Zanotto Stefano, Ruttink Tom, Pégard Marie, Skøt Leif, Grieder Christoph, Kölliker Roland, Ergon Åshild
Faculty of Biosciences, Norwegian University of Life Sciences (NMBU), Ås, Norway.
Plant Sciences Unit, Flanders Research Institute for Agriculture, Fisheries and Food (ILVO), Melle, Belgium.
Front Plant Sci. 2023 May 10;14:1189662. doi: 10.3389/fpls.2023.1189662. eCollection 2023.
Improvement of persistency is an important breeding goal in red clover ( L.). In areas with cold winters, lack of persistency is often due to poor winter survival, of which low freezing tolerance (FT) is an important component. We conducted a genome wide association study (GWAS) to identify loci associated with freezing tolerance in a collection of 393 red clover accessions, mostly of European origin, and performed analyses of linkage disequilibrium and inbreeding. Accessions were genotyped as pools of individuals using genotyping-by-sequencing (pool-GBS), generating both single nucleotide polymorphism (SNP) and haplotype allele frequency data at accession level. Linkage disequilibrium was determined as a squared partial correlation between the allele frequencies of pairs of SNPs and found to decay at extremely short distances (< 1 kb). The level of inbreeding, inferred from the diagonal elements of a genomic relationship matrix, varied considerably between different groups of accessions, with the strongest inbreeding found among ecotypes from Iberia and Great Britain, and the least found among landraces. Considerable variation in FT was found, with LT50-values (temperature at which 50% of the plants are killed) ranging from -6.0°C to -11.5°C. SNP and haplotype-based GWAS identified eight and six loci significantly associated with FT (of which only one was shared), explaining 30% and 26% of the phenotypic variation, respectively. Ten of the loci were found within or at a short distance (<0.5 kb) from genes possibly involved in mechanisms affecting FT. These include a caffeoyl shikimate esterase, an inositol transporter, and other genes involved in signaling, transport, lignin synthesis and amino acid or carbohydrate metabolism. This study paves the way for a better understanding of the genetic control of FT and for the development of molecular tools for the improvement of this trait in red clover through genomics assisted breeding.
提高持久性是红三叶草（Trifolium pratense L.）育种的一个重要目标。在冬季寒冷的地区，持久性差往往是由于冬季存活率低，其中耐寒性低是一个重要因素。我们进行了一项全基因组关联研究（GWAS），以在393份红三叶草种质资源（大多来自欧洲）中鉴定与耐寒性相关的基因座，并进行了连锁不平衡和近亲繁殖分析。利用简化基因组测序（pool-GBS）将种质作为个体池进行基因分型，在种质水平上生成单核苷酸多态性（SNP）和单倍型等位基因频率数据。连锁不平衡被确定为SNP对的等位基因频率之间的平方偏相关，发现其在极短距离（<1 kb）内衰减。从基因组关系矩阵的对角元素推断出的近亲繁殖水平在不同种质组之间差异很大，在伊比利亚和英国的生态型中近亲繁殖最强，而在地方品种中近亲繁殖最少。发现耐寒性存在相当大的变异，半致死温度（LT50，即50%的植株被冻死时的温度）范围为-6.0°C至-11.5°C。基于SNP和单倍型的GWAS分别鉴定出8个和6个与耐寒性显著相关的基因座（其中只有一个是共同的），分别解释了30%和26%的表型变异。其中10个基因座位于可能参与影响耐寒性机制的基因内部或距离其很近（<0.5 kb）的位置。这些基因包括一个咖啡酰莽草酸酯酶、一个肌醇转运蛋白，以及其他参与信号传导、转运、木质素合成和氨基酸或碳水化合物代谢的基因。这项研究为更好地理解耐寒性的遗传控制以及通过基因组辅助育种开发改善红三叶草这一性状的分子工具铺平了道路。
