Repeated loss of function at HD mating-type genes and of recombination suppression without mating-type locus linkage in anther-smut fungi

A wide diversity of mating systems occur in nature, with frequent evolutionary transitions in mating-compatibility mechanisms. Basidiomycete fungi typically have two mating-type loci controlling mating compatibility, HD and PR, usually residing on different chromosomes. In Microbotryum anther-smut fungi, there have been repeated events of linkage between the two mating-type loci through chromosome fusions, leading to large non-recombining regions. By generating high-quality genome assemblies, we found that two sister Microbotryum species parasitizing Dianthus plants, M. superbum and M. shykoffianum, as well as the distantly related M. scorzonarae, have their HD and PR mating-type loci on different chromosomes, but with the PR mating-type chromosome fused with part of the ancestral HD chromosome. Furthermore, progressive extensions of recombination suppression have generated evolutionary strata. In all three species, rearrangements suggest the existence of a transient stage of HD-PR linkage by whole chromosome fusion, and, unexpectedly, the HD genes lost their function. In M. superbum, multiple natural diploid strains were homozygous, and the disrupted HD2 gene was hardly expressed. Mating tests confirmed that a single genetic factor controlled mating compatibility (i.e. PR) and that haploid strains with identical HD alleles could mate and produce infectious hyphae. The HD genes have therefore lost their function in the control of mating compatibility in these Microbotryum species. While the loss of function of PR genes in mating compatibility has been reported in a few basidiomycete fungi, these are the first documented cases for the loss of mating-type determination by HD genes in heterothallic fungi. The control of mating compatibility by a single genetic factor is beneficial under selfing and can thus be achieved repeatedly, through evolutionary convergence in distant lineages, involving different genomic or similar pathways.

Methods
Read mapping and quantification

Reference genome matching reads were obtained with bbduk from bbmap [Ref:https://sourceforge.net/projects/bbmap/] with deffault options for quality and adapter trimming. Matching reads were mapped back to the reference genome with STAR 2.7.11a [Ref:https://doi.org/10.1093/bioinformatics/bts635] with the deffault options for paired end reads. Reads with inserts larger than 5 kbp were filtered out. Read counts by transcript feature were obtained with featureCounts from subread 2.0.2 suite [Ref:https://doi.org/10.1093/bioinformatics/btt656]. 

Trans-specific polymorphism

Gene trees were obtained with IQ-TREE 2.2.6 based on macse v2.05 codon-based alignments. Gene trees were midpoint rooted with gotree [Ref:https://doi.org/10.1093/nargab/lqab075] and subtrees containing the M. shykoffianum and M. superbum A1 alleles recovered with nw_clade from Newick utils [Ref: https://doi.org/10.1093/bioinformatics/btq243]. If the subtree contains a single branch connecting only two tips it means that A1 alleles were recovered as monophyletic (i.e. displaying transpecific polymorphism), we repeated the procedure for the A2 alleles and counted the number trees showing full (both alleles recovered as monophyletic clades) or partial (either A1 or A2 were recovered as monophyletic) transpecific polymorphism. Gene trees in which neither A1 nor A2 were recovered as monophyletic were collated as "Other" topologies, often following the species phylogeny (i.e. alternative alleles from the same species clustered together, M. superbum and M. shykoffianum being recovered as sister clades).

自然界中存在着极为多样的交配系统，交配相容性机制常发生进化转变。担子菌（Basidiomycete）通常拥有两个控制交配相容性的交配型位点HD与PR，二者通常位于不同的染色体上。在花药黑粉菌属（Microbotryum）的花药黑粉菌中，两类交配型位点已通过染色体融合发生了多次连锁事件，进而形成了大型非重组区域。
通过构建高质量基因组组装序列，我们发现两种寄生于石竹属（Dianthus）植物的近缘花药黑粉菌物种——超级花药黑粉菌（M. superbum）与希氏花药黑粉菌（M. shykoffianum），以及亲缘关系较远的斯科尔佐纳拉花药黑粉菌（M. scorzonarae），其HD与PR交配型位点虽仍位于不同染色体上，但PR交配型染色体已与祖先HD染色体的部分区域发生融合。此外，重组抑制的逐步延伸形成了进化层（evolutionary strata）。在这三个物种中，基因组重排均提示存在过通过全染色体融合实现HD-PR连锁的过渡阶段，且令人意外的是，HD基因已丧失其功能。在超级花药黑粉菌中，多个自然二倍体菌株呈纯合状态，且受损的HD2基因几乎不表达。交配试验证实，单一遗传因子（即PR）即可控制交配相容性，携带相同HD等位基因的单倍体菌株仍可交配并产生侵染性菌丝。因此，在这些花药黑粉菌物种中，HD基因已不再参与交配相容性的调控。
尽管已有少数担子菌中PR基因在交配相容性调控中功能丧失的报道，但这是首次在异宗配合真菌（heterothallic fungi）中记录到HD基因丧失交配型决定功能的案例。由单一遗传因子控制交配相容性在自交情境下具有进化优势，因此这一性状可通过远缘类群的趋同进化反复演化，涉及不同的基因组机制或相似的调控通路。

方法
读段比对与定量分析
使用bbmap套件中的bbduk工具，按照默认参数对测序读段进行质量修剪与接头去除，得到匹配参考基因组的读段。将过滤后的读段通过STAR 2.7.11a工具，以双端读段默认参数比对至参考基因组，过滤掉插入片段长度大于5 kbp的读段。使用subread 2.0.2套件中的featureCounts工具，获取每个转录本特征的读段计数。

跨物种多态性（trans-specific polymorphism）分析
基于macse v2.05的密码子比对结果，使用IQ-TREE 2.2.6构建基因树。使用gotree工具将基因树以中点为根节点，再通过Newick utils的nw_clade工具提取包含希氏花药黑粉菌与超级花药黑粉菌A1等位基因的子树。若该子树仅包含一条连接两个末端节点的分支，则说明A1等位基因被鉴定为单系群（即存在跨物种多态性）。随后对A2等位基因重复上述流程，并统计显示完全跨物种多态性（A1与A2等位基因均被鉴定为单系进化枝）与部分跨物种多态性（仅A1或A2等位基因被鉴定为单系进化枝）的基因树数量。未检测到A1与A2等位基因均为单系群的基因树归类为“其他”拓扑结构，这类树通常遵循物种系统发育关系（即同一物种的等位基因聚为一支，超级花药黑粉菌与希氏花药黑粉菌被鉴定为姐妹进化枝）。