Table_6_Factors Affecting Targeted Sequencing of 353 Nuclear Genes From Herbarium Specimens Spanning the Diversity of Angiosperms.xlsx

The world’s herbaria collectively house millions of diverse plant specimens, including endangered or extinct species and type specimens. Unlocking genetic data from the typically highly degraded DNA obtained from herbarium specimens was difficult until the arrival of high-throughput sequencing approaches, which can be applied to low quantities of severely fragmented DNA. Target enrichment involves using short molecular probes that hybridise and capture genomic regions of interest for high-throughput sequencing. In this study on herbariomics, we used this targeted sequencing approach and the Angiosperms353 universal probe set to recover up to 351 nuclear genes from 435 herbarium specimens that are up to 204 years old and span the breadth of angiosperm diversity. We show that on average 207 genes were successfully retrieved from herbarium specimens, although the mean number of genes retrieved and target enrichment efficiency is significantly higher for silica gel-dried specimens. Forty-seven target nuclear genes were recovered from a herbarium specimen of the critically endangered St Helena boxwood, Mellissia begoniifolia, collected in 1815. Herbarium specimens yield significantly less high-molecular-weight DNA than silica gel-dried specimens, and genomic DNA quality declines with sample age, which is negatively correlated with target enrichment efficiency. Climate, taxon-specific traits, and collection strategies additionally impact target sequence recovery. We also detected taxonomic bias in targeted sequencing outcomes for the 10 most numerous angiosperm families that were investigated in depth. We recommend that (1) for species distributed in wet tropical climates, silica gel-dried specimens should be used preferentially; (2) for species distributed in seasonally dry tropical climates, herbarium and silica gel-dried specimens yield similar results, and either collection can be used; (3) taxon-specific traits should be explored and established for effective optimisation of taxon-specific studies using herbarium specimens; (4) all herbarium sheets should, in future, be annotated with details of the preservation method used; (5) long-term storage of herbarium specimens should be in stable, low-humidity, and low-temperature environments; and (6) targeted sequencing with universal probes, such as Angiosperms353, should be investigated closely as a new approach for DNA barcoding that will ensure better exploitation of herbarium specimens than traditional Sanger sequencing approaches.

全球植物标本馆（herbaria）馆藏着数百万份多样的植物标本，涵盖濒危、已灭绝物种及模式标本（type specimens）。此前，从植物标本馆标本中提取的通常高度降解的DNA里获取遗传数据极具挑战，直至高通量测序（high-throughput sequencing）技术问世——该技术可针对微量且严重碎片化的DNA开展实验。目标富集技术（target enrichment）通过短分子探针杂交并捕获目标基因组区域，以用于高通量测序。本项植物标本组学（herbariomics）研究采用该靶向测序策略，并结合Angiosperms353通用探针组，从435份跨度达204年、涵盖被子植物（angiosperm）全部类群的植物标本馆标本中，成功获取了最多351个核基因序列。研究结果显示，平均可从植物标本馆标本中获取207个基因；不过硅胶干燥标本（silica gel-dried specimens）的平均获基因数与目标富集效率显著更高。针对1815年采集的极危物种圣赫勒拿黄杨（Mellissia begoniifolia）的一份植物标本馆标本，我们成功获取了47个目标核基因序列。相较于硅胶干燥标本，植物标本馆标本的高分子量DNA含量显著更低；且基因组DNA质量随标本存放年限下降，这与目标富集效率呈显著负相关。气候、类群特异性性状以及采集策略也会对目标序列获取产生影响。此外，针对深度研究的10个被子植物大科，我们在靶向测序结果中发现了分类学偏差。我们提出如下建议：（1）对于分布于湿润热带气候区的物种，应优先选用硅胶干燥标本；（2）对于分布于季节性干旱热带气候区的物种，植物标本馆标本与硅胶干燥标本的实验结果相近，二者均可选用；（3）应探索并明确类群特异性性状，以优化基于植物标本馆标本的类群特异性研究；（4）未来所有植物标本馆标本台（herbarium sheets）均应标注所用的标本保存方法细节；（5）植物标本馆标本的长期保存应置于稳定、低湿度且低温的环境中；（6）应深入研究采用通用探针（如Angiosperms353）的靶向测序技术作为DNA条形码（DNA barcoding）的新方法，相较于传统桑格测序（Sanger sequencing）技术，该方法可更高效地开发利用植物标本馆标本资源。