The brush is usually good for two to three years. One- and two-inch chicken wire will also suffice. Unlike beans, peas aren’t a heavy plant or fruit, thus they don’t need as strong a fence. In fact, garden twine run vertically or woven between horizontal 2x4s makes a biodegradable/compostable trellis. String on a wooden A-frame also works. The important thing is to install the trellis prior to planting and to rotate it around the garden so as not to be tempted to repeat the crop in the same bed before two to three years have passed. Crop Establishment. Unless peas are ridiculously oversown, thinning is unnecessary. Spacing plants farther than 3–4 inches apart makes no sense, nor increases yield per foot. One weeding at the 3-inch stage usually keeps the peas ahead of the weeds. Because peas are so succulent, the less the crop is handled the less the physical damage. Even micro-breaks in the foliage can lead to an “invasion” of powdery mildew. Mulch. Mulching helps protect the surface roots from heat and desiccation, thus prolonging cropping as summer approaches. Harvesting. This is usually not a problem on a garden scale. To avoid harming the plants as you pick, hold the stem in one hand and pinch the pod off the vine just behind the calyx with the other hand.In this genome release, raspberry container size we report on the first assembled genome of a member of the genus Arctostaphylos. 

Our genome assembly is part of the California Conservation Genomics Project , the goal of which is to establish patterns of genomic diversity across the state of California and its many habitats. The CCGP will sequence the complete genomes of approximately 150 carefully selected species projects. Many of these taxa are threatened or endangered, and therefore in need of conservation management in the face of rapidly accelerating biodiversity decline. The combined reference genome plus landscape genomics approach of the CCGP, based on the resequencing of many individuals of each target species across the state, will allow the identification of hotspots of diversity across California and provide a framework for informed conservation decisions and management plans. Manzanitas are among the most conspicuous and dominant native chaparral species in the California Floristic Province , a biodiversity hotspot characterized by a Mediterranean-type climate with hot, dry summers and cool, wet winters. These plants comprise the most diverse woody genus in the CFP , and their diversity has long fascinated taxonomists. Manzanitas serve essential roles in their native ecosystems, including rapidly regenerating in fired-disturbed areas, and providing food resources for pollinators and fruit-eating animals . In addition, these plants are of great importance for conservation management: over half of the more than 100 morphologically defined manzanita species and subspecies are narrow endemics with highly restricted distributions and are considered rare and/or endangered . In contrast to their importance in ecology, evolution, and conservation studies, genomic resources for manzanitas are nearly nonexistent beyond investigations into karyotypes of diploid and tetraploid species . In this study, we present the first genome sequence of a manzanita.

Big berry manzanita, Arctostaphylos glauca , is a widespread diploid species common in northern Baja California and across southern and coastal central California that is hypothesized to be the progenitor of several potential hybrid manzanita species . With funding and support from the CCGP, we created this scaffold-level assembly using a hybrid de novo assembly approach that combines Hi-C chromatin-proximity and PacBio HiFi long-read sequencing data. This genome assembly will provide a robust basis for studying the diversification and evolutionary history of Arctostaphylos in the CFP.A Dovetail Hi-C library was prepared in a similar manner as previously described . For each library, chromatin was fixed in place with formaldehyde in the nucleus. Extracted, fixed chromatin was digested with DpnII, the 5′ overhangs were filled in with biotinylated nucleotides, and free blunt ends were ligated. After ligation, crosslinks were reversed, and the DNA purified from protein. Purified DNA was treated to remove biotin that was not internal to ligated fragments. The DNA was then sheared to ~350 bp mean fragment size and sequencing libraries were generated using NEBNext Ultra enzymes and Illumina-compatible adapters. Biotin-containing fragments were isolated using streptavidin beads before PCR enrichment of each library. The libraries were prepared and sequenced on an Illumina HiSeq X by Dovetail Genomics .High molecular weight genomic DNA was extracted from a 750 mg sample of young floral buds following the protocol described in Workman et al. with the minor modification of using the nuclear isolation buffer supplemented with 350 mM Sorbitol to resuspend the ground tissue and during the first wash of the nuclei pellet. The integrity of the HMW DNA was evaluated using the Femto Pulse system . Purity of the DNA was assessed by 260/280 and 260/230 absorbance ratios on a NanoDrop spectrophotometer.

