Quantitative Trait Loci (QTL) Associated With Leaf Development in Winter Triticale (× Triticosecale Wittmack) Seedlings

The vitality and the development in the seedling stage is crucial in winter cereal’s life cycle, especially before and during winter. It has been reported that rapid seedling establishment and early growth may lead to higher crop yield. Localization of cereal genome regions is not often analysed in the seedling stage. The aim of this study was to identify winter triticale genome regions (QTL) associated with seedling leaves development. Based on ‘Hewo’ x ‘Magnat’ DH lines population genetic map composed of 3539 molecular markers assigned to 20 linkage groups with 4997.4 cM map length, in total 40 loci were identied by a composite interval mapping (CIM). Among them, 22 loci appeared in at least two experiments, were common for all analyzed traits as well as were identied on wheat chromosome 4B and on rye chromosomes 1R, 4R, 5R and 6R. Those loci explained up to 21.7% of phenotypic variation (Qwsl.hm.4R.2) and had LOD value up to 31.1 (Qlsl.hm.5R.1). The results of QTLs of seedling leaves development could be successively associated with QTLs of the freezing and fungal infection seedlings tolerance identied in this mapping population.


Introduction
Triticale (x Triticosecale Wittmack) is a man-made cereal developed by crossing wheat (Triticum aestivum L.) and rye (Secale cereale L.) with a genomic constitution of 2n = 6x = 42 ( Plant development is a process controlled by plant genetic information together with environment conditions e.g. light, temperature, water and soil (Von Arnim and Deng 1996; Hochholdinger et al. 2004; Chen et al. 2008). The cereal seedling development is controlled by metabolite and hormonal signals which participate in gene expression, growth, and metabolism (Thomas and Rodriguez, 1994). This process is very crucial in cereal life cycle because the health of seedlings affects the size and quality of the grain (Stratonovitch and Semenov 2015; Maulana et al. 2018). In winter cereals, the low temperature period during seedling development (hardening) provides to increasing crop's low-temperature and fungal pathogens tolerance (Tronsmo et al. 2001; Hudec and Bokor 2002). Also, our previous studies on triticale, especially 'Hewo' x 'Magnat' DH population showed that plant's hardening leads to increased tolerance to Microdochium nivale infection (Gołębiowska and Wędzony, 2009;Gołębiowska et al. 2011;Szechyńska-Hebda et al. 2011, 2013Dyda et al. 2019). The studies on the genetic control of morphological and yieldrelated traits in cereals have been mainly focused on adult plants of wheat, barley, maize and rice (Peng et

Plant growth conditions and experiment design
Analysis of seedlings morphological traits were performed in two-years' time period in controlled conditions in completely randomized blocks design (CRBD). Analysis were divided in three series of independent experiments. Experiments 1 and 2 (Exp. 1, Exp. 2) were performed on cold-hardened 7-weeks old seedlings. Expriment 3 (Exp. 3) was performed on 4-weeks old unhardened seedlings. In all experiments, plants were cultivated as described in Gołębiowska and Wędzony (2009) with detailed modi cations. All DH lines, together with parental cultivars, were grown in multi-pots in an isolated chamber with 67% of humidity. Three replicates were performed in a randomized complete block design in order to limit the error resulting from the marginal position of an individual genotype. Kernels were sawn in 10 rows of 6 kernels/ genotype/ box. One row per each parental line was sawn in variable positions in every box as a control.
In Experiments 1 (1st year) and 2 (2nd year), plants were grown in temperature 21°C/16°C, at 8h/16h (day/night) photoperiod for one week and were watered and supplemented with Hoagland and Arnon's (1938) sterile medium. Then seedlings were pre-hardened in 12°C/12°C, at 8h/16h (day/night) photoperiod for next 14 days and subsequently hardened in 4°C/4°C, at 8h/16h (day/night) photoperiod for the next 28 days. In the Experiment 3 (2nd year), plants were grown in 21°C/16°C, at 8h/16h (day/night) photoperiod for three weeks then were transferred into a greenhouse for one week and watered and supplemented with Hoagland and Arnon's (1938) sterile medium.

