Reddy et al.

Biological Research (2024) 57:94 Biological Research

https://doi.org/10.1186/s40659-024-00553-7

RESEARCH ARTICLE Open Access

A single‑base deletion in exon 2

of Hd1 delineates monogenic recessive
photoperiod insensitivity in aromatic Joha rice:
a novel allele for seasonal adaptability
Bodeddula Jayasankar Reddy1, Shreekant M. Baradkar2, Tamma V. S. S. Manogna1, Dibosh Bordoloi1,
Subhash C. Bihani3,4, Nagendra Sarma Barua1, Akhil Ranjan Baruah5, Bikram Kishore Das2,4,
Suvendu Mondal2,4*   and Debojit Sarma1*   

Abstract
Background Assam’s aromatic Joha rice is a unique rice class famous for its aroma, taste, and nutritional benefits,
which fetch high market prices in domestic and international markets. Joha landraces are inherently poor yield-
ers due to their strong aroma and predominantly photoperiod sensitivity. Hybridization involving non-aromatic
HYVs improves yield with concomitant loss of quality. In this context, mutation breeding, a sustainable approach
where genetic mutations are induced to create desirable traits, often provides useful allelic variation in specific mor-
pho-agronomic traits. The present study delves into the genetic characterization of a photoperiod-insensitive mutant.
As part of our mutation breeding programme, this mutant was isolated from a gamma ray-induced M ­ 2 population
of a Joha rice landrace, Kon Joha.
Results The mutant was unique, and a single recessive gene conditions the induced photoperiod insensitiv-
ity. Mutant gene tagging involved 402 SSR and InDel markers, and later polymorphic markers were used for bulk
segregant analysis (BSA) in the F2 population of ‘mutant × Kalijeera (distant parent)’. BSA revealed an association
between the SSR marker RM527 and this mutant trait. This marker is present on chromosome 6 of the rice genome.
Using chromosome 6-specific SSR markers in polymorphic screening and BSA revealed another associated marker,
RM19725, for the mutant trait. The genomic interval between RM527 and RM19725 harbors a photoperiod-insensitive
gene, Hd1, on chromosome 6. Cloning and sequencing of Hd1 genomic fragments from the parents and mutants
revealed a single-base deletion in exon 2, leading to a frameshift mutation in the Hd1 protein. This mutation in exon
2 leads to severe structural abnormalities in the CCT domain of the Hd1 protein that is critical for the interaction
of the repressing complex with conserved response elements in the florigen gene under long-day conditions,
thereby causing photoperiod insensitivity.
Conclusions The mutant’s pleasant aroma and other quality characteristics, comparable to those of the parent culti-
var, hold significant promise. They expand its potential use in a structured breeding programme aimed at developing

*Correspondence:
Suvendu Mondal
[email protected]
Debojit Sarma
[email protected]
Full list of author information is available at the end of the article

© The Author(s) 2024. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which
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Reddy et al. Biological Research (2024) 57:94 Page 2 of 13

high-value aromatic Joha rice. This rice, resilient to winter- and summer-growing environments and with broad
seasonal adaptability, could revolutionize the rice market. The practical value of our research is underscored by this
exciting possibility.
Keywords Joha rice, Allele-specific marker, Bulk segregant analysis, Gene tagging, Heading date 1, Oryza sativa L.,
Photoperiod insensitivity, Point mutation, SSR marker

