Panicle Traits in the CSSL population
In order to identify new genetic factors governing the panicle branching diversity observed between O. sativa cv. Caiapó (referred to as Os_Caiapó hereafter) and O. glaberrima cv. MG12 (referred to as Og_MG12 hereafter), phenotypic measurements were performed on a population of 60 BC3DH CSSLs and their parents (Additional file1: Table S1) (Gutiérrez et al. 2010).
Six quantitative traits were evaluated per panicle: rachis length (RL); primary, secondary and tertiary branch numbers (PBN, SBN, TBN respectively); primary branch length average (PBL); and spikelet number (SpN) (Table 1, Additional file1: Table S2). The two parents showed contrasting panicle phenotypes with a higher panicle complexity observed in Os_Caiapó compared to Og_MG12. More specifically, the Os_Caiapó parent displayed panicles with more PBN and SBN leading to a higher SpN compared to Og_MG12 (Fig. 1A). In contrast, the Og_MG12 parent produced a panicle with longer PBs compared to Os_Caiapó. Since the trials were conducted as two repeated experiments, we computed broad sense heritability for each experiment (Additional file1: Table S3). All the measured variables showed broad sense heritability values higher than 0.8 except for TBN. For all traits, the mean of the CSSL population was similar to the mean of the recurrent Os_Caiapó parent (Additional file1: Table S4). The coefficient of variation for the SBN/PBN ratio was higher in the CSSL population than in Os_Caiapó (26.10% versus 18.26%). Histograms of the CSSLs for panicle traits showed a continuous distribution for all traits except TBN (Fig. 1B). The observed distributions were similar between the two repeated experiments. The abnormal distribution of the TBN trait was associated with a high value of CV (Coefficient Variation) and a low heritability value. This observation probably results from the rare and unstable nature of this trait which is dependent on environmental conditions and does not appear frequently in the Os_Caiapó parent. Moreover, the formation of tertiary branches has not been described in O. glaberrima populations (Cubry et al., 2020), nor was it observed in our earlier experiments.
By analyzing relationships between the different panicle traits, we observed a strong correlation (0.95) between SpN and SBN (Fig. 2A). Other traits were moderately correlated with each other, notably SBN and PBN. We also observed a low correlation between PBL and SBN. Principal component analysis showed a separate distribution of Os_Caiapó and Og_MG12 parents on the PC1 and PC2 axes. In contrast, we observed an overlapping of the BC3DH lines with the Os_Caiapó recurrent parent (Fig. 2B). Analysis of variable contributions showed that SpN is the main trait contributing to the diversity observed in this population.
Detection of QTLs associated with panicle architecture traits
The Dunnett's test revealed a total of 37 lines with significant differences compared to the Os_Caiapó recurrent parent for all traits combined (Additional file1: Table S5). In general, these lines bear at least two Og_MG12 introgression fragments. For this reason, assigning the underlying QTL to a single chromosome segment was not straightforward. The CSSL Finder software was therefore used to detect genomic regions associated with panicle phenotype variation with a F-test value at each marker (Gutiérrez et al., 2010). Graphical genotype representations were also produced in which the CSSLs were ordered by trait value for each evaluated trait (Additional file 2: Figure S1). We considered the F-test as significant when its value was higher than 10.0. QTLs were assigned to the Og_MG12 introgressed regions of these CSSLs if the F-test was significant and confirmed by the graphical genotype logical analysis.
In total, 15 QTLs were detected for all combined traits analyzed with the exception of rachis length (Fig. 3). The F-test value of the markers allowing the detection of each QTL are reported in Table 2 along with their positions in the O. sativa and O. glaberrima reference genomes (cv. Nipponbare IRGSP-1.0 and cv. CG14 OglaRS2 respectively).
