Identification of quantitative trait loci for seedling cold tolerance using RILs derived from a cross between japonica and tropical japonica rice cultivars

Cold tolerance at the seedling stage of rice is an important phenotypic trait that causes normal plant growth and stable rice production in temperate regions as well as tropical high-lands in Asia and Africa. In order to find quantitative trait loci (QTLs)/genes associated with cold tolerance, we constructed a linkage map using 153 recombinant inbred lines (RILs) derived from a cross between a cold-tolerant temperate japonica cultivar, Geumobyeo, and a cold-sensitive tropical japonica breeding line, IR66160-121-4-4-2. The RILs were phenotyped for cold tolerance or sensitivity based on the degrees of cold tolerance as cold tolerance indices at the seedling stage. The seedlings for cold-tolerance/-sensitive traits were scored on the 7th day of the recovery period at 25°C after cold treatment at 10°C. Two QTLs (qCTS4a and qCTS4b) associated with cold tolerance at the seedling stage were identified on the long and short arms of chromosome 4 with an LOD score of 2.89 and 2.75, respectively, using composite interval mapping. The QTLs were flanked by simple sequence repeat (SSR) markers RM3648-RM2799 and RM3375a-RM558 that explained 8.3 and 7.8% of the total phenotypic variation, respectively. Seven of the selected RILs expressed cold tolerance at both the seedling and reproductive stages. The SSR markers associated with the QTLs will be useful for tracking favorable QTLs/genes into cold-sensitive elite cultivars and may have potential for pyramiding different QTLs for the improvement of cold tolerance in rice.     

Deployment of cold tolerance loci from Oryza sativa ssp. Japonica cv. ‘Nipponbare’ in a high‐yielding Indica rice cultivar ‘93‐11’

Cold tolerance is a complex trait, and QTL pyramiding is required for rice breeding. In this study, a total of seven QTLs for cold tolerance in the Japonica rice variety ‘Nipponbare’ were identified in an F_2:3 population. A stably inherited major QTL, called qCTS11, was detected in the region adjacent to the centromere of chromosome 11. In a near‐isogenic line population, the QTL was further dissected into two linked loci, qCTS11.1 and qCTS11.2. Both of the homozygous alleles of qCTS11.1 and qCTS11.2 from ‘Nipponbare’ showed major positive effects on cold tolerance. Through pyramiding the linked QTLs in the cold‐sensitive Indica rice cultivar ‘93‐11’, we have developed a new elite, high‐yielding Indica variety with cold tolerance.     

Identification of quantitative trait loci for cold tolerance during the germination and seedling stages in rice (Oryza sativa L.)

Low temperature is a serious abiotic stress affecting rice production in subtropical and temperate areas. In this study, cold tolerance of rice at the germination and seedling stages were evaluated using one recombinant inbred line (RIL) population derived from a cross between Daguandao (japonica), with highly cold-tolerant at the seedling stage, and IR28 (indica), with more cold-tolerant at the germination stage, and the quantitative trait loci (QTL) mapping was conducted using the multiple interval mapping (MIM) approach. Continuous segregation in low temperature germinability (LTG) and cold tolerance at the seedling stage (CTS) were observed among the RIL populations. Most RILs were moderately susceptible or tolerant at the germination stage, but were susceptible at the seedling stage. No significant relationship was found in cold tolerance between the germination and seedling stages. A total of seven QTLs were identified with limit of detection (LOD) >3.0 on chromosomes 3, 8, 11 and 12, and the amount of variation (R ²) explained by each QTL ranged from 5.5 to 22.4%. The rice LTG might be regulated by two minor QTLs, with the CTS controlled by one major QTL [qCTS8.1 (LOD = 16.1, R ² = 22.4%)] and several minor loci. Among these loci, one simultaneously controls LTG (qLTG11.1) and CTS (qCTS11.1). Several cold-tolerance-related QTLs identified in previous studies were found to be near the QTLs detected here, and three QTLs are novel alleles. The alleles from Daguandao at six QTLs increased cold tolerance and could be good sources of genes for cold tolerance. In addition, only one digenic interaction was detected for CTS, with a R ² value of 6.4%. Those major or minor QTLs could be used to significantly improve cold tolerance by marker-assisted selection (MAS) in rice.     

