study gene function and its regulation mechanism. But identification of reference genes (RGs) stability under cold stress have not been reported in famous medicinal mushroom-forming fungi Cordyceps militaris.Results: Herein, 12 candidate RGs had been systematically validated under cold stress in C. militaris.Three different algorithms, geNorm, NormFinder and BestKeeper were applied to evaluate the expression stability of the RGs. Our results showed that UBC and UBQ were the most stable RGs for cold treatments in short and long time, respectively. 2 RGs (UBC and PP2A) and 3 RGs (UBQ, TUB and CYP) were the suitable housekeeping genes for cold treatments in short and long time, respectively.Moreover, target genes, two-component-system histidine kinase genes, were selected to validate the most and least stable RGs under cold treatment, which indicated that use of unstable expressed genes as housekeeping genes leads to biased results. Conclusions:Our results provide a good starting point for accurate reverse transcriptase quantitative polymerase chain reaction normalization by using UBC and UBQ in C. militaris under cold stress and better support for understanding the mechanism of response to cold stress and fruiting body formation in C. militaris and other mushroom-forming fungi in future research. BackgroundCordyceps militaris, a famous traditional Chinese medicine, has been used as a healthy food for a long time in China. The C. militaris fruiting body has anti-inflammatory [1], anti-tumor [2], antiinfluenza virus [3] and radio-protection [4] functions. Methods for commercial production of fruiting bodies of this fungus have been established in artificial media [5,6] or with insects, such as silkworm Bombyx mori pupae [7]. The molecular mechanism of mushroom fruiting body formation is a basic biological problem. The essential role of light in fruit body development in C. militaris was demonstrated in previous study [8]. A blue-light receptor gene white collar-1 (wc-1) inactivation

“…M10 (mycelial and sclerotial tissues grown on CYM liquid medium for 3 days, 7 days and 14 days) was used for RNA-sequencing. The DNA and cDNA were prepared as reported by Liu et al and Zhang et al [12,40]. The information of the strains has been deposited in the NCBI with bioSample number SAMN12905432 (M10) and SAMN12916226 (M10M26).…”

Section: Strain Selection and Materials Preparationmentioning

confidence: 99%

“…The DNA and cDNA were prepared as previously reported [12,40], which were used to amplify the monospore population with mating type primer (Mat1-1F/R CATGTCACTYCGYCCRGTTTA/ CCACATRGCTTTCCTCTTCTC, Mat1-2F/R AACAGATGCTSGAAGAAGC/CTTATAYCCAGGAT GCTTTAC) or used to confirm the intron boundaries of mitochondrial genome or the accuracy of sequence assembling results. The amplification system (20 µL) consisted of 0.3 µL of 4× dNTPmix, 2 µL 10× PCR buffer, 10 nM each of primer, 50 ng DNA template, and 1.5 U of rTaq (Takara, Minamikusatsu, Japan), under the conditions of 95 • C for 3 min, followed by 35 cycles of 95 • C for 30 s, 60 • C for 30 s and 72 • C for 60 s. The amplified products were detected by 1.5% agarose gel electrophoresis and sequenced by pyrosequencing technology.…”

Section: Dna and Rna Extraction And Pcr Amplificationmentioning

confidence: 99%

Subchromosome-Scale Nuclear and Complete Mitochondrial Genome Characteristics of Morchella crassipes

Cai

Zhang

et al. 2020

IJMS

Self Cite

Morchella crassipes (Vent.) Pers., a typical yellow morel species with high economic value, is mainly distributed in the low altitude plains of Eurasia. However, rare research has been performed on its genomics and polarity, thus limiting its research and development. Here, we reported a fine physical map of the nuclear genome at the subchromosomal-scale and the complete mitochondrial genome of M. crassipes. The complete size of the nuclear genome was 56.7 Mb, and 23 scaffolds were assembled, with eight of them being complete chromosomes. A total of 11,565 encoding proteins were predicted. The divergence time analysis showed that M. crassipes representing yellow morels differentiated with black morels at ~33.98 Mya (million years), with 150 gene families contracted and expanded in M. crassipes versus the two black morels (M. snyderi and M. importuna). Furthermore, 409 CAZYme genes were annotated in M. crassipes, containing almost all plant cell wall degrading enzymes compared with the mycorrhizal fungi (truffles). Genomic annotation of mating type loci and amplification of the mating genes in the monospore population was conducted, the results indicated that M. crassipes is a heterothallic fungus. Additionally, a complete circular mitochondrial genome of M. crassipes was assembled, the size reached as large as 531,195 bp. It can be observed that the strikingly large size was the biggest up till now, coupled with 14 core conserved mitochondrial protein-coding genes, two rRNAs, 31 tRNAs, 51 introns, and 412 ncORFs. The total length of intron sequences accounted for 53.67% of the mitochondrial genome, with 19 introns having a length over 5 kb. Particularly, 221 of 412 ncORFs were distributed within 51 introns, and the total length of the ncORFs sequence accounted for 40.83% of the mitochondrial genome, and 297 ncORFs had expression activity in the mycelium stage, suggesting their potential functions in M. crassipes. Meanwhile, there was a high degree of repetition (51.31%) in the mitochondria of M. crassipes. Thus, the large number of introns, ncORFs and internal repeat sequences may contribute jointly to the largest fungal mitochondrial genome to date. The fine physical maps of nuclear genome and mitochondrial genome obtained in this study will open a new door for better understanding of the mysterious species of M. crassipes.