Inheritance of photoperiod insensitivity
Inheritance of mutant trait in PPIS mutant was confirmed in both F2 (Fig. 1) and F3 generation of the crosses ‘PPIS mutant x Kon Joha’ and ‘PPIS mutant x Kalijeera’. Of the 514 plants in the F2 population of ‘PPIS mutant x Kon Joha', 381 were photoperiod sensitive, and 133 were photoperiod insensitive (χ2 = 0.21; P = 0.647). Similarly, in the 502 F2 plants of ‘PPIS mutant x Kalijeera’, 364 and 138 plants were photoperiod-sensitive and -insensitive, respectively (χ2 = 1.66; P = 0.197). When 250 F2 progenies (66 photoperiod-insensitive: 184 photoperiod-sensitive) of ‘PPIS mutant x Kon Joha' were tested in the F3 generation, all the 66 photoperiod-insensitive mutant progenies were found to be true breeding. While 122 F2 plant progenies were heterozygous (3:1), and the remaining 62 plants were true-breeding for photoperiod sensitivity (χ2 (1:2:1) = 0.272; P = 0.873). Thus, F2 and F3 segregation confirmed the single recessive gene inheritance pattern for this photoperiod-insensitive trait in the PPIS mutant. Plotting the graphs using data on days to flowering of parents and F2 individuals of both the crosses, namely PPIS mutant/Kon Joha and PPIS mutant/Kalijeera, resulted in a bimodal distribution curve (Supplementary Figs. 1 & 2). Two peaks were observed for the flowering of F2 individuals in response to photoperiod - one is for photoperiod-insensitive group and another for photoperiod-sensitive group (Supplementary Figs. 1 & 2). These results clearly indicated the monogenic inheritance pattern for photoperiod insensitivity.
Genome integrity analysis and genome sequencing identify more point mutations than repeat length/indel variation.
Based on genome integrity analysis of mutant, parent (Kon Joha), Kalijeera and photoperiod insensitive Ahu Joha rice genotypes with 150 SSR and InDel markers (Supplementary Table S1), none of the markers could detect any polymorphism between parent and PPIS mutant. In contrast, the PPIS mutant was distinct from the already available photoperiod-insensitive Ahu Joha rice cultivars; this established the novelty of the isolated mutant (Fig. 2). Fifty-four descriptors comprising traits at seedling, vegetative, flowering and maturity phases were recorded (Supplementary Table S4) between the PPIS mutant and Kon Joha. The PPIS mutant was monomorphic and similar to Kon Joha for fifty-two traits. Polymorphism was evident for the other two traits: time of heading and maturity (Supplementary Table S4). However, the mutant showed polymorphism with Kon Joha for filled grains panicle-1, plant height (cm) and panicle length (cm) (Supplementary Table S5). These morphological differences may arise due to varied environments during flowering in mutant and parent.
Genome sequencing of parent and mutant detected as many as 3,57,562 nucleotide variations between Kon Joha and PPIS mutant. Genome sequencing generated 161,333,716 reads (29.5% duplicated data) in Kon Joha and 165,632,496 reads (33.7% repeated data) in the PPIS mutant. The overall GC content was 44.4 per cent. Of the 3,57,562 nucleotide variants, the proportion of silent, missense and nonsense mutations were 23.17%, 72.42% and 4.41%, respectively. Of such missense and nonsense mutation, the mutation in the exon region remained at 8.68 per cent. The average mutation density was 1 in 1046 bases. The Ts/Tv ratio for all the point mutations was 2.38.
BSA identified two closely spaced SSRs in chromosome 6 linked to the mutant trait.
Towards quick mapping of the mutant trait in PPIS mutant, it was hybridized with a distant parent (Kalijeera) to develop a segregating F2 population. Screening of 402 SSR and InDel markers revealed 57 (15.17%) polymorphic markers between mutant and Kalijeera. BSA of these polymorphic markers in photoperiod-sensitive bulk (PS bulk) and photoperiod-insensitive bulk (PIS bulk) identified a close association of RM527 with mutant trait in chromosome 6. Meanwhile, other SSR markers (RM204, RM225, RM586, RM587, RM588, and RM589) in chromosome 6 were not associated with the mutant trait. The RM527 marker lies between 9,862,291 and 9,862,523 bp in chromosome 6 of rice. While other markers remained at the proximal end of chromosome 6 (beyond 5.0 Mbp). We then synthesized 40 more SSR markers between 5.0 and 13.2 Mbp of chromosome 6 (Supplementary Table S2). While screening for polymorphism between Kalijeera and PPIS mutant, five more SSR markers (RM3794, RM19592, RM19725, RM5850 and RM19902) were found polymorphic (Supplementary Table S2). Further, BSA revealed a close association between RM19725 (8,134,068 to 8,134,111 bp in chromosome 6) with the mutant trait. Thus, BSA delineated a marker interval (RM19725 - RM527) in chromosome 6 that is tightly associated with the photoperiod insensitivity in the PPIS mutant (Figs. 3 & 4). To confirm the same, we genotyped all the 10 individual mutant-type plants (constitute the PIS bulk) with polymorphic SSR markers (RM589, RM225, RM3794, RM19592, RM19725, RM527, RM5850 and RM19902) in chromosome 6. The genotyping assay in these individual 10 PIS plants revealed no crossing over for RM527 and RM19725, whereas all the other polymorphic SSR markers in chromosome 6 showed recombination and proved their incomplete linkage with a photoperiod-insensitive gene in PPIS mutant (Fig. 5).
