First Report of Tobacco Streak Virus in Cannabis sativa in New York.

In 2022, virus-like symptoms were observed in a field of diverse hemp (Cannabis sativa L.) germplasm in Ontario County, New York. Less than 1% of plants exhibited stunting and curled leaves (Figure S1), consistent with tobacco streak virus (TSV) symptoms on other plants (Liu et al. 2022). Most typically, the plants were considerably reduced in overall size, with upwards, adaxial curling along the leaf margin with newer leaves appearing to be the most affected. Fifteen symptomatic plants representing nine accessions were tested for 12 viruses and viroids through Agdia Testing Services (Elkhart, IN). Of these, eight plants representing five accessions including: G 33204 21UO SD ('Cherry Wine S1'), G 33211 21UO SD ('Wife'), G 33225 22CL01 CL ('Candida #2'), G 33270 22UO SD ('Falkowski CBD Mix'), and G 33365 22UO SD ('Queen Dream'), were positive for TSV, a type of Ilarvirus in the Bromoviridae family. Presence of TSV was confirmed through enzyme-linked immunosorbent assay testing. TSV is a positive-sense, single-stranded RNA virus with a wide host range that can be transmitted by thrips, mechanical injury, seed, and pollen (Zambrana-Echevarría et al. 2021). To confirm the presence of TSV, two putatively TSV-infected samples were subjected to RNA-Seq analysis. RNA was extracted using the RNeasy Plant Mini Kit (Qiagen, Aarhus, Denmark) per manufacturer's direction. Stranded RNA libraries were prepared using the Illumina TruSeq Stranded Total RNA with Ribo-Zero Plant kit (San Diego, California, USA). Paired-end 2x150bp sequencing was performed on an Illumina NovaSeq6000 sequencer. RNA-Seq data was trimmed using the fastp program (Chen et al. 2018) with default parameters to remove adapter sequences and low-quality bases. After filtering, 49,696,041 and 56,126,804 paired-end reads were retained from 'Wife' and 'Falkowski CBD Mix' samples, respectively. Filtered RNA-seq reads were mapped to TSV genome accession GCF_000865505.1 using the bowtie2 (Langmead & Salzberg 2012) aligner with default parameters. From 'Wife' and 'Falkowski CBD Mix' samples, 153 and 139 reads mapped to the TSV reference genome. To further validate the presence of TSV reads, RNA-Seq data was analyzed using the Kraken2 pipeline (Wood et al. 2019). Using the Kraken2 virus database, reads associated with TSV (NCBI taxonomy ID: 12317) were identified. This analysis identified 172 and 151 TSV reads from 'Wife' and 'Falkowski CBD Mix,' respectively. Higher numbers of reads identified using the Kraken2 analysis is due to the more permissive k-mer matching approach implemented in Kraken2. Furthermore, we identified several other virus taxa in the samples. Of note, both samples had a high number of reads associated with Amazon lily mild mottle virus with 254,493 and 116,150 reads from 'Wife' and 'Falkowski CBD Mix,' respectively. Among other virus species belonging to Ilarviruses, Cassava Ivorian bacilliform virus and Cowpea chlorotic mottle viruses were detected from both samples. To further validate infection by TSV, samples from both ELISA-positive and ELISA-negative plants were subjected to PCR using the primers and protocol described in Zambrana-Echevarría et al. 2021. Amplification of an approximately 700 base-pair product was observed in the putatively ELISA-positive samples, but not in the ELISA-negative samples. The amplicons were further cloned into the pGEM-T Easy vector (Promega, Madison, WI, U.S.A) using the manufacturer's protocol and sequenced using M13 forward and M13 reverse primers (Functional Biosciences, Madison, WI, U.S.A). Sequencing results indicated considerable similarity to TSV genomes available in GenBank, between 88% and 99%. Raw sequence data generated from this study was deposited in NCBI under the bioproject ID PRJNA1009441. Though it cannot be ruled out that the observed symptoms were caused exclusively by TSV infection due to the high number of other viral reads, the results contribute to the literature that indicates hemp can host TSV and should be considered a potential source of TSV inoculum (Chiginsky et al. 2021). This new inoculum source could cause significant crop damage and economic loss when grown with TSV susceptible row and specialty crops.

