*2.3. InDel Based Estimation of the Allelic Distribution of Fifteen Genes in the Determination of Grain Size and Weight*

A number of genes and their alleles in different combinations have been found to be involved in determining the ultimate size, shape, and weight of rice grains (also detected by previous researchers) [7–20]. An amplification profile of 14 Insertion/Deletion (InDel) markers of nine studied genes related to grain size and weight in 204 rice germplasms is given in Figure 5. A functional insertion–deletion (InDel) marker (sequences of reverse and forward primers are given in Table 8) was used to determine the allelic frequency of the *qPE9~1* gene in the germplasm. The InDel marker produced 270 bp and 350 bp fragments, which corresponded to the A- and B-allele, respectively (Figure 5). The results (Table 3) showed that A- and B-alleles were distributed in the germplasm with frequencies of 0.8922 and 0.1078 (i.e., ~89% and ~11% of the whole germplasm) in 364 and 44 genotypes, respectively. The frequencies of alleles in the genotypes are given in Table 3. The germplasm with an A-allele of this gene was observed to have a lower grain length (7.94 ± 0.920 mm), whereas the B-allele was found to control grain length (Table 4) in rice grains, as suggested by the longer grain length (8.24 ± 0.961 mm) in the germplasm possessing the allele. Entries with lower grain widths (2.95 ± 0.275 mm) retained the A-allele, whereas the B-allele was retained by germplasm with higher values of gain width (3.05 ± 0.209 mm). For the *GW2* gene, another InDel marker (Table 8) was used to determine its allelic distribution in the germplasm. The A-allele (labelled for the fragment at 500 bp) was present in 290 rice genotypes (with 0.7108 allelic frequency), whereas the B-allele (labelled for the fragment at 520 bp) was present in 116 entries, with an allelic frequency of 0.2843 (Table 3). The A-allele was observed in the germplasm having grains with a lower grain width (2.95 ± 0.273 mm), whereas the B-allele was found in the germplasm with more grain width (3.02 ± 0.253 mm). However, the results for the gene associations with any traits were non-significant.

The InDel marker *SLG7*-InDel (Table 8) was used to determine the allelic distribution of *SLG7* in the 204 rice germplasms. Two alleles (A and B, designated for bands at 450 bp and 500 bp, respectively) successfully divided all the germplasms into two groups: the A-allele group (308 entries) and the B-allele group (100 entries), with allelic frequencies estimates of 0.7549 (~75% of the total population) and 0.2451 (~24% of the total population), respectively (Table 3). It was further shown that both the alleles have no effect in regulating grain shape (i.e., GL, GT, and GW) (Table 4). The results depicted in Table 4 show that the gene is significantly (*p* ≤ 0.05) associated with TGW. Two alleles of the *GW5*

gene, the A- and B-alleles (DNA fragments at 450 bp and 500 bp, respectively), were distinguished by another insertion–deletion (InDel) marker, *GW5*-InDel (Table 8), used to investigate the allelic frequencies of both alleles in the target germplasms of 204 rice lines. Both alleles were found in germplasm with different frequencies (Table 3). The A-allele was present in 294 genotypes, and the B-allele was found in 114 genotypes, with estimated frequencies of 0.7206 and 0.2794, respectively. Notably, the gene was found to have a significant (*p* ≤ 0.05) contribution in controlling GW and TGW. Two alleles (the A-allele labelled for the DNA fragment at 650 bp and the B-allele at 750 bp) were observed for the *GS3* gene by using an InDel marker, *GS3*-InDel (Table 8), which distinguished all the germplasms into groups, with A- and B-alleles in 204 germplasms with the frequencies of 69.6% and 30.4%, respectively, in 284 and 124 rice genotypes (Table 3). The gene was also found to have a significant (*p* ≤ 0.05) association (Table 4) with GL and GT, as indicated in Table 4, which shows that the A-allele of this gene was found in the germplasm with a less average GL (7.90 ± 0.926 mm) and thicker (2.14 ± 0.166 mm) grains, while the B-allele was present in genotypes with longer grains (8.16 ± 0.917 mm) and a low average GT (2.11 ± 0.159). The *GS7*-InDel marker (Table 8) was used to determine allelic frequencies in the subject population for the *GS7* gene. This InDel marker divided all germplasms into three groups: a group containing A-alleles and B-alleles only and a third one possessing both A- and B-alleles, which were distributed among the germplasms with different frequencies (0.4412, 0.3088, and 0.2451 respectively), present in 90, 63, and 50 entries, respectively (Table 3). It was further observed that the A-allele (DNA band labelled at 200 bp) was present in 56.4% of the total germplasm, whereas the B-allele (at 250 bp) was present in 43% of the total germplasm under study (Figure 5). Interestingly, both the A- and B-alleles showed a significantly higher mean grain length (8.19 ± 0921 and 8.08 ± 0.828 respectively) separately. However, in combination (AB), both alleles showed a lower mean grain length (7.57 ± 0.974). These studies, based on 204 germplasms, also showed that the gene has significant (*p* ≤ 0.05) associations with two grain size traits (i.e., GL and GT), indicating its contribution in regulating grain size in rice.

