Salinity stress limits the growth and productivity of agricultural crops in many regions of the world. Whole plant tolerance to soil salinity involves numerous processes in many different tissues and cell types. For many cereals, sensitivity to salinity is due to the accumulation of sodium (Na⁺) to toxic concentrations in the leaves. This thesis investigates a mechanism of control of Na⁺ accumulation in leaves of wheat. Bread wheat excludes sodium from the leaves better than durum wheat. Bread wheat is hexaploid (AABBDD) whereas durum wheat is tetraploid (AABB). The D-genome in bread wheat carries a major locus for sodium exclusion, Kna1, which may contribute to the differences in sodium exclusion between bread wheat and durum wheat. An unusual durum wheat, Line 149, excludes sodium to a similar degree as bread wheat. Line 149 was derived from a cross between a Triticum monococcum (accession C68-101; AA) and a durum wheat (T. turgidum ssp. durum cv. Marrocos; AABB). Line 149 had been found to contain two major genes for sodium exclusion, named Nax1 and Nax2, which appeared to retrieve sodium from the xylem sap in the roots and so prevent it reaching the leaves. Line 149 had been crossed with the durum wheat cv. Tamaroi, which accumulates high concentrations of Na⁺ in the leaves, and near-isogenic single-gene mapping populations had been developed for Nax1 and Nax2. Nax1 had been located on chromosome 2A. The objective of this thesis was to map Nax2 and identify a candidate gene. Nax2 mapped to chromosome 5AL based on linkage to microsatellite markers. A high-affinity potassium (K⁺) transporter (HKT)-like gene, HKT1;5 was considered as a candidate gene for Nax2, based on similarity of the phenotype to a rice orthologue. Sequence information from a wheat HKT1;5-like expressed sequence tag in the public database was used to develop a probe for use in Southern hybridsation. A HKT1;5-like fragment was identified in Line 149 and T. monococcum C68-101, but was absent in Tamaroi. The HKT1;5-like gene, named TmHKT1;5-A, co-segregated with Nax2 in the Nax2 single-gene mapping population. The HKT1;5 probe identified three putative HKT1;5-like genes on the long arm of chromosome 4B, and one HKT1;5-like gene on the long arm of chromosome 4D, in Langdon (T. turgidum ssp. durum) substitution lines, and in Chinese Spring (T. aestivum) ditelomeric lines. No A-genome HKT1;5 like gene was identified in Langdon or Chinese Spring. The D-genome HKT1;5 gene, named TaHKT1;5-D, was found to co-locate with Kna1, the gene for sodium exclusion in bread wheat, in Chinese Spring chromosome 4D deletion lines. Nax2 (TmHKT1;5-A) was found to be homoeologous with the gene for sodium exclusion in bread wheat, Kna1 (TaHKT1;5-D). TmHKT1;5-A and TaHKT1;5-D, and their promoters, were 94% identical, and both were expressed in the roots of wheat plants. This is consistent with the genes being located in the stele of the roots and retrieving Na⁺ from the xylem sap as it flows towards the shoot, and so excluding Na⁺ from the leaves. A marker for TmHKT1;5-A was developed to track this gene in durum wheat breeding programs. A study of the HKT1;5 gene in diploid ancestors of wheat indicated that this gene is present in most Triticum monococcum accessions, some T. boeoticum accessions, but not present in any T. urartu accessions. T. urartu is the likely A genome ancestor of modern wheat. This may explain the absence of HKT1;5 in the A genome of modern wheat. The protein encoded by TaHKT1;5-D transported sodium when expressed in Xenopus laevis oocytes. The inward currents were specific to Na⁺, but at particular mole fractions of Na⁺ and K⁺ outward currents were observed that were consistent with outward K⁺ transport. These data were consistent with the putative physiological function, of retrieving Na⁺ from the xylem sap as it flows to the leaves, and resulting in a net exchange with K⁺. A construct designed to silence the expression of TaHKT1;5-D was introduced to bread wheat cv. Bob White. Nineteen putative transgenic plants were developed. The leaf Na⁺ concentrations and genotype of the T1 individuals were assayed. The data from two of the transgenic plants indicated that TaHKT1;5-D may have been silenced and that this may have lead to the increase in Na⁺ accumulation in the leaves. However, this data is not conclusive at this time. The information gained from this study will assist the introduction of the Na⁺ exclusion trait into current durum cultivars, which are poor at excluding Na⁺ and are salt sensitive. This information will also contribute to the body of knowledge of ion transport in plants and salinity tolerance in wheat. / Thesis (Ph.D.) - University of Adelaide, School of Agriculture, Food and Wine, 2008