Development of salt tolerant IR64 near isogenic lines through marker-assisted breeding

Salt stress causes considerable damage to rice with a consequent reduction in grain yield, however, conventional breeding for this stress is time-consuming and costly. Recently, marker-assisted breeding has shown enormous potential to accelerate breeding of stress tolerant varieties because of its precision, time saving, and cost effectiveness. The present study was carried out to transfer Saltol, a major QTL on chromosome 1 associated with salinity tolerance, from FL478, a tolerant genotype, into IR64, a popular lowland variety through marker-assisted backcrossing (MABC). This technique considerably enhanced the recovery rate of the recurrent parent genome within three backcross generations, which could have saved several backcrosses compared with conventional schemes to achieve the same results. By using this technique, up to 99.7% of the recurrent parent genome was recovered at BC₃F₂ generation, saving at least three backcrosses compared with conventional breeding schemes. Salinity tolerance of IR64-Saltol lines was evaluated using saline culture solution adjusted to electrical conductivity of 12 dS m^-1 using NaCl. Based on selected physiological and growth parameters, the new Saltol introgression lines showed a significantly higher tolerance of salinity than their recurrent parent IR64. The results of this study confirm the benefits of using molecular markers in plant breeding to enhance tolerance of abiotic stresses.     

Identification and validation of cold responsive microRNAs of <Emphasis Type="Italic">Hevea brasiliensis</Emphasis> using high throughput sequencing

Cold stress is one of the major abiotic factors that influence the productivity and geographical distribution of many agriculturally important crops like Hevea brasiliensis. Cultivation of H. brasiliensis in India is being extended to northeastern regions, where low temperature during winter adversely affects its survival, growth, and productivity. Developing cold-tolerant genotypes is a primary requisite to maximize the productivity under such challenging environmental conditions. However, lack of methods for early evaluation of cold tolerance in the newly developed clones and the extensive time required for assessing their tolerance in the field are major constraints for clonal selection. The present study was initiated with an objective to identify and characterize cold stress responsive miRNAs from H. brasiliensis that show stronger association with cold tolerance. Next generation sequencing using Illumina HiSeq method revealed the expression of 21 and 29 conserved miRNA (from clone RRIM 600) families in cold-stressed and control samples, respectively. Forty-two novel miRNAs were identified from this study. Upon differential expression analysis, eight conserved miRNAs were found commonly expressed in both the samples. When expression analyses were performed subsequently with six selected miRNAs in two Hevea clones (viz. RRII 105 and RRIM 600), miR169 showed a strong association with cold tolerance. miRNAs such as miR482 and miR159 also exhibited association with cold tolerance. This study suggests the possibility of employing these miRNAs as markers for cold tolerance after validation in more number of genotypes with varying levels of cold tolerance.     

Evaluation of seedling cold tolerance in rice cultivars: a comparison of visual ratings and quantitative indicators of physiological changes

Rice (Oryza sativa L.) is sensitive to prolonged exposure to low temperature, which at the seedling stage can result in significant chilling injury and mortality. The objective of this study was to quantify physiological and biochemical changes in rice seedlings undergoing chilling stress and compare those changes with visual evaluation of tolerance. Seedlings from the cultivars M-202 (tolerant) and IR50 (sensitive) were subjected to 9°C for 14 days in a controlled environment chamber. Leaf tissues were harvested at various time points for determination of electrolyte leakage, proline, malondialdehyde, ascorbic acid and reduced glutathione. Significant differences between M-202 and IR50 were detected in electrolyte leakage, proline, and ascorbic acid starting at 7 days with IR50 exhibiting higher levels of these indicators. Most IR50 seedlings were dead at 14 days. A set of fifty rice accessions including M-202 and IR50 was evaluated at 10 days of cold treatment to examine the correlation of visual ratings with the physiological indices. Visual ratings were most highly correlated with electrolyte leakage and least correlated with proline content. Based on visual ratings and the physiological indices, we identified several cultivars that outperformed M-202 in cold tolerance while IR50 had the lowest tolerance of the cultivars tested.     

