Genetic map of the DH ‘Hewo’ x ‘Magnat’ population with SSR and DArT markers
A set of 92 DH lines derived from F1 triticale plants that originated from a cross between cv. ‘Hewo’ and cv. ‘Magnat’ were used to create a new and unique genetic linkage map. Upon the multiple-mapping approaches, a total of 41 SSR and 680 diversity array technology (DArT) markers were ordered into 22 linkage groups assigned to the A, B, and R genomes (Table 1; Fig. S1 A, B, R; Table S1). Additionally, three chromosomes: 7A, 2B and 3B were represented by double linkage groups. All mapped DArT markers belonged to three groups rPt, tPt, and wPt, that were developed respectively from rye, triticale and wheat. Additionally, during the map construction a small number of markers were eliminated, mainly owing to a high percentage of missing data or a lack of linkage with established markers clusters at LOD value of 2.0. Finally, all remaining markers have covered a total of 1367.7 cM with a mean distance between two markers of 4.7 cM. Microsatellite markers allowed assignment of linkage groups to chromosomes of the A and B genomes of wheat and to five chromosomes of rye (1R, 3R, 4R, 5R, and 6R) (Table 1). Comparative analyses with triticale maps (Tyrka et al. 2011, 2015) and additional information on distribution of wheat DArT markers (series wPt) were provided by Diversity Arrays Technology Pty Ltd. and validated the assignment of linkage groups to rye chromosomes. The order of DArT and SSR markers that were used to develop this linkage map are presented in Supplementary Fig. S1 A, B and R and Supplementary Table S1. Details on the distribution of SSR and DArT markers across triticale genomes of Hewo x Magnat population showed the highest saturation of R genome with unique markers, whereas lower densities were identified for the A and B genomes. R genome was covered by 326 markers with total length of 290,4 cM and a mean distance between two unique markers of 2.7 cM, whereas 140 and 255 markers were distributed within genome A and B respectively, with total length of 460.8 and 616.5 cM and density 6.4 and 5.5 cM (Table 1; Table S1).
Freezing tolerance of plants cold acclimated under field conditions
From October to January of winter 2011/2012, plants of the Experiment 1 grew under an average temperature about +4 °C, mostly above 0 °C with few days decrease below (minimum in the average daily temperature to -6.3 °C in December) (Fig. 1A). The winter 2012/2013 was more severe. Plants of Experiment 2 grew under large fluctuations of the average temperature which was mostly above 0 °C in October and November, but below 0 °C in December and January, with minimum -12.3 °C (Fig. 1B). The period with an average temperature of approximately -2 °C started in December and continued until March, when temperature raised to about +2 °C just before the Experiment 3 measurements were performed (Fig. 1B).
The results of the freezing tolerance testing
The mean percentage of survived plants from three independent freezing test performed during winter 2011/2012 and 2012/2013 varied from 0 to 85% (Fig. 2). Transgressive segregation of plant recovery was observed in the evaluated population of DH lines. The smallest – 0% percentage of survived plants was observed for DH lines named HM DH 59 and the highest for DH lines - HM DH 22 and was equal 85%. Relatively high standard deviation for most DH lines clearly indicates that plants recovery strongly depend on weather conditions. Parent Hewo characterized of higher percentage of survived plants after freezing (56%) than parents named Magnat (48.5%).
PSII photochemical efficiency and membrane stability after freezing
Changes in photochemical efficiency under different stresses can be expressed by the chlorophyll a fluorescence parameters (Kurepin et al., 2013; Rapacz et al., 2004). In our study the photosynthesis efficiency during cold hardening of triticale plants was evaluated based on the maximum quantum yield of photosystem II (PSII) (Fv/Fm), the overall performance indexes of PSII (PI) and particular OJIP-test parameters which were recommended by Rapacz at al. (2015a) as a good tools to evaluate freezing tolerance (RC/CSm, RC/CS0, TR0/CS and ET0/CS). For all evaluated chlorophyll fluorescence parameters transgressive segregation was observed in our mapping population. The values of each parameters were different and depend on DH lines. Detailed information were described in Table 2. Relatively high standard deviation of chlorophyll fluorescence parameters for parents may result from the high impact of various weather conditions. In general, the photosynthetic apparatus for Hewo was more active and functioned better than that for Magnat, resulting in significantly higher ABS/CS (characterizing light energy absorption), RC/CSm (number of active reaction centers). Fv/Fm (maximum quantum yield of PSII) and PI (overall performance index of PSII photochemistry) also had trends for higher means in Hewo, though similar amounts of energy used for electron transport and amount of excitation energy trapped in PSII reaction centers (ET0/CS and TR0/CS, respectively) were for both parents. However, the dissipation of energy in PSII reaction centers (DI0/CS and DI0/RC) was higher for Magnat than Hewo.