Salinity stress limits the growth and productivity of agricultural crops in many regions of the world. Whole plant tolerance to soil salinity involves numerous processes in many different tissues and cell types. For many cereals, sensitivity to salinity is due to the accumulation of sodium (Na⁺) to toxic concentrations in the leaves. This thesis investigates a mechanism of control of Na⁺ accumulation in leaves of wheat. Bread wheat excludes sodium from the leaves better than durum wheat. Bread wheat is hexaploid (AABBDD) whereas durum wheat is tetraploid (AABB). The D-genome in bread wheat carries a major locus for sodium exclusion, Kna1, which may contribute to the differences in sodium exclusion between bread wheat and durum wheat. An unusual durum wheat, Line 149, excludes sodium to a similar degree as bread wheat. Line 149 was derived from a cross between a Triticum monococcum (accession C68-101; AA) and a durum wheat (T. turgidum ssp. durum cv. Marrocos; AABB). Line 149 had been found to contain two major genes for sodium exclusion, named Nax1 and Nax2, which appeared to retrieve sodium from the xylem sap in the roots and so prevent it reaching the leaves. Line 149 had been crossed with the durum wheat cv. Tamaroi, which accumulates high concentrations of Na⁺ in the leaves, and near-isogenic single-gene mapping populations had been developed for Nax1 and Nax2. Nax1 had been located on chromosome 2A. The objective of this thesis was to map Nax2 and identify a candidate gene. Nax2 mapped to chromosome 5AL based on linkage to microsatellite markers. A high-affinity potassium (K⁺) transporter (HKT)-like gene, HKT1;5 was considered as a candidate gene for Nax2, based on similarity of the phenotype to a rice orthologue. Sequence information from a wheat HKT1;5-like expressed sequence tag in the public database was used to develop a probe for use in Southern hybridsation. A HKT1;5-like fragment was identified in Line 149 and T. monococcum C68-101, but was absent in Tamaroi. The HKT1;5-like gene, named TmHKT1;5-A, co-segregated with Nax2 in the Nax2 single-gene mapping population. The HKT1;5 probe identified three putative HKT1;5-like genes on the long arm of chromosome 4B, and one HKT1;5-like gene on the long arm of chromosome 4D, in Langdon (T. turgidum ssp. durum) substitution lines, and in Chinese Spring (T. aestivum) ditelomeric lines. No A-genome HKT1;5 like gene was identified in Langdon or Chinese Spring. The D-genome HKT1;5 gene, named TaHKT1;5-D, was found to co-locate with Kna1, the gene for sodium exclusion in bread wheat, in Chinese Spring chromosome 4D deletion lines. Nax2 (TmHKT1;5-A) was found to be homoeologous with the gene for sodium exclusion in bread wheat, Kna1 (TaHKT1;5-D). TmHKT1;5-A and TaHKT1;5-D, and their promoters, were 94% identical, and both were expressed in the roots of wheat plants. This is consistent with the genes being located in the stele of the roots and retrieving Na⁺ from the xylem sap as it flows towards the shoot, and so excluding Na⁺ from the leaves. A marker for TmHKT1;5-A was developed to track this gene in durum wheat breeding programs. A study of the HKT1;5 gene in diploid ancestors of wheat indicated that this gene is present in most Triticum monococcum accessions, some T. boeoticum accessions, but not present in any T. urartu accessions. T. urartu is the likely A genome ancestor of modern wheat. This may explain the absence of HKT1;5 in the A genome of modern wheat. The protein encoded by TaHKT1;5-D transported sodium when expressed in Xenopus laevis oocytes. The inward currents were specific to Na⁺, but at particular mole fractions of Na⁺ and K⁺ outward currents were observed that were consistent with outward K⁺ transport. These data were consistent with the putative physiological function, of retrieving Na⁺ from the xylem sap as it flows to the leaves, and resulting in a net exchange with K⁺. A construct designed to silence the expression of TaHKT1;5-D was introduced to bread wheat cv. Bob White. Nineteen putative transgenic plants were developed. The leaf Na⁺ concentrations and genotype of the T1 individuals were assayed. The data from two of the transgenic plants indicated that TaHKT1;5-D may have been silenced and that this may have lead to the increase in Na⁺ accumulation in the leaves. However, this data is not conclusive at this time. The information gained from this study will assist the introduction of the Na⁺ exclusion trait into current durum cultivars, which are poor at excluding Na⁺ and are salt sensitive. This information will also contribute to the body of knowledge of ion transport in plants and salinity tolerance in wheat. / Thesis (Ph.D.) - University of Adelaide, School of Agriculture, Food and Wine, 2008