Characterization and identification of cold tolerant near-isogenic lines in rice

To exploit the genetic mechanism of cold tolerance in rice, cold tolerant near-isogenic lines (NILs) were developed by backcrossing Kunmingxiaobaigu (KMXBG), reported to be the most cold-tolerant variety at the booting stage, as donor, with the cold sensitive Japanese commercial japonica variety, Towada. Comparisons of cold tolerance-related traits between five BC₆F₅ NILs and recurrent parent Towada under cold treatment and normal temperatures at the booting stage showed that the differences between the NILs and Towada were significant only for spikelet fertility-related traits. Analyses of cold tolerance in the NILs at the budding (germination), seedling and booting stages indicated both correlated effects and differences. Lines 1913-4 and 1916-1 showed strong and stable tolerance at all three stages. Whole genome marker screening showed that the proportion of genetic background recovery was more than 98%. Seventeen markers from KMXBG were introgressed in two or more NILs, and cold tolerance genes were possibly present in these marker regions. The NILs should be excellent materials for both rice improvement and map-based cloning of cold tolerance QTLs.     

Assessment of cold tolerance at early developmental stages and allelic variation at candidate genes in South American rice germplasm

Rice is susceptible to cold during early developmental stages. Most tolerant cultivars have been developed for other conditions than those occurring in South America’s rice growing areas, or their grain type is not suitable for the local markets. If locally adapted varieties were available, growers could anticipate sowing date, making flowering time coincide with the moment of maximum solar radiation and increasing yields. In this work, 116 rice inbred lines and varieties of diverse origin within the South American gene pool were tested for seedling survival and germination percentage under low temperature in controlled conditions. As a result, lines used as controls responded as expected, whereas lines with similar behavior to controls, intermediate between susceptible and tolerant controls and more extreme than controls were detected at both seedling and germination stages. Allelic variation at candidate genes OsGSTZ1, OsGSTZ2 and OsCDPK13 was analyzed in a subset of ten contrasting lines. Ten out of thirty-four polymorphisms detected in all three genes were associated with cold tolerance in these lines. A functional marker, designed from an aminoacid-changing SNP found in gene OsGSTZ2, was tested in the 116 lines. As a result, a tight association was observed between cold tolerance and marker score. In conclusion, wide variability for cold tolerance at early developmental stages has been detected in rice advanced breeding materials that are adapted to local growing conditions. Also, a functional marker tightly associated with the trait is available for performing marker assisted selection.     

Analysis of decision-making coefficients of the lint yield of upland cotton (Gossypium hirsutum L.)

Cold water irrigation and growth in low temperature phytotron are two commonly used methods to evaluate cold tolerance of rice at the seedling stage and the cold sensitive seedlings exhibit different injury symptoms, respectively. However, so far no one has systematically dissected the differences of cold tolerance at seedling stage in rice under the two cold environments. We used a recombinant inbred line (RIL) population derived from a cross of a cold-tolerant japonica cultivar, Lijiangxintuanheigu and a cold sensitive indica cultivar, Sanhuangzhan-2 for this study. The cold sensitive seedlings exhibited leaf yellowing after cold water irrigation and leaf rolling during growth in the low temperature phytotron. Leaf yellowing and leaf rolling in RILs was significantly correlated, but the correlation coefficient was low. A total of four quantitative trait locus (QTLs) on chromosomes 1, 6, 9 and 12 were detected using leaf yellowing and percent seedling survival as indicators of cold tolerance after cold water irrigation, while five QTLs on chromosomes 7, 8, 9, 11 and 12 were detected using leaf rolling and percent seedling survival as indicators of cold tolerance during growth in the low temperature phytotron. The two QTLs, qCTS-9 and qCTS-12 were detected using different evaluation indicators under the two cold environments. Our results suggest that rice cold tolerance mechanisms at the seedling stage differ between the two environments, but the detection of common QTL implies the existence of overlap in the metabolic pathways for cold tolerance. The two common QTLs have potential value in rice breeding.     

