Previous studies investigated the effects of leaf removal on grape berry skin anthocyanin and flavonol concentration, and some studies focused on scrutinizing the various outcomes from the different timings of leaf removal. However, in our case, PFLR was not effective in increasing berry skin anthocyanin concentration in either season. Previous work indicated that berry exposure to solar radiation late in the season might make the berries more prone to negative effects of radiation exposure and higher air temperature. As for flavonols, they are generally reported to be sensitive to solar radiation, but we did not notice a difference in flavonols in 2014 even though the LAI was significantly reduced by PFLR. The berry weight might have been the determining factor, where PFLR did not significantly reduce berry weight, hence it did not increase the concentration either. When comparing the first season to the second, the anthocyanins and flavonols were generally lower, although the TSS at harvest in both years were at the same level. Previous studies reported that <0.8–1.2 m2 of leaf area per kg of fruits could inhibit berry maturation. The second season had a lower leaf area to fruit ratio—the plants didnot have sufficient canopies as source tissues to reach the same maturity in both TSS and flavonoids, which might have contributed to the lower flavonoid accumulation in 2015. RDI significantly reduced plant water status and increased berry anthocyanin concentrations in 2014. Previous studies had shown that deficit irrigation could increase berry anthocyanin concentration. In our case, strawberry gutter system most of the anthocyanin derivatives were greater with RDI. As for flavonols, our study did not indicate that reduction of applied water amounts with RDI had noticeable influences on berry flavonols in either year.
This agreed with previous work, that flavonols are relatively insensitive towards water deficits. In our study, only a portion of the significant treatment effects on anthocyanins and flavonols were carried into wine. We attributed these discrepancies to differences in berry skin extractability affected by both treatments. Previous work indicated that higher permeability of the skin cell walls would lead to more advanced maturity, eventually increasing the extractability of flavonoids. PBLR showed the ability to promote berry maturity in 2014, and the more advanced maturity might have diminished flavonoid concentration benefiting from the leaf removal treatment. Some research attributed this observance to the warm and hot climate, where the impacts of leaf removal and deficit irrigation might be unhelpful in such regions, to a point that berry flavonoids are not increased, or are even decreased. Among the three classes of flavonoids, proanthocyanidins are most the chemically stable and less easily manipulated by cultural practices or grapevine physiological status. In our study, there were minimal effects from the treatments, where only EC terminal subunits were significantly affected. Some previous studies were able to see significant effects, mainly positive, of sun exposure and water deficits on berry or wine proanthocyanidin concentration. However, in warm/hot climates, environmental stresses could be sufficiently severe to degrade berry proanthocyanidins. This might have contributed to the lower total proanthocyanidin concentrations in the second season compared to the first due to the higher air temperatures in 2015. Among the proanthocyanidin subunits, the EGC extension subunits were the most drastically reduced in 2015.
This was corroborated by previous work, where EGC extension subunits were sensitive towards air temperature.In this genome release, 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. 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 bio-tinylated 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, hydroponic nft gully 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, 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. These mitochondrial reads were used as input in HiFiasm [Version 0.13-r308] to generate the mitochondrial assembly. Given the circularity of themitochondrial 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 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 of22Mb, 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 genomeincludes 57.71% repetitive elements.