Hong Phuong Le

Natural history and agricultural collections contain hundreds of millions of specimens classified in terms of time, space and taxonomy. Since the first success of ancient DNA (aDNA) isolation in the 1980s, these repositories, including herbaria for plants, have been intensively used to support studies in taxonomy, macroevolution and genetic responses to anthropogenic activities. For herbarium specimens, aDNA is usually extracted from leaf samples and is highly fragmented (typically length of 50–100 bp). To optimise the amount of data retrieved and minimise destructive sampling, we isolated DNA from an unconventional plant tissue type—seed embryos. We carried out whole‐genome sequencing and compared sequenced DNA quality between embryo and leaf tissue from the same specimen. We evaluated endogenous DNA proportion, median fragment length, damage fraction per site ( λ ), decay rates ( k ), nucleotide misincorporations and library complexity for three species: cultivated rice Oryza sativa , wild rice O. rufipogon and wild barley Hordeum spontaneum . In O. sativa , embryos exhibited significantly higher endogenous content and median fragment length than leaves. The superior DNA preservation was likely due to the protective role of the seed husk, which might play an important role in DNA preservation in plants collected in the tropics. By contrast, in temperate H. spontaneum , tissue type had minimal impact on DNA quality. Despite the minuscule size of the embryos, all derived genomic libraries were highly complex, sufficient for deep whole genome sequencing. These results highlight seed embryos as a promising alternative aDNA source for herbarium specimens, economic botany and anthropological museum collections.

Natural history and agricultural collections, which contain hundreds of millions of specimens classified in terms of time, space, and taxonomy, are valuable resources for diverse fields of research. Since the first success of ancient DNA (aDNA) isolation in the 1980s, these repositories, including herbaria for plants, have been intensively used to support studies in taxonomy, macroevolution, and genetic responses to anthropogenic activities over the past centuries. Two major challenges of aDNA research are environmental contamination and DNA degradation. For herbarium specimens, aDNA is usually extracted from leaf samples. It is highly fragmented (typically length of 50 to 100 bp) with a higher breakdown rate than that in most bone remains. To optimise the amount of data retrieved and minimise destructive sampling, we isolated DNA from an unconventional plant tissue type – seed embryos. We carried out whole-genome sequencing and compared sequenced DNA quality between embryo and leaf tissue. We evaluated endogenous DNA proportion, median fragment length, damage fraction per site ( λ ), decay rates, nucleotide misincorporations, and library complexity for three species: cultivated rice Oryza sativa , wild rice O. rufipogon , and wild barley Hordeum spontaneum . In O. sativa , embryos exhibited significantly higher endogenous content and median fragment length than leaves, while in O. rufipogon only median fragment length was higher. The superior DNA preservation was likely due to the protective role of the seed husk, which might play an important role in DNA preservation in plants collected in the tropics. By contrast, in temperate H. spontaneum , tissue type had minimal impact on DNA quality. Despite the minuscule size of the embryos, all derived genomic libraries were highly complex, sufficient for deep whole genome sequencing. These results highlight seed embryos as a promising alternative aDNA source for millions of herbarium specimens, and enable effective genomic analyses of other historical plant collections, such as economic botany and anthropological museum collections.

Herbarium collections are a vast but underutilized resource for ancient DNA research, containing over 400 million specimens with detailed metadata and spanning centuries of global biodiversity. Understanding patterns of DNA preservation in natural collections is crucial for optimizing ancient DNA studies and informing future curation practices. We analysed genomic data for 573 herbarium specimens from six plant species from the genera Hordeum and Oryza collected from the Americas and Eurasia over 220 years. Using standardized laboratory protocols and shotgun sequencing, we quantified DNA degradation and elucidated factors that accelerate it. We find significant age-dependent DNA fragmentation rates, indicating temporal degradation processes not detected in prehistoric samples. In our analysis, DNA decay rates in herbarium specimens were almost eight times faster than in moa bones, reflecting fundamental differences in tissue composition and preservation environments. Environmental conditions at the time of specimen collection emerged as the major determinants of post-mortem damage rates, with the interaction term between temperature and genus being the dominant driver of cytosine deamination. We find no effect of sample storage on DNA damage and degradation. These findings provide insights into how climatic origin, preservation environment, taxonomic identity and age influence DNA preservation while highlighting opportunities for improving institutional preservation practices. Due to standardised preservation conditions, museum collections can provide better insights into DNA damage and degradation over time than archaeological and paleontological samples.

Herbarium collections are a vast but underutilized resource for ancient DNA research, containing over 400 million specimens with detailed metadata and spanning centuries of global biodiversity. Understanding patterns of DNA preservation in natural collections is crucial for optimizing ancient DNA studies and informing future curation practices. We analysed genomic data for 573 herbarium specimens from six plant species from the genera Hordeum and Oryza collected from the Americas and Eurasia over 220 years. Using standardized laboratory protocols and shotgun sequencing, we quantified DNA degradation and elucidated factors that accelerate it. We find significant age-dependent DNA fragmentation rates, indicating temporal degradation processes not detected in prehistoric samples. In our analysis, DNA decay rates in herbarium specimens were almost eight times faster than in moa bones, reflecting fundamental differences in tissue composition and preservation environments. Environmental conditions at the time of specimen collection emerged as the major determinants of post-mortem damage rates, with the interaction term between temperature and genus being the dominant driver of cytosine deamination. We find no effect of sample storage on DNA damage and degradation. These findings provide insights into how climatic origin, preservation environment, taxonomic identity and age influence DNA preservation while highlighting opportunities for improving institutional preservation practices. Due to standardised preservation conditions, museum collections can provide better insights into DNA damage and degradation over time than archaeological and paleontological samples.

CRISPR/Cas9 gene editing in the model green alga Chlamydomonas reinhardtii relies on the use of selective marker genes to enrich for non-selectable target mutations. This becomes challenging when many sequential modifications are required in a single cell line, as useful markers are limited. Here, we demonstrate a cyclical selection process which only requires a single marker gene to identify an almost infinite sequential series of CRISPR-based target gene modifications. We used the NIA1 (Nit1, NR; nitrate reductase) gene as the selectable marker in this study. In the forward stage of the cycle, a stop codon was engineered into the NIA1 gene at the CRISPR target location. Cells retaining the wild-type NIA1 gene were killed by chlorate, while NIA1 knockout mutants survived. In the reverse phase of the cycle, the stop codon engineered into the NIA1 gene during the forward phase was edited back to the wild-type sequence. Using nitrate as the sole nitrogen source, only the reverted wild-type cells survived. By using CRISPR to specifically deactivate and reactivate the NIA1 gene, a marker system was established that flipped back and forth between chlorate- and auxotrophic (nitrate)-based selection. This provided a scarless cyclical marker system that enabled an indefinite series of CRISPR edits in other, non-selectable genes. We demonstrate that this ‘Sequential CRISPR via Recycling Endogenous Auxotrophic Markers (SCREAM)’ technology enables an essentially limitless series of genetic modifications to be introduced into a single cell lineage of C. reinhardtii in a fast and efficient manner to complete complex genetic engineering.

The University of Queensland's institutional repository, UQ eSpace, aims to create global visibility and accessibility of UQ’s scholarly research.

Carbendazim is one of the most commonly used fungicides in Vietnam in recent decades. The intensive application of this pesticide has led to the increase of its presence in soil, surface water, and ground