水稻新质型不育系幼苗期耐冷性及其生理生化特性的质核效应

为了探明水稻新质型不育系幼苗期耐冷性特点,以一套同质异核不育系（G软香A,G华香1A,G华香2A）和一套同核异质不育系（G软香A,Y软香A,WA软香A）为材料,采用光照培养箱培养的方法,鉴定和评价了3~4叶期幼苗的耐冷性,并分析了低温胁迫后不育系幼苗丙二醛(MDA)含量、过氧化物酶(POD)活性以及幼苗生物量的变化。结果表明：供试不育系的耐冷性分级可分为4级,其中G华香2A的耐冷性最强（1级）,Y软香A的耐冷性最差（9级）。就同质异核不育系而言,G华香2A耐冷性最强,G华香1A次之,G软香A中等,表现出细胞核对耐冷性的效应。就同核异质不育系而言,WA软香A耐冷性强,G软香A耐冷性中等,Y软香A耐冷性极弱,表现出细胞质对耐冷性的效应。因此,水稻不育系的耐冷性受到细胞质和细胞核基因的共同影响。幼苗MDA含量、POD的活性以及幼苗期水稻苗高、根数、鲜重等生物量等性状都与耐冷性有关。耐冷性越强的不育系,经低温处理后其幼苗MDA相对含量越低,POD相对活性越低,生物量相对鲜重越大,即说明耐冷性越强的不育系,经低温处理后较之对照幼苗的MDA含量增加量较小,POD活性增强程度小,生物量下降较少。     

Improved Cold and Drought Tolerance of Doubled Haploid Maize Plants Selected for Resistance to Prooxidant tert‐Butyl Hydroperoxide

To improve the abiotic stress tolerance of maize (Zea mays L.), doubled haploid (DH) plants were produced by in vitro selection of microspores exposed to tert‐butyl hydroperoxide (t‐BuOOH) as a powerful prooxidant This study investigated the tolerance of the progenies of t‐BuOOH‐selected DH lines to oxidative stress, cold and drought in controlled environment pot experiments by analyses of photosynthetic electron transport and CO₂ assimilation processes, chlorophyll bleaching and lipid peroxidation of leaves. Our results demonstrated that the t‐BuOOH‐selected DH plants exhibited enhanced tolerance not only to oxidative stress‐induced by t‐BuOOH but also to cold and drought stresses. In addition, they showed elevated activities of antioxidant enzymes such as superoxide dismutase, ascorbate peroxidase, catalase, glutathione reductase and glutathione S‐transferase when compared with the DH lines derived from microspores that were not exposed to t‐BuOOH and to the original hybrid plants. The results suggest that the simultaneous up‐regulation of several antioxidant enzymes may contribute to the oxidative and cold stress tolerance of the t‐BuOOH‐selected DH lines, and that the in vitro microspore selection represents a potential way to improve abiotic stress tolerance in maize.     

