Effect and Mechanism of Cold Tolerant Seed-Coating Agents on the Cold Tolerance of Early Indica Rice Seedlings

To better understand the effect and mechanism of cold tolerant seed-coating agents on the cold tolerance of rice seedlings, the physiological and biochemical effects of four cold tolerant seed-coating agents （HET, YKJ, YKZYJ, and the ABA seed coating agents） on two early indica rice varieties were studied under chilling stress. The results showed that the rice seedlings treated with cold tolerant seed-coating agents under chilling stress maintained dramatically higher root vigor, POD, CAT and SOD activities, and chlorophyll content, had lower MDA content and electrolyte leakage, and accumulated more soluble sugar and free proline, when compared with the control without the treatment, and finally showed lower plant injury rate. It was indicated that the cold tolerant seed coating agent improved the ability of rice seedlings in resisting to chilling stress. YKZYJ was ranked the first in terms of the efficiency in cold tolerance among the four cold tolerant seed-coating agents tested.

Differential Response of Grain Quality to Cold Water Irrigation in Cold Tolerant and Sensitive Lines of Rice

Three rice varieties and several F3 lines with high and low cold tolerance, selected from F3 segregation lines of two crossesMilyang 23/Tong 88-7 and Milyang 23/TR22183 were used to analyze the effects of cold tolerance on the response of grainquality to cold water irrigation. The result showed that cold water irrigation led to the decrease of rice grain size. Thelength, length-width ratio and weight of brown rice grain were more sensitively affected by cold water irrigation than thewidth and thickness. The shape of brown rice grain was not significantly affected by the selection for cold tolerance at theseedling stage (CTS). The gel consistency, amylose content, peak viscosity, cool viscosity, breakdown viscosity andconsistency viscosity were decreased, while alkali digestibility value and protein content were increased by cold waterirrigation. Under normal irrigation condition the physicochemical properties of milled rice and viscogram components ofmilled rice flour were not significantly different between lines with high and low cold tolerance. Under cold water irrigationthe amylose content, peak viscosity, hot viscosity, final viscosity of rice lines with high CTS or high cold tolerance at thebooting stage (CTB) were higher, while the protein content, setback viscosity, breakdown ratio and setback ratio werelower, than those of rice lines with low cold tolerance. This implied that the cold water response of rice grain quality wasless sensitive in the lines with high cold tolerance than in the lines with low cold tolerance, and the varietal improvementfor cold tolerance would be important for grain quality improvement at the same time.

Heterogeneous expression of stearoyl-acyl carrier protein desaturase genes SAD1 and SAD2 from Linum usitatissimum enhances seed oleic acid accumulation and seedling cold and drought tolerance in Brassica napus

Flax(Linum usitatissimum L.)is a versatile crop and its seeds are a major source of unsaturated fatty acids.Stearoyl-acyl carrier protein desaturase(SAD)is a dehydrogenase enzyme that plays a key role in oleic acid biosynthesis as well as responses to biotic and abiotic stresses.However,the function of SAD orthologs from L.usitatissimum has not been assessed.Here,we found that two LuSAD genes,LuSAD1 and LuSAD2,are present in the genome of L.usitatissimum cultivar‘Longya 10’.Heterogeneous expression of either LuSAD1 or LuSAD2 in Arabidopsis thaliana resulted in higher contents of total fatty acids and oleic acid in the seeds.Interestingly,ectopic expression of LuSAD2 in A.thaliana caused altered plant architecture.Similarly,the overexpression of either LuSAD1 or LuSAD2 in Brassica napus also resulted in increased contents of total fatty acids and oleic acid in the seeds.Furthermore,we demonstrated that either LuSAD1 or LuSAD2 enhances seedling resistance to cold and drought stresses by improving antioxidant enzyme activity and nonenzymatic antioxidant levels,as well as reducing membrane damage.These findings not only broaden our knowledge of the LuSAD functions in plants,but also offer promising targets for improving the quantity and quality of oil,and the abiotic stress tolerance of oil-producing crops,through molecular manipulation.