Table 2
QTLs for morphological panicle traits detected in the CSSL population
Traits | ID_QTL | Chromosome | SSR Marker | F-Test value | significance F-Test | Position (bp) in O. sativa Caiapo | Position (bp) in O. glaberrima MG12 | Position (bp) in O. sativa MSU 7 | Position (bp) in O. glaberrima OMAPv2 | Representative CSSL | Positive allele | Known genes related to branching and flowering (Locus_id, Gene Symbol Annotation and doi) |
PBN | qPBN1 | 1 | RM472 | 3.48 | NS | 38 918 631 | 35 483 127 | 37 890 275 | 34 691 456 | L_10 | Og | LOC_Os01g66030, OsMADS2;10.1186/s12864-015-1349-z |
| | | RM165 | 12.56 | * | 41 165 563 | 37 610 087 | 40 107 107 | 36 797 582 | L_46 | Og | LOC_Os01g66290, OsMADS21;10.1105/tpc.111.087007 |
| | | RM104 | 7.92 | NS | 41 226 405 | 37 667 912 | 40 167 993 | 36 855 255 | | | LOC_Os01g67430, EG1;10.1111/j.1365-313X.2008.03710.x |
| qPBN2 | 2 | RM191 | 0.01 | NS | 13 357 683 | 13 118 242 | 13 367 915 | 13 091 268 | L_10 | Og | LOC_Os02g32950, RCN2; 10.1046/j.1365-313x.2002.01255.x |
| | | RM341 | 15.49 | * | 19 822 576 | 19 710 485 | 19 342 082 | 19 060 676 | L_46 | Og | LOC_Os02g34260, ERF98 |
| | | RM262 | 7.87 | NS | 21 260 857 | 21 173 898 | 20 800 911 | 20 552 457 | | | |
| qPBN7 | 7 | RM10 | 8.88 | NS | 22 227 559 | 20 124 022 | 22 189 175 | 19 801 671 | L_10 | Og | LOC_Os07g39820, OsSHR1; doi: 10.1016/j.ydbio.2017.03.001 |
| | | RM351 | 16.44 | * | 23 991 049 | 21 742 091 | 23 926 002 | 21 416 312 | L_46 | Og | LOC_Os07g39320, OsHOX14 |
| | | RM234 | 7.08 | NS | 25 545 695 | 23 016 334 | 25 473 749 | 22 717 218 | | | LOC_Os07g38130, OsFOR1;10.1023/B:PLAN.0000006940.89955.f1 |
| | | | | | | | | | | | LOC_Os07g41370, OsMADS18; doi: 10.1093/jxb/erz198 |
| | | | | | | | | | | | LOC_Os07g42410, EP2/DEP2; doi: 10.1038/cr.2010.69 |
PbL | qPBL1 | 1 | RM315 | 3.91 | NS | 37 748 125 | 34 409 891 | 36 735 198 | 33 646 637 | L_10 | Og | LOC_Os01g66030, OsMADS2 |
| | | RM472 | 12.23 | * | 38 918 631 | 35 483 127 | 37 890 275 | 34 691 456 | L_46 | Og | LOC_Os01g66290, OsMADS21 |
| | | RM165 | 22.55 | * | 41 165 563 | 37 610 087 | 40 107 107 | 36 797 582 | | | LOC_Os01g67430, EG1;10.1111/j.1365-313X.2008.03710.x |
| | | RM104 | 14.08 | * | 41 226 406 | 37 667 913 | 40 167 994 | 36 855 255 | | | LOC_Os01g63510, QHB;10.1046/j.1365-313X.2003.01816.x |
| | | RM3362 | 11.51 | * | 44 150 041 | 40 246 380 | 43 044 090 | 39 436 568 | | | LOC_Os01g64660, MOC2;10.5511/plantbiotechnology.12.1210a |
| | | | | | | | | | | | LOC_Os01g66100, SD1;10.1038/416701a |
| | | | | | | | | | | | LOC_Os01g69210, OsMADS51;10.1104/pp.107.103291 |
| qPBL3 | 3 | RM55 | 1.08 | NS | 30 677 384 | 28 677 802 | 29 052 298 | 28 518 740 | L_10 | Og | LOC_Os03g51690, OsH1; 10.1105/tpc.5.9.1039–10.1006/dbio.2000.9624 |
| | | RM3525 | 14.51 | * | 32 028 705 | 30 028 933 | 30 393 958 | 29 873 318 | L_46 | Og | LOC_Os03g51970, OsGRF;10.1104/pp.114.235564 |
| | | RM7000 | 12.89 | * | 35 524 810 | 33 277 548 | 33 802 689 | 33 128 938 | | | LOC_Os03g54160, OsMADS14;10.1105/tpc.112.097105 |
| | | RM227 | 1.08 | NS | 36 679 424 | 34 296 048 | 34 932 380 | 34 146 855 | | | LOC_Os03g54170, OsMADS34/PAP2;10.1093/pcp/pcp166–10.1104/pp.110.156711 |
| | | | | | | | | | | | LOC_Os03g59550, OsY14b;10.1104/pp.114.237958 |
