**3. Discussion**

Before this study, the size and weight related traits of rice grains, including grain length, grain width, grain thickness, and thousand grain weight, had not been explored at the same time using a large and diverse germplasm group with the help of InDel markers. New Generation Sequencing (NGS) tools have yielded advanced, cheaper, and more efficient methods for developing such markers. This investigatory research efficaciously shows the capable utilization of deletion / insertion variations (DIV) that naturally accrue in the rice genome. InDel polymorphisms are the second most abundant (after SNPs) forms of genetic variations in animals and plants, with great diversity. Moreover, previous studies only showed the contributions of the reported genes in regulating these grain traits separately [11,15,16,19,20,59,60]. However, recent studies have shown the modes of InDel based allelic contributions in the expression of grain size, as well as weight related traits, the allelic combination of all the InDel marker loci involved in the final texture of the rice grains, and the potential of each loci in regulating these traits in rice. This research can assist in selecting or deselecting genes for rapid breeding strategies. The correlation coefficient values suggest that the thousand grain weight (TGW) was positively influenced by the other studied grain size traits but with a different aptitude. The TGW can be improved by using all three studied grain traits—most importantly GT, which contributes 49%, followed by GW and GL, which contribute 37.4% and 24.9% of the final grain weight, respectively. These findings are in agreement with those of previous studies [19,54,61,62], which showed that grain weight is significantly correlated with grain size. The present study further showed that the thickness of rice grains was most strongly correlated with grain weight, followed by width, whereas the length of the grains was the least associated, suggesting that selecting for grain thickness is more fruitful for heavier grains (Table 2, Figure 4).

In the present study, two hundred and four rice genotypes were used to investigate alleles in nine different genes that regulate size and weight in rice grains, using InDel markers. In the past few years, PCR based InDel markers have gained popularity in variation studies because of their reproducibility, easy to use nature, and co-dominant inheritance [40]. Dendrograms were used to separate the germplasms according to their grain size and weight and divide the InDel marker data into distinguishable clusters that could be used for breeding preferential grain appearances and weights. Results of the genetic diversity (D) analysis and InDel based polymorphism information content (PIC) values (Table 6) indicate that the InDel markers (*GW8*-InDel, *GS5*-InDel1A, *GW8*-InDel2B, *GS2*-InDel1A) are highly informative (D ≥ 0.5; PIC ≥ 0.4) for the studied traits, whereas the rest of the markers were found to be moderately (D 0.3–0.5; PIC 0.2–0.4) to slightly/less (D ≤ 0.3; PIC ≤ 0.2) informative. These InDel markers show potential to be efficiently used to study the genetic variations (DIVs) in rice germplasm. Only two InDel markers showed very low values (gene diversity ≤0.3; PIC ≤ 0.2) for D and PIC, indicating that the deployed markers were fairly informative [20]. Furthermore, this investigatory research also showed the capability of InDel markers to distinguish a diverse rice germplasm into distinctive groups (Figure 6) with different combinations of grain lengths, widths, thicknesses, and weights that could be used to breed desirable genotypes with better potential for higher market value rice grains and heavier grains for a better yield.

This study successfully identified 25 InDel marker derived loci highly associated (*p* ≤ 0.05) with grain size and weight in rice (Table 4). A total of 38 alleles were identified, out of which 27 alleles were major and were found in more than 20 genotypes. In the case of GL, five markers (*GW8*-InDel, *GW8*-InDel1A, *GW8*-InDel2B, *GS2*-InDel1A, and *GS2*-InDel2B) corresponding to two genes (*GW8* and *GS2*) were found to have a highly significant association with GL at *p* ≤ 0.01. Similarly, two markers (*GS3*-InDel and *GS5*-InDel1A) corresponding to two genes (*GS3* and *GS5*) were found to have a significant association with GL at *p* ≤ 0.05. In the case of GT, five markers (*GW8*-InDel, *GW8*-InDel1A, *GW8*-InDel2B, *GS2*-InDel1A, and *GS2*-InDel2B), corresponding to two genes (*GW8* and *GS2*), were found to have a significant association with GT (at *p* ≤ 0.01). Similarly, two markers (*GS3*-InDel and *GS7*-InDel), corresponding to genes *GS3* and *GS7*, were found to have a significant association with GT (at *p* ≤ 0.05).