The degree of damage to cell membranes under frost was determined by the measurement of electrolyte leakage (EL) from leaf tissues. The statistically significant differences between parents were not observed. The value of the electrolyte leakage for parental forms: Hewo and Magnat was 69.51% and 77.05%, respectively. The DH lines differed from values of electrolyte leakage. The transgression segregation was clearly highlighted: 65.2% of the DH line of the whole population was characterized by a lower statistically significant EL in relation to both Magnat and Hewo parental forms, while 10.9% of the DH line examined showed a statistically significant EL value in relation to both parent forms. The min and max values of EL was equal 18.98% and 90.02%, respectively (Table 2).
Correlation between plant recovery, electrolyte leakage and chlorophyll a fluorescence parameters
A relationship was established between the average percentage of survived plants from three independent experiments and the mean values of the electrolyte leakage and JIP parameters. For that purpose, the Pearson r correlation was calculated between the individual parameters (Table 3). There was no statistically significant correlation between plant recovery and electrolyte leakage. In contrast, a relatively high positive correlation (0.34 to 0.62) was found between plant recovery (REC) and JIP parameters: Fv/Fm, Ψ0, φE0, PI, PICSo, PICSm, ABS/CS, TR0/CS, ET0/CS, ET0/RC, RC/CS0, RC/CSm and negative statistically significant correlation (-0.32 to -0.35) for ABS/RC, DI0/RC, DI0/CS. The obtained statistical significance correlations confirmed the appropriateness of the photochemical analysis of the photosystem II activity selection and its applicability in breeding practice for the selection of frost resistant lines. EL was significantly negatively correlated with the individual chlorophyll a fluorescence parameters: Fv/Fm, Ψ0 and φE0. Positive correlation was statistically significant from 0.36 to 0.48 for the parameters: ABS/CS, DI0/CS, TR0/RC and DI0/RC.
Quantitative trait loci (QTL) analysis
QTL analysis identified several genomic regions involved in regulating plants recovery, chlorophyll fluorescence parameters and electrolyte leakage under three different winter conditions of cold acclimation (Fig. 1). Analysis performed with SMA and CIM method showed similar results for all traits, though more significant QTLs described in this paper were identified only by CIM calculations.
Quantitative trait loci (QTL) for plants recovery
Using CIM analysis, QTL regions for recovery (REC) expressed as percentage of survived plants were identified on chromosomes 7A.1, 1B, 2B.1, 4R and 5R (Table 4; Fig. 3). Additionally, all QTL regions identified by SMA method are shown in Table S2. The phenotypic variation explained by those QTLs ranged from 4.4 to 15.8 % depending on winter conditions and method used (Table 4; Table S2). The QTLs for percentage of survived plants evaluated in two experiments were co-located on chromosome 7A.1 and 1B.1 and named Qfr.hm-7A.1 and Qrec.hm-1B.1, respectively (Table 4; Fig. 3). Those loci covered 27.5-57.2 cM distance for Qfr.hm-7A.1 and explained up to 9.5 % of phenotypic variation. For second locus, Qrec.hm-1B.1 CIM analysis showed only one marker wPt-2725(4) in Experiment 2 and one marker wPt-5899(3) in Experiment 3 (Table 4; Fig. 3). Those loci explained up to 8.8 % of phenotypic variation with the LOD value 2.5 for Experiment 3 (Table 4).
