Gene expression in xylem tissue on an Eucalyptus pseudo-testcross population: discovery array probes

Technological advances are progressively increasing the application of genomics to a wider array of economically and ecologically important species. High-density maps enriched for transcribed genes facilitate the discovery of connections between genes and phenotypes. We report the construction of a high-density linkage map of expressed genes for the heterozygous genome of Eucalyptus using Single Feature Polymorphism (SFP) markers. SFP discovery and mapping was achieved using pseudo-testcross screening and selective mapping to simultaneously optimize linkage mapping and microarray costs. SFP genotyping was carried out by hybridizing complementary RNA prepared from 4.5 year-old trees xylem to an SFP array containing 103,000 25-mer oligonucleotide probes representing 20,726 unigenes derived from a modest size expressed sequence tags collection. An SFP-mapping microarray with 43,777 selected candidate SFP probes representing 15,698 genes was subsequently designed and used to genotype SFPs in a larger subset of the segregating population drawn by selective mapping. A total of 1,845 genes were mapped, with 884 of them ordered with high likelihood support on a framework map anchored to 180 microsatellites with average density of 1.2 cM. Using more probes per unigene increased by two-fold the likelihood of detecting segregating SFPs eventually resulting in more genes mapped. In silico validation showed that 87% of the SFPs map to the expected location on the 4.5X draft sequence of the Eucalyptus grandis genome. The Eucalyptus 1,845 gene map is the most highly enriched map for transcriptional information for any forest tree species to date. It represents a major improvement on the number of genes previously positioned on Eucalyptus maps and provides an initial glimpse at the gene space for this global tree genome. A general protocol is proposed to build high-density transcript linkage maps in less characterized plant species by SFP genotyping with a concurrent objective of reducing microarray costs. HIgh-density gene-rich maps represent a powerful resource to assist gene discovery endeavors when used in combination with QTL and association mapping and should be especially valuable to assist the assembly of reference genome sequences soon to come for several plant and animal species. Two biological replication of 28 genotypes were hybridized to the custom microarray, labeled with Cy3 and Cy5. Genotypes are fullsib progenies of the cross E. urophylla X E. grandis. A loop design was employed for the hybridization using xylem tissue. In total the microarray comprised 103,000 probes representing 20,726 unigenes with an average of five probes per unigene. Twenty-six negative control probes were also included in the microarray.

技术进步正逐步推动基因组学在更多兼具经济与生态价值的物种中得到应用。富集转录基因的高密度遗传图谱，有助于解析基因与表型之间的关联机制。本研究报道了利用单特征多态性（Single Feature Polymorphism, SFP）标记，构建桉树杂合基因组表达基因高密度连锁图谱的工作。研究采用拟测交筛选与选择性作图策略，同步优化连锁作图与微阵列（microarray）使用成本，实现了SFP的发掘与定位。SFP基因分型实验通过将取自4.5年生树木木质部的互补RNA，与包含103000条25聚体寡核苷酸探针的SFP芯片进行杂交完成；该芯片覆盖了来自小规模表达序列标签（expressed sequence tags, EST）集合的20726个单基因簇（unigene）。后续研究设计了一款包含43777条经筛选的候选SFP探针的SFP作图微阵列，这些探针对应15698个基因，用于对通过选择性作图筛选出的更大规模分离群体子集进行SFP基因分型。最终共定位1845个基因，其中884个基因在以180个微卫星（microsatellite）为锚定标记的框架图谱上得到高置信度排序，图谱平均密度为1.2 cM。每个单基因簇使用更多探针，可使分离型SFP的检出率提升一倍，最终实现更多基因的定位。计算机模拟验证（in silico validation）结果显示，87%的SFP定位结果与巨桉（Eucalyptus grandis）基因组4.5倍覆盖度草图中的预期位置相符。这款包含1845个基因的桉树图谱，是目前所有林木树种中转录信息富集度最高的遗传图谱。相较以往桉树遗传图谱上定位的基因数量，本研究实现了大幅提升，并首次初步解析了这一全球重要林木树种的基因空间。本研究提出了一套通用实验方案，可在研究基础较为薄弱的植物物种中通过SFP基因分型构建高密度转录连锁图谱，同时兼顾降低微阵列使用成本。高密度基因富集型图谱与数量性状位点（quantitative trait locus, QTL）定位、关联分析结合使用时，可成为助力基因发掘的高效工具；对于即将开展参考基因组组装的多种动植物物种而言，这类图谱尤其具有应用价值。研究将28份基因型材料的两个生物学重复样本，分别用Cy3与Cy5荧光标记后与定制微阵列进行杂交。这些基因型材料来自尾叶桉（Eucalyptus urophylla）与巨桉（Eucalyptus grandis）杂交得到的全同胞后代群体。本次杂交实验采用环状设计（loop design），使用的组织为木质部。该定制微阵列总计包含103000条探针，覆盖20726个单基因簇，平均每个单基因簇对应5条探针；同时芯片中还设置了26个阴性对照探针。