Mapping freezing tolerance QTL in alfalfa: based on indoor phenotyping

Abstract

Background: Winter freezing temperature impacts alfalfa (Medicago sativa L.) persistence and seasonal yield and can lead to the death of the plant. Understanding the genetic mechanisms of alfalfa freezing tolerance (FT) using high-throughput phenotyping and genotyping is crucial to select suitable germplasm and develop winter-hardy cultivars. Several clones of an alfalfa F₁ mapping population (3010 x CW 1010) were tested for FT using a cold chamber. The population was genotyped with SNP markers identified using genotyping-by-sequencing (GBS) and the quantitative trait loci (QTL) associated with FT were mapped on the parent-specific linkage maps. The ultimate goal is to develop non-dormant and winter-hardy alfalfa cultivars that can produce extended growth in the areas where winters are often mild.

Results: Alfalfa FT screening method optimized in this experiment comprises three major steps: clone preparation, acclimation, and freezing test. Twenty clones of each genotype were tested, where 10 samples were treated with freezing temperature, and 10 were used as controls. A moderate positive correlation (r ~ 0.36, P < 0.01) was observed between indoor FT and field-based winter hardiness (WH), suggesting that the indoor FT test is a useful indirect selection method for winter hardiness of alfalfa germplasm. We detected a total of 20 QTL associated with four traits; nine for visual rating-based FT, five for percentage survival (PS), four for treated to control regrowth ratio (RR), and two for treated to control biomass ratio (BR). Some QTL positions overlapped with WH QTL reported previously, suggesting a genetic relationship between FT and WH. Some favorable QTL from the winter-hardy parent (3010) were from the potential genic region for a cold tolerance gene CBF. The BLAST alignment of a CBF sequence of M. truncatula, a close relative of alfalfa, against the alfalfa reference showed that the gene's ortholog resides around 75 Mb on chromosome 6.

Conclusions: The indoor freezing tolerance selection method reported is useful for alfalfa breeders to accelerate breeding cycles through indirect selection. The QTL and associated markers add to the genomic resources for the research community and can be used in marker-assisted selection (MAS) for alfalfa cold tolerance improvement.

Keywords: Alfalfa; CBF; Cold tolerance; Freezing tolerance; GBS; Germplasms; SNP; Winter survival.

Fig. 1

Regrowth pattern of indoor freeze-tested alfalfa plants from the treatment group (left) and the control group (right) after two weeks of freezing test. Some tested genotypes in the treatment group had good regrowth while some of them could not survive

Fig. 2

Distribution of mean of regrowth ratio (RR) computed for treated vs. a control group of plants among indoor tested alfalfa bi-parental (3010 x CW 1010) F₁ progenies. The data is available for 179 progenies and the trait values exhibited near to normal distribution

Fig. 3

Linkage map of homolog 6B for the maternal parent (3010) where two maps 6B [1] and 6B [2] indicate two portions of a single linkage map 6B. The map shows a QTL (dft4) for freezing tolerance. The rectangle of the QTL bar represents an inner (1-LOD support) interval, and the line of the bar indicates an outer (2-LOD support) interval

Fig. 4

Winter frost tolerant (left) and sensitive (right) alfalfa F₁ progenies. The sensitive alfalfa has very distinct frost damage symptoms. The image was taken after frost occurrence in the first week of March 2017 at Watkinsville, GA. Before the frost, the winter was mild, and the alfalfa had gained sufficient regrowth