Five hotpots were found in chloroplast genome of Veroniceae , and two universal marker, trnH-psbA and matK were identified, respectively. Then highly variable regions were selected as potential molecular markers for Fritillaria, including ycf1, which was also selected in this study. Sequences of these variable regions founded in this study could be regarded as potential molecular markers for species identification and evolutionary studies and have been shown to be valuable for studies in other groups.Oligonucleotide repeats play an important role for generating indels, inversion and substitutions. Repeat sequences in the chloroplast genome could provide valuable information for understanding not only the sequence divergence but the evolutionary history of the plants. We have detected five types of large repeats in the seven Pulsatilla cp genomes. Among them, the most common repeat types are forward and palindromic repeats, followed by reverse repeats, and only little complement repeats were found in Pulsatilla cp genomes . Most of the repeats were short, ranging from 30–49 bp . We also identified multiple microsatellite repeats, also known as simple sequence repeats or short tandem repeats. Due to their codominant inheritance and high variability, SSRs are robust and effective markers for species identification and population genetic analyses. Most of the mononucleotide repeats were composed of A/T. The other microsatellites types were also dominated by AT/TA, with very little G/C . In this study, plentiful microsatellite loci were found through the comparative analysis of Pulsatilla cp genome sequences. In total,raspberry grow in pots we detected six types of microsatellite based on the comparison of seven Pulsatilla cp genomes . Each Pulsatilla cp genome had 69–87 microsatellites. The lengths of repeat motifs of these microsatellites ranged from 10 to 21 bp .
Among the four structural regions in the cp genomes, most of the repeats and microsatellites were distributed in LSC, followed by SSC, and fewest in IRa/IRb , which were also reported in other studies in angiosperms. These SSRs and repeat sequences are uncorrelated with genome size and phylogenetic position of the species, but will provide important information for further studies of phylogenetic reconstruction and infra- and inter-specifc genetic diversity.Chloroplast genomes have been widely used and have made significant contributions to phylogeny reconstruction at different taxonomic levels in plants. To better clarify the evolutionary relationships within Pulsatilla, we used each data set to construct phylogenetic trees using the ML analytical methods. We also construct phylogenetic trees with those eight highly variable regions using the ML, MP analytical methods. All tree topology structures were identical. Therefore, here we presented the phylogenetic studies using the ML tree with the support values from the MP analyses recorded at the corresponding nodes . The phylogenetic tree based on all data sets from the complete plastid genome sequences yielded the same topology. The phylogenetic tree based on chloroplast genome differed from that of the DNA barcode combination rbcL+matK+trnH-psbA, but with higher support values. The phylogenetic trees based on data from complete plastid genome sequences showed that the species of Pulsatilla formed a monophyletic group which in turn includes two strongly supported clades. One clade comprised P. alpina and P. occidentalis, members of subg. Preonanthus. The other comprised two subclades: members of P. hirsutissima, P. ludoviciana, P. multifdi, P. patens and P. vernalis, and species of P. chinensis, P. dahurica, P. grandis and P. pratensis. All the species of the two subclades are members of the subg. Pulsatilla. These results were congruent with our former results based on universal markers.
In phylogenetic analyses, compared to the combination of barcodes, the full chloroplast genome sequence data formed distinct clades with high bootstrap support, improving the inadequate resolution of barcodes combination. The LSC regions and coding regions have the same topology structures with robust support. However, sequencing of genomic DNA is still expensive. It is necessary to utilize variation within chloroplast regions for rapid species-specific assay. Here we found that phylogenetic inference based on highly variable regions yielded a tree with the same topology as the one recovered based on complete chloroplast genome sequences, demonstrating the high utility of hot spots of variability for species identification and phylogenetic analysis. More samples and laboratory works are needed in the future to increase the number of these variable regions available for study.High frequency irrigation systems involve fastidious planning and complex designs, so that timely and accurate additions of water and fertilizer can result in sustainable irrigation. At the same time these production systems are becoming more intensive, in an effort to optimise the return on expensive and scarce resources such as water and nutrients. Advanced fertigation systems combine drip irrigation and fertilizer application to deliver water and nutrients directly to the roots of crops, with the aim of synchronising the applications with crop demands , and maintaining the desired concentration and distribution of ions and water in the soil . Hence a clear understanding of water dynamics in the soil is important for the design, operation, and management of irrigation and fertigation under drip irrigation . However, there is a need to evaluate the performance of these systems, because considerable localised leaching canoccurnear the driplines, evenunderdeficitirrigation conditions .