Most research to date has focused on non-pregnant sheep grazing saltbush to fill the summer/autumn feed gap in temperate regions of southern Australia. However, the summer/autumn period coincides with late pregnancy for autumn- or winter-lambing ewes, and feeding saltbush may reduce the amount and cost of supplementary feed that is required to meet the energy demands of late pregnancy. The challenge of dealing with a high-salt diet may be exacerbated during pregnancy since pregnancy is a salt-retaining physiological state, yet a high-salt intake requires an increase in mechanisms to excrete salt. The effect of high dietary salt on the developing foetus(es) has been studied in rodent models, but less so in sheep. Hence the aims of this thesis were to determine whether pregnant ewes can manage a high dietary salt content resembling that found in saltbush, and whether there are consequences to the offspring’s physiological responses to ingested salt. Merino ewes were synchronized for ovulation and artificially inseminated. To mimic the concentration of salt in animals grazing saltbush-based pastures in summer and autumn, a diet of 13% NaCl was fed from insemination through to parturition. It was found that pregnant ewes can be fed a 13% NaCl diet and manage the physiological conflict of high salt and pregnancy by decreasing their aldosterone concentrations and increasing their water consumption. There was no effect of high dietary salt on pregnancy rates, lamb birth weights, lamb survival or milk composition (fat and protein percentages). A series of experiments were conducted to test if the high-salt intake of ewes during pregnancy was associated with a change in the dietary preference for salt and/or changes in physiological responses to ingested salt in the offspring (‘S lambs’ vs. control, ‘C lambs’). C lambs and S lambs were exposed to short- and long-term preference testing to determine if there were differences in their voluntary selection for salt in their diet. There were no significant differences in dietary salt preference between C and S lambs. The lambs were subjected to salt 'challenges' (oral dose of 40 g NaCl in 25% w/v solution) from 3-10 months of age and their water intake, urinary output, sodium excretion and hormone concentrations were measured over the ensuing 23 hours, and compared against counterparts dosed with an equal volume of water without salt. Following the initial salt challenge further experiments were conducted with slight alterations; water intake was manipulated immediately following the salt challenge; two consecutive salt challenges, 8 hours apart, were administered; and C and S lambs were offered salty water (1.5% NaCl) over a period of two days. The results of these salt challenge experiments showed that C and S lambs excreted a salt load at a similar rate, but they differed in the magnitude of changes in water intake and hormone concentrations required to achieve sodium homeostasis. S lambs were able excrete sodium at the same rate as C lambs but without decreasing aldosterone concentrations to the same extent and whilst consuming 400 mL less water in the first two hours post challenge. The aldosterone results suggested a lowered responsiveness to aldosterone and the lower water consumption suggested an altered thirst threshold. The experiment in which water consumption was manipulated suggested that when the supply or access to fresh water is limited, the capacity to remove a salt load is likely to be less impaired in S lambs than C lambs; S lambs were able to excrete the salt load faster than the C lambs when the availability of drinking water was limited. From the experiment in which lambs were treated with two consecutive salt challenges, the rate of sodium excretion increased after the second dose, but there remained no difference in the rate of excretion between C and S lambs; all animals were able to excrete 95% of the administered dose of sodium within 23 hours. The final experiment in which animals were given salty water (1.5% NaCl) for a period of two days showed consistent results with the previous experiments for water consumption and aldosterone concentrations between C and S lambs. There was no difference in sodium excretion between C and S lambs. A novel finding was a markedly lower voluntary feed intake in S lambs than C lambs. Although mechanisms for this are unknown, it may have profound effects on the productivity of the animals. The experiments reported in this thesis provide new information of relevance to pregnant ewes grazing halophytic forages. It is apparent that they can withstand a high NaCl content typical, of a saltbush-based pasture. Further work is warranted to conclude whether high salt during pregnancy is (i) beneficial to the offspring in regards to a higher capacity to deal with excess salt under farming conditions and (ii) consistently associated with a lower voluntary feed intake of the offspring. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1290752 / Thesis (Ph.D.) -- University of Adelaide, School of Agriculture, Food and Wine, 2007.