An InDel marker, *GW8*-InDel (Table 8), was applied to distinguish all germplasms into two groups carrying A- and B-alleles, as designated by DNA fragments/bands at 350 bp and 450 bp, respectively (Figure 5). The A-allele was found to be present in 29.9% (122 genotypes) and the B-allele was present in the remaining 62.8% (256 genotypes) of the total germplasms (Table 3). Remarkably, the B-allele had a significantly higher mean grain length (8.41 ± 0.890), a significantly lower grain width (2.92 ± 0.259), and a lower grain thickness (2.09 ± 0.118) compared to the A-allele, which showed a lower mean grain length (7.34 ± 0.578), a significantly higher grain width (3.05 ± 0.203), and a higher thickness (2.27 ± 0.133) in the studied 204 germplasms. Based on the *GW8*-InDel marker loci in the *GW8* gene, the gene was found to be strongly associated (*p* ≤ 0.01) with three grain size traits (i.e., GL, GW, and GT) (Table 4). However, no allelic associations were observed in the case of TGW in this study. Likewise, another InDel marker, *GW8*-InDel1A was used for the same germplasm. It also separated the whole germplasm into two groups carrying A- (350 bp) and B-alleles (450 bp), with a frequency of 31.1% (127 genotypes) and 66.4% (271 genotypes), respectively. The results show that the B-allele has a significantly higher average gain length (8.40 ± 0.901) and a lower grain width (2.91 ± 0.260) and grain thickness (2.09 ±0.118) compared to the A-allele, which showed a lower average grain length (7.42 ± 0.667), a significantly higher grain width (3.06 ± 0.231), and an average grain thickness (2.27 ± 0.169) in the studied genotypes. TGW showed no changes due to these alleles. A third InDel marker, *GW8*-InDel2B, was also used for further studies, as it separated the whole germplasm into A-allele (at 270 bp) with a lower frequency (46%) and the B-allele (at 300 bp) with frequency (51.9%) that was found in 94 and 106 genotypes out of the 204 genotypes (Figure 5). These results show that B-allele has a significantly higher grain length (8.35 ± 0.852), a lower grain width (2.92 ± 0.274), and a lower grain thickness (2.10 ± 0.129) compared to the A-allele, which showed a lower average grain length (7.64 ± 0.965) and a higher average grain width (3.00 ± 0.238) in the studied genotypes. Unlike other InDel markers, *GW8*-InDel2B also distinguished the germplasm into two groups for the thousand grain weight, with the A-allele having a significantly higher TGW (26.0 ± 2.824) and the B-allele with a

lower average TGW (25.0 ± 2.611). For all three InDel markers, the same results were observed for GL, GT, and GW, showing a very strong association (*p* ≤ 0.01), whereas, in the case of TGW, only one marker (*GW8*-InDel2B) showed a strong and highly significant (*p* ≤ 0.01) association, as depicted in Table 4. A-alleles of this gene should be considered while improving the GW, GT, and TGW in rice, whereas the B-alleles should be considered when breeding for longer grains.

Another QTL *GS5* has already been reported to regulate grain size in rice via grain filling and weight [32]. In this study, two InDel markers, *GS5*-InDel1A and *GS5*-InDel1B (Table 8), were applied to determine the contribution of the affective alleles for the *GS5* gene in the germplasm. The InDel1A marker yielded two alleles, A (500 bp) and B (550 bp), with a frequency of 181 (44.3%) and 215 (52.7%), respectively (Table 3). The other InDel marker, *GS5*-InDel2B, also yielded two alleles, A (500 bp) and B (550 bp), with a frequency of 101 (24.5%) and 295 (72.3%), respectively. Both these markers had no observable association with the grain size traits in this study, except the thousand grain weight (TGW). The B-alleles of both InDel markers (i.e., *GS5*-InDel1A and *GS5*-InDel1B) were found to control the heavier grains (Table 4), as indicated by the significantly higher average TGW values (27.0 ± 2.535 and 26.2 ± 2.899, respectively) of the genotypes possessing B-alleles, whereas the germplasm with the other allele (A-allele) showed a lower average TGW for both markers (24.0 ± 3.180 and 23.0 ± 2.729, respectively). Likewise, in the case of the *GS5*-InDel1A makers, the A-allele was found to be present in 90 germplasms with a lower average grain length (7.79 ± 0.921 mm), and B-allele was found in 107 genotypes with a longer grain length (8.16 ± 0.925 mm). For the marker *GS5*-InDel2B, the A-allele was present in only seven genotypes with shorter grains (7.40 ± 0.900 mm) and in the B-allele in the larger portion of germplasm (185 genotypes) with a longer grain length (8.24 ± 0.934 mm), as represented in Table 4. The study further revealed that there is a strong (*p* ≤ 0.05) association of the *GS5* gene for both the InDel markers with TGW.

