Approaches to breeding for salinity tolerance - a case study on Porteresia coarctata

Cereals are the world's major source of food for human nutrition. Among these, rice (Oryza sativa) is the most prominent and represents the staple diet for more than two-fifths (2.4 billion) of the world's population, making it the most important food crop of the developing world (Anon., 2000a). Rice production in vast stretches of coastal areas is hampered due to high soil salinity. This is because rice is a glycophyte and it does not grow well under saline conditions. In order to increase rice production in these areas there is a need to develop rice varieties suited to saline environments. Research has shown that Porteresia coarctata, a highly salt tolerant wild relative of rice growing in estuarine soils, is an important material for transferring salt tolerant characteristics to rice. It is quite possible that Porteresia may be used as a parent for evolving better and truly salt resistant varieties. The inadequate results and the difficulties associated with conventional breeding techniques necessitate the use of the tools of crop biotechnology in unravelling some of the characteristics of Porteresia that have been highlighted in this report. In view of the limited resources available for increasing salinity tolerance to the breeders to wild rice germplasm, Porteresia is undoubtedly one of the key source species for elevating salinity tolerance in cultivated rice.     

Genome-wide association mapping of salinity tolerance in rice (Oryza sativa)

Salinity tolerance in rice is highly desirable to sustain production in areas rendered saline due to various reasons. It is a complex quantitative trait having different components, which can be dissected effectively by genome-wide association study (GWAS). Here, we implemented GWAS to identify loci controlling salinity tolerance in rice. A custom-designed array based on 6,000 single nucleotide polymorphisms (SNPs) in as many stress-responsive genes, distributed at an average physical interval of <100 kb on 12 rice chromosomes, was used to genotype 220 rice accessions using Infinium high-throughput assay. Genetic association was analysed with 12 different traits recorded on these accessions under field conditions at reproductive stage. We identified 20 SNPs (loci) significantly associated with Na⁺/K⁺ ratio, and 44 SNPs with other traits observed under stress condition. The loci identified for various salinity indices through GWAS explained 5–18% of the phenotypic variance. The region harbouring Saltol, a major quantitative trait loci (QTLs) on chromosome 1 in rice, which is known to control salinity tolerance at seedling stage, was detected as a major association with Na⁺/K⁺ ratio measured at reproductive stage in our study. In addition to Saltol, we also found GWAS peaks representing new QTLs on chromosomes 4, 6 and 7. The current association mapping panel contained mostly indica accessions that can serve as source of novel salt tolerance genes and alleles. The gene-based SNP array used in this study was found cost-effective and efficient in unveiling genomic regions/candidate genes regulating salinity stress tolerance in rice.     

Unlocking Allelic Diversity for Sustainable Development of Salinity Stress Tolerance in Rice

Rice is a major cereal crop, negatively impacted by soil-salinity, both in terms of plant growth as well as productivity. Salinity tolerant rice varieties have been developed using conventional breeding approaches, however, there has been limited success which is primarily due to the complexity of the trait, low yield, variable salt stress response and availability of genetic resources. Furthermore, the narrow genetic base is a hindrance for further improvement of the rice varieties. Therefore, there is a greater need to screen available donor germplasm in rice for salinity tolerance related genes and traits. In this regard, genomics based techniques are useful for exploring new gene resources and QTLs. In rice, the vast allelic diversity existing in the wild and cultivated germplasm needs to be explored for improving salt tolerance. In the present review, we provide an overview of the allelic diversity in the Quantitative Trait Loci (QTLs) like Saltol, qGR6.2, qSE3 and RNC4 as well as genes like OsHKT1;1, SKC1 (OsHKT1;5/HKT8) and OsSTL1 (salt tolerance level 1 gene) related to salt tolerance in rice. We have also discussed approaches for developing salt-tolerant cultivars by utilizing the effective QTLs or genes/alleles in rice.     