Background Both Hd1 and Ghd7 contain CCT domains. The CCT
Flowering involves the transition of the apical meris- domain of Hd1 interacts with the B and C subunits of the
tem from vegetative to reproductive growth. The timing nuclear factor Y (NF-Y) complex, such as in OsNF-YB11
of flowering in rice is called the heading date. It is one (also named GHD8) and OsNF-YC7. This trimeric com-
of the critical factors considered for its adaptation in plex binds to a conserved response element (OsCORE2,
different agroecological situations as well as crop sea- containing the CCACA motif ) in the Hd3a promoter [11,
sons across the globe. Rice flowering is generally pro- 12].
moted by short photoperiods; thus, rice is considered The vast collection of rice landraces in India is pre-
a short-day plant. However, it can be grown under long served. Short-grained aromatic rice is a small rice
photoperiod conditions where the adaptation of spe- subgroup belonging to the Indica group [13], gain-
cific genetic backgrounds due to the accumulation of ing popularity among global consumers. Joha rice is a
mutations in different essential genes is considered the unique, fragrant rice class grown as winter rice in Assam,
primary evolutionary force. Flowering is promoted by India. It is trendy and highly valued due to its quality. It
heading date 3a (Hd3a) and rice flowering locus T (RFT) possesses a superfine kernel, distinctive aroma, better
under both short-day (SD) and long-day (LD) condi- cooking qualities, and excellent palatability. The aroma
tions [1, 2]. Hd3a and RFT1 are expressed diurnally in of Joha rice cultivars is due to the presence of a non-
leaves under SDs and LDs. Both Hd3a and RFT1 pro- functional betaine aldehyde dehydrogenase 2 (BADH2),
teins move to the shoot apical meristem (SAM), where which also lowers grain yield [15, 16]. Joha rice’s low
they interact with the basic leucine zipper (bZIP) yield, late maturity, and tall stature generally make it a
transcription factor OsFD (Oryza sativa flowering poor competitor to modern high-yielding varieties, occu-
delay) to regulate the expression of two rice orthologs pying approximately 5 per cent of the Sali rice area in
(OsMADS14 and OsMADS15) of the Arabidopsis floral Assam, with an average yield of 1.0 -1.5 metric tons per
meristem identity gene APETALA1, thus provoking the ha [14]. However, its aroma and unique cooking quality
initiation of primary panicle branch primordia [3, 4]. allowed a global market for Joha rice to enter the Euro-
Furthermore, rice FT-INTERACTING PROTEIN1 pean market in 2007. Joha rice got the Geographical Indi-
(OsFTIP1) is required to export RFT1 from companion cations (GI) tag from the Union Ministry of Commerce,
cells to sieve elements for further movement to the SAM India in 2017 (http://​ipind​iaser​vices.​gov.​in). Photoper-
[5]. The ubiquitin-like domain kinase γ4 (OsUbDKγ4) iod-insensitive aromatic Joha rice cultivars would enable
interacts with OsFTIP1 to modulate its degradation in the farmers to increase the cropping intensity by growing
leaves through the 26S proteasome. This dynamic mod- two crops per year, offering a viable option to expand and
ulation of OsFTIP1 abundance in leaves by the negative sustain its cultivation for family income and health. The
regulator OsUbDKγ4 is essential for regulating florigen mutant can improve Joha rice through crossbreeding and
transport in rice under LD conditions [6]. act as pre-breeding material. Furthermore, they can help
Hd3a and RFT are generally positively regulated by preserve the unique genetic wealth of this rice class for
early heading date 1 (Ehd1) [7]. However, the upstream future generations.
regulation of Ehd1 is different under both SD and LD Kon Joha is a Joha rice type that is typically grown in
conditions. Under SD conditions, heading date 1 (Hd1) the Sali season (July to November) and flowers under
promotes Ehd1 and flowering at night. While Hd1 SD conditions. This type of rice genotype will not flower
represses Ehd1 during the day under SD conditions normally if grown during LD (February to June) and thus
[8], the scenario differs under LD conditions. In LDs, takes a very long period for its maturation in these off-
the monocot-specific CCT domain-containing protein season periods. Gamma-ray mutagenesis was used to
’grain number, plant height and heading date7’ (Ghd7) broaden the genetic variability of this Kon Joha rice vari-
represses Ehd1 and thus flowering [8, 9]. This repression ety. Seven fertile photoperiod-insensitive mutants were
is supported by complex formation among Ghd1, DTH8 isolated while growing a subset of the ­M2 population
and Hd1 during the day under LD conditions [8, 10]. (approx. 10,000 plant population) during the off-season
Reddy et al. Biological Research (2024) 57:94 Page 3 of 13