Quantitative trait loci (QTL) for electrolyte leakage
Electrolyte leakage (EL) was measured two times during winter 2012/2013. QTLs controlling EL after freezing, identified by CIM method were on chromosomes 7A.1, 7A.2, 4R and 5R (Table 4; Fig. 3). Additionally, all QTL regions identified by SMA method are shown in Table S3. The phenotypic variation explained by those QTLs ranged from 3.3 to 22.8 % depending on Experiment and method used (Table 4; Table S3). The locus Qfr.hm-7A.1, which was identified as associated with EL in Experiment 3 was also co-located with QTL identified for plants recovery on chromosome 7A.1. Locus Qfr.hm-7A.1 explained 9.5 % of phenotypic variation observed for EL (Table 4) with LOD value 3.1 and was in similar position in cM to loci identified for plants recovery (Table 4). Another locus identified on chromosome 7A, Qel.hm-7A.2 was located in 8.9-19.9 cM distance and explained up to 13 % of phenotypic variation; it contained loci found in Experiment 2 and 3 (Table 4; Fig. 3). Locus Qel.hm-4R.1 located in 9.8-16.5 cM distance on chromosome 4R was also identified in Experiment 2 and 3 (Table 4; Fig. 3). It explained up to 22.8 % of phenotypic variation with the LOD value 6.5 in Experiment 2 (Table 4). One loci identified in Experiment 3 was located on chromosome 5R and covered by wmc289 to rPt-506350(10) markers (Table 4; Fig. 3). This loci explained 6.8 % of phenotypic variation (Table 4).
Quantitative trait loci (QTL) for chlorophyll a fluorescence parameters
QTLs for chlorophyll a fluorescence parameters identified by CIM method were located on chromosomes 4A, 5A, 5B, 4R and 5R (Table 5; Fig. 3). Additionally, QTLs identified by SMA method are shown in Tables S4 – S6. All identified QTLs contain loci identified for different chlorophyll a fluorescence parameters.
Locus Qchl.hm-4A.1 identified in a chromosome 4A 21.1 cM to 56.6 cM distance consisted QTLs for DI0/RC and ET0/RC and explained up to 10.6 % of phenotypic variation with the LOD value 2.5 (Table 5; Fig. 3). Locus Qchl.hm-5A.1 covered by wmc327.1 to wPt-1370(2) markers was identified for Fv/Fm, PI, ABS/CS, ET0/CS and TR0/CS in Experiment 2 and 3 (Table 5; Fig. 3). It explained up to 19.6 % of phenotypic variation with the LOD value up to 5.4 for TR0/CS (Table 5). On chromosome 5B, locus Qchl.hm-5B.1 was identified for PI, TR0/CS and ABS/CS with the LOD value up to 3.9 for ABS/CS (Table 5; Fig. 3). Locus Qchl.hm-4R.1 was identified on chromosome 4R for TR0/CS measured in Experiment 2 and ABS/CS for Experiment 2 and 3 (Table 5; Fig. 3). Loci identified for ABS/CS and TR0/CS measured in Experiment 2 were located in the same position (32.9 – 33.9 cM; Table 5). On chromosome 5R, locus Qchl.hm-5R.1 was identified for PI, TR0/CS and ABS/CS measured in Experiment 2 and TR0/CS measured in Experiment 1 (Table 5; Fig. 3). This QTL explained up to 10.8 % of phenotypic variation with LOD value up to 3.2 (Table 5).
Candidate genes for analyzed traits
Seven candidate genes were in silico identified within QTLs found for plants recovery (REC), electrolyte leakage (EL) as well as trapped energy flux in PSII reaction centers per leaf cross-section (Tr0/CS), the energy flux for electron transport per leaf cross-section (ET0/CS) and absorbed energy flux per leaf cross-section (ABS/CS) traits (Tab. 6). Gene coding PPR protein involved in chloroplast RNA processing, modification and splicing was identified as associated with plants recovery (Tab. 6). Genes identified as associated with electrolyte leakage encode mRNA-binding protein BTR1-like, involved in regulation of gene expression as well as transmembrane cyclic nucleotide-gated ion channel (Tab. 6). In turn, genes associated with chlorophyll a fluorescence parameters were chloroplastic uridine kinase-like protein 1 involved in the pyrimidine salvage pathway, uncharacterized LOC119301557 and phosphoinositide phosphatase SAC9 involved in stress signaling (Tab. 6).