From landrace to modern hybrid broccoli: the genomic and morphological domestication syndrome within a diverse B. oleracea collection.

Worldwide, broccoli (Brassica oleracea var. italica) is among the most economically important, nutritionally rich, and widely-grown vegetable crops. To explore the genomic basis of the dramatic changes in broccoli morphology in the last century, we evaluated 109 broccoli or broccoli/cauliflower intermediates for 24 horticultural traits. Genotype-by-sequencing markers were used to determine four subpopulations within italica: Calabrese broccoli landraces and hybrids, sprouting broccoli, and violet cauliflower, and to evaluate between and within group relatedness and diversity. While overall horticultural quality and harvest index of improved hybrid broccoli germplasm has increased by year of cultivar release, this improvement has been accompanied by a considerable reduction in allelic diversity when compared to the larger pool of germplasm. Two landraces are the most likely founding source of modern broccoli hybrids, and within these modern hybrids, we identified 13 reduction-in-diversity genomic regions, 53 selective sweeps, and 30 (>1 Mbp) runs of homozygosity. Landrace accessions collected in southern Italy contained 4.8-fold greater unique alleles per accessions compared to modern hybrids and provide a valuable resource in subsequent improvement efforts. This work broadens the understanding of broccoli germplasm, informs conservation efforts, and enables breeding for complex quality traits and regionally adapted cultivars.

Complex Horticultural Quality Traits in Broccoli Are Illuminated by Evaluation of the Immortal BolTBDH Mapping Population.

Improving horticultural quality in regionally adapted broccoli (Brassica oleracea var. italica) and other B. oleracea crops is challenging due to complex genetic control of traits affecting morphology, development, and yield. Mapping horticultural quality traits to genomic loci is an essential step in these improvement efforts. Understanding the mechanisms underlying horticultural quality enables multi-trait marker-assisted selection for improved, resilient, and regionally adapted B. oleracea germplasm. The publicly-available biparental double-haploid BolTBDH mapping population (Chinese kale × broccoli; N = 175) was evaluated for 25 horticultural traits in six trait classes (architecture, biomass, phenology, leaf morphology, floral morphology, and head quality) by multiple quantitative trait loci mapping using 1,881 genotype-by-sequencing derived single nucleotide polymorphisms. The physical locations of 56 single and 41 epistatic quantitative trait locus (QTL) were identified. Four head quality QTL (OQ_C03@57.0, OQ_C04@33.3, OQ_CC08@25.5, and OQ_C09@49.7) explain a cumulative 81.9% of phenotypic variance in the broccoli heading phenotype, contain the FLOWERING LOCUS C (FLC) homologs Bo9g173400 and Bo9g173370, and exhibit epistatic effects. Three key genomic hotspots associated with pleiotropic control of the broccoli heading phenotype were identified. One phenology hotspot reduces days to flowering by 7.0 days and includes an additional FLC homolog Bo3g024250 that does not exhibit epistatic effects with the three horticultural quality hotspots. Strong candidates for other horticultural traits were identified: BoLMI1 (Bo3g002560) associated with serrated leaf margins and leaf apex shape, BoCCD4 (Bo3g158650) implicated in flower color, and BoAP2 (Bo1g004960) implicated in the hooked sepal horticultural trait. The BolTBDH population provides a framework for B. oleracea improvement by targeting key genomic loci contributing to high horticultural quality broccoli and enabling de novo mapping of currently unexplored traits.

Genotyping-by-sequencing of Brassica oleracea vegetables reveals unique phylogenetic patterns, population structure and domestication footprints.

