Biology subjects

Tan, E. H.

Publications and source records attributed to Tan, E. H..

7 recordsLinked to original sources

bioRxiv2025

Two-Year Field Trial of Genetically Engineered American Chestnut Reveals Greater Fungal Blight Tolerance Compared to Wild-Type Full-Sibling Trees

The American chestnut (Castanea dentata [Marsh.] Borkh.) was a foundational forest canopy species in eastern North America until an accidentally imported fungal blight (caused by Cryphonectria parasitica [Murr.] Barr.) rendered it functionally extinct across its native range. Biotechnological approaches, such as the bioengineered Darling 54 line, have potential for future restoration of American chestnut, but field-based evaluations of blight tolerance have been limited. Progress has been slowed by the many years it takes for seedlings to grow to saplings, then to full-fledged trees. Current regulatory restrictions also constrain the testing of transgenic chestnuts to within permitted orchards. This research reports on a two-year field trial of Darling 54, their non-transgenic wild-type siblings and Chinese chestnut (Castanea mollissima Blume) controls deployed a randomized block design to test for blight tolerance. In the two years, three replicates each of 261 trees were branch-inoculated with EP-155, a highly virulent isolate of the fungal blight. Based on canker length, Darling 54 trees consistently outperformed their non-transgenic wild-type siblings and Chinese chestnut. To our knowledge, this is the first report of a multi-year field trial of fungal blight inoculations comparing advanced generation Darling 54 families. This field-based evaluation suggests that reintroduction programs using Darling 54 American chestnuts, which can commence after federal approval, may offer a promising path to success.

synthetic biology↗

bioRxiv2025

Potato dihaploids uncover diverse alleles to facilitate diploid potato breeding

Commercial potato in North America is a clonal autotetraploid crop, which complicates breeding. Efforts are underway to convert potato to a diploid inbred-hybrid crop that is amenable to additional breeding strategies and allows breeders to more quickly respond to demands for crop improvement. With the goal of preserving haplotypes developed over the last 200 years of selection, diploid potato breeding in the US started with the creation of diploids from tetraploid commercial varieties and advanced breeding lines through prickle pollination. This is an effective but slow method which presents a barrier to entry for individual breeding programs. Therefore, we developed 97 publicly available dihaploids (diploids from prickle pollination of tetraploids) as a resource for diploid breeding in the U.S. These clones contain the majority of alleles in the US breeding population for three market classes: chips, russets, and fresh market reds. To facilitate genomic informed breeding, all clones have been resequenced using short read sequencing technology, and we have developed de novo assemblies based on PacBio HiFi long reads for 20 individuals. As an illustration of how these data will be used in breeding, we explored the maturity locus (StCDF1) and identified 15 different alleles. The majority of dihaploids were heterozygous for early and late alleles, resulting in intermediate maturity. Beyond informing breeding, this data facilitates investigations into potato genomics. The dihaploid population is both highly heterozygous and incredibly diverse on a population level. In particular, there is extensive structural diversity, including copy number variation, segregating within the population. This contrasts with a relatively low genome-wide historical recombination rate ({rho}). Taken together, these findings indicate that potato is highly diverse, with much of that diversity found within long linkage blocks.

plant biology↗

bioRxiv2025

Speed breeding transgenic American chestnut trees toward restoration

The American chestnut (Castanea dentata) was a dominant, foundational forest canopy tree in eastern North America until an imported chestnut blight (caused by Cryphonectria parasitica) rendered it functionally extinct across its native range. Biotechnological approaches have the potential to help restore the species, but field-based breeding advances are hampered by long generation times, [≤]50% transgene inheritance, and regulatory restrictions on outdoor breeding of transgenic trees. Self-incompatibility and flowering phenology further limit generational advances and field testing of chestnuts. Our work here demonstrates that long generational times and field constraints can be circumvented by producing both male and receptive female flowers in controlled indoor environments. Additionally, we developed an embryo rescue protocol for both indoor and field conditions, in which developing embryos can be extracted and micropropagated from immature seeds between 6- and 8-weeks post pollination. These advances have enabled production of the first homozygous transgenic American chestnuts, which have produced pollen that was used for outdoor controlled pollinations and yielded nearly 100% transgene inheritance by offspring. This work also provides event-specific DNA markers to differentiate transgenic chestnut lines and identify homozygous individuals. We demonstrate that an obligate outcrossing forest tree can reach sexual maturity rapidly in controlled, indoor environments. When coupled with genomic analyses and other biotechnological advances, this procedure could facilitate the reintroduction of this iconic species.