| | | | | | | | | | | | LOC_Os03g60430, OsIDS1;10.1111/j.1365-313X.2011.04804.x |
| qPBL7 | 7 | RM10 | 8.14 | NS | 22 227 559 | 20 124 022 | 22 189 175 | 19 801 671 | L_10 | Og | LOC_Os07g39820, OsSHR1; 10.1016/j.ydbio.2017.03.001 |
| | | RM351 | 15.78 | * | 23 991 049 | 21 742 091 | 23 926 002 | 21 416 312 | L_46 | Og | LOC_Os07g39320, OsHOX14 |
| | | RM234 | 8.8 | NS | 25 545 695 | 23 016 334 | 25 473 749 | 22 717 218 | | | LOC_Os07g38130, OsFOR1;10.1023/B:PLAN.0000006940.89955.f1 |
| | | | | | | | | | | | LOC_Os07g41370, OsMADS18;10.1093/jxb/erz198 |
| | | | | | | | | | | | LOC_Os07g42410, EP2/DEP2;10.1038/cr.2010.69 |
SBN | qSBN/PBN11 | 11 | RM21 | 1.14 | NS | 20 161 293 | 19 279 722 | 19 639 289 | 18 598 655 | L_42 | Os | LOC_Os11g38270, FON2;10.1093/pcp/pcl025 |
| | | RM206 | 13.74 | * | 22 961 364 | 21 790 549 | 22 480 895 | 21 092 821 | L_55 | Og | |
| | | RM254 | 15.09 | * | 24 651 269 | 23 182 998 | 24 230 511 | 22 477 748 | L_56 | Og | |
| | | RM224 | 9.2 | NS | 29 246 818 | 25 784 047 | 27 673 312 | 25 499 087 | | | |
| qSBN/PBN12 | 12 | RM28130 | 9.09 | NS | 16 565 178 | 15 215 344 | 16 706 323 | 15 275 476 | L_42 | Og | LOC_Os12g31748, OsMADS20 |
| | | RM277 | 14.84 | * | 21 403 653 | 20 209 650 | 22 364 793 | 20 270 371 | L_55 | - | LOC_Os12g41060, ERF112 |
| | | RM235 | 2.69 | NS | 25 324 296 | 23 473 458 | 26 141 561 | 23 604 232 | L_56 | - | LOC_Os12g40900, IAA31;10.1111/j.1365-313X.2006.02693.x |
SpN | qSpN3 | 3 | RM60 | 10.8 | * | 109 164 | 86 928 | 106 972 | 77 908 | L_55 | Og | |
| | | RM22 | 9.45 | NS | 1 561 917 | 1 493 255 | 1 520 630 | 1 485 562 | L_11 | Og | |
| | | | | | | | | | L_26 | Os | |
| qSpN11 | 11 | RM21 | 1.14 | NS | 20 161 293 | 19 279 722 | 19 639 289 | 18 598 655 | L_55 | Og | LOC_Os11g38270, FON2;10.1093/pcp/pcl025 |
| | | RM206 | 13.74 | * | 22 961 364 | 21 790 549 | 22 480 895 | 21 092 821 | L_11 | Os | |
| | | RM254 | 15.09 | * | 24 651 269 | 23 182 998 | 24 230 511 | 22 477 748 | L_26 | Og | |
| | | RM224 | 9.2 | NS | 29 246 818 | 25 784 047 | 27 673 312 | 25 499 087 | | | |
TBN | qTBN1 | 1 | RM226 | 0.31 | NS | 35 105 017 | 31 957 160 | 34 034 101 | 31 200 058 | L_10 | Og | LOC_Os01g61480, LAX1;10.1073/pnas.1932414100 |
| | | RM265 | 22.67 | * | 36 243 983 | 33 029 536 | 35 197 671 | 32 275 011 | L_4 | Og | LOC_Os01g62920, qSH1|RIL1;10.1126/science.1126410 |
| | | RM315 | 6.09 | NS | 37 748 125 | 34 409 891 | 36 735 198 | 33 646 637 | L_46 | | LOC_Os01g63510, QHB;10.1046/j.1365-313X.2003.01816.x |
| | | RM472 | 31.11 | * | 38 918 631 | 35 483 127 | 37 890 275 | 34 691 456 | | | LOC_Os01g64660, MOC2;10.5511/plantbiotechnology.12.1210a |
| | | RM165 | 4.62 | NS | 41 165 563 | 37 610 087 | 40 107 107 | 36 797 582 | | | LOC_Os01g66030, OsMADS2;10.1186/s12864-015-1349-z |
| | | | | | | | | | | | LOC_Os01g66290, OsMADS21;10.1105/tpc.111.087007. |
| | | | | | | | | | | | LOC_Os01g67430, EG1;10.1111/j.1365-313X.2008.03710.x |
| qTBN3-1 | 3 | RM175 | 1.93 | NS | 3 911 091 | 3 633 683 | 3 866 833 | 3 625 881 | L_10 | Og | LOC_Os03g10620, d88;10.1007/s00344-011-9228-6 |
| | | RM7576 | 21.91 | * | 6 107 599 | 5 772 156 | 6 079 319 | 5 767 071 | L_4 | Og | LOC_Os03g11614, OsMADS1/LHS1;10.1111/j.1365-313X.2009.04101.x − 10.1105/tpc.12.6.871 |