Transcription factor CabHLH035 promotes cold resistance and homeostasis of reactive oxygen species in pepper

Plant basic helix-loop-helix(bHLH)transcription factors(TFs)play central roles in various abiotic stresses.However,its role in plant cold resistance is largely unknown.Previously,we characterised CaNAC035 in pepper,which positively regulates tolerance to cold,salt and drought stresses tolerance.Here,we identified CabHLH035,a CaNAC035-interacting protein in pepper.To explore its functions in cold stress tolerance,we silenced the gene in pepper via virus-induced gene silencing(VIGS)and overexpressed the gene in Arabidopsis.The results showed that CabHLH035 expression was induced by cold treatment,and silencing of CabHLH035 decreased cold stress tolerance.Conversely,overexpression of CabHLH035 in Arabidopsis increased cold stress tolerance.To investigate homologs genes of C-repeat binding factor(CBF)pathway proteins and reactive oxygen species(ROS)marker gene expression blocking by CabHLH035,we performed yeast one-hybrid(Y1H),dual luciferase and electrophoretic mobility shift assay experiments.The results showed that CabHLH035 bound to the region upstream of the CaCBF1A and CaAPX promoters.Additionally,CaCBF1A bound to the CaDHN4 promoter.Taken together,our results showed that CabHLH035 plays a crucial role in cold stress tolerance and its potential as a target for breeding cold-resistant crops.The findings provide a basis for studying the functions and regulatory network of cold stress tolerance in pepper.

The UDP-glycosyltransferase OsUGT706D2 positively regulates cold and submergence stress tolerance in rice

In a genome-wide association study,we identified a rice UDP-glycosyltransferase gene,OsUGT706D2,whose transcription was activated in response to cold and submergence stress and to exogenous abscisic acid(ABA).OsUGT706D2 positively regulated the biosynthesis of tricin-4’-O-(syringyl alcohol)ether-7-O-glucoside at both the transcriptional and metabolic levels.OsUGT706D2 mediated cold and submergence tolerance by modulating the expression of stress-responsive genes as well as the abscisic acid(ABA)signaling pathway.Gain of function of OsUGT706D2 increased cold and submergence tolerance and loss of function of OsUGT706D2 reduced cold tolerance.ABA positively regulated OsUGT706D2-mediated cold tolerance but reduced submergence tolerance.These findings suggest the potential use of OsUGT706D2 for improving abiotic stress tolerance in rice.

MIR1868 negatively regulates rice cold tolerance at both the seedling and booting stages

Low temperature causes rice yield losses of up to 30%–40%,therefore increasing its cold tolerance is a breeding target.Few genes in rice are reported to confer cold tolerance at both the vegetative and reproductive stages.This study revealed a rice-specific 24-nt miRNA,miR1868,whose accumulation was suppressed by cold stress.Knockdown of MIR1868 increased seedling survival,pollen fertility,seed setting,and grain yield under cold stress,whereas its overexpression conferred the opposite phenotype.Knockdown of MIR1868 increased reactive oxygen species(ROS)scavenging and soluble sugar content under cold stress by increasing the expression of peroxidase genes and sugar metabolism genes,and its overexpression produced the opposite effect.Thus,MIR1868 negatively regulated rice cold tolerance via ROS scavenging and sugar accumulation.

Effects of Silicon Formulations on Cold Tolerance of Rice Seedlings

To investigate the effects of silicon formulations on the cold tolerance of rice seedlings,Song Japonica 16(not cold tolerant)and Dongnong 427(cold tolerant)rice varieties were used as test materials and four different types of silicon formulations,Si-50-G,Si-60-G,Si-T-G,and Si-E-G,were applied as foliar sprays at the seedling stage,and a control group CK(equal amount of distilled water)was set up.One week after the first silicon spray,two types of rice were subjected to low-temperature stress treatments at day/night temperatures of 12℃/10℃for 2,4,6,and 8 days.The effects of different silicon formulations on the chlorophyll,proline(Pro)and soluble sugar contents as well as superoxide dismutase(SOD),peroxidase(POD)and catalase(CAT)activities of rice seedlings under low-temperature stress were compared to find out the effects of silicon formulations on the cold tolerance of rice seedlings.The results showed that silicon formulations could significantly increase the chlorophyll content of rice seedling leaves,with Si-50-G being the most effective,with a significant increment of 40.17%compared to the CK at 2 days of low temperature.Four silicon formulations significantly increased the proline content and soluble sugar content of rice leaves at low temperature for 4-8 days.For Song Japonica 16,the most significant increment in leaf POD activity was observed in Si-E-G treatment at 2,4 and 8 days of temperature stress,with 73.58%,20.95%and 217.24%increases compared to the CK,respectively.For 4 and 6 days of temperature stress,the most significant increase in CAT activity was observed in Si-E-G treatment,with 25.70%and 75.78%increases compared to the CK,respectively.For Dongnong 427,the Si-60-G treatment showed the highest increase in leaf SOD activity for 4 and 8 days of temperature stress,with significant increases of 58.15%and 82.76%compared to the CK,respectively,and the Si-E-G treatment showed the highest increase in leaf POD activity for 2 and 8 days of temperature stress,with significant increases of 97.75%and 245.10%compared to the CK,respectively.It showed that the spraying of silicon formulations could significantly enhance the cold tolerance of rice.This study provided a scientific basis for the rational use of silicon formulations to enhance cold tolerance in rice and had important theoretical and practical significance for ensuring sustainable high and stable rice yields in Heilongjiang Province,as well as for the development of silicon fertilizers.