Salt stress severely constrains plant performance and global agricultural productivity. 5% of arable land, 20% of irrigated areas and 98% of water reserves worldwide are saline. Improving the salt tolerance of major crop species could help attenuate yield losses and expand irrigation opportunities and provide in situ relief in areas where poverty, food and water scarcity are prevalent. Increasing the salt tolerance of crops with high commercial and nutritional value, such as tomato (Solanum lycopersicum L.), would provide particularly significant economic and health benefits. However, salt tolerance is a complex trait with a limited genetic repertoire in domesticated crop varieties, including tomato, frustrating attempts to breed and engineer tolerant crop varieties. Here, a genome-wide association study (GWAS) was undertaken, leveraging the rich genetic diversity of the wild, salt tolerant tomato Solanum pimpinellifolium and the latest phenotyping technologies to identify traits that contribute to salt tolerance and the genetic basis for variation in those traits. A panel of 220 S. pimpinellifolium accessions was phenotyped, focusing on image-based high-throughput phenotyping over time in controlled and field conditions in young and mature plants. Results reveal substantial natural variation in salt tolerance over time across many traits. In particular, the use of unmanned aerial vehicle (UAV)-based remote sensing in the field allowed high-resolution RGB, thermal and hyperspectral mapping that offers new insights into plant performance in the field, over time. To empower our GWAS and facilitate the identification of candidate genes, a new S. pimpinellifolium reference genome was generated, 811Mb in size, N50 of ~76kb, containing 25,970 annotated genes. Analysis of this reference genome highlighted potential contributors to salt tolerance, including enrichments in genes with stress response functions and a high copy number of the salt tolerance-associated gene inositol- 3-phosphate synthase (I3PS). A recently completed full genome re-sequencing of the panel, along with a newly available pseudomolecule-level assembly of the S. pimpinellifolium genome with N50 of ~11Mb, will serve to drive a GWAS to identify loci associated with traits that contribute to salt tolerance. Further research including gene validation, breeding, genetic modification and gene editing experiments will drive the development of new salt tolerant tomato cultivars.

Salinity is a major abiotic stress in plants that causes significant reductions in crop yield. The need for improvement of food production has driven research to understand factors underlying plant responses to salt and mechanisms of salt tolerance. The aim of improving tolerance in traditional crops has been initiated but most crops can only tolerate a limited amount of salt in their systems to survive and produce biomass. Studies of naturally occurring high salt-tolerant plants (halophytes) are now being promoted for economic interests such as food, fodder or ecological reasons. Suaeda fruticosa, a member of the family Chenopodiaceae, belongs to a potential model halophyte genus for studying salt tolerance. However, published reports on the identification of genes, expression patterns and mechanisms of salinity tolerance in succulent halophytes are very limited. Next generation RNA-sequencing techniques are now available to help characterize genes involved in salinity response, along with expression patterns and functions of responsive genes. In this study, we have optimized the assembly of the transcriptome of S. fruticosa. We have annotated the genes based on their gene ontology characteristics and analyzed differential expression to identify genes that are up- and down-regulated in the presence of salt and have grouped the genes based on their putative functions. We also have provided evidence for groups of transcription factors that are involved in salt tolerance of this species and have identified those that may affect the regulation of salt tolerance. This work elucidates the characterization of genes involved in salinity tolerance to increase our understanding of the regulation of salt in a succulent halophyte.

