Sex chromosomes are generally derived from a pair of classical type-A chromosomes, and relatively few alternative models have been proposed up to now.^1,2 B chromosomes (Bs) are supernumerary and dispensable chromosomes with non-Mendelian inheritance found in many plant and animal species^3,4 that have often been considered as selfish genetic elements that behave as genome parasites.^5,6 The observation that in some species Bs can be either restricted or predominant in one sex7, 8, 9, 10, 11, 12, 13, 14 raised the interesting hypothesis that Bs could play a role in sex determination.¹⁵ The characterization of putative B master sex-determining (MSD) genes, however, has not yet been provided to support this hypothesis. Here, in Astyanax mexicanus cavefish originating from Pachón cave, we show that Bs are strongly male predominant. Based on a high-quality genome assembly of a B-carrying male, we characterized the Pachón cavefish B sequence and found that it contains two duplicated loci of the putative MSD gene growth differentiation factor 6b (gdf6b). Supporting its role as an MSD gene, we found that the Pachón cavefish gdf6b gene is expressed specifically in differentiating male gonads, and that its knockout induces male-to-female sex reversal in B-carrying males. This demonstrates that gdf6b is necessary for triggering male sex determination in Pachón cavefish. Altogether these results bring multiple and independent lines of evidence supporting the conclusion that the Pachón cavefish B is a “B-sex” chromosome that contains duplicated copies of the gdf6b gene, which can promote male sex determination in this species.

性染色体一般来源于一对经典的A型染色体，迄今为止提出的替代模型相对较少。^{1 , 2 B 染色体 (Bs) 是在许多植物和动物物种3 , 4}中发现的具有非孟德尔遗传的多余和可有可无的染色体，它们通常被认为是自私的遗传元件，表现为基因组寄生虫。^{5 , 6}观察到在某些物种中，Bs 可以在一种性别中受到限制或占优势7, 8, 9, 10, 11, 12, 13, 14 提出了一个有趣的假设，即 Bs 可以在性别决定中发挥作用。¹⁵然而，尚未提供推定的 B 主性别决定 (MSD) 基因的特征来支持这一假设。在这里，在源自 Pachón 洞穴的Astyanax mexicanus Cavefish 中，我们表明 Bs 以雄性为主。基于携带 B 的雄性的高质量基因组组装，我们对 Pachón 洞穴鱼 B 序列进行了表征，发现它包含假定的 MSD 基因生长分化因子 6b ( gdf6b ) 的两个重复基因座。支持其作为 MSD 基因的作用，我们发现Pachón 穴居鱼gdf6b基因在分化的雄性性腺中特异性表达，并且其敲除诱导携带 B 的雄性中的雄性到雌性的性别逆转。这表明gdf6b是触发 Pachón 洞穴鱼的雄性性别决定所必需的。总之，这些结果带来了多个独立的证据支持这一结论，即 Pachón 洞穴鱼 B 是一个“B 性”染色体，其中包含gdf6b基因的重复拷贝，可以促进该物种的雄性性别决定。