| | | RM7 | 0.27 | NS | 9 810 142 | 9 300 611 | 9 829 641 | 9 280 539 | L_46 | | |
| qTBN3-2 | 3 | RM227 | 0.72 | NS | 36 679 424 | 34 296 048 | 34 932 380 | 34 146 855 | L_10 | Og | LOC_Os03g61760, OsSPL6;10.1631/jzus.B2000519 |
| | | RM148 | 15.58 | * | 37 552 877 | 35 129 500 | 35 843 050 | 34 980 430 | L_4 | Og | |
| | | RM85 | 0.72 | NS | 38 025 225 | 35 613 693 | 36 348 204 | 35 464 999 | | | |
| qTBN5 | 5 | RM159 | 6.76 | NS | 496 697 | 433 400 | 488 155 | 404 817 | L_4 | Og | LOC_Os05g03040, RSR1; 10.1104/pp.110.159517 |
| | | RM267 | 25.12 | * | 2 871 237 | 2 517 535 | 2 881 410 | 2 485 558 | | Os | LOC_Os05g03884, OSH71|Oskn2;10.1006/dbio.2000.9624 |
| | | RM194 | 25.12 | * | 5 179 705 | 4 653 443 | 5 329 987 | 4 472 981 | | | LOC_Os05g11414, OsMADS58;10.1105/tpc.111.087262 |
| | | RM169 | 0.96 | NS | 7 361 060 | 6 805 643 | 7 498 060 | 6 637 671 | | | |
| qTBN7 | 7 | RM11 | 0.1 | NS | 19 315 836 | 17 468 875 | 19 257 970 | 17 138 872 | L_10 | Og | LOC_Os07g38130, OsFOR1;10.1023/B:PLAN.0000006940.89955.f1 |
| | | RM10 | 12.61 | * | 22 227 559 | 20 124 022 | 22 189 175 | 19 801 671 | L_4 | Og | LOC_Os07g39820, OsSHR1; 10.1016/j.ydbio.2017.03.001 |
| | | RM351 | 17.42 | * | 23 991 049 | 21 742 091 | 23 926 002 | 21 416 312 | L_46 | | LOC_Os07g39320, OsHOX14 |
| | | RM234 | 66.31 | ** | 25 545 695 | 23 016 334 | 25 473 749 | 22 717 218 | | | LOC_Os07g41370, OsMADS18;10.1093/jxb/erz198 |
| | | RM18 | 62.09 | ** | 25 725 478 | 23 182 832 | 25 653 583 | 22 884 015 | | | LOC_Os07g42410, EP2/DEP2;10.1038/cr.2010.69 |
| | | RM134 | 26.55 | * | 26 708 250 | 24 069 776 | 26 637 574 | 23 770 479 | | | LOC_Os07g47330, FZP;10.1186/1471-2229-3-6–10.1242/dev.00564 |
| | | RM118 | 33.95 | * | 26 708 384 | 24 069 876 | 26 637 674 | 23 770 550 | | | LOC_Os07g48560, WOX11; 10.1105/tpc.108.061655 |
| | | RM420 | 0.09 | NS | 29 538 599 | 26 687 759 | 29 432 340 | 26 361 122 | | | |
RL trait: 13 CSSLs showed significant changes in RL value in comparison to the Os_Caiapó recurrent parent (Additional file1: Table S5). However, no QTLs were detected for this trait (Additional file2: Figure S1).
SBN/PBN trait: a total of 16 CSSLs showed a significant difference in secondary branch number per primary branch (SBN/PBN ratio) compared to the Og_MG12 parent (Additional file 1: Table S5). Two QTLs associated with a decreased SBN/PBN ratio were detected (qSBN/PBN11 and qSBN/PBN12, maximum F-test scores of 15.09 and of 14.94 respectively) (Table 2; Additional file2: Figure S1). The average SBN/PBN ratio values were 3.0 for Os_Caiapó and of 1.7 for Og_MG12, corresponding to a decrease of 42% in Og_MG12 relative to Os_Caiapó. The CSSLs L_42, L_55 and L_56 showed a decrease of 44.7%, 42.4% and 36.3% respectively relative to the Os_Caiapó parent, suggesting a high effect of the Og_MG12 introgression(s) in these lines (Additional file1: TableS5). The phenotyping of these lines was repeated for a second year to confirm panicle trait variation (Additional file3: FigureS2). The L_55 and L_56 lines contain an Og_MG12 segment in chromosome 11, but with missing marker information at the position of qSBN/PBN12. In contrast, the CSSL L_42 displays only an Og_MG12 segment in chromosome 12 (Additional file3: Figure S2).