For more than half of the world’s population, rice (Oryza sativa L.), the most saltsensitive cereal, is a dietary staple. Soil salinity is a major constraint to rice production worldwide. Thus, to feed 9 billion people by 2050, we need to increase rice production while facing the challenges of rapid global environmental changes. To meet some of these challenges, there is a vital requirement to significantly increase rice production in salinized land and improve photosynthetic efficiency. Exposure of plants to soil salinity rapidly reduces their growth and transpiration rates (TRs) due to the ‘osmotic component’ of salt stress (sensu Munns and Tester), which is hypothesized to be related to sensing and signaling mechanisms. Over time, toxic concentrations of Na+ and Cl− accumulate in the cells of the shoot, known as the ‘ionic component’ of salt stress, which causes premature leaf senescence. Both osmotic and ionic components of salinity stress are likely to impact yield. Despite significant advances in our understanding of the ionic components of salinity tolerance, little is known about the early responses of plants to salinity stress. In my PhD project, the aim was to analyze naturally occurring variation in salinity tolerance of rice and identify key genes related to higher salinity tolerance using high-throughput phenomics and field trials. I used a forward genetics approach, with two rice diversity panels (indica and aus) and recently published sequencing data (McCouch et al., 2017). Indica and aus were phenotyped under controlled conditions, while the indica diversity panel was also further studied under field conditions for salinity tolerance. I also examined previously unexplored traits associated with salinity tolerance, in particular the effects of salinity on transpiration and transpiration use efficiency. The non-destructive high-throughput experiments conducted under controlled conditions gave insights into the understudied shoot ion-independent component of salinity tolerance. In parallel, the field experiments increased our understanding of the genetic control of further components of salinity tolerance, including the maintenance of yield under saline conditions. Importantly, this project also aimed to improve the current association methods of GWAS by exploring and testing novel Mixed Linear Models. One major benefit of this Ph.D. project was the development of a more holistic approach that recognizes the complexity of the genotype–phenotype interaction. The purpose of my work was to shed more light on the genetic mechanisms of salinity tolerance in rice and discover genes associated with traits contributing to higher photosynthetic activity under both controlled and field conditions. This will ultimately lead to further exploration of the genetic diversity present in the PRAY indica panel, in order to develop higher yielding rice varieties.

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Thesis (MSc (Genetics))--Stellenbosch University, 2008. / Thinopyrum distichum (2n = 4x = 28; J1dJ1dJ2dJ2d) is a hardy, salt-tolerant maritime wheatgrass indigenous to southern Africa. In order to transfer its salt-tolerance to cultivated cereals, the Thinopyrum chromosomes involved must first be characterized with molecular markers. Thinopyrum distichum chromosomes 2J1d, 3J1d, 4J1d and 5J1d have previously been found to be major determinants of salt-tolerance. A genotype panel consisting of two triticale/Th. distichum allopolyploids, two Th. distichum/2*triticale doubled-haploids, eight triticale addition-lines (for chromosomes 2J1d; 2J1dβ; 3J1d; 3J1dL; 4J1d; 4J2d; 5J1d and 7J2d, respectively) and two triticale translocation-lines (involving chromosome arms 3J1dS and 3J1dL, respectively) were used for fluorescence-based, semi-automated AFLP-analyses and to a lesser extent for EST-SSR microsatellite marker-development, to identify molecular markers specific to the critical Th. distichum chromosomes. Thirteen EST-SSR primer pairs produced four putative Th. distichum-specific microsatellite-markers, one of which was specific for critical chromosome 5J1d. AFLP-analysis with 60 selective EcoRI/MseI and 18 Sse8387I/MseI primer combinations produced 159 AFLP-fragments specific for Th. distichum. These included seven putative markers for chromosome 2J1d, 15 for 3J1d, one marker for 4J1d and two for 5J1d. A salt-tolerance experiment was done to determine which chromosome 2J1d and 3J1d regions may carry genes for salt-tolerance. Plants were selected that had a monosomic addition of a chromosome 2J1d variant (either the complete chromosome or a modified version referred to as 2J1dβ) in addition to one of four chromosome 3J1d variants (the complete 3J1d chromosome; a 3J1dL-telosome; a 3J1dS-translocation or a 3J1dL-translocation). The results suggested that Th. distichum chromosome-arms 2J1dL and 3J1dS are probably involved in salt-tolerance. A group of 93 trigeneric (Triticum/Secale/Thinopyrum) F2 secondary hybrids were then analyzed in