Selective footprints and genes relevant to cold adaptation and other phenotypic traits are unscrambled in the genomes of divergently selected chicken breeds

Background The genomes of worldwide poultry breeds divergently selected for performance and other phenotypic traits may also be affected by,and formed due to,past and current admixture events.Adaptation to diverse environments,including acclimation to harsh climatic conditions,has also left selection footprints in breed genomes.Results Using the Chicken 50K_CobbCons SNP chip,we genotyped four divergently selected breeds:two aboriginal,cold tolerant Ushanka and Orloff Mille Fleur,one egg-type Russian White subjected to artificial selection for cold tolerance,and one meat-type White Cornish.Signals of selective sweeps were determined in the studied breeds using three methods:(1)assessment of runs of homozygosity islands,(2)F_(ST) based population differential analysis,and(3)haplotype differentiation analysis.Genomic regions of true selection signatures were identified by two or more methods or in two or more breeds.In these regions,we detected 540 prioritized candidate genes supplemented them with those that occurred in one breed using one statistic and were suggested in other studies.Amongst them,SOX5,ME3,ZNF536,WWP1,RIPK2,OSGIN2,DECR1,TPO,PPARGC1A,BDNF,MSTN,and beta-keratin genes can be especially mentioned as candidates for cold adaptation.Epigenetic factors may be involved in regulating some of these important genes(e.g.,TPO and BDNF).Conclusion Based on a genome-wide scan,our findings can help dissect the genetic architecture underlying various phenotypic traits in chicken breeds.These include genes representing the sine qua non for adaptation to harsh environments.Cold tolerance in acclimated chicken breeds may be developed following one of few specific gene expression mechanisms or more than one overlapping response known in cold-exposed individuals,and this warrants further investigation.

More than a simple egg:Underlying mechanisms of cold tolerance in avian embryos

Avian embryos,which develop within eggs,exhibit remarkable tolerance to extremely low temperatures.Despite being a common trait among all birds,the mechanisms underlying this cold tolerance in avian embryos remain largely unknown.To gain a better understanding of this phenomenon and the coping mechanisms involved,we reviewed the literature on severe cold tolerance in embryos of both wild and domestic birds.We found that embryos of different bird orders exhibit tolerance to severe cold during their development.In response to cold stress,embryos slow down their heartbeat rates and metabolism.In severe cold temperatures,embryos can suspend these processes,entering a torpid-like state of cardiac arrest.To compensate for these developmental delays,embryos extend their regular incubation periods.Depending on their embryonic age,embryos of all bird species can tolerate acute severe cold regimes;only a few tolerate chronic severe cold regimes.We also discussed various extrinsic and intrinsic factors that affect the tolerance of bird embryos to low temperatures before and after incubation.Cold tolerance appears to be a heritable trait shared by wild and domestic embryos of all bird classes,regardless of egg size or development(altricial/precocial).Driven by environmental variability,cold tolerance in avian embryos is an optimal physiological and ecological strategy to mitigate the adverse effects of cold conditions on their development in response to fluctuating environmental temperatures.