Statistical analysis and QTL mapping
All data were analyzed using Statistica 13.0 PL software (Statsoft, Tulsa, OK, USA). Distribution of the data was checked using histograms and analyzed with a Shapiro-Wilk test, together with skewness and kurtosis. Additionally, one-dimensional variance analysis was performed at p < 0.05. Linear correlation coe cients (Pearson's) were calculated for each of three experiments separately on the basis of mean value of replicates. The regression line was presented with a 95% coe cient interval.
QTL regions were identi ed with Windows QTLCartographer 2.5 software according to Wang et al. (2012) using composite interval mapping (CIM) with signi cance at the p = 0.05 level and 1000 permutations with LOD threshold ≥ 3.0. The percentage of the phenotypic variation covered by each QTL was calculated with a single factor regression (R 2 ). Favourable alleles in each QTL region were selected on the basis of the additive effect (Add): negative additive effect referred to cv. 'Magnat'; positive additive effect referred to cv. 'Hewo'. Results of QTL analysis were visualized using CorelDRAW9 software and the label of each QTL was created from the short name of each morphological trait; Hewo × Magnat (hm), chromosome names (wheat A and B group, rye group R) and QTL number on the chromosome (1-3).

Results
The cv. 'Hewo' seedlings had an upright habit, while cv. 'Magnat' seedlings -lying, with leaves spread close to the soil surface. Additionally, the dimensions of the leaves differed between parental cultivars at the same 7-weeks old cold-hardened seedlings stage (Fig. 1S). The rst leaf was on average 69 mm longer and 0.94 mm narrower in cv. 'Hewo' than in cv. 'Magnat' seedlings (p < 0.05). The second leaf sheath and the lamina were on average 7.9 mm and 55.5 mm longer in 'Hewo' than in 'Magnat' (p < 0.05) seedlings. The second leaf blade was almost 1/3 narrower in 'Hewo' than in 'Magnat' (p < 0.05) seedlings.
All independent factors (plant genotype and experiment number) as well as the interaction between them had signi cant in uence on the studied traits (p < 0.05). For that reason QTLs were calculated for the data of each experiment separately. The Shapiro-Wilk test as well as skewness and kurtosis results con rmed the normal distribution of values for every experiment and each trait (Table 1), what allowed to perform further QTL analysis. Table 1 The values range of the analyzed traits: number of leaves (NL), length of rst (LFL) and second (LSL) leaf, width of rst (WFL) and second (WSL) leaf, second leaf sheath length (LSSL), second leaf blade length (LSLB) and fresh mass of leaves (LFM). Experimental mean value, together with standard deviation as well as the results of the Shapiro-Wilk test, skewness and kurtosis were presented. QTL analyses revealed 22 loci identi ed by a composite interval mapping (CIM) with LOD value ≥ 3.0 which appeared in at least two experiments with range common for all analyzed traits (in cM). They included: 2 loci for the rst leaf length (LFL), 3 loci for the rst leaf width (WFL), 5 loci for the second leaf length (LSL), 3 loci for the second leaf width (WSL), 4 loci for the second leaf sheath length (LSSL) and 5 loci for second leaf blade length (LSLB) ( Table 3). Additionally, 18 loci of QTL regions with LOD value ≥ 3.0 are presented for all analyzed traits (Table 1S)

First leaf length (LFL)
The rst leaf length ranged from 8.5 cm (Exp. 1) to 16.9 cm (Exp. 3). The longest rst leaf was observed in unhardened, 4-weeks-old seedlings (Table 1). Five QTL regions were found for this trait (