order to: (i) Evaluate some (ten) of the newly developed putative AFLP-markers; and (ii) attempt to find translocations, telosomes or substitutions involving the critical Thinopyrum chromosomes. Five (50 %) of the ten putative AFLP-markers could be reproduced, but only four proved to be chromosome-specific. It was also possible to assign hese four markers to chromosome arms: E32M49.118 (2J1dS); E41M49.103 (2J1dS); E35M49.137 (3J1d); and E41M49.188 (3J1dL). The selective primer combination that produced marker E41M49.103 (2J1dS), also amplified a fragment of the same size on chromosome 4J1d. These markers will be useful for further mapping and selection of the salt-tolerance genes. The fact that only four of the ten putative AFLP-markers evaluated proved to be repeatable implies that the remaining untested markers need to be confirmed against larger genotype panels as well. Probable reasons for the relatively low frequency of markers that turned out to be reliable are discussed. The marker-association study also revealed that visual examination of all electropherograms produced by AFLP-fragment analysis is necessary to correctly identify all AFLP-fragments. Use of the AFLP- and STS-/SCAR-markers in conjunction with the group of 93 F2 secondary hybrids showed that 18 of these probably carried a 3J1dL-translocation. Several hybrids possibly had translocations involving the 4J1d and 5J1d chromosomes. However, these results need to be confirmed. Various hybrids also appeared to have critical Th. distichum substitutions, although this still requires further confirmation. The identified plant material could prove useful for further characterization of salt-tolerance in Thinopyrum, and its eventual utilization in cereal crops.

The possibility of manipulating calcium (Ca2+) and potassium (K+) levels in seeds of Brassica rapa by altering parent plant nutrition and investigating the potential for increased salinity tolerance during germination, given that considerable amounts of literature imply that greater amounts of available exogenous Ca2+ and K+ can ameliorate the effects of salinity on both whole plant growth and germination, was evaluated. The investigation consisted of four growth trials. Two preliminary growth trials suggested that seed ion manipulation was possible without affecting the overall growth and vigour of the plant. After developing suitable high and low Ca2+ and K+ nutrient solutions for growth, a trial was carried out in a growth room and greenhouse, with various substrates and the seed of a certain size category was collected for subsequent ion and salinity tolerance analysis. Seed Ca2+ and K+ was significantly affected by growth substrate and nutrient solution and data showed that a significant negative regression relationship existed between seed Ca2+, K+ and Ca2+ + K+ levels and salinity tolerance. Further experimentation using hydroponic culture attempted to remove any possible effects of substrate and also to compare size categories of seed with a view to elucidating localisation of Ca2+ and K+. Seed Ca2+ was found to be significantly altered by nutrient solution in the two different sizes tested and higher Ca2+ nutrient solution was found to increase salinity tolerance in daughter seed. One significant negative regression correlation between salinity tolerance and seed K+ concentration existed in smaller seed, but disregarding seed size in a regression analysis of seed ion content and salinity tolerance, a significant negative relationship existed between seed Ca2+, K+ and Ca2++ K+. The results, especially in terms of Ca2+ nutrition, contradict much previous research that suggests increased salinity tolerance at germination can arise with the increased presence of Ca2+ and/or K+. Salinity tolerance was greater in seeds of larger size across all nutritional treatments and the smaller size range exhibited increased Ca2+ and K+ per μg seed. Ca2+ concentration in smaller seeds with greater surface area:volume ratios provided a clue to the potential localisation of Ca2+. Cross sectional staining showed that a greater proportion of seed Ca2+ may reside in the coat. This was confirmed by analysis which showed an approximate 50% split of total extractable seed Ca2+, regardless of size, between coat and embryo within a seed; the majority of which, per μg, resides in the coat. Further work looked at the relative solubility of the Ca2+ and K+ in these tissues and whole seed to look at the potential bioavailability of Ca2+ during germination from various parts of the seed. Most water soluble Ca2+ exists in the embryo and most insoluble Ca2+ exists in the coat, but coat Ca2+ was found to be ionically exchangeable and therefore bioavailable. K+ appeared mostly water soluble in embryo and coat. In line with previous whole plant research in this species, most Ca2+ is readily water soluble or ionically exchangeable in form and the possible negative effects of how increasing bioavailable Ca2+ may reduce salinity tolerance was discussed.