Development of a New Cold-Tolerant Maize(Zea mays L.)Germplasm Using the ICE1 Gene from Arabidopsis thaliana

To develop cold-tolerant maize germplasms and identify the activation of INDUCER OF CRT/DRE-BINDING FACTOR EXPRESSION(ICE1)expression in response to cold stress,RT-PCR was used to amplify the complete open reading frame sequence of the ICE1 gene and construct the plant expression vector pCAMBIA3301-ICE1-Bar.Immature maize embryos and calli were transformed with the recombinant vector using Agrobacterium tumefaciens-mediated transformations.From the regenerated plantlets,three T1 lines were screened and identi-fied by PCR.A Southern blot analysis showed that a single copy of the ICE1 gene was integrated into the maize(Zea mays L.)genomes of the three T1 generations.Under low temperature-stress conditions(4°C),the relative conductivity levels decreased by 27.51%–31.44%,the proline concentrations increased by 12.50%–17.50%,the malondialdehyde concentrations decreased by 16.78%–18.37%,and the peroxidase activities increased by 19.60%–22.89%in the T1 lines compared with those of the control.A real-time quantitative PCR analysis showed that the ICE1 gene was ectopically expressed in the roots,stems,and leaves of the T1 lines.ICE1 positively regulates the expression of the CBF genes in response to cold stress.Thus,this study showed the successful transformation of maize with the ICE1 gene,resulting in the generation of a new maize germplasm that had increased tolerance to cold stress.

Elevation difference of Ca^(2+) levels in young leaf cells of bromegrass and induced cold-tolerant enhancement under different controlled chilling temperatures

The changes of Ca2＋ levels in young leaf cells of bromegrass under different controlled chilling temperatures were inves- tigated by an antimonite precipitation cytochemical method. The main results were as follows： under 25/20℃ （day/night） tempera- ture and 14 h photoperiod, electron-dense Ca2＋ antimonite precipitates, indicators of Ca2＋ localization, were mainly localized in the vacuoles, cell walls and intercellular spaces; few Ca2＋ deposits were observed in the cytosol and nuclei. After a 3℃ chilling treatment for 3 h, many Ca2＋ precipitates appeared in the cytosol and nuclei, indicating that Ca2＋ influx had occurred in the cytosol and nuclei. When the 3℃ treatment was prolonged to 8 h, more Ca2＋ deposits appeared in the nuclei and cytosol, but the amount of Ca2＋ deposits in both the cytosol and nuclei decreased markedly after a 24 h treatment and most Ca2＋ deposits were returned to the vacuoles and intercellular spaces after an 8 d treatment. When bromegrass was exposed to 7℃ for 3 h, the Ca2＋ distribution in the cells had no visible changes, compared with that of the 25/20℃ grown control plants. However, when the chilling treatment of 7℃ was prolonged to 8 h, a Ca2＋ influx occurred, where many Ca2＋ deposits were observed in the nuclei and cytosol. More Ca2＋ deposits appeared in the nuclei and cytosol after a 24 h treatment, but the amount of Ca2＋ deposits in the cytosol and nuclei was reduced markedly after an 8 d treatment. After a 14 d treatment, the remaining low level of Ca2＋ was recovered in both the cytosol and nuclei and the Ca2＋ deposits were again located in the vacuoles and the intercellular spaces. The dynamics of subcellular Ca2＋ localization in young leaf cells of bromegrass during a 12℃ chilling treatment were similar to those of the 7℃ treatment. Besides, the results showed that the frost tolerance ofbromegrass exposed to 3℃ for 8 d increased by 6℃, for 7℃ and 8 d by 4℃ and for 12℃ and 14 d by 3℃, compared with the controls. Finally, the relationship between different Ca2＋ dynamics and induced frost tolerance was also explored.

MdWRKY40is directly promotes anthocyanin accumulation and blocks MdMYB15L, the repressor of MdCBF2, which improves cold tolerance in apple

Cold stress is an important factor that limits apple production. In this study, we examined the tissue-cultured plantlets of apple rootstocks ‘M9T337' and ‘60-160', which are resistant and sensitive to cold stress, respectively. The enriched pathways of differentially expressed genes(DEGs) and physiological changes in ‘M9T337' and ‘60-160' plantlets were clearly different after cold stress(1°C) treatment for 48 h, suggesting that they have differential responses to cold stress. The differential expression of WRKY transcription factors in the two plantlets showed that MdWRKY40is and MdWRKY48 are potential regulators of cold tolerance. When we overexpressed MdWRKY40is and MdWRKY48in apple calli, the overexpression of MdWRKY48 had no significant effect on the callus, while MdWRKY40is overexpression promoted anthocyanin accumulation, increased callus cold tolerance, and promoted the expression of anthocyanin structural gene MdDFR and cold-signaling core gene MdCBF2. Yeast one-hybrid screening and electrophoretic mobility shift assays showed that MdWRKY40is could only bind to the MdDFR promoter. Yeast twohybrid screening and bimolecular fluorescence complementation showed that MdWRKY40is interacts with the CBF2inhibitor MdMYB15L through the leucine zipper(LZ). When the LZ of MdWRMY40is was knocked out, MdWRKY40is overexpression in the callus did not affect MdCBF2 expression or callus cold tolerance, indicating that MdWRKY40is acts in the cold signaling pathway by interacting with MdMYB15L. In summary, MdWRKY40is can directly bind to the MdDFR promoter in order to promote anthocyanin accumulation, and it can also interact with MdMYB15L to interfere with its inhibitory effect on MdCBF2, indirectly promoting MdCBF2 expression, and thereby improving cold tolerance.These results provide a new perspective for the cold-resistance mechanism of apple rootstocks and a molecular basis for the screening of cold-resistant rootstocks.

