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Standardized protocols for absolute quantification of potato virus Y (PVY) from potato tissue is critical for host-virus dynamic studies. Here, we developed a standardized protocol using a cloned viral sequence as standards to detect and quantify PVY. Starting with total RNA, concentrated via column-based kit, this protocol is able to detect approximately 50 viral copies/reaction from multiple PVY strains. Validation of this protocol confirmed linearity across 8 orders of magnitude with high repeatability, reproducibility and statistical robustness across three independent runs. This protocol offers reliable PVY quantification to manage potato crop health and enables comparative studies with other viral systems. 

The use of true potato seed (TPS) is fundamental to potato breeding and research, but can be hindered by poor germination and seed dormancy. TPS germination studies had focused mainly on seed treatment methods after seed extraction and not in combination with the seed extraction methods used. In potato, TPS extraction using water, using yeast fermentation or using sodium bicarbonate are common, but TPS extraction using dilute aqueous hydrochloric acid (HCl) followed by bleach treatment (3% sodium hypochlorite) had never been tested in potato even though this is standard practice for tomato seeds. Therefore, three seed extraction methods (water, 0.1 M HCl, and 0.8% yeast fermentation) in combination with three seed treatment methods (water, 1500 ppm GA3, and 3% sodium hypochlorite) were tested on diploid TPS at 1 week and 1 month after seed extraction. TPS treated with GA3 improved germination for both 1 week- and 1 month-old seeds, while TPS treated with 3% sodium hypochlorite only improved germination for 1 month-old seeds. This study shows that TPS extraction using water, yeast or HCl had no effect on germination, but supports the use of GA3 or bleach to promote TPS germination. 

Abstract The American chestnut (Castanea dentata) was a dominant, foundational forest canopy tree in eastern North America until an imported chestnut blight (caused byCryphonectria 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. 

Abstract In cultivated tetraploid potato (Solanum tuberosum), reduction to diploidy (dihaploidy) allows for hybridization to diploids and introgression breeding and may facilitate the production of inbreds. Pollination with haploid inducers yields maternal dihaploids, as well as triploid and tetraploid hybrids. Dihaploids may result from parthenogenesis, entailing the development of embryos from unfertilized eggs, or genome elimination, entailing missegregation and the loss of paternal chromosomes. A sign of genome elimination is the occasional persistence of haploid inducer DNA in some dihaploids. We characterized the genomes of 919 putative dihaploids and 134 hybrids produced by pollinating tetraploid clones with three haploid inducers: IVP35, IVP101, and PL-4. Whole-chromosome or segmental aneuploidy was observed in 76 dihaploids, with karyotypes ranging from 2n=2x-1=23 to 2n=2x+3=27. Of the additional chromosomes in 74 aneuploids, 66 were from the non-inducer parent and 8 from the inducer parent. Overall, we detected full or partial chromosomes from the haploid inducer parent in 0.87% of the dihaploids, irrespective of parental genotypes. Chromosomal breaks commonly affected the paternal genome in the dihaploid and tetraploid progeny, but not in the triploid progeny, correlating instability to sperm ploidy and to haploid induction. The residual haploid inducer DNA discovered in the progeny is consistent with genome elimination as the mechanism of haploid induction. 

Abstract 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 have described aneuploid and euploid progeny that, in some cases, displayed genetic markers from the haploid inducer (HI). Here, we surveyed a population of 167 presumed dihaploids for large-scale structural variation that would underlie chromosomal addition from the HI, and for small-scale introgression of genetic markers. In 19 progeny, we detected 10 of the 12 possible trisomies and, in all cases, demonstrated the noninducer parent origin of the additional chromosome. Deep sequencing indicated that occasional, short-tract signals appearing to be of HI origin were better explained as technical artifacts. Leveraging recurring copy number variation patterns, we documented subchromosomal dosage variation indicating segregation of polymorphic maternal haplotypes. Collectively, 52% of the 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.