SpN trait: a total of 12 CSSLs showed a significant difference compared to the Os_Caiapó recurrent parent (Additional file 1: Table S5). Two QTLs (qSpN3 and qSpN11) were associated with a decreased SpN with maximum F-test scores of 10.8 and 15.1 respectively (Table 2; Additional file2: Figure S1). CSSLs L_55, L_11 and L_26 showed decreases of 37.9%, 32.3% and 30.8% respectively compared to the Os_Caiapó parent (Additional file1: Table S5). Lines L_55 and L_26 contain Og segments in both chromosomes 3 and 11 in the positions of the detected QTLs (Additional file3: Figure S2). Line L_11 contains only one Og_MG12 introgression in chromosome 11, related to qSpN11 (Additional file 3: Figure S2).
PBN trait: a total of 13 CSSLs had significantly different PBN values compared to the Os_Caiapó recurrent parent (Additional file1: Table S5). Three QTLs corresponding to a decreased PBN (qPBN1, qPBN2 and qPBN7) with maximum F-test scores of 12.56, 15.49 and 16.44 were detected on chromosomes 1, 2 and 7 respectively (Table 2; Additional file2: Figure S1). Average PBN values were 11.9 for Os_Caiapó and 8.2 for Og_MG12 (Additional file1: Table S5). The PBN values of two CSSLs (L_10 and L_46) differed significantly from the Os_Caiapó recurrent parent with a reduction of 24.1% and 20.9% respectively (Additional file 1: Table S5). These 2 CSSLs harbor similar Og_MG12 segments in chromosomes 1, 2 and 7 (Additional file3: Figure S2).
PBL trait: Fourteen CSSLs exhibited significant differences with the Os_Caiapó recurrent parent for the PBL trait. Three QTLs corresponding to an increased PBL (qPBL1, qPBL3 and qPBL7) with maximum F-test scores of 22.55, 14.51 and 15.78 were detected on chromosomes 1, 3 and 7 respectively (Table 2; Additional file2: Figure S1). Average PBL values were 11.1 for Os_Caiapó and 12.3 for Og_MG12 (Additional file1: Table S4). Two CSSLs (L_10 and L_46) showed increased PBL values of 19% and 12.8% respectively in comparison to the Os_Caiapó recurrent parent and harbored similar Og_MG12 segments in chromosomes 1, 3 and 7 (Additional file1: Table S5; Additional file3: Figure S2).
TBN trait: Surprisingly, several CSSLs showed a significantly increased TBN value in our field conditions (Additional file1: Table S5) and five QTLs were detected: qTBN1, qTBN3-1, qTBN3-2, qTBN5 and qTBN7 with maximum F-test scores of 31.11, 21.91, 15.98, 25.12 and 66.09 respectively (Table 2; Additional file2: Figure S1). The L_10 and L_4 lines have similar Og_MG12 segments in chromosomes 1, 3 and 7. Within chromosome 5, only the L_4 line contains a segment from the Og_MG12 parent (Additional file3: Figure S2). As the L_46 line contains otherwise similar Og_MG12 introgressions in chromosome 1, 3 and 7, this CSSL was included in the second phenotyping campaign, in which it revealed the presence of tertiary branches on its panicles in contrast to L_4 (Additional file3: Figure S2). Overall, these results support an association of Og_MG12 introgressions with the presence of tertiary branches.
For the PBN, PBL and TBN panicle traits, the same BC3DH CSSLs (L_10 and L_46) led to the detection of several QTLs. The 2 lines in question display a similar panicle phenotype with a higher number of tertiary branches associated with a decreased PBN and increased PBL and SBN/PBN ratio values compared to the Os_Caiapó recurrent parent (Fig. 4A, B). The L_10 and L_46 BC3DH lines contain a complex association of Og_MG12 introgressions in their genomes (Fig. 4C). For clarification, the regions corresponding to colocalized QTLs, associated with different traits, were renamed without specifying the associated traits: q_1 (for qPBL1, qPBN1 and qTBN1); q_2 (for qPBN2), q_3 − 1 (for qTBN3-1); q_3 − 2 (for qPBL3 and qTBN3-2); q_5 (for qTBN5); and q_7 (for qTBN7, qPBN7 and qPBL7).
Substitutions of Os_Caiapó genomic regions by corresponding Og_MG12 segments can result in an added value panicle phenotype
As the altered phenotype observed in lines L_10 and L_46 was associated with multiple substitution segments (Fig. 4C), it was critical to determine whether this phenotype, and notably the formation of tertiary branches, involved one or several QTLs. Thus, different BC4F3:5 lines were obtained from the BC3DH lines by backcrossing and self-pollination (Fig. 4C), after which homozygosity was checked using the SSR markers carried by the introgressed Og_MG12 segments present in the BC3DHs. Panicle traits were phenotyped for five BC4F3:5 lines containing the different individual O. glaberrima introgressed segments affecting TBN, together with Os_Caiapó and Og_MG12 (Fig. 4B; Additional file1: Table S1). A second phenotypic campaign was carried out for validation of the BC4 lines L_B, L_D and L_E (Additional file4: Figure S3).