A single base deletion mutation in Hd1 between RM19725 and RM527
Several flowering-related genes were located in rice chromosome 6. Hd17 (OsELF3), Hd3a, and RFT1genes are between 2.23 Mbp and 3.0 Mbp. Another three genes, Os06g40080/Se5, Os06g41090/OsFTIPand Os06g45640/OsNF-YC4, present at 23.85 Mbp to 27.64 Mbp. While heading date 1 (Hd1) is present at 9,336,359 to 9,338,643 bp. Hd1 lies perfectly between the identified markers RM19725 and RM527 in chromosome 6. After cloning, we amplified the Hd1 from Kon Joha and PPIS mutant and sequenced four gene fragments. After aligning the complete sequence of Kon Joha (OR113693) and PPIS mutant (OR113694), a single base deletion (G to -) was identified in exon 2 of Hd1(Fig. 6).
Validating the relationship between mutant trait and candidate gene mutation in two segregating populations
From the above pattern of mutations in Hd1, we could visualize the non-synonymous nature of single base deletion in exon 2. The single base deletion leads to a frameshift mutation phenomenon and the possible rise of non-functional proteins. Using the Web-SNAPPPER tool, two allele-specific forward primers were designed for parent (wild type) and mutant (PPIS mutant) along with a standard reverse primer. Amplification of both these allele-specific primer pairs was first confirmed in Kon Joha and PPIS mutant (Figs. 7 & 8). The wild allele-specific primer pairs amplified a 200 bp band in Kon Joha but no amplification in the PPIS mutant. Mutant-allele-specific primer pairs amplified the band in mutants but did not have amplicons in Kon Joha. Towards validation, two different populations were used. Initially, individual gDNA of bulk components of BSA study in F2 of ‘PPIS mutant x Kalijeera (distant parent)' was used. All 10 individual mutant-type plants amplified the band with mutant-allele-specific primer pairs but no amplicons with parental-allele-specific primer pairs (Figs. 9 & 10). Seven out of 10 wild-type plants were amplified band with mutant-allele specific primer pairs, and all 10 were amplified 200 bp amplicon with parental-allele specific primer pair (Figs. 9 & 10). Amplification of the band in seven wild-type plants in the F2 population of 'PPIS mutant x Kalijeera' was due to their heterozygous nature, as revealed by the amplification of the RM19725 marker (Fig. 4). The heterozygosity shown through the allele-specific SNP marker was well matched with the RM19725 amplification due to the close physical proximity with Hd1. Using allele-specific SNP primer pairs could further confirm the heterozygosity and homozygosity of wild-type plants in the F2 of ‘mutant x Kalijeera'. Of the 10 plants, 7 were heterozygous wild type, and 3 were homozygous. Thus, the segregation ratio in the wild-type pool (10 plants) for heterozygous and homozygous plants maintains the genotypic segregation 2:1 (χ2 = 0.13, P value = 0.937). For the next level of validation, we used true breeding (deduced from F3 segregation), wild-type (10 plants), and mutant-type (10 plants) plants from 'PPIS mutant x Kon Joha'. Due to the homozygosity nature, all the wild-type plants amplified bands with parental-allele-specific primer pairs but no bands in mutant-allele-specific primer pairs (Fig. 7). All 10 mutant-type plants amplified bands with mutant-allele specific primer pairs but no bands with parental-allele specific primers (Fig. 8).
Frameshift in exon 2 of Hd1 leads to drastic changes in the C-terminal part of the Hd1 protein.
A single base deletion in exon 2 of Hd1 leads to a frameshift mutation at the C-terminal of theHd1 protein. The C-terminal of Hd1 contains a CCT {Constant (CO), CO-LIKE (COL) and TIMING OF CAB EXPRESSION1 (TOC1)} domain. Due to this frameshift, the mutant Hd1 protein has an altered primary sequence at the C-terminal part, which includes the CCT domain (Supplementary Fig. 3). To gain insight into the structural changes due to frameshift mutation, three-dimensional models of wild-type and mutant Hd1 proteins were constructed using AlphaFold2 and only the CCT domain was used for the structural analysis (Supplementary Fig. 4). The modelled structure of the wild-type CCT domain was compared to the crystal structure of the CCT domain. Very high structural similarity shows that the AlphaFold can correctly predict the structure of the CCT domain of the Hd1 protein (Supplementary Fig 4). Wild-type Hd1 protein shows the typical CCT domain containing two ɑ-helices (ɑ1 and ɑ2) and 2 loops (Fig. 11 and Supplementary Fig. 4). However, mutant type Hd1 protein showed changes in the secondary structural elements, as illustrated in Figure 11.