03 DNA barcode technology

The concept of DNA barcode is similar to supermarket commodity scanning: identifying species by analyzing sequences in specific regions of biological DNA.For fungi, the internationally recognized standard barcode region is ITS (internal transcriptional spacer).This technology not only solves the problem that has plagued mycologists for decades, but also provides unprecedentedly accurate tools for field collectors, chefs and researchers.

ITS is located between the 18S-5.8S-28S gene of ribosomal DNA and contains two spacers: ITS1 and ITS2.Selecting ITS as the fungal standard barcode region is based on four key advantages:

Ideal Variation: The ITS region has a large enough difference between species to distinguish between relative species, while the intraspecies variation is relatively small, ensuring that the individual sequences of the same species are basically consistent.Actual data show that the ITS region varies between species by 3-15% in most fungal populations, while intraspecies variation is usually less than 1-2%.

General Primers: Researchers have developed multiple pairs of universal primers (such as ITS1/ITS4, ITS5/ITS4) that can amplify the ITS regions of the vast majority of fungal populations without the need to design specific primers for each species.

Domain rich: Mycologists around the world have jointly built databases containing millions of ITS sequences, such as UNITE, GenBank and BOLD systems, providing a solid foundation for comparison and identification.

Easy to amplify: The ITS region has a moderate length (usually 500-700 base pairs), and the PCR amplification success rate is high, and available sequences can be obtained even from degraded samples.

In the Pacific Northwest, collectors used a white amanita as an edible species for many years until the DNA barcode revealed that it was actually a new species close to the death cap (Amanita phalloides).This discovery may have prevented numerous poisoning incidents.Through ITS sequence analysis, the researchers found that this mushroom was 8.7% different from the known edible Amanita, which was much higher than the species-grade distinction threshold (usually 3%).

Collection tool preparation:

Experts recommend: Be sure to keep the complete fruiting body during collection, including the base of the stem – many key identification features are located here.Ideally, individuals from multiple developmental stages are collected, never opening an umbrella until they are fully mature.

Sample saving method:

-Cryo-storage: -20°C for long-term storage, -80°C best

Basic Home Laboratory Program:

1. Take 50-100mg of dried bacterial cap tissue and grind it into fine powder with liquid nitrogen.

2. Add CTAB extraction buffer and bathe in 65°C for 30 minutes

3. Chloroform-isoamyl alcohol extraction to remove protein

4. Isopropanol precipitates DNA

5. Washed with 70% ethanol and dissolved in TE buffer

Commercial kit selection: For beginners, it is recommended to use Qiagen DNeasy Plant Mini Kit or MP Biomedicals FastDNA SPIN Kit, with a success rate of up to 95%, and the whole process only takes 1-2 hours.

Common error avoidance:

Standard ITS amplification scheme:

Sequencing selection:

Basic Process:

1. Sequence quality control: remove low-quality areas and ensure Q value >30

2. BLAST search: Comparison in NCBI or UNITE databases

3. Similarity Assessment: >97-99% Similarity usually indicates the same species

4. Phylogenetic analysis: Confirm the identification results by constructing evolution tree

Practical skills: Don’t blindly believe in the highest matching result.Check multiple high-matching sequences to see their origins and annotation quality.In UNITE databases, sequences with "Species Hypothesis" numbers are preferred, which are professionally annotated and have higher reliability.

UNITE database (https://unite.ut.ee)

GenBank (https://www.ncbi.nlm.nih.gov)

BOLD system (http://www.boldsystems.org)

Multi-database verification: Important authentication should be confirmed in at least two independent databases.If UNITE and BOLD give the same results, the credibility is greatly improved.

Evaluate sequence quality:

LSU (Large Subunit Ribosomal RNA):

TEF1-α (translation extension factor):

RPB2 (second largest subunit of RNA polymerase):

For difficult identification, combining multiple gene markers greatly improves accuracy.Standard multigene analysis includes ITS+LSU+RPB2+TEF1-α, and this combination has a success rate of nearly 100% in the distinction between relative species.

Traditionally, North American collectors view all black morels as the same species.DNA barcode reveals that this is actually a complex of 12 different species, some of which have limited distribution ranges and collection stress may lead to local extinction.Responsible and professional collectors now conduct DNA validation of high-value species to ensure sustainable collection.

