It has been estimated that soil salinity has affected 80 million hectares of the world’s cultivated land [1
]. Millions of hectares of land are too saline to produce economically valuable crop yields, and this trend is on the rise every year, making the land nonproductive [2
]. The problem of soil salinization is worsening in countries, such as the USA, China, Australia, Hungary and is threatening to become more intense in North and East African countries, and Middle Eastern and East Asian countries. Salt-affected areas in China constitute 4.88% of the country’s total land area, which equates to 3.6 × 107
ha of land nationwide [3
]. Excessive salinity of the soil can cause ion toxicity, osmotic stress, water, and nutrient scarcity and, thus, promptly decrease crop growth due to reduced photosynthesis [4
]. Ionic homeostasis, balanced root water intake, and leaf transpiration coupled with increased nutrient uptake are crucial for plants to deal with salinity stress [5
]. In addition to arid and semiarid areas, which account for approximately 30% of global saline areas, 20% of irrigated land has saline soils, and this proportion continues to increase [6
]. Due to environmental degradation caused by climate change, the salinity conundrum will also become more severe in areas with low and mid-latitudes. Cotton is a global industrial crop grown to meet the demand of 7.7 billion people worldwide. The exponential increase in the global population is expected to reach almost 9 billion by 2050 [7
], consequently increasing the demand for food and fiber equally. In order to combat future challenges, breeders are trying to improve cotton varieties especially for marginal lands, such as saline and drought-affected soils.
The genetic causes of phenotypic variations aimed at improving crop productivity and biotic and abiotic stress tolerance have been major focuses of plant studies. Quantitative trait locus (QTL) mapping has been successfully used in plants for mapping biparental crosses to detect sections of genomes that co-segregate with a certain trait either in F2 populations or in recombinant inbred line (RIL) families [8
]. Nevertheless, QTL mapping has two major pitfalls: it is constrained by both allelic diversity and narrow genomic resolution caused by relatively few recombination events that occur during the creation of the RIL population [10
]. Genome-wide association studies (GWASs) have the potential to overcome possible major limitations of QTL mapping by providing relatively high resolution even at the gene level, and the use of samples from previously well-studied populations with naturally occurring genetic variants can be concomitant with phenotypic variation. The basic approach in GWASs is to evaluate the association between each genotyped single-nucleotide polymorphism (SNP) marker and a phenotype of interest that has been recorded across a RIL population or a large number of individuals of a natural population. Recent developments in sequencing techniques have enabled GWASs to emerge as the most successful tool for identifying genetic causes of complex quantitative traits in plant species [11
]. GWASs have been successfully used to dissect the underpinnings of yield traits [12
], salinity, water deprivation, and heavy metal stress tolerance [13
]; fiber quality traits, and disease resistance [15
]. GWASs have been successfully used in cotton crops to trace genetic signatures associated with yield parameters and stress tolerance traits [17
], but there is a lack of extensive study in the cotton crop for stress tolerance at the seedling stage [20
]. In a previous study conducted by Sun et al. [21
], the authors found twenty-three SNPs on seven chromosomes associated with two salt tolerance related traits in cotton.
Cotton (Gossypium hirsutum
L.) is an important fiber crop species and the only commercial crop where the fiber is converted to fabric at a commercial scale [22
]. Salt stress can affect plant growth and development throughout plant ontogeny, but the seedling stage is considered one of the most vulnerable stages [23
]. Moreover, although cotton is considered moderately salt-tolerant with a cut-off of 7.7 dS/m, its growth is severely affected at the seedling stage, which reduces the yield [24
]. The current study was designed to perform a GWAS of salt tolerance traits associated with 17,264 SNPs in a core collection of 419 diverse natural populations of G. hirsutum
L. at the seedling stage. The present study pinpoints associated SNPs and probable genes to decipher the complex genetic background of salt stress tolerance in cotton. The principal theoretical implication of this study is the development of molecular markers that will foster salt stress tolerance breeding programs in cotton.
In the current study, we performed a GWAS of salinity stress tolerance traits at the seedling stage with a core collection of 419 cotton accessions selected from genetically diverse backgrounds and SNPs from the high-throughput Illumina sequencing platform. The findings of this study complement the understanding of the complex nature of salt stress tolerance mechanisms and the scouring of novel alleles and candidate genes. One of the implications of the present study is the possibility of accelerating the progress of cotton stress tolerance breeding.