Divergent panicle traits compared to the Os_Caiapó parent were observed in all BC4 lines except for L_C (Fig. 5B). The latter contains an introgression at the beginning of q_7 (RM11 to RM10), suggesting that this region does not influence panicle architecture. All BC4 lines except L_C showed longer primary branches compared to the Os_Caiapó parent, suggesting that introgressions in chromosomes 1, 3 and 7 could independently influence primary branch length (Fig. 5A). The L_A line, containing Og_MG12 regions corresponding to q_1, q_2 and q_3 − 2, also showed a decreased PBN value associated with a higher SBN/PBN ratio. However, no tertiary branches were observed. This suggests that the association of the q_1, q_2 and q_3 − 2 regions could induce a reduction in PBN associated with an increased SBN.
Line L_B containing only one Og_MG12 introgression (from RM60 to RM7) in place of q_3 − 1 showed increased values for four panicle traits (PBN, PBL, SBN and SpN) but not for TBN. However, these results were not observed during the second phenotyping of this line, except for an increase in PBL. Thus, it can be deduced that the q_3 − 1 region positively influences primary branch length in a manner that is independent of the other detected QTLs.
Finally, the BC4 lines L_D and L_E were the only ones observed to develop tertiary branches (Fig. 5A). In both lines, this phenotype is associated with an increased SBN/PBN and a longer PBL. The higher SBN/PBN ratio is associated with a decreased PBN in L_D, meaning that in this line more secondary branch meristems are established during panicle development. Similar results for SBN/PBN and TBN were observed in the second round of phenotyping, with a comparable decrease in PBN in the L_E line (Additional file4: FigureS3).
The two aforementioned lines contain contiguous introgression fragments in chromosome 7, from RM10 to RM18 for L_D and from RM134 to RM118 for L_E. Since the exact recombination positions of the introgressed segments for each BC4 is not known, the regions between SSRs with different genotypes are included in the QTL intervals. In Fig. 5A, it can be observed that lines L_D and L_E, which share the same phenotypes except for PBN, may harbor introgressions that overlap over only a small region between RM18 and RM134. Based on these observations, two different hypotheses can be proposed with regard to QTL position(s) in the q_7 region (Fig. 5A). The first one postulates the existence of a common QTL located between RM18 and RM134 that controls PBL, SBN/PBN and TBN. The second hypothesis is that two different QTLs, q_7 − 1 between RM10 and RM134, and q_7 − 2 between RM18 and RM420, exert a similar effect on PBL, SBN/PBN and TBN but act differently on PBN.
Taken together, the above results suggest that panicle architecture is a complex trait controlled by various different genomic regions which positively or negatively influence branching. An association of q_1, q_2 and q_3 − 2 produces opposing effects on PBN and SBN while only the q_3 − 1 region, comprised between RM175 and RM7, may impact positively upon PB length. Finally, the region in q_7, which negatively influences the number of primary branches and may be associated with the formation of tertiary branches and an increase in SBN per primary branch, may be defined as being either between the RM18 and RM134 marker positions or associated with two QTLs positioned between RM10 and RM134 for q_7 − 1 and from RM18 to RM420 for q_7 − 2.
Co-location of QTLs with known genes and QTLs detected with other populations.
Many studies have reported QTLs associated with panicle architecture and/or yield traits or both, using bi-parental (QTL mapping) or diversity panels GWAS (Crowell et al., 2014; Ya-fang et al., 2015; Bai et al., 2016b; Crowell et al., 2016; Rebolledo et al., 2016; Ta et al., 2018; Zhang et al., 2019; Cubry et al., 2020; Bai et al., 2021; Zhong et al., 2021). We found 70 common sites between the QTLs detected in this study and those reported earlier (Additional file 1: TableS6). For instance, co-located sites relating to similar traits (SBN and SpN) were found on chromosomes 11 and 12. Nevertheless, despite the significant number of common sites identified in the different studies, the traits that they have been reported to affect are not always strictly comparable or even similar, even if they relate in some way to panicle architecture or yield.
