Wheat grain yield has improved exponentially since the green revolution and continues to improve, although the pace of increment is decreasing. Despite achieving a reasonable level of global production of wheat, work for improving yields has always remained at the forefront, one to fulfill demands of ever-increasing mouths and other to answer the scientific curiosity of “how much yield potential can be improved?” Wild species of wheat encompass a wide diversity of alleles, and T. dicoccoides is one of the important species, housing a vast variation in grain size and novel alleles for grain size-related component traits (Gegas et al. 2010; Nevo 2001). For allowing more targeted selection in wheat breeding, the characterization of grain yield components ought to be exploited (Wurschum et al. 2018). TGW is one of the incredibly important yield components in hexaploid wheat, composed of different individual grain morphometric components like GL, GW, and GA (Fuller 2007; Kuchel et al. 2007). For any improvement in TGW, insight into grain size component traits is required to achieve the targeted improvement. In the present study, we used T. dicoccoides accessions showing variations in grain size and weight to understand the effect of different genes on the GL, GW, and GA during the grain development from 4DPA to 28DPA and maturity. The selected T. dicoccoides accessions included two accessions with higher TGW (33.74g and 31.72g) and the other two with low TGW (17.83g and 14.58g), with slight variations in grain size parameters.
In this study, we tried to gain insight into the effect of multiple genes during grain development across the wheat spikes and within the individual grains. Focusing on specific grain morphometric components at different grain development stages will provide a genetic dissection of TGW along with the mechanistic understanding of genes involved in it and their level of expression. This knowledge plays a vital role in modulating grain yield components to ensure the improvement in wheat yield by understanding the molecular mechanism underlying (Guan et al. 2019; Yu et al. 2019).
In wheat, anthesis begins in the central part of the spike and continues bidirectionally towards the basal and apical parts. Furthermore, the proximal/primary florets of the central spikelet are fertilized two to four days earlier than the distal florets; hence grains from these florets usually have higher weight (Bonnett 1936; Kirby 1974; Peterson 1965; Simmons and Crookston 1979). In the present study, the primary floret of the central three spikelets was selected to assess the different grain parameters to maintain uniformity in the experimental material.
The grain development in wheat has been divided into three phases after anthesis, grain enlargement (0-14DPA), grain filling (15-35DPA), and physiological maturity (36-50DPA). There is a significant increase observed in the length of the developing grain after an initial period of isotropic growth, and this would become maximum at around 15DPA, which contributed more towards grain area, compared to width (Brinton et al. 2017; Xie et al. 2015), suggests that initially effect of GL is more on TGW (Hasan et al. 2011; Lizana et al. 2010). After this, endosperm expands, and grain filling starts with a higher rate at 14-28DPA (Shewry et al. 2012). In the present study, a similar pattern of the rate of increase of GL, GW, and GA was observed. We observed that the increase in GL and GA in the tetraploid T. dicoccoides is more prominent between 4DPA to 8DPA (5.44-8.92mm in LG and 4.50-7.33mm in SG accessions), while the maximum gain in GW is between 8DPA to 12DPA (2.10-3.05mm in LG and 1.71-2.48mm in SG accessions). After that, there is a gradual increase in GL, GW, and GA, with the rate of gain decreasing from 16DPA to 28DPA (Brinton et al. 2017). The temporal differentiation of developmental stages of the selected lines from the previous studies may indicate the differences in the genetic background of modern-day bread wheat and wild emmer wheat
Grain size is a complex trait with multiple subcomponents under independent genetic control (Brinton and Uauy 2019; Gegas et al. 2010), and grain development plays an important role in the final TGW. The transition between grain formation and maturation in cereal seed development involves a number of genes that initiate seed size development and decide the ultimate grain yield of wheat. We tried to identify the molecular mechanism behind this process by expression analysis of genes involved in grain size increment (Fig. 4; Supplementary fig S1 and S2). In the present study, we shortlisted eight genes involved in determining different grain size components in rice and wheat. GL7 (Wang et al. 2015b), TaGL3 (Qi et al. 2012; Yang et al. 2019), SRS3 (Kitagawa et al. 2010; Si et al. 2016; Yu et al. 2019), and TAGASR7 (Huang et al. 2012; Zhang et al. 2014) are well known to affect the grain length, GS3 (Fan et al. 2006) and TGW6 (Hanif et al. 2016; Ishimaru et al. 2013) for grain length and weight, TaGS5 (Ma et al. 2016) for grain size, and TaGW2 (Simmonds et al. 2016; Su et al. 2011; Wang et al. 2018; Yang et al. 2012; Zhang et al. 2018) is known to affect grain width and weight. The orthologue identification of these genes in T. dicoccoides and validation of their expression profiles will open up a new avenue of novel genetic resources to improve the grain size and weight.
GL7 in rice is a major QTL for grain length that controls the grain size and shape through cell elongation and by decreasing cell expansion in terms of grain width direction to produce longer grains (Wang et al. 2015a; Wang et al. 2015b; Zhou et al. 2015). We observed higher expression of GL7 in LG accessions of T. dicoccoides than short grain accessions, confirming that this gene is expressed to produce longer and heavier grains. Due to the increased copy number or mutations in the promoter, the expression of GL7 was higher in long grain varieties of rice (Wang et al. 2015b). The sequence analysis of this gene in long grain accessions of T. dicoccoides might lead to the identification of novel alleles associated with longer grains.