A systematic identification of cold tolerance genes in peanut using yeast functional screening system

Peanut(Arachis hypogaea L.)is a thermophilic crop,and low temperature leads to a significant reduction in annual yields.Despite a few cold tolerant germplasms or cultivars have been discovered and developed,molecular mechanisms governing peanut cold tolerance is poorly understood.Identification of keys genes involved in cold tolerance is the first step to address the underlying mechanism.In this study,we isolated and characterized 157 genes with potentials to confer cold tolerance in peanut by using a yeast functional screening system.GO(Gene ontology)and KEGG(Kyoto encyclopedia of genes and genomes)enrichment analysis of these genes revealed that ribosome and photosynthesis proteins might play essential roles in peanut cold response.Transcriptome results indicated that 60 cold tolerance candidate genes were significantly induced or depressed by low temperature.qRT-PCR analysis demonstrated that several candidate genes could be also regulated by salt or drought stress.Individual overexpression of two UDP-glycosyltransferases(AhUGT2 and AhUGT268)in transgenic yeast cells could enhance their tolerance to multiple abiotic stress.In conclusion,this study advances our understanding of the mechanisms associated with the cold stress responses in peanut,and offers valuable gene resources for genetic improvement of abiotic stress tolerance in crops.

Effect of Rapid Cold Hardening on the Cold Tolerance of the Larvae of the Rice Stem Borer,Chilo suppressalis(Walker)

The effects of rapid cold hardening （RCH） on the cold tolerance of the last instar larvae of Chilo suppressalis （Walker） were evaluated for the first time. The discriminating temperature, induction, detection, duration and extent of RCH of the larvae in the laboratory were tested, and the supercooling points （SCPs） and the contents of water and lipid of the larvae after RCH treatment were determined, respectively. The results showed that the discriminating temperature of the larvae was about -21℃. Mean survival rates of the larvae which exposed to either 0 or 5℃ for 2 and 4 h before exposure to the discriminating temperature for 2 h were significantly higher than those of the control groups （P 〈 0.05）. Moreover, the highest survival rate appeared in the larvae after 0℃ for 4 h treatment. The protection against low temperature gained by RCH at 0℃ for 4 h was rapidly lost on return to 28℃. Mean survival rates of RCH larvae were significantly higher than those of non-acclimated （NACC） larvae and acclimation （ACC） larvae when they were exposed to the discriminating temperature for 2 or 4 h （P〈 0.05）. Moreover, the rates of NACC, ACC, RCH and ACC ＋ RCH larvae from 2 to 6 h to the discriminating temperature resulted in a significant decline. The values of SCPs and the contents of lipid of the larvae which exposed to either 0 or 5℃ for 2 and 4 h showed no significant difference at 0.05 level compared to those of the control groups. But the contents of water in the larvae were obviously decreased. Therefore, it could be concluded that RCH could enhance cold tolerance and affect partly physiological and biochemical components of the larvae of C. suppressalis, but the underlying mechanisms needs to be further explored.

Genetic Analysis of Cold Tolerance at Seedling Stage and Heat Tolerance at Anthesis in Rice (Oryza sativa L.)