For GW, five markers (*GW8*-InDel, *GW8*-InDel1A, *GW8*-InDel2B, *GS2*-InDel1A, and *GS2*-InDel2B), corresponding to genes *GW8* and *GS2*, respectively, were found to have a significant association with GW at *p* ≤ 0.01. Similarly, one marker (*GW5*-InDel), corresponding to gene *GW5*, was found to have a significant association with GW at *p* ≤ 0.1. For TGW, one marker (*GW8*-InDel2B), corresponding to gene *GW8* was found to have a significant association with GW at *p* ≤ 0.01. Similarly, three markers (*GW5*-InDel, *GS5*-InDel1A, and *GS5*-InDel2B), corresponding to two genes (*GW8* and *GS5*) were found to have a significant association with GW at *p* ≤ 0.05.

The *SLG7* gene is known to regulate the grain size in rice via increased cell division, longitudinally resulting in longer grains [21]. In the present study, the *SLG7*-InDel marker showed a significant (*p* ≤ 0.05) association with the thousand grain weight (TGW), which is also in agreement with the results of other researchers. This gene encodes the TONNEAU1-recruiting motif protein, which was found by many researchers [37] to be responsible for grain appearance by altering cell divisions, thus having significant effects in regulating grain weight, as well. Notably, GW5 gene was found to have a significant contribution in controlling GW and TGW (*p* ≤ 0.05), also revealed by previous studies [23–26], which showed that this gene encodes a calmodulin-binding protein and *GW5* physically interacts with calmodulin AsCaM1-1, which is responsible for grain width in rice. Recent studies have also demonstrated that this gene is responsible for regulating TGW (a significant correlation with *p* ≤ 0.05), as TGW is directly and highly associated with GW (Table 4), thereby confirming its utilization in grain yield improving objectives in rice breeding programs. Previous studies also identified this gene for controlling seed width and weight in rice [23,24,63].

Previous studies showed that *GS3* was among the first reported genes to have minor effects on grain thickness and width. The domains on its encoded protein have been reported to regulate cell divisions in the upper epidermis of the glume inside the rice seed, causing minor effects on cell size [29]. In the present study, the *GS3*-InDel marker was found to be significantly associated (*p* ≤ 0.05) with grain length and grain thickness, which is consistent with previous reports [20,28,31]. Another InDel marker for the *GS7* gene (*GS7*-InDel) was also found to be significantly (*p* ≤ 0.05) related with grain length and thickness. Our studies showed that the investigated alleles for both genes *GS3* and *GS7* affectively regulated grain length and thickness in the rice (Table 3). Previous studies [20,30] also reported that the germplasm carrying different alleles of the *GS3* gene with different allele combinations of *GS7* produced different grain lengths and thicknesses. Shao et al. [30] also reported that *GS7* is a strong QTL known to regulate grain size and controls grain length, roundness (thickness), and area (size) in rice. Ngangkham et al. [20] also found this gene to be associated with GL and GT, thereby playing a significant role in regulating grain size. For *GW2* gene, the results for gene associations with any trait were non-significant, but in previous studies, the gene was found to control the grain width in rice grains [12]. Ngangkham et al. [20] also found no association of this gene with any of these traits using STS (Sequence-Tagged Sites) markers, thus emphasizing the ineffectuality of the markers used. This might be due to inter- and/or intra-allelic interactions that may be subjected to further studies.

Among all the studied genes for grain size and weight, *GW8* was detected to represent a highly significant (*p* ≤ 0.01) association with all the grain size related traits, thereby suggesting its great importance in regulating grain size in rice. The *GW8* bearing genotypes were reported to have a higher grain length and grain length-width ratio [20]. The scanning results of the electron microscopy analysis

