The evolution of spinable cotton fiber entailed prolonged development and a novel metabolism. The evolution of spinable cotton fiber entailed prolonged development and a novel metabolism

exceptional example of evolutionary innovation is the single-celled seed trichome in Gossypium ("cotton fiber"). We have used fiber development in Gossypium as a system to understand how morphology can rapidly evolve. Fiber has undergone considerable morphological changes between the short, tightly adherent fibers of G. longicalyx and the derived long, spinable fibers of its closest relative, G. herbaceum, that facilitated cotton domestication. We conducted comparative gene expression profiling across a developmental time-course of fibers from G. longicalyx and G. herbaceum using microarrays with ~ 22,000 genes. Expression changes between stages were temporally protracted in G. herbaceum relative to G. longicalyx, reflecting a prolongation of the ancestral developmental program. Gene expression and GO analyses showed that many genes involved with stress responses were up-regulated early in G. longicalyx fiber development. Several candidate genes up-regulated in G. herbaceum have been implicated in regulating redox levels and cell elongation processes. Three genes previously shown to modulate hydrogen peroxide levels were consistently expressed in domesticated and wild cotton species with long fibers but expression was not detected by qRT-PCR in wild species with short fibers. Hydrogen peroxide is important for cell elongation, but at high concentrations it becomes toxic, activating stress processes that may lead to early onset of secondary cell wall synthesis and the end of cell elongation. These observations suggest that the evolution of long spinable fibers in cotton was accompanied by novel expression of genes assisting in the regulation of reactive oxygen species levels. Our data suggest a model for the evolutionary origin of a novel morphology through differential gene regulation causing prolongation of an ancestral developmental program. Keywords: Cotton, fiber, evolution, time-point, comparative genomic hybridization, stress response genes, H2O2 Overall design: A balanced developmental loop design for microarray analysis was performed. For G. herbaceum (A1) and G. longicalyx (F), four fiber developmental time-points, 5, 10, 20 and 25 DPA were sampled. Within each species, hybridizations were performed between each pair of consecutive developmental stages by labeling one with Cy5 and the other with Cy3, and by closing the loop with a comparison of 25 and 5 dpa. In addition, 2 hybridizations were done between species at each time-point, using a dye swap for each pair. With three biological replications and 16 slides each, we generated gene expression data from a total of 48 microarrays.

进化创新的一个杰出范例，便是棉属（Gossypium）植物的单细胞种子表皮毛——棉纤维（cotton fiber）。本研究以棉属植物的纤维发育为研究体系，旨在解析形态性状如何实现快速演化。长萼棉（G. longicalyx）的纤维短小且紧密附着，而其近缘姊妹种草棉（G. herbaceum）则演化出可纺的长纤维，二者纤维形态差异显著，这一演化事件推动了棉花的驯化进程。本研究针对长萼棉与草棉的纤维发育时序开展比较基因表达谱分析，所用微阵列（microarray）包含约22000个基因。相较于长萼棉，草棉各发育阶段间的基因表达变化在时间维度上更为持久，这反映出其祖先发育程序的延长。基因表达与基因本体（Gene Ontology, GO）富集分析显示，长萼棉纤维发育早期，大量参与应激响应的基因被上调表达。草棉中上调表达的若干候选基因，被证实参与调控氧化还原水平与细胞伸长过程。此前被证实可调控过氧化氢（hydrogen peroxide, H₂O₂）水平的3个基因，在拥有长纤维的栽培棉与野生棉种中均稳定表达，但在拥有短纤维的野生棉种中，实时定量反转录PCR（qRT-PCR）未检测到其转录产物。过氧化氢对细胞伸长至关重要，但浓度过高时会产生毒性，激活应激通路，进而可能引发次生细胞壁合成提前启动，并终止细胞伸长过程。上述结果表明，棉花可纺长纤维的演化过程，伴随了调控活性氧（reactive oxygen species, ROS）水平相关基因的新型表达模式。本研究数据提出了一个演化模型：通过差异基因调控延长祖先发育程序，进而催生全新的形态性状。关键词：棉花，纤维，演化，时序样本，比较基因组杂交（comparative genomic hybridization），应激响应基因，过氧化氢（H₂O₂）。实验设计：本研究采用平衡发育环路设计开展微阵列分析。针对草棉（G. herbaceum，基因组类型A1）与长萼棉（G. longicalyx，基因组类型F），分别采集其纤维发育的4个时序样本：开花后5、10、20、25天（days post anthesis, DPA）。在每个物种内，对每对连续发育阶段的样本进行杂交：分别用Cy5和Cy3标记两个样本，并通过25 DPA与5 DPA的杂交完成环路闭环。此外，在每个时序节点开展2组种间杂交，每组均进行荧光染料互换。本实验设置3次生物学重复，每个物种对应16张芯片，最终共获得48张微阵列的基因表达数据。