The need for additional replication may also have been reduced by using elevated amounts of genomic DNA and the use of end-labeling rather than BioPrime may have increased the reliability of calls. The protocol used for hybridization of lettuce genomic DNA was also subsequently highly effective for pepper and other Solanaceae. Furthermore, the use of genomic DNA is a desirable target because SFPs identified using cDNA may be a result of alternate splicing or gene expression differences. Rostoks et al. indicated that 40% of the SFPs they identified may have been falsely called and partially explained them as being mRNA structural variants. They also reported a high predicted false positive rate of 22% for SFPs detectedusing genomic DNA. We concluded that fragmented, end-labeled genomic DNA provided a suitable target for detection of polymorphisms while reducing false positive sequence polymorphism . The overlapping tile design increases the likelihood of detecting polymorphisms due to redundancy at individual positions, coverage along the contigs and optimal position of the SNP within a probe. Furthermore, the number of probes and hence the possible genome coverage was increased by substituting mismatch probes with AG probes for background correction and normalization of data. Because the peripheral 1 to 6 bases of a 25 bp oligonucleotide are less sensitive than the central bases, in terms of detecting sequence polymorphisms, procona system the tiling strategy reduces the loss of coverage due to probe position. The number and reliability of SPP calls in our experiments demonstrates that the overlapping tiling array design has improved coverage, sensitivity and specificity to detect polymorphisms.
SPP calls were validated using several approaches. The data from the two pair-wise comparisons yielded 20 to 41 thousand and 27 to 40 thousand SPPs respectively, depending on the criteria used for specificity and sensitivity. When SPPs from MSA and SFPdev were compared to the 51,552 SNPs detected between RNAseq reads of Salinas and US96UC23, 61.5% and 57.8% were found in or within at least 8 bp of the SPP range respectively, similar to that described by Gresham et al.. However, because of the high FDR associated with duplicated sequences, SPPs that were found to have a duplicated locus within the chip assembly, the gene space assembly or the genome assembly were removed from consideration; one third of the SPPs called that had duplicated loci did not contain a SNP in any of our validation tests. These identified SPPs likely were due to differences between paralogs rather than alleles at a single locus. Due to the increased redundancy provided by the mapping population of 213 RILs compared to the pair-wise comparison of the parents, SPPs in the SFPdev and MSA pair-wise comparisons that coincided with SPP mapped by Truco et al. but were absent of a SNP were considered real. Removal of duplicated loci and inclusion of mapped SPPs provided a balance between false positive and false negative rates and allowed us to optimize FDR while still discovering a high number of SPPs. Taking into consideration the lower observed FDR we concluded that the MSA method performed best as a pairwise comparison; however using multiple detection methods would yield a higher confidence in the subset of SPPs identified by both methods. The SPPs identified in the diversity panel that were polymorphic between L. sativa cv. Salinas and L. serriola acc. US96UC23 showed a low FDR. However, as a result of the filtering, sensitivity of this analysis was reduced compared to the two-genotype analyses by MSA and SFPdev. Specific analysis of the DP data for regions containing known SNPs showed that SFPdev values would have been significant in a pair-wise comparison, between SAL and SER but due to inclusion of data from all genotypes in the DP, the two were not called as polymorphic . The lack of some called SPPs in the DP may be due to larger genetic differences between L. perennis, L. virosa, or L. saligna relative to L. serriola and L. sativa. As a result of smaller hybridization differences between the more closely related genotypes, genotypes differing at a locus may have been grouped together reducing the number of SPPs called between the two genotypes.
Consequently, the DP analysis showed a lower false positive rate, but a higher false negative rate when comparing SAL and SER to sequence and mapping data. As part of our goal was to investigate the diversity and relationships of the genotypes in the DP, SPPs identified by the DP analysis were evaluated. Removal of SPPs in duplicated regions with inconsistent data or missing data was a reasonable method of removing unreliable data as these data may be from poorly performing probes in one or all replicates, heterozygous loci, paralogous genes or deleted genes. There was not a large difference in the observed FDRs for the three SPFdev cutoff values for the DP analysis; so in order to maximize the number of markers used in our phylogenetic analysis and principal component analysis, we used the least stringent cutoff value of 1.2. As the assumptions for analysis with the PHYLIP [21] package were not violated with the large number of markers, they were left as independent. To meet the constraints of the PC analysis software, markers were limited to those that were mapped. The markers discovered in our DP analysis were used to generate a phylogenetic tree showing species separation with 100% boot strap support. L. virosa and L. saligna are sexually incompatible species with L. sativa and appear to be more closely related to each other than to other species in the DP. Our data supports the conclusion by Kesseli et al., that these two species are not progenitors of L. sativa. By limiting markers to those polymorphic within cultivated lettuce we are able to separate most genotypes into classes representing each of the plant types. The butterhead type formed a distinct clade from the iceberg and cos types with 100% bootstrap support. However, the leafy type and the Batavia type both showed a wide distribution across the L. sativa clade. This is not unexpected and may reflect admixture between types during breeding programs. Alternatively, this distribution may indicate that these types are artificial polyphyletic groups based on loose morphological criteria. The leafy types are non-heading with a broad range of leaf morphology. Batavia types vary from heading to non-heading phenotypes. Batavia and iceberg cultivars are both considered crisphead types; however our phylogenetic and PC analyses showed that the two did not cluster together and are significantly different from each other . Rapid advancements in sequencing technology today are changing the methods for genetic analyses. Microarray technology presented in this paper yielded an in depth analysis of diversity for lettuce germplasm separating even closely related lines such as the crisp head class. It also has potentially several other uses including: detection of copy number variants, splice site identification, expression analysis or use with other species within the Compositae. TheSPPs identified in this study were highly reproducible and showed similar false positive results to current sequencing methods in the literature. This technology has also been used to create an ultra-dense, inter-specific genetic map between L. sativa cv. Salinas and L. serriola acc. US96UC23 to dissect phenotypic traits as well as validate and align genomic assemblies of lettuce into chromosomal linkage groups.Managing urban runoff and its associated pollutants is one of the most challenging environmental issues facing urban landscape management. The conversion of naturally pervious land surfaces to buildings, roads, parking lots, and other impervious surfaces results in a rapid surface runoff response for both time of concentration and peak flow. Impervious land surfaces adversely impact the quantity and quality of surface runoff because of their effects on surface water retention, infiltration, and contaminant fate and transport.