Salt stress tolerance is a complex trait regulated by polygenes [36
]. GWASs provide an opportunity to explore genes responsible for quantitative trait variation in plants and animals [37
]. Relative to forward genetic approaches, GWASs have the potential to identify genes with smaller phenotypic effects [38
]. GWASs have become an obvious general methodology for studying the effects of natural variations and traits of agricultural and economic importance [39
]. A handful of research papers are available on association studies, particularly on fiber, yield, disease and their respective component traits, in cotton and several other crop species, but little work has been done on association analyses for stress tolerance in general in other crop species, particularly cotton [15
]. To date, few studies have investigated salt stress tolerance with natural variation and genome-wide markers by means of GWAS approaches. Jia et al. [41
] identified three SSR markers associated with salt tolerance by employing a mixed linear model and a panel of 323 cotton accessions and using 106 SSR markers. Using 179 polymorphic SSR markers in 503 upland cotton accessions, the researchers identified 15 SSR and 3119 SNP markers associated with relative germination rate under salt stress and ultimately found four differentially expressed candidate genes in tolerant and sensitive accessions under salt stress. Sun et al. [21
], screened 713 accessions and identified 23 SNPs representing seven genomic regions that were significantly associated with salt tolerance level (STL) and relative survival rate (RSR). Furthermore, 280 putative genes showing different expression levels were screened, and six apparent putative genes were validated with qRT-PCR in salt-tolerant and sensitive varieties.
Lectin receptor-like kinases (LecRKS) play an important role in plant innate defense mechanisms. L-type LecRKs, one of the three types of LecRKs, are considered to play an important role in abiotic stress signaling in Arabidopsis
]. In our study of fresh weight under salt stress, we found that the expression of the RNA of Gh_D10G2298, which encodes an LeCRK, under salt stress was high in salt-tolerant genotypes compared to salt-sensitive genotypes, as shown in Figure 7
. Rubisco activase (RCA) is an important enzyme involved in the carboxylation and oxygenation of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) and participates in the photosynthetic carbon reduction cycle. In a study conducted by Chen et al. [43
], rubisco activase responded to abiotic stress in multiple ways. An investigation was carried out with respect to the RCA gene’s 2.0 kb 5′-upstream promoter region, some cis-elements related to certain stress-related components were identified in the RCA promoter. Multiple species comparisons with respect to the RCA protein revealed conserved regions among different species; their extent and nature varied. This finding might reveal the various transcription and translation splicing stages of the two RCA isoforms during adaptation to various abiotic stresses. These findings suggest that RCA, particularly RCAL, is a multiple responder to abiotic stresses.
In our study of leaf fresh weight under salinity stress, the gene Gh_D10G2299 on chromosome D10 was found to encode (RCA2) protein. This protein has gained much attention as a regulator of a number of biotic and abiotic stress tolerances. Bi et al. [44
] studied the overexpression of rubisco activase in cucumber (Cucumis sativus
L.); it was found that CsRCA overexpression resulted in increased leaf area, plant height, and dry matter content, with a reduced root/shoot ratio in transgenic cucumber plants, compared to wild-type plants.
Salinity stress causes all types of root damage in all crops. A study conducted by Robin et al. [45
] showed that adventitious root length and density of wheat crops decreased by 25% and 40%, respectively, under salt stress. Lateral organ boundary (LOB) proteins are expressed in lateral and adventitious roots in plants [45
]. Interestingly, we found the lateral organ boundary protein-coding gene, Gh_D10G2300, on chromosome D10 under salt stress. The relative expression of Gh_D10G2300 under salt stress, as shown in Figure 7
, is higher in salt-tolerant genotypes than in salt-sensitive genotypes. Chlorophyll contents are reduced under salt stress, as shown in our study, so if chloroplast performance is improved, plant performance could increase under salt stress. In our study, we found that the Gh_D10G2302 gene, which encodes a 15 kDa thylakoid lumen protein, can enhance stress tolerance in crop plants. Chaperones and chaperonins play an important role in nascent protein folding, stabilization, and assistance to obtain a particular function [46
]. In a study conducted by Rodríguez et al. [47
], PFD5, a chaperone protein, was found to play an important role in Arabidopsis thaliana
L. salt stress tolerance. We found a gene for RWC_150 on chromosome A10, Gh_A10G1885, coding for a probable prefoldin subunit 2 chaperone. Differential gene expression was detected between the salt-tolerant genotypes and salt-sensitive genotypes under salt stress. Splicing factor 3B subunit 3 belongs to the ion channel family and participates in RNA modulation in plants, which involves an inverse resistance response in plants [48
]. In our study, the gene Gh_A10G1886, which encodes the splicing factor 3b subunit3 SF3B3, was found on chromosome A10 in RWC_150. Its relative expression was high in salt-sensitive genotypes and vice versa. Dirigent and dirigent-like family (DIR) proteins are a group of proteins responsible for lignification, pathogen infection responses, and abiotic stress tolerance in plants. DIR genes play a vital role in augmenting stress tolerance in different crop species. Yang et al. [49
], studied a dirigent-like gene in sugarcane designated ScDir with a full-length cDNA sequence. The expression of ScDir in an E. coli
system indicated that ScDir protein improved the host cell’s tolerance to polyethylene glycol (PEG) and NaCl. The ScDir expression level increased in sugarcane seedlings under H2
, PEG, and NaCl stress. ScDir expression was significantly upregulated under PEG stress, and the highest level of expression was observed at 12 hours post-stress application. Thus, both the ScDir-hosted cell performance and the enhanced expression in sugarcane suggest that the ScDir gene provides responses to abiotic stresses, such as drought, salt, and oxidation.
