Auricularia heimuer is a valuable traditional Chinese fungus used as food and medicine. Hei29 is a strain derived from wild A. heimuer through systematic domestication and selection. It has been the primary A. heimuer variety in Northeast China for 30 years and offers the advantages of high yield, good commercial property, and stable traits. This study used protoplast nucleation on Hei29 to produce two amiable and paired monokaryons, Hei29-D1 and Hei29-D2. The genome of Hei29 was sequenced utilizing the Illumina PE150 and PacBio Sequel sequencing platforms. Hei29-D1 and Hei29-D2 had genomic sizes of 47.54 Mb and 47.49 Mb, GC contents of 56.95% and 56.99%, and an N50 of 2.37 Mb and 4.28 Mb, respectively. Hei29’s genome possessed two phytoene synthase (PSY) protein genes, one of which—PSY encoded by g894—has a transmembrane domain. The phylogenetic tree showed that Hei29 shared the closest evolutionary relationship with Auricularia subglabra TFB-10046 SS5. Collinearity analysis showed that the correlation between the two monokaryons was as high as 90.81%. Cluster analysis revealed that Hei29 contains 12,362 core genes, 223 unique genes in Hei29-D1, and 228 unique genes in Hei29-D2. This study is the first to sequence two related and paired monokaryons from A. heimuer, which is critical for fully understanding the genetic composition and information of the characteristic strain of A. heimuer in Northeast China. It establishes the data and theoretical foundation for gene mining, usage, and molecular breeding. It further promotes the genetic breeding and active substance utilization of A. heimuer.

Auricularia heimuer is an important cultivated edible mushroom in China. This study used the early maturity, chrysanthemum-shaped, high yield strain “8808” and the late-maturity, single-shaped, high-quality strain “HW15” as parental strains. The objective was to develop a new strain of A. heimuer combining early maturity with excellent quality through protoplast fusion. Protoplasts from the parental strains were fused using a polyethylene glycol (PEG)-mediated method. The obtained hybrids were identified through clamp connection, antagonism tests, esterase isozyme analysis, and Inter-Simple Sequence Repeat (ISSR) molecular marker techniques, followed by ultimate selection through cultivation. Among the evaluated hybrids, strain R33 exhibited desirable agronomic traits: primordium formation occurred 4 days earlier than the parental strain HW15, harvest time was advanced by 6 days, yield increased by 14.7%, and the ratio of absorbed water improved by 29.8%. Transcriptomic analysis revealed that starch and sucrose metabolism as well as amino acid metabolism pathways were significantly upregulated in R33, which may be associated with its early maturity and high-yield phenotype. Overall, protoplast fusion technology can be used to create promising candidate strains that combine early maturity, high yield, and excellent quality.