Large volumes of storm runoff from urbanized areas cause flooding, sewer system overflows, water pollution, groundwater recharge deficits, habitat destruction, beach closures, toxicity to aquatic organisms, procona valencia buckets and groundwater contamination. Traditional urban runoff management focuses on removing the surface runoff from urban areas as soon as possible to protect public safety. However, as excess surface water is quickly drained from urban areas, it is no longer available for recharging groundwater, irrigating urban landscapes, sustaining wildlife habitat and other uses. Green infrastructure uses natural or engineered systems that mimic natural processes to control storm water runoff. For example, traditional detention ponds have been widely used to treat storm runoff and permeable paving promotes infiltration of rain where it falls. Importantly, decentralized green infrastructure strategies control runoff and contaminants at their source. Vegetation is a green infrastructure strategy that can play an important role in surface runoff management . Large-scale tree planting programs have been established in many cities to mitigate the urban heat island effect, improve urban air quality, and reduce and treat urban runoff . There are municipal storm water credit programs in a growing number of cities that promote retaining existing tree canopy, as well as planting new trees . Although these programs encourage planning and management of urban forests to reduce runoff impacts , fertilizer is required to promote plant growth, and these added nutrients may contribute to contamination of surface runoff. Thus, reducing nutrients in storm runoff is a challenging task for landscape and water managers. Bioswales are shallow drainage courses that are filled with vegetation, compost, and/or riprap. As a part of the surface runoff flow path, they are designed to maximize the time water spends in the swale, which aids in the trapping and breakdown of certain pollutants. Bioswales have been widely recognized as an effective decentralized stormwater BMP to control urban runoff . Their effects are threefold; vegetation intercepts rainfall reducing net precipitation; plant uptake of water via transpiration reduces soil moisture, thereby increasing subsurface water storage capacity, and root channels improve infiltration . New bioswales are being developed for harvesting surface runoff and supporting urban tree growth. Bioswales that integrate engineered soil mixes and vegetation are being used to enhance treatment and storage of surface runoff . The composition of ESMs varies widely, from simple mixtures of stones and native soil to patented commercial products . Highly porous ESM mixes provide ample infiltration and pore space for temporary storage of surface runoff. Also, they support tree growth by providing more water and aeration to roots than compacted native soil alone. ESMs can reduce conflicts between surface roots and sidewalks by promoting deeper rooting systems. In California alone, over $70 million is spent annually to remediate damage by shallow tree roots to sidewalks, curbs and gutters, and street pavement . In Davis, California, a bioswale installed next to a parking lot reduced runoff from the parking lot by 88.8% and the total pollutant loading by 95.4% during the nearly two year monitoring period . Furthermore, a bioswale installed next to a turf grass patch at the University of California-Davis campus eliminated dry weather runoff from an irrigated urban landscape. The ESM used in these studies offered several advantages over other ESMs because the main structural element was locally quarried and relatively inexpensive lava rock . This ESM had a high porosity, high infiltration rate, and a high water storage capacity . The lava rock had many interstitial pores and a high surface area to volume ratio. It effectively fostered the growth of biofilms that retain nutrients and degrade organic pollutants. Because vegetated bioswale research is in its infancy, very few studies have monitored vegetation growth and its impacts on bioswale performance. Moreover, evaluation of system performance is generally conducted before vegetation is fully established . In contrast, this study evaluated the effectiveness of two bioswales on surface runoff reduction, pollutant reduction, and tree growth eight years after construction. The control bioswale contained native soil and the treatment contained an ESM. At the time of this study, the trees in the control and treatment bioswales were fully established and approaching mature size. Measurements recorded the differences in surface runoff dynamics and pollutant reduction rates, as well as tree and shrub growth. This study provides new information on the long-term effectiveness of engineered bioswales in a region with a Mediterranean climate. The water collection system was installed in 2007 to collect composited samples from natural runoff . In this study, surface runoff samples from the control site were collected at a high frequency using grab samples to better observe pollutant concentration dynamics for each experiment throughout a storm hydrograph.