plant biology↗

bioRxiv2021

Accurate Direct PCR with Arabidopsis and rice

Plant sampling methods for PCR that avoid costly and time-consuming plant DNA purification can be accurate but have not been widely adopted, partly because the efficacy of the methods is unclear. Here, we introduce new sampling methods for Direct PCR in Arabidopsis and rice and compare them to previously published methods. CutTip, stabbing a pipette tip into a plant and depositing the tip of the pipette tip into the reaction buffer, yielded high accuracy for genotyping Arabidopsis and rice. This did not require visible tissue fragments in the reactions. We found that using short thin segments of fishing line, called Line-PCR, to be even more practical and highly accurate. Compared to traditional and other Direct PCR preparation methods, Line-PCR is faster, inexpensive, easier, uses less plastic and chemicals, and causes the least tissue damage. CutTip and Line-PCR require no modified PCR reagents. Line-PCR particularly lacks any complicated steps. Probably, the most difficult step in Line-PCR is a wipe of the tweezer between samples. These methods can help address the challenge of genotyping at different scales with high accuracy. CutTip and Line-PCR also simplify the application of PCR in the field.

molecular biology↗

bioRxiv2020

Rare instances of haploid inducer DNA in potato dihaploids and ploidy-dependent genome instability

In cultivated tetraploid potato, reduction to diploidy (dihaploidy) allows hybridization to diploid germplasm, introgression breeding, and may facilitate the production of inbreds. Pollination with haploid inducers yields maternal dihaploids, as well as triploid and tetraploid hybrids. It is not known if dihaploids result from parthenogenesis, entailing development of embryos from unfertilized eggs, or genome elimination, entailing missegregation and loss of paternal chromosomes. A sign of genome elimination is the occasional persistence of haploid inducer DNA in some of the dihaploids. We characterized the genomes of 1,001 putative dihaploids and 134 hybrids produced by pollinating tetraploid clones with three haploid inducers, IVP35, IVP101, and PL4. We detected inheritance of full or partial chromosomes from the haploid inducer parent in 0.87% of the overall dihaploid progeny, irrespective of the combination of parental genotypes. Chromosomal breaks commonly affected the paternal genome in the dihaploid and tetraploid progeny, but not in the triploid progeny. Residual haploid inducer DNA is consistent with genome elimination as the mechanism of haploid induction. Further, the fact that paternal chromosome breaks are specific to dihaploids and tetraploid progeny suggests that they may be specific to 2x sperms, and supports the hypothesis that 2x sperms facilitate genome elimination.

plant biology↗

bioRxiv2019

Genomic Outcomes of Haploid Induction Crosses in Potato (Solanum tuberosum L.)

The challenges of breeding autotetraploid potato (Solanum tuberosum) have motivated the development of alternative breeding strategies. A common approach is to obtain uniparental dihaploids from a tetraploid of interest through pollination with S. tuberosum Andigenum Group (formerly S. phureja) cultivars. The mechanism underlying haploid formation of these crosses is unclear, and questions regarding the frequency of paternal DNA transmission remain. Previous reports described aneuploid and euploid progeny, which, in some cases, displayed genetic markers from the haploid inducer. Here, we surveyed a population of 167 presumed dihaploids for large-scale structural variation that would underlie chromosomal addition from the haploid inducer, and for small-scale introgression of genetic markers. In 19 progeny, we detected ten of the twelve possible trisomies and, in all cases, demonstrated the non-inducer parent origin of the additional chromosome. Deep sequencing indicated that occasional, short-tract signals appearing of haploid inducer origin were better explained as technical artifacts. Leveraging recurring CNV patterns, we documented sub-chromosomal dosage variation indicating segregation of polymorphic maternal haplotypes. Collectively, 52% of assayed chromosomal loci were classified as dosage variable. Our findings help elucidate the genomic consequences of potato haploid induction and suggest that most potato dihaploids will be free of residual pollinator DNA.

genomics↗

bioRxiv2018

Translocation and duplication from CRISPR-Cas9 editing in Arabidopsis thaliana

Cut DNA ends in plants may recombine to form novel molecules. We asked whether CRISPR-Cas9 expression in plants could induce nonhomologous recombination between diverse and heterologous broken DNA ends. We induced two breaks separated by 2.3 or by 8.5 kilobases leading to duplication of the intervening DNA and meiotic transmission of the 2.3kb duplication. Two or more dsDNA breaks in nonhomologous chromosomes led to ligation of breakpoints consistent with chromosome arm translocations. Screening 881 primary transformants we obtained 195 PCR products spanning independent, expected translocation junctions involving ends produced by cutting different loci. Sequencing indicated a true positive rate of 84/91 and demonstrated the occurrence of different junction alleles. A majority of the resulting structures would be deleterious and none were transmitted meiotically. Ligation of interchromosomal, heterologous dsDNA ends suggest that the CRISPR-Cas9 can be used to engineer plant genes and chromosomes in vivo.\n\nSignificance StatementWe explored how genome editing tools such as CRISPR-Cas9 could provide new ways to tailor novel genomic combinations and arrangements. We show that distant cut ends often precisely come together, that cuts in different chromosomes can result in translocations, and that two cuts within a chromosome often result in the duplication of the intervening segment. Formation of multiple structures with precise junctions will enable engineered rearrangements that can be predicted with accuracy.

genetics↗