Impact of wheat-Leymus racemosus added chromosomes on wheat adaptation and tolerance to heat stress

Adaptation of wheat (Triticum aestivum L.) to high temperatures could be improved by introducing alien genes from wild relatives. We evaluated the responses of wheat-Leymus racemosus chromosome introgression lines to high temperature to determine their potentiality for developing improved wheat cultivars. Introgression lines and their parent Chinese Spring were evaluated in a growth chamber at the seedling stage and in the field at the reproductive stage in two heat-stressed environments in Sudan. Optimum and late planting were used to ensure exposure of the plants to heat stress at the reproductive stage. The results revealed the impact of several Leymus chromosomes in improving wheat adaptation and tolerance to heat. Three lines possessed enhanced adaptation, whereas two showed high heat tolerance. Two addition lines showed a large number of kernels per spike, while one possessed high yield potential. Grain yield was correlated negatively with the heat susceptibility index, days to heading and maturity and positively with kernel number per spike and triphenyl tetrazolium chloride assay under late planting. The findings suggest that these genetic stocks could be used as a bridge to introduce the valuable Leymus traits into a superior wheat genetic background, thus helping maximize wheat yield in heat-stressed environments.     

Overexpression of cold-inducible wheat galactinol synthase confers tolerance to chilling stress in transgenic rice

Galactinol synthase (GolS) is considered to be a key regulator of the biosynthesis of Raffinose family oligosaccharides (RFOs). Accumulation of RFOs has been reported to play a role in protection against abiotic stresses. We identified two cDNAs encoding galactinol synthase from wheat (Triticum aestivum L.), which we designated as TaGolS1 and TaGolS2. Expression of the two TaGolS genes was induced by cold stress but not by drought, heat stress or ABA treatment in wheat. We generated transgenic lines of rice (Oryza sativa L.) constitutively overexpressing TaGolS1 or TaGolS2. These transgenic plants accumulated significantly higher levels of galactinol and raffinose than did wild-type plants and exhibited enhanced cold-stress tolerance. The results demonstrate the involvement of galactinol and raffinose in the development of chilling stress in rice and indicate that the genetic modification of the biosynthesis of RFOs by transformation with GolS genes could be an effective method for enhancing chilling-stress tolerance in rice.     

Transformation of tobacco with an Arabidopsis thaliana gene involved in trehalose biosynthesis increases tolerance to several abiotic stresses

Summary Trehalose (a non-reducing disaccharide) plays an important role in abiotic stress protection. It has been shown that using trehalose synthesis genes of bacterial origin, drought and salt tolerance could be achieved in several plants. A cassette harboring the AtTPS1 gene under the control of the CaMV35S promoter and the Bialaphos resistance gene was inserted in the binary plasmid vector pGreen0229 and used for Agrobacterium-mediated transformation of tobacco (Nicotiana tabacum). T0 plants obtained were analyzed by PCR for the presence of AtTPS1 gene. Thirty lines were positive and seeds were germinated on media with 6 mg/l PPT to obtain T1 plants that were grown in the greenhouse to obtain T2 seeds that were germinated on selective media. Lines which seeds showed a 100 % survival rate were considered homozygous transgenic T1 lines. Three lines were selected and gene expression confirmed by northern and western blots. Transgenic seeds were germinated on media with different concentrations of mannitol (0, 0.25, 0.5 and 0.75 M) and sodium chloride (0, 0.07, 0.14, 0.2, 0.27 and 0.34 M) to score their tolerance to osmotic stress. Assays were conducted to test the tolerance of transgenic plants to drought (measurement of water percentage as a consequence of water withdrawal), desiccation (measurement of water loss as a consequence leaf detaching) and temperature stresses (germination at 15 ^∘C and 35^∘C). Transgenic tobacco plant lines registered higher germination rates under osmotic and temperature stress situations than did wild-type plants. Responses to drought and desiccation stresses were similar for all plant lines. It can hence be suggested that the heterologous expression of TPS1 gene from Arabidopsis can be used successfully to increase abiotic stress tolerance in model plants and probably in other crops.     