A set of 240 introgression lines derived from the advanced backcross population of a cross between a japonica cultivar,Xiushui 09,and an indica breeding line,IR2061,was developed to dissect QTLs affecting cold tolerance （CT） at seedling stage and heat tolerance （HT） at anthesis.Survival rate of seedlings （SRS） and spikelet fertility （SF）,the index traits of CT and HT,showed significant differences between the two parents under stresses.A total of four QTLs （qSRS1,qSRS7,qSRS11a and qSRS11b） for CT were identified on chromosomes 1,7,11,and the Xiushui 09 alleles increased SRS at all loci except qSRS7.Four QTLs for SF were identified on chromosomes 4,5,6,and 11.These QTLs could be classified into two major types based on their behaviors under normal and stress conditions.The first was QTL expressed only under normal condition;and the second QTL was apparently stress induced and only expressed under stress.Among them,two QTLs （qSF4 and qSF6） which reduced the trait difference between heat stress and normal conditions must have contributed to HT because of their obvious contribution to trait stability,and the IR2061 allele at the qSF6 and the Xiushui 09 allele at the qSF4 improved HT,respectively.No similar QTL was found between CT at seedling stage and HT at anthesis.Therefore,it is possible to breed a new variety with CT and HT by pyramiding the favorable CT-and HT-improved alleles at above loci from Xiushui 09 and IR2061,respectively,through marker-assisted selection （MAS）.

Genetic parameters for cold tolerance and body weight of Chinese fleshy prawn,Fenneropenaeus chinensis

The inability of Fenneropenaeus chinensis to tolerate low temperatures is of major economic concern in temperate climates,as it reduces their growing season and leads to over-winter mortality.In this study,the heritability of body weight under low grow-out temperature and cold tolerance in F.chinensis were first investigated and estimated using 88 ful-sib families,which might provide crucial information in Chinese fleshy prawn breeding programs.The heritability for body weight under suitable and low temperature of F.chinensis were both moderate（0.158 0±0.307 5 and 0.132 0±0.026 9 respectively）;the large coefficient of variation（approximately 21%） and moderate estimate of heritability for body weight indicated substantial potential for selective breeding.The heritability estimate for cold tolerance was low（0.019 2±0.023 5）,and showed no significant differences from zero（P〉0.05）.A weak genetic correlation between cold tolerance and body weight was also estimated in the present study,also showing no significant differences from zero（P〉0.05）.Thus,more research needs to be conducted on the more accurate heritability estimate of cold tolerance and genetic correlations between traits in F.chinensis to further improve the achievement of breeding goals.

Identification of Cold Tolerance of Rice Germplasm Resource at Germinating Stage in Cold Region

Total 75 rice varieties （lines） in Heilongjiang Province （or cold region） as germplasm resources were identified for cold tolerance at germinating stage by controlling temperature in artificial incubator. The results showed that the shooting seed rate at the germinating stage could be used as the evaluation index of cold tolerance. The cold tolerance was recorded on 1-9 scale and could be identified by the criteria of five indexes such as highly tolerant （HT）, tolerant （T）, moderately tolerant （MT）, susceptible （S）, highly susceptible （HS）.

Identification of QTLs for Rice Cold Tolerance at Plumule and 3-Leaf-Seedling Stages by Using QTLNetwork Software

A doubled haploid （DH） population consisted of 120 lines, derived from a cross between an indica vadety, TN1, and a japonica variety, Chunjiang 06, was used to identify QTLs controlling rice cold tolerance at the plumule and 3-leaf-seedling stages by using the QTLNetwork software. The percentages of normal plumules after 4~C treatments for 7 d, 9 d, 11 d, and 14 d, as well as the cold stress tolerance index （CSTI） and the withering index （WI） of rice seedling were investigated. A total of five single-effect QTLs, each for percentages of normal plumules after 4℃treatments for 9 d, 11 d and 14 d, and CSTI and WI, respectively were identified. The QTLs for the percentages of normal plumules after low temperature treatments for g d, 11 d and 14 d were on chromosomes 4, 2 and 11, accounting for 14.1%, 17.3% and 21.5% of the phenotypic variation, respectively. QTLs for CSTI and WI were on chromosomes 10 and 1, respectively. Two pairs of epistatic loci were identified, but none of the epistatic loci involved the single-effect QTLs. The RM528-RM340 interval on chromosome 6 interacted with the RM278-RM3919B interval on chromosome 9 for CSTI, and the epistatic interaction accounted for 17.7% of the phenotypic variation. A pair of epistatic loci was identified for WI, the RM246-RM5461 interval on chromosome 1 interacted with the ISA-RM447 interval on chromosome 8, which accounted for 22.6% of the phenotypic variation.