Arguments for the use of physiological criteria for improving the salt tolerance in crops

Efforts to develop new crop varieties with improved salt tolerance have been intensified over the past 15–20 years. Despite the existence of genetic variation for salt tolerance within species, and many methods available for expanding the source of genetic variation, there is only a limited number of varieties that have been developed with improved tolerance. These new varieties have all been based upon selection for agronomic characters such as yield or survival in saline conditions. That is, based upon characters that integrate the various physiological mechanisms responsible for tolerance. Yet over the same time period, knowledge of physiological salt responses has increased substantially.Selection and breeding to increase salt tolerance might be more successful if selection is based directly on the physiological mechanisms or characters conferring tolerance. Basic questions associated with using physiological selection criteria are discussed in the paper. These are centred around the need for genetic variation, the importance of the targeted mechanism, the ease of detection of the physiological mechanism (including the analytical requirements) and the breeding strategy. Many mechanisms, including ion exclusion, ion accumulation, compatible solute production and osmotic adjustment have been associated with genetic variation in salt tolerance. Yet their successful use in improving salt tolerance, via physiological selection criteria, is largely non-existent. Consideration is given to the role of physiological criteria in the short and long term in improving salt tolerance. In several glycophytic species, particularly legumes, physiological selection based on ion exclusion from the shoots shows promise. Recent results for white clover indicate the potential for using a broad physiological selection criterion of restricted Cl accumulation in the shoots, with scope for future refinement based upon the specific physiological characters that combined result in ion exclusion.     

Salt‐Treated Roots of Oryza australiensis Seedlings are Enriched with Proteins Involved in Energetics and Transport

Salinity is a major constraint on rice productivity worldwide. However, mechanisms of salt tolerance in wild rice relatives are unknown. Root microsomal proteins are extracted from two Oryza australiensis accessions contrasting in salt tolerance. Whole roots of 2‐week‐old seedlings are treated with 80 mM NaCl for 30 days to induce salt stress. Proteins are quantified by tandem mass tags (TMT) and triple‐stage Mass Spectrometry. More than 200 differentially expressed proteins between the salt‐treated and control samples in the two accessions (p‐value <0.05) are found. Gene Ontology (GO) analysis shows that proteins categorized as “metabolic process,” “transport,” and “transmembrane transporter” are highly responsive to salt treatment. In particular, mitochondrial ATPases and SNARE proteins are more abundant in roots of the salt‐tolerant accession and responded strongly when roots are exposed to salinity. mRNA quantification validated the elevated protein abundances of a monosaccharide transporter and an antiporter observed in the salt‐tolerant genotype. The importance of the upregulated monosaccharide transporter and a VAMP‐like protein by measuring salinity responses of two yeast knockout mutants for genes homologous to those encoding these proteins in rice are confirmed. Potential new mechanisms of salt tolerance in rice, with implications for breeding of elite cultivars are also discussed.     

不同类型盐生植物适应盐胁迫的生理生长机制及Na+逆向转运研究进展

盐生植物是指能在离子浓度至少200 mmol/L以上的生境中生长并完成生活史的植物。盐生植物可分为稀盐盐生植物、泌盐盐生植物、拒盐盐生植物三类。本文从生长形态、生理和分子3个方面总结三类盐生植物响应盐胁迫的不同策略及研究进展,发现盐生植物在分子水平上主要通过Na⁺转运蛋白和为其提供能量的两类基因应对体内过高Na⁺,这可能是引起盐生植物生理和生长形态异于非盐生植物的重要因素。其中稀盐盐生植物主要通过液泡离子区隔化应对盐胁迫,并表现出肉质化生长形态;泌盐盐生植物通过将体内盐分排出体外应对盐胁迫,并进化出特有的生理结构——盐腺或盐囊泡;拒盐盐生植物通过将盐离子积累在皮层细胞液泡和根部木质部薄壁细胞中减少向上运输Na⁺,同时根部多栓质化减少Na⁺吸收。本综述旨在为今后研究盐生植物及其耐盐机制提供相关依据,为植物耐盐分子育种奠定基础。     

耐盐转基因植物研究进展

高盐是限制作物生长、发育和产量的最严重的非生物胁迫之一。长期以来,改善作物的耐盐性一直是一个伟大的目标。然而,由于耐盐反应是一个极为复杂的过程,过去,通过传统的育种和遗传工程取得的成功有限。近十年来,由于分子生物学的发展,发现了一些与耐盐相关的新基因,对于这些基因的表达方式及其在耐盐反应中的作用已逐步得到了解,这为转基因工程提供了新的材料。通过控制耐盐相关基因在植物体内的表达,已获得了一些提高耐盐性的转基因植物,展示了诱人的前景,但该领域研究仍然存在许多困难和问题,文章重点讨论耐盐转基因植物的进展。     