To evaluate the synteny of the QTL regions between the genomes of the two CSSL parents, sequences from the assemblies of the Os_Caiapó and Og_MG12 genomes obtained by ONT sequencing (https://doi.org/10.23708/QMM2WH; https://doi.org/10.23708/1JST4X; Additional file1: TableS7) were used to carry out local alignment for each QTL region. The assembled genomes are of high continuity and completeness, with BUSCO score of 98.4% allowing precise detection of structural variations. For the majority of the QTL regions, no major structural variation was observed between the two genomes (Additional file5: Figure S4). However, some notable differences were detected at the qPBN2, qTBN5, qTBN7, qSpN3 and qSBN12/PBN12 sites. Within the QTLs qPBN2 and qTBN7, inverted segments of 1,296,899 and 230,762 bp were observed between Os_Caiapó and Og_MG12 respectively (Additional file1: Table S8). The genomic regions for QTLs qTBN5, qSpN3 and qSBN/PBN12 displayed InDels between Os_Caiapó and Og_MG12 (Additional file1: Table S8). With the exception of qSBN11, we observed a high sequence similarity in the QTL regions between the two genomes, suggesting similar content in terms of coding sequences. Synteny analysis was extended by the alignment of the Os_Caiapó and Og_MG12 QTL regions with those of the O. sativa cv. Nipponbare reference genome (Additional file5: Figure S4). Based on the high conservation of the corresponding regions between Os_Caiapó and Os_Nipponbare, we then used as a reference, for subsequent analyses, the O. sativa cv. Nipponbare (IRGSPv1.0) functional gene annotation databases (i.e., MSU7 and RAP_db) for candidate gene prioritization in relation to panicle architecture and flowering.
For all QTLs identified in the present study, we found several known genes with relevant functions that related to flowering and/or panicle development (Table 2; Additional file6: Figure S5). Genes such as LAX PANICLE1 (LAX1), OsMADS1/LEAFY HULL STERILE1 (LHS1), OsMADS14, OsMADS34/PANICLE PHYTOMER2 (PAP2), OsINDETERMINATE SPIKELET 1 (OsIDS1), OsMADS18, DENSE AND ERECT PANICLE2 (DEP2) and FRIZZY PANICLE (FZP) are known to be involved in the control of the panicle development and were suggested as candidates that might contribute to the panicle branching diversity observed between the two parents Os_Caiapó and Og_MG12 (Chongloi et al., 2019; Wang et al., 2021; Yin et al., 2021).
q_7 genetic variations between Os_Caiapó and Og_MG12
We paid particular attention to the q_7 region, as it was found to be associated with several panicle morphological traits and to span a genomic region that included several panicle-associated genes. To understand if specific genetic modifications present in the q_7 QTL region could be associated with panicle architecture variation, we further compared the constituent genes in the region between the RM10 and RM420 markers in the Os_Caiapó and Og_MG12 genomes on chromosome 7 (22.23–29.59 Mbp in Os_Caiapó vs 20.12–26.69 Mbp in Og_MG12). For this purpose, gene annotation comparison and BLAST analyses were performed to explore in detail gene synteny and presence/absence of genes between the Os_Caiapó and Og_MG12 genomes within this region (Fig. 5B; Additional file1: Table S9). Between the RM10 and RM420 marker positions, we observed a variation in the number of genes between the two genomes (Additional file1: Table S9). This variation is due to several factors: the differential presence of corresponding orthologs between the two genomes; differential gene duplication within the region; and different locations of a given gene within the two genomes (i.e., genomic rearrangement) (Fig. 5B; Additional file1: Table S9). Among the genes that differ between the two genomes in this region, many are related to transposable elements (TEs) or are hypothetical protein-encoding genes. None of them has a known function relating to panicle development or flowering control, nor to any other developmental processes.
Special attention was paid to common genes present in the q_7 region that were known to be related to inflorescence development and/or meristem activity and maintenance. Based on a bibliographic survey, no candidate genes of special interest were identified within the region between the RM18 and RM134 markers. In contrast, several interesting candidate genes were identified in the q_7 − 1 and q_7 − 2 regions: the OsHOX14, OsMADS18 and DEP2 genes in q_7 − 1 and the FZP and WOX11 genes in q_7 − 2 (Fig. 5C). SNP and InDel sites associated with the aforementioned genes that were polymorphic between the Os_Caiapó and Og_MG12 genomes were analyzed in order to detect amino acid modifications, open reading frame alterations or transcription factor binding site (TFBS) variations within promoter regions.
OsHOX14 (LOC_Os07g39320/Os07g0581700) encodes a protein of the Homeodomain-leucine zipper (HD-Zip) TF family and is involved in the regulation of panicle development (Beretta et al., 2023; Shao et al., 2018). Two of the identified SNPs cause amino acid variations in the coding sequence, including one in the homeobox domain (Fig. 5C; Additional file1: Table S10). Comparison of the OsHOX14 and OgHOX14 promoter regions revealed InDel and SNP variations leading to the loss of 9 TFBSs and a gain of three new TFBSs in the CSSLs bearing Og_MG12 q_7 − 1 introgression (Fig. 5C; Additional file1: Table S11).
