Wheat (Triticum aestivum L.) is the most widely cultivated major food crop, providing staple food of approximately 40% of the world's population (Li et al., 2019). Grain hardness often correlates with yield traits, such as thousand-grain weight and bulk density, and is one of the key traits contributing to wheat processing quality (Li et al., 2020). Grain hardness is affected by certain physical and chemical properties of the seed, determines the flour yield and milling quality, and influences a wide range of end-use quality parameters. Because of the importance of this trait to wheat end-use quality, the genetic basis of kernel hardness has been studied extensively for several decades.
Wheat researchers have concluded that grain hardness differences between cultivars are caused by varying degrees of interaction between starch grains and the protein matrix within the endosperm (Tu and Li, 2020). Hard wheat kernels exhibit high resistance to crushing, as a result of strong adhesion between starch particles and the protein matrix. Flour from these hard grains is coarse-textured with more damaged starch and able to absorb more water than flour from soft grains. Hard wheat is best suited for making breads and noodles. In contrast, soft wheat is less resistant to crushing, and the flour has a finer texture with less damaged starch, making it better for cookies, cakes, and pastries. Thus, grain hardness has been an important breeding target, with various degrees of hardness needed for different end-uses. For example, soft and waxy pasta is preferred in Southern China and these products require soft wheat grains (Liu et al., 2003). People in the north of China prefer products that need stronger gluten flour and consequently wheat cultivars from the north have higher grain hardness (Chen et al., 2006a).
Many studies have demonstrated that grain hardness is mainly controlled by multiple genes and is less affected by environmental conditions and other grain characteristics (Tu and Li, 2020). In a landmark paper of Philip Greenwell and J. David Schofield (1986), discovered that the Mr 15 000 protein Friabilin, was found on the surface of water-washed starch granules. Subsequently, a series of biochemical separation and amino acid sequencing studies identified the two major proteins, puroindoline a and b (PINA and PINB, respectively), and a minor protein, grain softness protein-1(Gsp-1), composed of friabilin (Turner et al., 1999). The Pina-D1, Pinb-D1, and Gsp-D1 genes are linked at the Ha site on chromosome 5DS and are associated with the expression of grain softness (Tranquilli et al. 1999). A PIN-like gene nearly identical to the Pinb gene sequence was first discovered in 2008 (Wilkinson et al.,2008). Among those genes, Pina and Pinb genes play a key role in grain hardness. Studies have been focused on identifying allelic variations for Pin and the resulting phenotypes. Up to now, resulting in the identification of 26 alleles of Pina, 33 alleles of Pinb, and a few double null alleles, and in their wild type alleles (Pina-D1a and Pinb-D1a) result in softness of grains (Chen et al., 2006b, 2012, 2013; Kumar et al., 2015; Li et al., 2019; Tu and Li, 2020). Although most of alleles related to grain hardness are not widely used, a few have shown good utility for breeding. For example, Pinb-D1p is a rare allele but is found in the cultivar Jinmai 47(Pina-D1a/Pinb-D1p) which has been unusually successful and grown on 1.33ⅹ107 hectares since its approval in 1995 (Li et al., 2019). Therefore, it is important to find other alleles with similar practical value.
In addition to the Puroindoline genes, grain hardness-related quantitative trait loci (QTLs) have been reported on all wheat chromosomes (Tu and Li, 2020). The most important locus is Ha on chromosome 5Ds, which can explain 60%~80% of the phenotypic variation for the trait (Sun et al., 2010; Wang et al.,2012; Li et al., 2013, 2016). Five QTLs for grain hardness were found using recombinant inbred lines (RILs), and the Pinb gene explained 76.8% of phenotypic variation while the other four QTLs explained only 2.8–6.5% (Sun et al., 2010). Two loci controlling soft wheat grain hardness, qkha.Orr4B and Qkha.OR4D explaining 20%~34% of the phenotypic variation, were found by using ultra-soft RILs (Wang et al., 2012). By using soft wheat RILs, a major QTL (Q.HI.scau-7D) was detected that accounted for ~ 30% of the phenotypic variation in kernel hardness (Li et al., 2013). Overall, these studies strengthen the idea that grain hardness was mainly controlled by PINs, but also other QTLs are also involved.
Linkage mapping studies have been used to identify genetic loci for grain hardness using a limited selection of parents. Populations selected for a genome-wide association study (GWAS) can avoid the limitations of bi-parental QTL studies. Only a few have used a GWAS to study the genetics of grain hardness (Chen et al., 2019; Muqaddasi et al., 2020; Navrotskyi et al., 2020; Aoun et al., 2021; Elhadi et al., 2021; Lou et al., 2021). Two studies used different materials and the 90K SNP array to do a GWAS for grain hardness and identified four (Lou et al., 2021) and nine (Navrotskyi et al., 2020) single-nucleotide polymorphisms (SNPs) associated with the trait. The phenotypic variation explanation rate (R2) ranged from 2.6 to 12.68% and the R2 of SNPs on the PIN gene were the highest. The Pinb gene on chromosome 5D has been located in association analysis of 372 European wheat varieties in up to eight environments (Muqaddasi et al., 2020). These studies all located SNPs in Pinb gene with the highest R2. Due to low phenotypic variation and low PIN gene locus diversity, some populations could not be associated with PIN gene or could be associated with SNPs in PIN gene without the highest R2. For example, 400 lines developed by crossing and backcrossing the Japanese wheat cultivar Norin61 were used to identify 47 significant hardness-related MTAs explaining 8.3%~22.6% of the phenotypic variation, yet the R2 of the MTA in the PIN gene was only 13% (Elhadi et al., 2021). Twenty SNPs were located using 172 advanced soft white winter wheat breeding lines in a GWAS (Aoun et al., 2021). Among these, QSKhard.wql-3A and QSKhard.wql-5A had the highest R2. Similarly, Chen et al. (2019) selected 299 American hard winter wheats and located nine SNPs significantly related to grain hardness using the 90K SNP array. The R2 of barc154-7D and wms130 on chromosome 7D was higher than that for the two SNPs located on the PIN gene. In addition to the Ha locus, grain hardness is also controlled by multiple genes. There are few studies on grain hardness using a GWAS approach and diverse wheat populations and, therefore, it is likely that other loci for this important trait have yet to be discovered.
Shanxi is located in the loess Plateau, accounting for more than 1/4 of the total area of the Loess Plateau. Shanxi Province has an ancient tradition of wheat cultivation going back more than 4000 years (Zheng et al., 2021). Within this environment over the last several millennia, Shanxi wheat has developed to representative for wheat research in semi-arid area. Shanxi has long been based on pasta, Pasta is prevalent in Shanxi and there are at least 300 kinds of pasta that have been invented in the province. The process of wheat breeding in this region can fully confirmed the theory of co-evolution of wheat quality resources and human environment. In the present study, Shanxi wheat germplasm and a 15K microarray are used for GWAS to assess the diversity of PIN alleles and search for novel loci for grain hardness. The experimental results provide a basis for molecular marker-assisted breeding for wheat hardness.