The loss of nutrients, particularly nitrogen, from irrigation systems can be expensive and pose a serious threat to receiving water bodies . Citrus is one of the important horticultural crops being grown under advanced fertigation systems in Australia. Fertigation delivers nutrients in a soluble form with irrigation water directly into the root-zone, thus providing ideal conditions for rapid uptake of water and nutrients. Scholberg et al. demonstrated that more frequent applications of a dilute N solution to citrus seedlings doubled nitrogen uptake efficiency compared with less frequent applications of a more concentrated nutrient solution. Delivery of N through fertigation reduces N losses in the soil-plant system by ammonia volatilisation and nitrate leaching . However, poor irrigation management, i.e., an application of water in excess of crop requirements, plus the storage capacity of the soil within the rooting depth, can contribute to leaching of water and nutrients below the root zone. Therefore, optimal irrigation scheduling is important to maximise the uptake efficiencies of water and nutrients . Most of the citrus production along the Murray River corridor is on sandy soils, which are highly vulnerable to rapid leaching of water and nutrients. Nitrogen is the key limiting nutrient and is therefore a main component of fertigation. An increasing use of nitrogenous fertilizers and their subsequent leaching as nitrate from the root zone of cropping systems is recognised as a potential source of groundwater contamination,30 planter pot because the harvested crop seldom takes up more than 25–70% of the total applied fertilizer . Several researchers have reported substantial leaching of applied N under citrus cultivation in field conditions . Similarly, in lysimeter experiments, Boaretto et al. showed 36% recovery of applied nitrogen by orange trees, while Jiang and Xia reported N leaching of 70% of the initial N value, and found denitrification and leaching to be the main processes for the loss of N. These studies suggest that knowledge of the nitrogen balance in cropping systems is essential for designing and managing drip irrigation systems and achieving high efficiency of N fertilizer use, thereby limiting the export of this nutrient as a pollutant to downstream water systems. Quantifying water and nitrogen losses below the root zone is highly challenging due to uncertainties associated with estimating drainage fluxes and solute concentrations in the leachate, even under well-controlled experimental conditions . Moreover, direct field measurements of simultaneous migration of water and nitrogen under drip irrigation is laborious, time-consuming and expensive . Hence simulation models have become valuable research tools for studying the complex and interactive processes of water and solute transport through the soil profile, as well as the effects of management practices on crop yields and on the environment . In fact, models have proved to be particularly useful for describing and predicting transport processes, simulating conditions which are economically or technically impossible to carry out in field experiments . Several models have been developed to simulate flow and transport processes, nutrient uptake and biological transformations of nutrients in the soil .
HYDRUS 2D/3D has been used extensively for evaluating the effects of soil hydraulic properties, soil layering, dripper discharge rates, irrigation frequency and quality, timing of nutrient applications on wetting patterns and solute distribution because it has the capability to analyse water flow and nutrient transport in multiple spatial dimensions . In the absence of experimental data we can use multidimensional models solving water flow and nutrient transport equations to evaluate the multi-dimensional aspect of nitrate movement under fertigation . However, earlier simulation studies have reported contradictory results on nitrate distribution in soils. For example, Cote et al. reported that nitrate application at the beginning of an irrigation cycle reduced the risk of leaching compared to fertigation at the end of the irrigation cycle. On the other hand, Hanson et al. reported that fertigation at the end of an irrigation cycle resulted in a higher nitrogen use efficiency compared to fertigation at the beginning or middle of an irrigation cycle. These studies very well outlined the importance of numerical modelling in the design and management of irrigation and fertigation systems, especially when there is a lack of experimental data on nutrient transport in soils. However, there is still a need to verify the fate of nitrate in soils with horticultural crops and modern irrigation systems. Therefore, a lysimeter was established to observe water movement and drainage under drip irrigated navel orange, and to calibrate the HYDRUS 2D/3D model against collected experimental data. The model was then used, in the absence of experimental data on nitrate, to develop various modelling scenarios to assess the fate of nitrate for different irrigation and fertigation schemes.The study was conducted on a weighing lysimeter assembled and installed at the Loxton Research Centre of the South Australian Research and Development Institute. The lysimeter consisted of a PVC tank located on 1.2 m × 1.2 m pallet scales fitted with 4 × 1 tonne load-cells, and connected to a computerised logging system which logged readings hourly. A specially designed drainage system placed at the bottom of the lysimeter consisted of radially running drainage pipes, which were connected to a pair of parallel pipes, which facilitated a rapid exit of drainage water from the lysimeter . These pipes were covered in a drainage sock and buried in a 25-cmlayer of coarse washed river sand at the base of the lysimeter, which ensured easy flushing of water through the drainage pipe. A layer of geo-textile material was placed over the top of the sand layer to prevent roots growing down into it, as this layer was intended to be only a drainage layer. A healthy young citrus tree was excavated from an orchard at the Loxton Research Centre and transplanted into the lysimeter. A soil profile approximately 85 cm deep was transferred to the tank with the tree and saturated to remove air pockets and to facilitate settling. The final soil surface was around 10 cm below the rim of the tank. Soil samples were collected from0 to 20, 20 to 40, 40 to 60, 60 to 85, and 85 to 110 cm depths to measure bulk density and to carry out particle size analysis. Two months after transplanting, the lysimeter was installed amongst existing trees in the orchard. Measurements were initiated after about six months, in order to enable the plant to adjust to the lysimeter conditions. The lysimeter was equipped with Sentek® EnviroSCAN® logging capacitance soil water sensors installed adjacent to the drip line at depths of 10, 20, 40, 60, and 80 cm to measure changes in the volumetric soil water content.The experimental site was approximately 240 m from an established weather station, which measured air temperature, relative humidity, wind speed , rainfall, and net radiation.Irrigation was applied using 3 pressure compensated emitters with a discharge rate of 4 L h−1. Emitters were located on a circle 25 cm away from the tree trunk at an equal distance from each other . The irrigation schedule was based on the average reference evapotranspiration during the last 10 years at the site, multiplied by the crop coefficient taken from Sluggett .