Second leaf width (WSL)
The mean value of second leaf width was similar in Exp. 2 and 3 (4.9 cm and 4.6 cm, accordingly) and higher in Exp. 1 (5.7 cm, Table 1). Five loci were identi ed but only three were considered as common for all other traits; they were found on rye chromosomes 4R and 6R (Table 3, Table 1S). Loci speci ed for WSL were identi ed on wheat chromosome 1A as well as rye chromosome 3R (Table 1S) Table 3). Positive allele effect of all those loci referred to cv. 'Hewo'. The highest explained phenotypic variation (12.2%) was found for Qlslb.hm.6R.2 ( Table 3).
Comparison of common QTL regions 22 out of total 40 loci identi ed in at least two experiments were common for all analyzed traits (Table 3). Those loci were identi ed on wheat chromosome 4B and on rye chromosomes 1R, 4R, 5R and 6R (Table 3).
On chromosome 4B, two loci were found for LSL and LSLB in Exp. 2 -Qlsl.hm.4B.1 and Qlslb.hm.4B.1, respectively (Table 3). Between those traits high correlation (0.992) was also observed ( Table 2). Those QTL regions were in the same position on this chromosome, from 0.0 cM to 17.6 cM. In both regions, the CIM peak showed two markers and one of them, 3044038 was common for both QTL regions. The maximum LOD position was 5.6 and 5.7 for Qlslb.hm.4B.1 (  Table 3). The correlation between WFL and WSL had high (0.689), and between WFL and LFL medium (0.428) value ( Table 2). The highest LOD value was observed for Ql .hm.4R.1 (4.4) and Qw .hm.4R.2 (5.8). Negative allele effect of those loci referred to cv. 'Magnat' ( Table 3). All of those loci explained 12.5% − 21.7% of phenotypic variation. For Qw .hm.4R.1 and Qwsl.hm.4R.1 maximum LOD peak was for two markers located near to each other on chromosome 4R (Table 3).

Discussion
The vitality and development in the seedling stage is very important in plant's life cycle, especially for winter cereals. It has been reported that rapid seedling establishment and early growth are important traits for improving yield (Von Arnim and Deng 1996, Aparicio et al. 2002). Localization of QTL regions in the cereal seedling stage is not often reported yet. In our study, analysed traits were selected on the basis of their potential role in seedlings ability to overwinter as well as potential use as an indicator of tillering and future yield early evaluation. According to our knowledge, this is the rst research which describes localization of triticale genomic regions associated with leaf development in the seedling stage.  Table 3, Tab. S1, Fig. 1). Among those 40 loci, we selected 22 loci on chromosomes 4B, 1R, 4R, 5R and 6R which were common for all analyzed traits (Table 3, Fig. 1).
On wheat chromosome 4B two loci Qlsl.hm.4B.1 and Qlslb.hm.4B.1 for second leaf length and second leaf blade length were identi ed, respectively. Both loci were on the same position on this chromosome ( Table 3 In present research, most of QTLs common for all analysed traits were located on rye chromosomes 1R, 4R, 5R and 6R (Table 3, Fig. 1 Fig. 1). All of those loci were characterized with very high LOD value, especially for Qlsl.hm.5R.1 (Table 3). Chromosome 5R was also described as a chromosome associated with rye preharvest sprouting resistance (Masojć et al. 2007  nivale. On chromosome 6R we detected loci of the rst and second leaf length, the second leaf width and the second leaf blade length (Table 3, Fig. 1). Three loci -Ql .hm.6R.2, Qlsl.hm.6R.3 and Qlslb.hm.6R.2 which explained up to 16.3% of phenotypic variation, were in the same cM position and were found in Exp. 3 in unhardened seedlings (Table 3 In conclusion, the most signi cant loci identi ed in this research were located on chromosomes 4B, 1R, 4R, 5R and 6R. All those regions had a high LOD value for Qlsl.hm.5R.1 and high phenotypic variation for Qwsl.hm.4R.2. Up to date this is the rst paper describing QTL regions associated with leaf development in winter triticale seedling after and without cold-hardening process. Based on our results, identi ed loci can be correlated in future with seedling freezing and fungal infection tolerance. Declarations Figure 1 The interval map (cM) for chromosomes 4B, 1R, 4R, 5R and 6R of the DH 'Hewo' x 'Magnat' lines mapping population of winter triticale (xTriticosecale), with QTLs identi ed by CIM method for seedling traits: the rst leaf length (LFL) and width (WFL), the second leaf length (LSL) and width (WSL), the second leaf sheath length (LSSL) and the second leaf blade length (LSLB). Additionally, black lines show marker closest to the LOD peak identi ed on each QTL.

Supplementary Files
This is a list of supplementary les associated with this preprint. Click to download. Wajdziketal.Tablessuplementarymaterials.docx