大豆耐盐相关QTLs鉴定和功能基因研究进展

大豆(Glycine max (L.) Merill)是重要的粮食作物和经济作物,盐胁迫能造成大豆产量的大幅度降低。本文综述了通过正向遗传学手段获得的大豆耐盐数量性状位点(Quantitative trait locus, QTL)以及通过反向遗传学方法获得的大豆耐盐功能基因方面的研究进展。目前,正向遗传学发掘基因主要有图位克隆(Map-based cloning)和全基因组关联分析(Genome-wide association study, GWAS)两种方案,其中通过图位克隆在大豆中已经获得了6个耐盐QTL位点并且定位了1个重要的耐盐基因;利用GWAS在大豆中获得了1个耐盐功能基因。利用反向遗传学在大豆中获得了大量的耐盐相关功能基因并在模式植物中验证了其功能,主要包括离子转运蛋白基因和转录因子基因。这些研究为揭示大豆耐盐分子机制以及通过分子标记辅助育种或转基因技术创制耐盐大豆奠定了基础。     

New allelic variants found in key rice salt‐tolerance genes: an association study

Salt stress is a complex physiological trait affecting plants by limiting growth and productivity. Rice, one of the most important food crops, is rated as salt‐sensitive. High‐throughput screening methods are required to exploit novel sources of genetic variation in rice and further improve salinity tolerance in breeding programmes. To search for genotypic differences related to salt stress, we genotyped 392 rice accessions by EcoTILLING. We targeted five key salt‐related genes involved in mechanisms such as Na⁺/K⁺ ratio equilibrium, signalling cascade and stress protection, and we found 40 new allelic variants in coding sequences. By performing association analyses using both general and mixed linear models, we identified 11 significant SNPs related to salinity. We further evaluated the putative consequences of these SNPs at the protein level using bioinformatic tools. Amongst the five nonsynonymous SNPs significantly associated with salt‐stress traits, we found a T67K mutation that may cause the destabilization of one transmembrane domain in OsHKT1;5, and a P140A alteration that significantly increases the probability of OsHKT1;5 phosphorylation. The K24E mutation can putatively affect SalT interaction with other proteins thus impacting its function. Our results have uncovered allelic variants affecting salinity tolerance that may be important in breeding.     

OsMPK4 promotes phosphorylation and degradation of IPA1 in response to salt stress to confer salt tolerance in rice

Salt stress adversely affects plant growth, development, and crop yield. Rice (Oryza sativa L.) is one of the most salt-sensitive cereal crops, especially at the early seedling stage. Mitogen-activated protein kinase (MAPK/MPK) cascades have been shown to play critical roles in salt response in Arabidopsis. However, the roles of the MPK cascade signaling in rice salt response and substrates of OsMPK remain largely unknown. Here, we report that the salt-induced OsMPK4-Ideal Plant Architecture 1 (IPA1) signaling pathway regulates the salt tolerance in rice. Under salt stress, OsMPK4 could interact with IPA1 and phosphorylate IPA1 at Thr180, leading to degradation of IPA1. Genetic evidence shows that IPA1 is a negative regulator of salt tolerance in rice, whereas OsMPK4 promotes salt response in an IPA1-dependent manner. Taken together, our results uncover an OsMPK4-IPA1 signal cascade that modulates the salt stress response in rice and sheds new light on the breeding of salt-tolerant rice varieties.     

Joint genetic and network analyses identify loci associated with root growth under NaCl stress in Arabidopsis thaliana

Plants have evolved a series of tolerance mechanisms to saline stress, which perturbs physiological processes throughout the plant. To identify genetic mechanisms associated with salinity tolerance, we performed linkage analysis and genome‐wide association study (GWAS) on maintenance of root growth of Arabidopsis thaliana in hydroponic culture with weak and severe NaCl toxicity. The top 200 single‐nucleotide polymorphisms (SNPs) determined by GWAS could cumulatively explain approximately 70% of the variation observed at each stress level. The most significant SNPs were linked to the genes of ATP‐binding cassette B10 and vacuolar proton ATPase A2. Several known salinity tolerance genes such as potassium channel KAT1 and calcium sensor SOS3 were also linked to SNPs in the top 200. In parallel, we constructed a gene co‐expression network to independently verify that particular groups of genes work together to a common purpose. We identify molecular mechanisms to confer salt tolerance from both predictable and novel physiological sources and validate the utility of combined genetic and network analysis. Additionally, our study indicates that the genetic architecture of salt tolerance is responsive to the severity of stress. These gene datasets are a significant information resource for a following exploration of gene function.     

