Map-based cloning of genes encoding key enzymes for pigment synthesis in Auricularia cornea

Color is an important quality attribute of fungi, and a useful marker for classification, genetic, and molecular research. However, there is much debate over which enzymes play key regulatory roles in pigment synthesis pathways among different fungi and even within the same species. Auricularia cornea is the most widely cultivated mushroom in the genus Auricularia; 1.834 million tons of this mushroom were produced in 2016 in China. Thus, systematic studies on its color inheritance and the genes encoding key enzymes for pigment synthesis have high scientific and economic value. In this study, the white strain ACW001 and the purple strain ACP004 of A. cornea were used as dikaryotic parents. Selfing populations of ACW001 and ACP004 were constructed with their monokaryotic strains. The fruiting body color of the two populations was consistent with that of their parents, confirming that the two parents were color homozygotes. All strains in the hybrid population of the two parents produced purple fruiting bodies. A robust hybrid strain (ACW001-33×ACP004-33) was selected from the hybrid population, and 87 monokaryotic strains of ACW001-33×ACP004-33 were obtained as a mapping population. Finally, a testcross population was constructed by crossing the mapping population with the test strain ACW001-9. The color genotype of each monokaryotic strain in the mapping population was identified by a fruiting test. The genomes of the two monokaryotic strains ACW001-33 and ACP004-33 were sequenced, and then simple sequence repeat (SSR) and sequence-related amplified polymorphism (SRAP) molecular marker primers were developed. Then, 88 pairs of primers that could distinguish the genotypes of the mapping population were used to construct a genetic linkage map. The genetic linkage map consisted of 12 linkage groups (LGs) spanning 1315.2 cM. The color control locus was preliminarily located at 24.5 cM of the 11th LG. Fine-mapping primers were designed based on sequence differences between ACW001-33 and ACP004-33 in the primary location region. Four color control candidate genes were located in an 8.2-kb region of ACW001-33_contig733 and a 9.2-kb region of ACP004-33_contig802. Homologous alignment and prediction of conserved domain analyses indicated that two of the color control candidate genes encoded proteins with unknown function, and the other two, ACP004_g11815 and ACP004_g11816, encoded glutamyl aminotransferases. These two genes were consecutively arranged on ACP004-33_contig802, and were likely to encode key enzymes in the γ-glutamine-4-hydroxy-benzoate (GHB) pigment synthesis pathway. Primers were designed from the flanking sequences of the two genes and used to analyze the testcross population. Products were amplified only from the 30 testcross strains with purple fruiting bodies, confirming the accuracy of the localization results. We discuss the deficiencies and advantages of map-based cloning in fungi vs. plants, and summarize the steps and requirements of the map-based cloning method for fungi. This study has provided novel ideas and methods for locating functional genes in fungi.