The frost tolerance of Miscanthus at the juvenile stage: Differences between clones are influenced by leaf-stage and acclimation

Miscanthus, a perennial, C₄ grass, has numerous advantages for biomass production, notably its high yield per hectare and low input requirements. However, establishment of this crop may be affected in Europe by frost damage when stem emergence occurs early in the year. The principal aim of this study was to quantify the impact of frost on young miscanthus shoots at different leaf-stages, and to characterise inter-species variations in frost tolerance. Four clones belonging to two species were tested at three leaf stages in a climate-controlled chamber simulating acclimation conditions and frost treatments. Frost tolerance was scored using a 0-6 visual assessment scale and analysed with nonparametric tests.We were thus able to show that more developed plants (6 or 7-leaf stage) were less frost tolerant than those at the 3 or 5-leaf stage. Plants at the 6 or 7-leaf stage also displayed differences in tolerance between clones. The leaf-stage of the plant is linked to apex height, and this appeared to play a role in frost tolerance. M. sinensis displayed variable frost tolerance (tolerance score of between 3.6 and 4.9), although the three clones observed were always more frost tolerant than M. x giganteus (with a score of 3). Moreover, the differences in frost tolerance were negatively correlated (r = −0.94) with the mean leaf surface area of clones at the time of frost exposure. Finally, we observed that acclimation at 12 °C under strong light intensity (600 μmol m⁻² s⁻¹) enabled an increase in the tolerance of young shoots in all the clones tested.     

Signatures of divergent selection for cold tolerance in maize

Divergently selected genotypes can be used for detecting the genomic regions affecting the selected trait (selection signature). Moreover, the genetic distances (GDs) among divergently selected lines can be correlated with the agronomic performances of the crosses among them. Using as source the maize F₂ of B73?×?IABO78, we previously conducted four cycles of divergent recurrent selection and three cycles of divergent selection in inbreeding for cold tolerance at germination. We finally obtained 10 lines selected for low (L) and 10 lines selected for high (H) cold tolerance, which exhibited a notable divergence for both the selected and associated traits. Herein, we investigated the 20 lines and the 28 single diallel crosses among eight random lines (four L and four H); the main objectives were to identify the putative regions controlling the selected and associated traits and to study the relationships between crosses performances and GDs among their parental lines. Allele frequencies at 932 recombination blocks based on 19,220 polymorphic SNPs were obtained for the two lines’ groups; the F _ST calculated across sliding windows indicated 18 regions highly divergent between groups. The increasing alleles for cold tolerance were contributed by both parents, consistently with the transgressive segregations previously found. Several regions associated to DG also affected various agronomic traits. The cross performances showed some relationships with the genetic distances among parental lines for traits affected by dominance, provided that all crosses were considered, while these relationships vanished when only L?×?H crosses were examined.     

Divergent recurrent selection for cold tolerance in two improved maize populations

Maize (Zea mays L.) production has significantly expanded into very short-season environments where germination and growth in cooler environments is essentially a pre-requisite. Therefore, an important goal for maize breeders is to improve local germplasm sources of inbred lines that are able to grow under these challenging conditions. The objective of this research was to evaluate direct and correlated responses in two improved early maturing maize populations [NDSCD(M)C10 and BS22(R)C7] after two cycles of S₁ and full-sib intra-population recurrent selection for cold tolerance. The S₁ and full-sib progenies were obtained by self-pollinating 100 random plants and by intercrossing 200 random plants, respectively. Ten percent of the families were selected, based on an index that included emergence percentage, seedling vigor, and root lodging percentage, and recombined at the same time in a summer nursery based upon data across northern North Dakota locations. The essential benefit of this breeding methodology was to achieve one year per cycle of selection based upon progenies. However, direct response to selection was not significant while some correlated responses were significant. We decided to report these results in order to encourage other scientists the evaluation of additional sources of germplasm, the screening at various dates, and the selection of target environments with more intensive cold stress before initiating long-term selection programs for cold tolerance. In addition, further research on the current and alternative long-term selection methods for cold tolerance is recommended for continuous genetic improvement of advanced cycles in the northern U.S. Corn Belt. Part of the thesis submitted by B. Sezegen in partial fulfillment of the requirements for a MS degree at North Dakota State University.