The 141-year period for Dr. Beal's seed viability experiment: A hybrid surprise.

PREMISE: In 1879, Dr. William Beal buried 20 glass bottles filled with seeds and sand at a single site at Michigan State University. The goal of the experiment was to understand seed longevity in the soil, a topic of general importance in ecology, restoration, conservation, and agriculture, by periodically assaying germinability of these seeds over 100 years. The interval between germination assays has been extended and the experiment will now end after 221 years, in 2100. METHODS: We dug up the 16th bottle in April 2021 and attempted to germinate the 141-year-old seeds it contained. We grew germinants to maturity and identified these to species by vegetative and reproductive phenotypes. For the first time in the history of this experiment, genomic DNA was sequenced to confirm species identities. RESULTS: Twenty seeds germinated over the 244-day assay. Eight germinated in the first 11 days. All 20 belonged to the Verbascum genus: Nineteen were V. blattaria according to phenotype and ITS2 genotype; and one had a hybrid V. blattaria × V. thapsus phenotype and ITS2 genotype. In total, 20/50 (40%) of the original Verbascum seeds in the bottle germinated in year 141. CONCLUSIONS: While most species in the Beal experiment lost all seed viability in the first 60 years, a high percentage of Verbascum seeds can still germinate after 141 years in the soil. Long-term experiments such as this one are rare and invaluable for studying seed viability in natural soil conditions.

Ppe.XapF: High throughput KASP assays to identify fruit response to Xanthomonas arboricola pv. pruni (Xap) in peach.

Bacterial spot, caused by Xanthomonas arboricola pv. pruni (Xap), is a serious peach disease with symptoms that traverse severe defoliation and black surface pitting, cracking or blemishes on peach fruit with global economic impacts. A management option for control and meeting consumer demand for chemical-free, environmentally friendly fruit production is the development of resistant or tolerant cultivars. We developed simple, accurate, and efficient DNA assays (Ppe.XapF) based on SNP genotyping with KASP technology to quickly test for bacterial spot resistance alleles in peach fruit that allows breeders to cull seedlings at the greenhouse stage. The objective of this research was to validate newly developed DNA tests that target the two major QTLs for fruit resistance in peach with diagnostic utility in predicting fruit response to bacterial spot infection. Our study confirms that with only two Ppe.XapF DNA tests, Ppe.XapF1-1 and Ppe.XapF6-2, individuals carrying susceptible alleles can be identified. Use of these efficient and accurate Ppe.XapF KASP tests resulted in 44% reduction in seedling planting rate in the Clemson University peach breeding program.

Genome-Wide Association Study Reveals Genomic Region Associated with Mite-Recruitment Phenotypes in the Domesticated Grapevine (Vitis vinifera).

Indirect defenses are plant phenotypes that reduce damage by attracting natural enemies of plant pests and pathogens to leaves. Despite their economic and ecological importance, few studies have investigated the genetic underpinnings of indirect defense phenotypes. Here, we present a genome-wide association study of five phenotypes previously determined to increase populations of beneficial (fungivorous and predacious) mites on grape leaves (genus Vitis): leaf bristles, leaf hairs, and the size, density, and depth of leaf domatia. Using a common garden genetic panel of 399 V. vinifera cultivars, we tested for genetic associations of these phenotypes using previously obtained genotyping data from the Vitis9kSNP array. We found one single nucleotide polymorphism (SNP) significantly associated with domatia density. This SNP (Chr5:1160194) is near two genes of interest: Importin Alpha Isoform 1 (VIT_205s0077g01440), involved in downy mildew resistance, and GATA Transcription Factor 8 (VIT_205s0077g01450), involved in leaf shape development. Our findings are among the first to examine the genomic regions associated with ecologically important plant traits that facilitate interactions with beneficial mites, and suggest promising candidate genes for breeding and genetic editing to increase naturally occurring predator-based defenses in grapevines.

The kinetics of ageing in dry-stored seeds: a comparison of viability loss and RNA degradation in unique legacy seed collections.

BACKGROUND AND AIMS: Determining seed longevity by identifying chemical changes that precede, and may be linked to, seed mortality, is an important but difficult task. The standard assessment, germination proportion, reveals seed longevity by showing that germination proportion declines, but cannot be used to predict when germination will be significantly compromised. Assessment of molecular integrity, such as RNA integrity, may be more informative about changes in seed health that precede viability loss, and has been shown to be useful in soybean. METHODS: A collection of seeds stored at 5 °C and 35-50 % relative humidity for 1-30 years was used to test how germination proportion and RNA integrity are affected by storage time. Similarly, a collection of seeds stored at temperatures from -12 to +32 °C for 59 years was used to manipulate ageing rate. RNA integrity was calculated using total RNA extracted from one to five seeds per sample, analysed on an Agilent Bioanalyzer. RESULTS: Decreased RNA integrity was usually observed before viability loss. Correlation of RNA integrity with storage time or storage temperature was negative and significant for most species tested. Exceptions were watermelon, for which germination proportion and storage time were poorly correlated, and tomato, which showed electropherogram anomalies that affected RNA integrity number calculation. Temperature dependencies of ageing reactions were not significantly different across species or mode of detection. The overall correlation between germination proportion and RNA integrity, across all experiments, was positive and significant. CONCLUSIONS: Changes in RNA integrity when ageing is asymptomatic can be used to predict onset of viability decline. RNA integrity appears to be a metric of seed ageing that is broadly applicable across species. Time and molecular mobility of the substrate affect both the progress of seed ageing and loss of RNA integrity.

Exploring the fate of mRNA in aging seeds: protection, destruction, or slow decay?