For PacBio library preparation, 11 ug of HMW gDNA were sheared to an average size distribution of ~16 kb mode using Diagenode’s Megaruptor 3 system . Sheared DNA was quantified by Quantus Fluorometer QuantiFluor ONE dsDNA Dye assay and the size distribution was checked by Agilent Femto Pulse . The sheared gDNA was concentrated using 0.45× of AMPure PB beads . Concentrated, sheared gDNA was quantified by Quantus Fluorometer QuantiFluor ONE dsDNA Dye assay . A HiFi library was constructed using the SMRTbell Express Template Prep Kit v2.0 according to the manufacturer’s instructions. 6 ug of sheared, concentrated DNA was used as input for the removal of single-strand overhangs at 37° for 15 min, followed by further enzymatic steps of DNA damage repair at 37° for 30 minutes, end repair and A-tailing at 20° for 10 min and 65° for 30 min, ligation of overhang adapter v3 at 20° for 1 h and 65° for 10 min to inactivate the ligase, and nuclease treatment of SMRTbell library at 37° for 1 h to remove damaged or non-intact SMRTbell templates . The SMRTbell library was purified and concentrated with 1X Ampure PB beads for size selection using the BluePippin system . The input of 2.2 ug purified SMRTbell library was used to load into the Blue Pippin 0.75% Agarose Cassette using cassette definition 0.75% DF Marer S1 3–10 kb Improved Recovery for the run protocol. Fragments >7 kb were collected from the cassette elution well. The size-selected SMRTbell library was purified and concentrated with 0.8× AMPure beads . The 17 kb average HiFi SMRTbell library was sequenced at UC Davis DNA Technologies Core using a single 8M SMRT Cell and Sequel II sequencing chemistry 2.0 on a PacBio Sequel II sequencer.We assembled the genome of the big berry manzanita following a protocol adapted from Rhie et al. as part of the CCGP assembly efforts. The CCGP assembly protocol version 1.0 uses PacBio HiFi reads and Hi-C chromatin capture data for the generation of high-quality and highly contiguous nuclear genome assemblies. The output corresponding to a diploid assembly consists of two pseudo haplotypes . The primary assembly is more complete and consists of longer phased blocks. The alternate consists of haplotigs in heterozygous regions and is not as complete and more fragmented. Given the characteristics of the latter, it cannot be considered on its own but as a complement of the primary assembly . To generate this assembly, raspberry plant container we removed remnant adapter sequences from the PacBio HiFi dataset using HiFiAdapterFilt [Version 1.0] and assembled the initial set of contigs with the filtered PacBio reads using HiFiasm [Version 0.13-r308] . Next, we identified sequences corresponding to haplotypic duplications and contig overlaps on the primary assembly with purge_dups [Version 1.0.1] and transferred them to the alternate assembly. We aligned the Hi-C data to both primary and alternate assemblies using the Arima Genomics Mapping Pipeline and scaffolded the genomes using SALSA [Version 2, options –e GATCGATC] . We closed the generated gaps in both assemblies using the PacBio HiFi reads and YAGCloser [commit 20e2769] . The primary assembly was manually curated by iteratively generating and analyzing Hi-C contact maps. To generate the contact maps, we aligned the Hi-C data against the corresponding reference with bwa mem [Version 0.7.17-r1188, options -5SP] , identified ligation junctions, and generated Hi-C pairs using pairtools [Version 0.3.0] . We generated a multi-resolution Hi-C matrix in binary form with cooler [Version 0.8.10] and balanced it with hicExplorer [Version 3.6] . We used HiGlass [Version 2.1.11] and the PretextSuite to visualize the contact maps. Assemblies were then checked for contamination using the BlobToolKit Framework [Version 2.3.3] , and trimmed for remnants of sequence adaptors and mitochondrial contamination.We identified a subset of mitochondrial reads from the PacBio HiFi dataset using BLAST+ [Version 2.10] by identifying regions of similarity between the reads and the mitochondrial database , National Center for Biotechnology Information 2004). These mitochondrial reads were used as input in HiFiasm [Version 0.13-r308] to generate the mitochondrial assembly.