of the lemma in *GW8*.1 carrying NILs showed that the inner epidermal cell length was higher than the lines without this gene, indicating that *GW8*.1 might be responsible for regulating cell elongation [64]. *GW8* (OsSPL16) encodes a protein that is positively associated with cell proliferation [31]. Its higher expression promoted cell division and grain filling, consequentially increasing grain width and yield in rice. Another study also suggested that *GW8* suppresses the expression of the GW7 gene and plays a significant role in controlling grain size [21]. In the present study, *GW8* was determined to regulate grain length, width, and thickness. All three InDel markers successfully distinguished the germplasm into two alleles: the A-allele, which is responsible for shorter, thicker, and wider grains, and the B-allele, which carries genotypes possessing longer but narrower grains. This is due to the fact that grain length (GL) is negatively correlated with grain width (GW) and grain thickness (GT), as depicted by correlation analysis in Table 2. Based on these results, the B-allele carrying germplasm may be selected to breed longer grains, and the A-alleles may be screened for broader, shorter, and thicker grains. However, all three traits (i.e., GL, GT, and GW) contributed to the thousand grain weight (as suggested by the positive correlation between GL, GT, and GW with thousand grain weight), assuming that both alleles contribute to an increased yield. Two markers (i.e., *GW8*-InDel and *GW8*-InDel1A) were shown to have highly significant (*p* ≤ 0.0001) associations with GL, GT, and GW, thus indicating the ample potential of InDel markers in variation studies and genome-wide association studies. The third marker (i.e., *GW8*-InDel2B) for the *GW8* gene was also identified to have a highly significant (*p* ≤ 0.01) association with GL and GW (Table 5). However, unlike the other two InDel markers for the *GW8* gene, *GW8*-InDel2B also showed a highly significant (*p* ≤ 0.0001) relationship with GT and the thousand grain weight (Table 4), indicating its potential to be used for all four studied traits to improve the grain size and grain weight in rice.

Two InDel based markers were used for the *GS5* gene, and both of these markers showed a significant (*p* ≤ 0.05) association with only the thousand grain weight. These findings are partially inconsistent with other studies [32,65], which suggested that the *GS5* gene is associated with grain width and grain weight in rice. Previously, Lee et al. [63] attained three types of alleles by applying the markers generated from the promoter-region of the *GS5* gene, thus demonstrating the relatedness of this gene with grain weight. However, in another study, this gene was reported to participate in the regulation of grain length and grain width [63]. This might be due to higher genetic and/or allelic interactions with other genes/alleles that must be studied more comprehensively. This gene has been reported to have significant importance in regulating grain yield, as concluded by Li et al. [32], who showed that this gene encoded proteins—i.e., the putative serine carboxypeptidase executes its function as a positive regulator of a subset of the transition genes (G1-to-S) of the cell cycle, thereby causing increased cell divisions and resulting in enhanced grain filling and grain weight.

The Present study further explored the previously reported gene *GS2* to be highly associated (*p* ≤ 0.001) with all three-grain size related traits. Out of the three markers, two markers for this gene (including *GS2*-InDel1A and *GS2*-InDel2B) showed the potential for GL, GT, and GW, whereas the marker *GS2*-InDel showed no association with any trait (Table 4). For both markers 1A and 1B for this gene, the germplasm was separated into two groups carrying A- and B-alleles with different grain size traits. In the case of InDel1A, the A-allele was associated (*p* ≤ 0.001) with a shorter grain length (7.66 ± 0.820 mm) with thicker and wider grains, whereas its B-allele had a germplasm with a longer (8.44 ± 0.908 mm) grain length and narrower and slander grains (Table 5). Conversely, for the other marker, *GS2*-InDel2B, the A-allele was detected to relate to the germplasm with the longest grain lengths (9.32 ± 0.951 mm) and narrowest grains (the GW average is 2.65 ± 0.303 mm) in the whole germplasm, whereas the genotypes carrying the B-allele possessed shorter grains (7.81 ± 0.742 mm) and wider grains (3.01 ± 201 mm), as depicted in Table 4. This finding suggests that these InDel markers can further be investigated to breed for >9 mm grain lengths. Previous studies also showed that this gene directly controls two important grain size related traits, including grain width and grain length in rice. Researchers showed that its overexpression increased cell enlargement and enhanced cell division in the grain, thus producing longer and wider rice grains [33–36].

This study further investigated the favorable alleles in the studied germplasm to improve the grain length (>8 mm) with heavier grains. Identifying the beneficial alleles of the target traits is one of the most important prerequisites to improve modern cultivars via introgressions of favorable alleles from a vast gene pool using marker assisted selection approaches. This investigatory research discovered seven favorable alleles for grain length that can be utilized to improve grain size, while keeping in mind the recent criterion for longer grains with improved grain sizes and weights.

This research explored 7 genes and 11 InDel marker associations with grain size and weight related traits in rice. The present study further showed that InDel markers may be used efficiently in research investigations related to genetic variations, genome-wide association studies, germplasm genetic characterization, gene mapping, and other studies to further develop the ease and efficiency of breeding procedures and create more desirable varieties to cope with climate change and food security risks.