Brassica oleracea forms a diverse and economically significant crop group. Improvement efforts are often hindered by limited knowledge of diversity contained within available germplasm. Here, we employ genotyping-by-sequencing to investigate a diverse panel of 85 landrace and improved B. oleracea broccoli, cauliflower, and Chinese kale entries. Ultimately, 21,680 high-quality SNPs were used to reveal a complex and admixed population structure and clarify phylogenetic relationships among B. oleracea groups. Each broccoli landrace contained, on average, 8.4 times as many unique alleles as an improved broccoli and landraces collectively represented 81% of all broccoli-specific alleles. Commercial broccoli hybrids were largely represented by a single subpopulation identified within a complex population structure. Greater allelic diversity in landrace broccoli and 96.1% of SNPs differentiating improved cauliflower from landrace cauliflower were common to the larger pool of broccoli germplasm, supporting a parallel or later development of cauliflower due to introgression events from broccoli. Chinese kale was readily distinguished by principal coordinate analysis. Genotyping was accomplished with and without reliance upon a reference genome producing 141,317 and 20,815 filtered SNPs, respectively, supporting robust SNP discovery methods in neglected or unimproved crop groups that lack a reference genome. This work clarifies the population structure, phylogeny, and domestication footprints of landrace and improved B. oleracea broccoli using many genotyping-by-sequencing markers. Additionally, a large pool of genetic diversity contained in broccoli landraces is described which may enhance future breeding efforts.

Quantitative trait loci mapping of heat tolerance in broccoli (Brassica oleracea var. italica) using genotyping-by-sequencing.

KEY MESSAGE: Five quantitative trait loci and one epistatic interaction were associated with heat tolerance in a doubled haploid population of broccoli evaluated in three summer field trials. Predicted rising global temperatures due to climate change have generated a demand for crops that are resistant to yield and quality losses from heat stress. Broccoli (Brassica oleracea var. italica) is a cool weather crop with high temperatures during production decreasing both head quality and yield. Breeding for heat tolerance in broccoli has potential to both expand viable production areas and extend the growing season but breeding efficiency is constrained by limited genetic information. A doubled haploid (DH) broccoli population segregating for heat tolerance was evaluated for head quality in three summer fields in Charleston, SC, USA. Multiple quantitative trait loci (QTL) mapping of 1,423 single nucleotide polymorphisms developed through genotyping-by-sequencing identified five QTL and one positive epistatic interaction that explained 62.1% of variation in heat tolerance. The QTL identified here can be used to develop markers for marker-assisted selection and to increase our understanding of the molecular mechanisms underlying plant response to heat stress.

Genetic Diversity and Population Structure of Collard Landraces and their Relationship to Other Brassica oleracea Crops.

Landraces have the potential to provide a reservoir of genetic diversity for crop improvement to combat the genetic erosion of the food supply. A landrace collection of the vitamin-rich specialty crop collard (Brassica oleracea L. var. viridis) was genetically characterized to assess its potential for improving the diverse crop varieties of B. oleracea. We used the Illumina 60K Brassica SNP BeadChip array with 52,157 single nucleotide polymorphisms (SNPs) to (i) clarify the relationship of collard to the most economically important B. oleracea crop types, (ii) evaluate genetic diversity and population structure of 75 collard landraces, and (iii) assess the potential of the collection for genome-wide association studies (GWAS) through characterization of genomic patterns of linkage disequilibrium. Confirming the collection as a valuable genetic resource, the collard landraces had twice the polymorphic markers (11,322 SNPs) and 10 times the variety-specific alleles (521 alleles) of the remaining crop types examined in this study. On average, linkage disequilibrium decayed to background levels within 600 kilobase (kb), allowing for sufficient coverage of the genome for GWAS using the physical positions of the 8273 SNPs polymorphic among the landraces. Although other relationships varied, the previous placement of collard with the cabbage family was confirmed through phylogenetic analysis and principal coordinates analysis (PCoA).