GS5 functions as a positive regulator of grain size and higher expression of GS5 is correlated with larger grain size (Li et al. 2011; Xu et al. 2015). We observed higher expression levels of TaGS5 at initial grain development stages in long grain accessions, whereas very low levels in all the developing stages of short grain accessions, indicating that higher expression of TaGS5 might be involved in the development of larger grains. Ma et al. (2016) investigated the temporal and spatial expression patterns of the TaGS5 homoeologues ortholog of rice gene OsGS5 in various tissues of wheat, which showed higher expression in seedlings, young spikes, and developing grains.
GS3 in rice encodes a putative transmembrane protein, and a major QTL for grain length, weight, and a minor QTL for grain width have been identified. A loss of function of allele in GS3 promotes cell proliferation and forms long grains, while gain of function produces short grains (Fan et al. 2006). The cause of mutation by premature stop-codons between grain size in rice suggests that orthologous genes and similar related regulatory processes for this type of traits may be conserved across a broad range of taxa ranging from monocot to dicot species. Our result showed higher expression of GS3 in mid-grain developmental stages in long grain accessions, indicating the role of this orthologue in regulating the grain length in wheat.
In rice, GL3 encodes a protein phosphatase with kletch-like repeat domains (OsPPKLs), restricting cell division in spikelet hulls that increase grain length, weight, and yield (Qi et al. 2012; Zhang et al. 2012). Our results showed higher expression of TaGL3 in both the LG accession at an early stage of grain development, whereas a low expression level was observed in all the stages of grain development in SG accession. Yang et al. (2019) cloned a wheat orthologous TaGL3-5A, its expression pattern was similar with increasing grain size at the early (8DPA) and middle stages (20DPA) of seed development, suggesting that TaGL3 play a role at an early phase of seed development. Association analysis revealed that the TaGL3-5A-G allele was significantly correlated with longer grains and higher TGW, and the frequency of the allele in hexaploid wheat was slightly lower than in T. dicoccoides.
SRS3, a kinesin 13 protein family gene, regulates seed length in rice and produces long grains by cell elongation (Kitagawa et al. 2010; Si et al. 2016). Yu et al. (2019) conducted a transcriptome profile study in wheat to unravel the genetic architecture of grain size, where the homolog of rice SRS showed successively higher expression across the early to middle stages of grain development. Our study also observed higher expression of this gene at an early and middle stage of grain development, which correlates with the phenotypic gain of grain size in LG accessions.
OsGASR7 in rice showed similarity to Arabidopsis GASA4 and was considered as a candidate gene determining grain length (Huang et al. 2012). The wheat ortholog of OsGASR was also reported to play the same role and was involved in grain length development (Dong et al. 2014). Zhang et al. (2014) studied expression patterns of TaGASR7 in immature seeds in a synthetic hexaploid wheat. GASR7B was highly expressed from 6 DPA till 14 DPA in developing seeds of the tetraploid accessions and began to decrease at 17 DPA. Our results also showed an increased expression till 12DPA, indicating that TaGASR7 was also involved in grain length increase during seed development in T. dicoccoides.
GW2 in rice and Arabidopsis affect grain size by suppressing cell proliferation (Song et al. 2007; Xia et al. 2013). Our result showed a negative correlation between TaGW2 and long grain in T. dicoccoides, as this gene was highly expressed in SG accessions than large ones. TaGW2 has been associated with kernel width and weight, which has been validated as a negative regulator of grain size in wheat by gene editing and mutant analysis. The association analysis indicated that the mutated TaGAW2 allele significantly increased kernel width (KW) and thousand-kernel weight (TKW) and slightly improved kernel length (KL) in tetraploid and hexaploid wheat. The increase in grain width and length was consistent across grains of different sizes, suggesting that the effect of the mutation is stable across the ear and within spikelets (Simmonds et al. 2016; Su et al. 2011; Wang et al. 2018; Yang et al. 2012; Zhang et al. 2018).
Ishimaru et al. (2013) identified a novel gene for grain length, and weight, TGW6, which encodes a novel protein related to indole-3-acetic acid (IAA) synthesis, loss of function of this allele enhances grain length and weight. TaTGW6-A1, an ortholog of rice TGW6, is associated with grain weight and yield in bread wheat (Hanif et al. 2016). Very low expression of TGW6 was found in all the accessions used in the present study across different grain developmental stages, indicating a functional allele in the selected T. dicoccoides accessions.
Notably, we observed the expression peak of six genes in long grain accession at around 8DPA-20DPA, corresponding to the change in grain size, suggesting that these genes play an important role in the early phase of grain development. There was also a correlation observed between gene expression and grain size traits at the early and middle stages during seed development. Cluster analysis illustrated by the heat map showed that the expression pattern of GL7, TaGL3, TaGS5, GS3, SRS3, and TaGASR7 has more similarity to grain size traits (Fig. 5). In contrast, TaGW2 and TGW6 showed a negative correlation with grain size traits in developing stages.
As there is a correlation between grain size-related traits and expression profiles of selected genes at the early stages of grain development, it depicts the scope of targeting the grain development process to improve yield. T. dicoccoides has a large variation in grain size; thus, we speculate that allelic variations of multiple genes involved in grain size are responsible for grain size variation in T. dicoccoides. Although other studies have reported these genes in wheat, but expression patterns of these genes in the present study indicate the presence of novel alleles in the T. dicoccoides germplasm. The long grain T. dicoccoides accessions studied in the present investigation and characterized genes can form a basis for systematic marker-assisted breeding for enhancing the grain size of the breeder’s germplasm.