OsMADS18 (LOC_Os07g41370/Os07g0605200) encodes a protein of the AP1/FUL-like MADS-box TF subfamily (Masiero et al., 2002). In addition to its role in flowering time promotion (Fornara et al., 2004), OsMADS18 is known to specify inflorescence meristem identity (Kobayashi et al., 2012). SNPs were detected in the coding sequence; one of the SNPs leads to a non-synonymous change in the OgMADS18 protein outside the known binding or functional domains (Fig. 5C; Additional file 1: Table S10). Scanning of the promoter regions of OsMADS18 and OgMADS18 in the Os_Caiapó and Og_MG12 genomes respectively revealed several SNPs and InDels that lead to variations between the two genomes (presence/absence) of TFBSs recognized by AP2, NAC or HB TFs (Fig. 5C, Additional file1: Table S11).
DEP2 (LOC_Os07g42410/Os07g061600) encodes a plant-specific protein of unknown function associated with panicle size (Li et al., 2010). Various SNPs and one InDel are observed between the coding sequences of OsDEP2 and OgDEP2 which result respectively in changed amino acid identities between the two protein orthologs (Fig. 5C; Additional file 1: Table S10) and an insertion of two amino acids in the OgDEP2 protein. Sequence variations were also observed between the promoters of OsDEP2 and OgDEP2; these variations lead to the loss of bZIP, B3, AT-Hook and AP2 TFBSs in the CSSLs carrying q_7. On the other hand, in comparison to O. sativa, the CSSLs carrying q_7 include NAC and bZIP TFBSs specific to the promoter of OgDEP2.
Moreover, among the genes contained in the q_7 − 2 region, FZP (LOC_Os07g47330/Os07g0669500) encodes an AP2/ERF TF known to play a role in the regulation of floral meristem determinacy in rice (Komatsu et al., 2003; Zhu et al., 2003). Several SNP and InDel variations observed in the coding region of FZP lead to coding sequence amino acid changes between the two genomes (Fig. 5C; Additional file 1: Table S10). An InDel of 9 bp in OgFZP results in the insertion of three histidine amino acids outside the single AP2 domain in the OgFZP protein. Several recent studies have shown that variations in the promoter region and 3' UTR (Untranslated Transcribed Region) of the OsFZP gene affect panicle architecture by through changes in the number of branches produced (Bai et al., 2016, 2017; Huang et al., 2018; Wang et al., 2020; Chen et al., 2022). Here, we observed several SNPs and InDels between the promoter of the two orthologs, which result in a differential presence of TFBSs (Fig. 5C, Additional file1: Table S11). No variation was observed in the copy number variant (CNV) motif of 18 bp described by Bai et al. (2017). Huang et al. (2018) revealed a deletion of 4 bp in the OsFZP 5' regulatory region in comparison to the sequence of O. rufipogon. This deletion is observed in Os_Caiapó and not in the genome of Og_MG12 (Fig. 5C). Recently, it has been shown that CU-rich elements (CUREs) present in the 3' UTR of the OsFZP mRNA are crucial for efficient OsFZP translational repression (Chen et al., 2022). Three CUREs are detected in the 3' UTR of OsFZP in the Os_Caiapó genome. In contrast, a deletion of the third CURE sequence is observed in the Og_MG12 genome (Fig. 5C).
Although the WUSCHEL-related homeobox WOX11 gene (LOC_Os07g48560/Os07g0684900) has been mainly described as playing an important role in root development (Zhao et al., 2009; Zhang et al., 2018), this gene is also required to regulate rice shoot development and panicle architecture (Cheng et al., 2018). An InDel in the coding region of WOX11 leads to an Asn amino acid insertion in the OgWOX11 protein in Og_MG12 without affecting the homeobox domain of the protein (Fig. 5C, Additional file1: Table S10). In the promoter region, variations between Os_Caiapó and Og_MG12 lead to the presence/absence of several TFBSs in the promoter of OgWOX11 (Fig. 5C; Additional file 1: Table S11). We also observed a large insertion of 3,760 bp, containing about 31 AP2 TFBs, in Og_MG12 compared to the Os_Caiapó genome.
Overall, our analysis revealed variations in synteny that demonstrate an absence of certain Os_Caiapó genes and the addition of other loci as a consequence of Og_MG12 genomic introgressions within the CSSLs: some of these changes can be hypothesized to play a role in determining panicle trait diversity. Moreover, our analysis of candidate genes revealed TFBS variations and protein coding sequence polymorphisms that may lead to variations in transcript levels and/or protein activity in CSSLs harboring the q_7 introgression.