Discovering new genes for alfalfa (Medicago sativa) growth and biomass resilience in combined salinity and Phoma medicaginis infection through GWAS

Wiem Mnafgui; Cheima Jabri; Nada Jihnaoui; Nourhene Maiza; Amal Guerchi; Nawres Zaidi; Gerhard Basson; Eden Maré Keyster; Naceur Djébali; Luciano Pecetti; Mohsen Hanana; Paolo Annicchiarico; Muhammet Sakiroglu; Ndiko Ludidi; Mounawer Badri

doi:10.3389/fpls.2024.1348168

Discovering new genes for alfalfa (Medicago sativa) growth and biomass resilience in combined salinity and Phoma medicaginis infection through GWAS

Front Plant Sci. 2024 May 2:15:1348168. doi: 10.3389/fpls.2024.1348168. eCollection 2024.

Authors

Wiem Mnafgui^#¹, Cheima Jabri^#¹, Nada Jihnaoui^{1

2}, Nourhene Maiza^{1

2}, Amal Guerchi^{1

2}, Nawres Zaidi^{1

2}, Gerhard Basson³, Eden Maré Keyster^{3

4}, Naceur Djébali⁵, Luciano Pecetti⁶, Mohsen Hanana¹, Paolo Annicchiarico⁶, Muhammet Sakiroglu⁷, Ndiko Ludidi^{4

8}, Mounawer Badri¹

Affiliations

¹ Laboratory of Extremophile Plants, Centre of Biotechnology of Borj Cedria, Hammam-Lif, Tunisia.
² Faculty of Sciences of Tunis, University of Tunis El Manar, El Manar Tunis, Tunisia.
³ Environmental Biotechnology Laboratory, Department of Biotechnology, University of the Western Cape, Bellville, South Africa.
⁴ Plant Stress Tolerance Laboratory, University of Mpumalanga, Mbombela, South Africa.
⁵ Laboratory of Bioactive Substances, Centre of Biotechnology of Borj Cedria, Hammam-Lif, Tunisia.
⁶ Council for Agricultural Research and Economics, Research Centre for Animal Production and Aquaculture, Lodi, Italy.
⁷ Department of Bioengineering, Adana Alparslan Türkeş Science and Technology University, Adana, Türkiye.
⁸ DSI-NRF Centre of Excellence in Food Security, University of the Western Cape, Bellville, South Africa.

^# Contributed equally.

Abstract

Salinity and Phoma medicaginis infection represent significant challenges for alfalfa cultivation in South Africa, Europe, Australia, and, particularly, Tunisia. These constraints have a severe impact on both yield and quality. The primary aim of this study was to establish the genetic basis of traits associated with biomass and growth of 129 Medicago sativa genotypes through genome-wide association studies (GWAS) under combined salt and P. medicaginis infection stresses. The results of the analysis of variance (ANOVA) indicated that the variation in these traits could be primarily attributed to genotype effects. Among the test genotypes, the length of the main stem, the number of ramifications, the number of chlorotic leaves, and the aerial fresh weight exhibited the most significant variation. The broad-sense heritability (H²) was relatively high for most of the assessed traits, primarily due to genetic factors. Cluster analysis, applied to morpho-physiological traits under the combined stresses, revealed three major groups of accessions. Subsequently, a GWAS analysis was conducted to validate significant associations between 54,866 SNP-filtered single-nucleotide polymorphisms (SNPs) and seven traits. The study identified 27 SNPs that were significantly associated with the following traits: number of healthy leaves (two SNPs), number of chlorotic leaves (five SNPs), number of infected necrotic leaves (three SNPs), aerial fresh weight (six SNPs), aerial dry weight (nine SNPs), number of ramifications (one SNP), and length of the main stem (one SNP). Some of these markers are related to the ionic transporters, cell membrane rigidity (related to salinity tolerance), and the NBS_LRR gene family (associated with disease resistance). These findings underscore the potential for selecting alfalfa genotypes with tolerance to the combined constraints of salinity and P. medicaginis infection.

Keywords: GWAS; Phoma medicaginis; alfalfa; biomass traits; genetic resources; salinity; stress tolerance.

Grants and funding

The authors declare financial support was received for the research, authorship, and/or publication of this article. This research was funded by the Tunisian-South African project (2019-2024) and the Tunisian Ministry of Higher Education and Scientific Research (LR15 CBBC02). The plant material was generated by the FP7-ArimNet project REFORMA funded by the Governments of Italy, Algeria, Morocco and Tunisia.