Seeds exist in the vulnerable state of being unable to repair the chemical degradation all organisms suffer, which slowly ages seeds and eventually results in death. Proposed seed aging mechanisms involve all classes of biological molecules, and degradation of total RNA has been detected contemporaneously with viability loss in dry-stored seeds. To identify changes specific to mRNA, we examined the soybean (Glycine max) seed transcriptome, using new, whole-molecule sequencing technology. We detected strong evidence of transcript fragmentation in 23-year-old, compared with 2-year-old, seeds. Transcripts were broken non-specifically, and greater fragmentation occurred in longer transcripts, consistent with the proposed mechanism of molecular fission by free radical attack at random bases. Seeds died despite high integrity of short transcripts, indicating that functions encoded by short transcripts are not sufficient to maintain viability. This study provides an approach to probe the asymptomatic phase of seed aging, namely by quantifying transcript degradation as a function of storage time.

A KASP Genotyping Method to Identify Northern Watermilfoil, Eurasian Watermilfoil, and Their Interspecific Hybrids.

The invasive aquatic plant Eurasian watermilfoil (Myriophyllum spicatum L.) can hybridize with the related North American native species northern watermilfoil (M. sibiricum Kom.). Hybrid watermilfoil (M. spicatum × M. sibiricum) populations have higher fitness and reduced sensitivity to some commonly used aquatic herbicides, making management more difficult. There is growing concern that management practices using herbicides in lakes with mixed populations of watermilfoil species may further select for hybrid individuals due to the difference in herbicide sensitivity. Accurate and cost-effective identification of rare hybrid individuals within populations is therefore critical for herbicide management decisions. Here we describe KASP assays for three SNPs in the ITS region to genotype individuals from both parental watermilfoil species and their hybrid, using synthesized plasmids containing the respective sequences as positive controls. Using KASP we genotyped 16 individuals from one lake and 23 individuals from a second lake, giving a highly accurate picture of Myriophyllum species distribution dynamics. We identified one hybrid individual among 16 samples from one lake, a discovery rate of <10%. Discriminant analysis showed that while a single SNP was generally sufficient for genotyping an individual, using multiple SNPs increased the reliability of genotyping. In the future, the ability to genotype many samples will provide the ability to identify the presence of rare individuals, such as a less common parental species or the inter-specific hybrid. Lakes with complex species distribution dynamics, such as a low proportion of hybrids, are where herbicide application must be carefully chosen so as not to select for the more vigorous and less herbicide-sensitive hybrid individuals.

Indaziflam: a new cellulose-biosynthesis-inhibiting herbicide provides long-term control of invasive winter annual grasses.

BACKGROUND: Indaziflam is a cellulose-biosynthesis-inhibiting (CBI) herbicide that is a unique mode of action for resistance management and has broad spectrum activity at low application rates. This research further explores indaziflam's activity on monocotyledons and dicotyledons and evaluates indaziflam's potential for restoring non-crop sites infested with invasive winter annual grasses. RESULTS: Treated Arabidopsis, downy brome, feral rye and kochia were all susceptible to indaziflam in a dose-dependent manner. We confirmed that indaziflam has increased activity on monocots (average GR50  = 231 pm and 0.38 g AI ha-1 ) at reduced concentrations compared with dicots (average GR50  = 512 pm and 0.87 g AI ha-1 ). Fluorescence microscopy confirmed common CBI symptomologies following indaziflam treatments, as well as aberrant root and cell morphology. Across five application timings, indaziflam treatments resulted in superior invasive winter annual grass control 2 years after treatment (from 84 ± 5.1% to 99 ± 0.5%) compared with imazapic (36% ± 1.2%). Indaziflam treatments significantly increased biomass and species richness of co-occurring species 2 years after treatment. CONCLUSION: Indaziflam's increased activity on monocots could provide a new alternative management strategy for long-term control of multiple invasive winter annual grasses that invade >23 million ha of US rangeland. Indaziflam could potentially be used to eliminate the soil seed bank of these invasive grasses, reduce fine fuel accumulation and ultimately increase the competitiveness of perennial co-occuring species. © 2017 Society of Chemical Industry.

Decline in RNA integrity of dry-stored soybean seeds correlates with loss of germination potential.

This study investigates the relationship between germination ability and damage to RNA in soybean seeds (cv 'Williams 82') stored dry at 5 °C for 1-27 years. Total germination of 14 age cohorts harvested between 2015 and 1989 ranged from 100% to 3%. Germination decline followed classic seed viability kinetics, with symptomatic seed aging beginning after 17 years of storage. RNA integrity was assessed in dry seeds by electrophoresis of total RNA, followed by calculation of the RNA integrity number (RIN, Agilent Bioanalyzer software), which evaluates RNA fragment size distributions. Analysis of RNA extracted from cotyledons, embryonic axes, plumules, and seed coats across the range of age cohorts showed consistent RNA degradation: older seeds had over-representation of small RNAs compared with younger seeds, which had nearly a 2:1 ratio of 25S and 18S rRNAs. RIN values for cotyledons and embryonic axes from the same seed were correlated. Decline in RIN tracked reduced germination, with a pronounced decrease in RIN after 17 years of storage. This led to a high correlation between the mean RIN of cotyledon RNA and the total germination percentage (R2=0.91, P<0.0001). Despite this relationship, germinable and non-germinable seeds within cohorts could not be distinguished unless the RIN was <3.5, indicating substantial deterioration. Our work demonstrates that seed RNA incurs damage over time, observable in fragment size distributions. Under the experimental conditions used here, RIN appears to be a promising surrogate for germination tests used to monitor viability of stored seeds.