For the *GS2* gene, three InDel markers (i.e., *GS2*-InDel, *GS2*-InDel1A, and *GS2*-InDel2B (Table 8)) were used to determine the different alleles for the *GS2* gene in the germplasm of 204 entries. Two alleles, A and B, were determined for the DNA fragments at 400 bp and 500 bp, respectively, which distinguished the whole germplasm into two groups: one having 16% (65 entries) germplasm of the total (with A-alleles) and other having 83% of the total (339 entries) (with B-alleles). However, no significant associations were detected for these alleles with regards to grain size and weight related traits, suggesting the inefficiency of this marker in distinguishing this gene in the rice germplasm under study. The other InDel marker, *GS2*-InDel1A, distinguished germplasm into two groups: one with A-alleles (designated for the band at 400 bp), comprising 52.7%, and one with B-alleles (at 500 bp), comprising 45.3% of the total germplasm. Genotypes possessing B-alleles showed a significantly higher average grain length (8.44 ± 0.908), a significantly lower grain width (2.84 ± 0.275), and a lower grain thickness (2.09 ± 0.126); whereas genotypes with A-alleles showed a significantly lower average grain length (7.66 ± 0.820), as well as wider and thicker grains compared to the rest of the germplasm. No associations were observed for the TGW marker in the present study. The third marker, *GS2*-InDel2B, also distinguished A- and B-alleles, labelled for the bands at 250 bp and 300 bp, respectively, and present in 24.7% and 72.3% of the total germplasm (Figure 5). This marker separated the genotypes into two groups: one with genotypes possessing A-alleles with a significantly higher average grain length (9.32 ± 0.951), a significantly lower grain width (2.65 ± 0.303), and a lower grain thickness (2.08 ± 0.153), and the other group consisting of genotypes with B-alleles with a significantly lower average grain length (7.81 ± 0.742) and wider and thicker grains with a higher average grain width (3.01 ± 0.201) compared to the rest of the germplasm (Table 3). This gene was found to have a strong and highly significant (*p* ≤ 0.01) association with GL, GT, and GW, thereby revealing its importance in regulating all these traits in rice grains (Table 4). *GS2* showed no association with TGW, indicating that this gene is responsible only for regulating the grain size in the studied germplasm. These results showed that, for longer grains, the B-allele should be taken into consideration for the InDel1A marker, and the A-allele should be considered for the InDel2B marker.


**Table 3.** Fragment lengths on electrophoresis gel, expected frequency estimates, allelic variance, and standard deviation (SD) estimates of each allele of the 14 InDel loci.



*Int. J. Mol. Sci.* **2019**, *20*, 4824

**Figure 5.** *Cont.*

**Figure 5.** Amplification profile of the 14 Insertion/Deletion (InDel) markers of nine genes related to grain size and weight in 204 rice germplasms. M1 represents a 2000 base pair (bp) DNA ladder, 1–24 represent the rice germplasm. The size of the DNA fragment is depicted on the right side of the gel picture.

#### *2.4. Favorable Alleles of Studied Genes for Gene Pyramiding*

In the present study, a total of 38 alleles were identified. Out of these, only 27 alleles were major alleles (genotype frequency ≥0.2). Alleles present in less than 20 genotypes were considered minor alleles. Major alleles were further classified into favorable and non-favorable categories. A total of seven alleles were found to be favorable/beneficial for improving the grain length (>8 mm), having a better TGW, followed by GT and GW, respectively (Table 5). The individual role of the nine studied genes for improving grain size (GL, GW, and GT) and weight (TGW) were estimated, and the combined impact of these favorable alleles was comprehensively analyzed. ANOVA was used to test the difference between the germplasms possessing favorable alleles and non-favorable alleles in the studied germplasms (Table 5). The frequency of the favorable alleles was recorded to be in the range of 24.7% to 66.4%, with the highest for the gene *GW8* (with marker InDel1A), followed by *GW8* (InDel), *GS7* (InDel), *GW8* (InDel2B), *GS2* (InDel1A), *GS3* (InDel), and *GS2* (InDel2B), as depicted in Figure S1. All the favorable alleles, except *GS3* (InDel) and *GW8* (InDel2B), were observed to contribute to the grain length (GL > 8 mm) and thousand grain weight (Table 5). As GL and TGW are positively correlated, these genotypes may be utilized in pyramiding the target genes for longer and heavier grains to simultaneously improve the yield and quality. Average GL of the cumulated FAs were found to be higher compared with the average GL of the cumulated N-FAs depicted in Table 5.


Pyramiding of the favorable alleles of the studied genes for Grain Length (GL).

**Table**

**5.**