Given the circularity of the mitochondrial genome, we carried out self-alignment of the sequence using lastz [Version 1.04.08] to manually identify and remove duplicated regions. We aligned the subset of mitochondrial reads to the assembly using raptor [Version 0.20.3-171e0f1] and polished it with racon [Version 1.14.] . We searched for matches of the resulting mitochondrial assembly sequence in the nuclear genome assembly using BLAST+ and filtered out scaffolds from the nuclear genome with a percentage of sequence identity >99% and size smaller than the mitochondrial assembly sequence. From the subset of mitochondrial reads used for the assembly, we analyzed the BLAST output and the species of the closest mitochondrial sequence available in the NCBI GenBank database, Vaccinium macrocarpon . We used the mitochondrial assembly of V. macrocarpon as a guide for the mitochondrial gene annotation generated with MitoFinder [Version 1.4] .We identified chloroplast reads from the PacBio HiFi dataset with BLAST+ using the plastids RefSeq genomes [v4.1] . From this subset, we analyzed the matches and identified the species of the closest chloroplast sequence available in the NCBI database as Camellia taliensis . Next, we found matches of the C. taliensis chloroplast genome sequence in the nuclear genome assembly with BLAST+ and filtered out scaffolds from the nuclear genome assembly with length smaller than the C. taliensis length, sequence identity >90%, and e-value <0.00001. We aligned the filtered scaffolds to the C. taliensis chloroplast genome with minimap2 and generated a consensus sequence with bcftools . We manually curated the sequence using lastz. Finally, we polished the last assembly version using raptor and racon and annotated it using the web platform GeSeq .We generated a de novo nuclear genome assembly of the big berry manzanita using 199 million read pairs of Hi-C data and 1.8 million PacBio HiFi reads. The latter yielded ~45- fold coverage . Calculation of coverage is based on a flow-cytometry estimated genome size of ~600 Mb reported in a previous study of Arctostaphylos uva-ursi . Assembly statistics are reported in tabular and graphical form in Table 2 and Figure 2, respectively. The primary assembly consists of 271 scaffolds spanning 547Mb with contig N50 of 8Mb, scaffold N50 of 31Mb, largest contig of 22Mb, and largest scaffold of 44Mb. The Hi-C contact map suggests that the primary assembly is highly contiguous . As expected, the alternate assembly, which consists of sequence from heterozygous regions, is less contiguous . Because the primary assembly is not fully phased, we have deposited scaffolds corresponding to the alternate haplotype in addition to the primary assembly. The final genome size is close to the estimated values from the Genomescope2.0 k-mer spectra . The k-mer spectrum output shows a bimodal distribution with two major peaks, at ~24- and ~47-fold coverage, where peaks correspond to homozygous and heterozygous states respectively. This pattern corresponds to a diploid genome. Based on PacBio HiFi reads, we estimated a 0.164% sequencing error rate and 2.51% nucleotide heterozygosity rate. The assembly has a BUSCO completeness score of 98.2% using the embryophyta gene set, and a per base quality of 66. RepeatModeler indicates that the genome includes 57.71% repetitive elements. The classification of repeat elements generated by RepeatMasker is shown in Table 3.We generated an initial mitochondrial genome assembly with HiFiasm using a subset of HiFi reads that matched to publicly available mitochondrial reference genomes. Following an initial gene annotation with MitoFinder, we manually curated the assembly and introduced 13 gaps to solve partial annotation of 12 genes and re-annotated the assembly. Final mitochondrial genome size was 592 049 bp, about average for a plant mitochondrial genome. The base composition of the final assembly version is A = 27.16%, C = 22.78%, G = 22.84%, T = 26.99%, and consists of 23 transfer RNAs and 33 protein coding genes .