Genetic analysis of developmental traits associated with enhanced winter survival in autumn-seeded rye (Secale cereale L.).

Abstract

Autumn seeded winter cereals are ecologically friendly and usually yield higher than spring seeded cereals. However, the low and unpredictable winter field survival (WFS) is a major factor limiting the widespread adoption of winter cereals on the Canadian prairies. In the autumn, winter cereals seedlings exposed to gradual low temperatures along with changes in day length and light quality, initiate cold acclimation and accumulate low temperature tolerance (LTT) to overcome the extreme low temperatures in the winter. Simultaneously, the plants undergo vernalization and initiate developmental changes at the shoot apical meristem to adjust to the low temperatures. The research in this thesis is based on the hypothesis that the complex WFS trait is largely affected by the LTT accumulated during cold acclimation and low-temperature induced plant developmental traits. Autumn seeded rye (Secale cereale ssp. cereale L.) is the most cold-hardy winter cereal among cultivars adapted to the Northern latitudes. A panel of 96 rye genotypes from diverse geographic regions and growth habits was assessed for WFS by five years of field trials (Saskatoon, SK, Canada). The plants were also assessed for LTT by whole plant freeze tests in a climate chamber, and six developmental traits during growth under controlled conditions in a growth room or greenhouse. From the Best Linear Unbiased Estimates (BLUEs) for five years of field tests, five WFS classes were defined for the rye panel (i) very high (64.5 - 92.5%; 19 genotypes), (ii) high (56.7 - 64.3%; 20 genotypes), (iii) moderate (46.2 - 56.6%; 19 genotypes), (iv) low (30.5 - 43.7%; 19 genotypes), and (v) very low (0.0 - 25.2%; 19 genotypes). The WFS BLUE values correlated strongly with LTT (r = 0.90, p <0.001); inferring that cold acclimation efficiency was the major contributor to WFS. Among the six plant developmental traits, WFS showed strongest correlations (p <0.001) with final leaf number (FLN, r = 0.80), prostrate growth habit (PGH, r = 0.61), and plant height (PHT. r = 0.34). The same panel of rye genotypes was also analyzed for anthocyanin (ANT) production in leaves and crown tissues during cold acclimation. With the use of a high-performance liquid chromatography system combined with quadrupole time-of-flight mass spectrometry (HPLC-QTOF MS/MS), a total of 18 different anthocyanidins (cyanidins, delphinidins, and pelargonidins) in glycosylated forms were identified. Among anthocyanins, seven compounds correlated with WFS, and Cya-3-Glc had the strongest correlation (r = 0.35, p <0.001). In this study, association genetic studies allowed identification of genes associated with WFS in rye, due to the outcrossing nature of rye that provided a low linkage disequilibrium (LD) and the availability of a near complete genome sequence in 2021. The 96 rye genotypes were genotyped by sequencing (GBS), resulting in a total of 357.1 million reads with an average read length of 108 bp. Alignment of the processed sequences to the scaffolds of rye inbred line Lo7 (version 2) produced 252,158 single nucleotide polymorphism (SNP) markers, which after data filtering yielded 10,244 SNP markers with no missing data. Genome wide association studies (GWAS) revealed 679 marker-trait-associations (MTAs; p <0.01). The ten most significant SNPs (p <1.49e-04) associated with WFS, corresponded to nine different genes. Seven candidate genes were also associated with LTT, and developmental traits (FLN, PGH), suggesting a close linkage between acquisition of LTT and plant development. Allelic variations for genes encoding Inducer of CBF Expression 1 (ICE1), Cold-regulated 413-Plasma Membrane Protein 1 (COR413-PM1), Ice Recrystallization Inhibition Protein 1 (IRIP1), Jasmonate-resistant 1 (JAR1), BIPP2C1-like protein phosphatase, and Chloroplast Unusual Positioning Protein-1 (CHUP1) were among the most significant candidate genes for WFS. Among the anthocyanins, the four most significant SNP markers associated with WFS (p < 2.76e-04), corresponded to phenylpropanoid pathway genes including, Chalcone Synthase 2 (CHS2), Anthocyanidin 3-O-Glucosyltransferase (3GT), and Phenylalanine Ammonia-Lyase 8 (PAL8). The winter-hardy rye genotypes generally carried additional allele variants for candidate genes, which suggested allele diversity was a major contributor to cold acclimation efficiency and consistent high WFS under varying field conditions. The present study identified candidate genes associated with WFS, LTT and developmental traits in rye. The results confirmed the complex nature of WFS and a close association with plant developmental traits affected by changes in shoot apical meristem during cold acclimation. Since rye chromosomes are syntenous with wheat and barley, the knowledge can be used to increase WFS in the ecologically friendly temperate winter cereals of interest in the Northern hemisphere.