水稻耐盐/碱性鉴定评价方法

土壤盐/碱化是盐/碱稻作区水稻生产稳定发展的主要限制因素.为了提高水稻耐盐/碱性,扩大水稻种植面积,减轻盐/碱胁迫导致的水稻减产,许多学者广泛开展了水稻耐盐/碱性的基因型差异、生理生化、遗传及定位等研究,并取得了显著成绩.但国内在耐盐/碱性鉴定评价方法方面还缺乏统一标准,这影响着水稻耐盐/碱性研究的深入开展.本文阐述了国内外至今所采用的水稻耐盐/碱性鉴定方法、耐盐/碱指标和分级标准等,以期为我国水稻耐盐/碱性鉴定评价技术规范的制定以及水稻耐盐/碱性种质资源鉴定、生理生化分析和遗传育种提供参考依据.     

Breeding for salt tolerance in crop plants — the role of molecular biology

Salinity in soil affects about 7 % of the land’s surface and about 5 % of cultivated land. Most importantly, about 20 % of irrigated land has suffered from secondary salinisation and 50 % of irrigation schemes are affected by salts. In many hotter, drier countries of the world salinity is a concern in their agriculture and could become a key issue. Consequently, the development of salt resistant crops is seen as an important area of research. Although there has been considerable research into the effects of salts on crop plants, there has not, unfortunately, been a commensurate release of salt tolerant cultivars of crop plants. The reason is likely to be the complex nature of the effect of salts on plants. Given the rapid increase in molecular biological techniques, a key question is whether such techniques can aid the development of salt resistance in plants. Physiological and biochemical research has shown that salt tolerance depends on a range of adaptations embracing many aspects of a plant’s physiology: one of these the compartmentation of ions. Introducing genes for compatible solutes, a key part of ion compartmentation, in salt-sensitive species is, conceptually, a simple way of enhancing tolerance. However, analysis of the few data available suggests the consequences of transformation are not straightforward. This is not unexpected for a multigenic trait where the hierarchy of various aspects of tolerance may differ between and within species. The experimental evaluation of the response of transgenic plants to stress does not always match, in quality, the molecular biology. We have advocated the use of physiological traits in breeding programmes as a process that can be undertaken at the present while more knowledge of the genetic basis of salt tolerance is obtained. The use of molecular biological techniques might aid plant breeders through the development of marker aided selection.     

Developing salt tolerant plants in a new century: a molecular biology approach

Soil salinity is a major abiotic stress in plant agriculture strongly, influencing plant productivity world-wide. Classical breeding for salt tolerance in crop plants has been attempted to improve field performance without success. Therefore, an alternative strategy is to generate salt tolerant plants through genetic engineering. Several species and experimental approaches have been used in order to identify those genes that are important for salt tolerance. Due to high level of salt tolerance, halophytes are good candidates to identify salt tolerance genes. However, other species such as yeast and glycophytes have also been employed. Three approaches are commonly used to identify genes important for salt tolerance. The first approach is to identify genes involved in processes known to be critical for salt tolerance (osmolyte synthesis, ion homeostasis, etc.). The second approach is to identify genes whose expression is regulated by salt stress. This is relatively simply and applicable to any plant species. Genetic amenability of some species allows the third approach, which consists in the identification of salt tolerance determinants based on functionality. At the moment, there is a large number of reports in the literature claiming that plants with increased salt tolerance have been obtained. The main problem is that different plant species, stage of development, organs, promoters and salt conditions used it is difficult to compare the degree of salt tolerance conferred by different genes. In this review, we discuss progress made towards understanding the molecular elements involved in salt stress responses that have been used in transgenic approaches to improve salt tolerance.     

大豆出苗期和苗期对盐胁迫的响应及耐盐指标评价

比较了4个大豆品种出苗期和苗期的耐盐性,测定150 mmol/L NaCl胁迫下的株高、下胚轴长、侧根数、地上干/鲜重、根干/鲜重、MDA含量、SOD活性、游离Pro含量,并将幼苗移栽到田间生长至成熟。结果表明:出苗期和苗期盐胁迫下4个品种的株高都显著降低、地上干/鲜重和根干/鲜重降低;出苗期胁迫侧根数减少,下胚轴长降低;而苗期胁迫侧根数增加,下胚轴长升高。未胁迫条件下,出苗期和苗期耐盐性强的品种22021-1的MDA含量和SOD活性高于耐盐性弱的品种22293-1。胁迫后,22021-1的MDA含量降低、SOD活性升高,其MDA含量分别比对照低51.03%和21.45%,SOD活性比对照高5.85%和45.77%;22293-1的MDA含量出苗期比对照高58.97%,苗期基本无变化,SOD活性出苗期和苗期升高都不显著。MDA和SOD可以作为大豆耐盐性筛选指标。早期的短时胁迫对不同耐盐性大豆品种的经济产量影响不同。