Laboratory tests found that 30% of the "wild-collected" mushrooms on the market are actually cultivars.Using DNA barcodes, we are able to verify the authenticity of the vendor's statement.Especially suitable for high-value species such as matsutake, morel and chanterelles.

Revolutionary Technology: Extract total DNA from soil, water or air, analyze fungal community composition through high-throughput sequencing without culturing or observing fruit entities.

Practical Application:

Outdoor Operation Guide:

1. Collect 100-200g of surface soil (removal of leaf layer)

2. Use sterile tools to avoid cross-contamination

3. Refrigerate immediately or add storage buffer

4. Record accurate GPS coordinates and environmental parameters

In cases of poisonous mushroom poisoning, DNA analysis can identify species from vomit, cooking residues, and even the digestive system, providing key information for medical interventions.In a 2019 California poisoning incident, Amanita phalloides were identified from the contents of the stomach through DNA barcodes, and doctors were guided to use experimental antidotes to successfully save patients' lives.

Oxford Nanopore MinION:

Fast PCR Device:

1. On-site sample collection and recording

2. Rapid DNA extraction (15 minutes)

3. Portable PCR amplification (30 minutes)

4. MinION sequencing and real-time analysis (1-4 hours)

5. Access database comparison through satellite network

Practical Experience: In a fungal survey in remote areas of Montana, we used this system to complete the on-site identification of 12 samples in 3 hours, and the traditional method took several weeks.

Business Service (Send Sample):

Self-service plan:

DNA sequencing costs have dropped by 1,000 times over the past decade, from $0.10 per megabase in 2008 to $0.0001 in 2023.This trend continues, and individual fungal identification costs are expected to drop below $5 per time in the next five years.

iNaturalist Platform:

1. Upload clear mushroom photos

2. Record detailed collection information

3. Obtain preliminary community appraisal

4. Select high-quality observations to submit DNA analysis

Professional Project Cooperation:

Sample Submission Agreement:

Through the joint efforts of citizen scientists, the project has collected more than 5,000 georeference samples over the past five years, discovered 23 new fungal species, revised the classification boundaries of 23 genera, and provided key data on the impact of climate change on fungal phenology.

Incomplete database:

Intra-type mutation problem:

Resource requirements:

Identification Trap:

Whole genome sequencing:

Artificial Intelligence Integration:

On-site technology:

Global Initiative:

Study Materials:

Laboratory Equipment:

Consumables:

Sustainable Practice:

Data Sharing:

Accurate identification:

DNA barcodes are not about replacing traditional mycology, but about forming a strong synergy with them.The most reliable identification comes from multiple evidence integration:

Comprehensive Appraisal Agreement:

1. Field macro observation and recording

2. Micro feature verification

3. Ecological and distributed data assessment

4. DNA barcode confirmation

5. Assisted with chemical analysis if necessary

**Experts suggest: Even with the most advanced DNA technology, don’t ignore cultivating your morphological identification skills.The best mycologists are those who can seamlessly combine field observation experience with laboratory data.

Action items of the week:

1. Choose 3 common local mushrooms and take detailed photos

2. Create an account in iNaturalist and upload an observation

3. Study the DNA barcode project of the local fungi society

4. Order books on basic fungi molecular biology

Target for this month:

1. Participate in online or offline DNA barcode seminars

2. Establish a sample collection and preservation system

3. Contact the local university mycology research group

4. Prepare your home lab budget and plan

Vision of the Year:

1. Complete at least 50 native species DNA barcodes

2. Publish a report on scientific discoveries of citizens

3. Create a personal reference sequence library

4. Guide at least one person to learn DNA barcode technology

DNA barcode technology has been democratized for fungi identification, giving the capabilities once limited to professional laboratories to everyone who takes myology seriously.This technology is developing rapidly, with costs continuing to decline and accessibility increasing.Now is the perfect time to learn in depth and integrate DNA barcode into your mycology practice.

Remember, the goal is not to replace the pleasure of natural observation with technology, but to deepen our connection with the fungal kingdom, enhance our identification confidence, and contribute to the global fungal knowledge system.Every sample carefully collected, carefully recorded and accurately identified is a valuable contribution to science.