Salinity stress induces osmolyte wavering in plant cells, consequently causing relative water imbalance. Relative water content (RWC) is considered the most suitable sign of plant water status in terms of the physiological concern of cellular water scarcity under water deficit and salt stress [50
With respect to relative water content under salt stress (RWC_150), a group of three dirigent genes, Gh_A10G1887, Gh_A10G1888, and Gh_A10G1889, was found: two genes (Gh_A10G1887 and Gh_A10G1889) coding for dirigent protein 25 and one gene (Gh_A10G1888) coding for dirigent protein 9 on chromosome A10. These speculative genes were homologous to Arabidopsis At1g07730.2 and At2g39430.1, which encode members of the disease resistance protein family.
A study conducted by Xu et al. [51
] confirmed the role of the glutathione S-transferase gene (GST) in genetically modified tobacco under drought and salt stress. Genetic transformation of the glutathione S-transferase gene GsGST from wild soybean (Glycine soja
L.) enhanced drought and salt tolerance in transgenic tobacco. Tobacco plants overexpressing the GsGST gene showed a six-fold increase of GST expression compared with that of wild-type (WT) plants, further revealing improved desiccation resistance and higher tolerance to salt and mannitol at the seedling stage than WT plants, as corroborated by longer root length and less growth obstruction in the former. Kumar et al. [52
] studied the role of a member of the lambda class of proteins, OsGSTL2, by checking the expression in a heterologous system—Arabidopsis
. Transgenic lines were analyzed to check their response to a number of abiotic stresses, such as heavy metal, cold, osmotic and salt stresses. Differential expression of OsGSTL genes was observed in arsenate-sensitive and arsenate-tolerant rice accessions. Heterologous expression of glutathione S-transferase gene 2 in Arabidopsis
provided tolerance to different heavy metal, salt, drought and other abiotic stresses during early germination stages.
On chromosome A10, we found Gh_A10G1891, a DHAR2 gene that is homologous to the Arabidopsis gene AT1G75270.1 and shares 76% identity with the encoded glutathione s-transferase DHAR2 protein. Glutathione s-transferases (GSTs) are thought to play major roles in oxidative stress metabolism. A number of studies have confirmed their role in stress tolerance.
Plant scientists consider sulfur an important constituent in plants to withstand abiotic stress [53
]. The level of sulfate in the xylem acts as a signal for abscisic acid-dependent leaf stomatal closure during the early onset stage of drought when ABA synthesis is limited to the leaves [54
]. Sulfur metabolism and ABA biosynthesis together ensure sufficient cysteine for ABA production under abiotic stress. Sulfate acts as a precursor of cysteine, which plays a crucial role in ABA synthesis. Gallardo et al. [55
], conducted a comparative study of the SULTR gene family under drought and salinity stress in Arabidopsis
and Medicago truncatula
. The SULTR genes in M. truncatula
were found to be similarly regulated, as in Arabidopsis
, they likely encode factors for improving sulfate transport dimensions. Group 3 SULTR genes were found to be abiotic stress-responsive genes common between Arabidopsis
and M. truncatula
Metal toxicity produces reactive oxygen species (ROS) in plants, leading to an imbalance in cell homeostasis, breakage of the DNA, protein denaturation, and damage to the cell membrane and photosynthetic machinery, leading to cell death [57
]. Plant metal tolerance proteins (MTPs) are divalent-cation/H+ antiporters and generally act to efflux metals from the cytoplasm [59
]. We found a probable role of the metal tolerance protein-coding gene Gh_A10G1895 in RWC_150; its expression was high in the salt-tolerant genotypes compared to the salt stress-sensitive genotypes. Therefore, this gene may play a vital role in water homeostasis. We also found two genes Gh_A10G1884 and Gh_A10G1890 that have no previously studied role for salt stress tolerance in any crop. Therefore, functional studies of these two genes may provide useful insights into their role in salinity stress tolerance in cotton.