(February to June). The photoperiod-insensitive mutants differentiated from outcrossing-derived contaminants

(PPISMs) flowered during May, while the other parental- through microsatellite marker analysis. The SSR band-
type plants flowered in the 3rd week of October. After ing patterns of true mutants should be identical to those
verifying its true breeding behavior in ­M3, a particu- of their immediate parent. Hence, the genome integrity
lar PPIS mutant (JKOJM-250-22-17-120) was used for of the isolated mutant in combination with the parent
molecular characterization. The present research will was tested, including two already available photoperiod-
report on the genetics and molecular nature of this muta- insensitive rice cultivars (Ahu Joha-1 & Ahu Joha-2) and
tion that controls photoperiod insensitivity in this Joha a distant parent, Kalijeera, with 150 SSR and InDel [21]
rice variety. markers (Supplementary Table S1). The morphological
similarity of the mutants and parents was also tested by
Methods observing 54 DUS characteristics and 12 morphometric
Induced mutagenesis for photoperiod insensitivity traits [22, 23].
A mutation breeding program was used to improve the
agronomic characteristics of Joha rice through 250 Gy
Inheritance study of induced photoperiod insensitivity
gamma-ray treatment. The parent Kon Joha is photo-
To assess the pattern of photoperiod insensitivity inher-
period sensitive (PS) and thus does not flower under LD
itance in the PPIS mutant, it was hybridized with the
conditions. Kon Joha is a small-grain aromatic traditional
parent cultivar (Kon Joha) and distant parent, Kalijeera,
rice genotype preferred by the Assamese population and
in the winter/Sali season of 2019. We chose the dis-
is used mainly in traditional foods. The PS aromatic Joha
tant parent to discover more polymorphic markers. ­F1
rice cultivars did not flower long (13 to 14 h.) May–June
plants were grown in winter/Sali, 2020, to obtain suf-
summer days at Assam. The commercial photoperiod-
ficient ­F2 seeds. To expose the plants to two different
insensitive (PI) genotypes flower in LD, and the crop
periods of daylight, i.e., long days (March to September)
matures by June–July. In 2017, 3000 dry uniform seeds of
and short days (October to February), ­F2 generations of
Kon Joha maintained by selfing for two generations were
both crosses were evaluated in two continuous seasons,
exposed to 250 Gy gamma rays from a Cobalt-60 source
referred to as early autumn/Ahu, 2021, to winter/Sali,
at Bhabha Atomic Research Centre (BARC), Trombay.
2021. A total of 250 F ­ 2-derived ­F3 ­(F2:3) progeny rows
The ­M1 generation and the parent were raised as a trans-
from the cross PPIS mutant × Kon Joha were studied in
planted crop in two adjacent blocks during early Ahu
early autumn, 2022, to further demonstrate the geno-
(February to May) 2017. The field emergence rate in the
typic ratio of photoperiod insensitivity by observing
nursery was 37.40% for the treated seeds and 41.13% for
photoperiod-insensitive plants in ­F2:3 progeny rows. The
the control seeds. However, the crop started flowering
resulting ­F2 and ­F2:3 data were subjected to the χ2 test
on October 17, 2017. All 390 surviving M ­ 1 plants bear-
for their goodness of fit with the predicted 3:1 and 1:2:1
ing panicles were grown in the plant to-rows along with
Mendelian segregation ratios, respectively. During the
intermittent controls during early Ahu, 2018. Flowering
confirmation of the genotypic ratio in F ­ 3, gDNAs from
occurred in nine plants of progeny no. 22 from May 21
an individual of 10 true bred wild-type and 10 true bred
to June 7, and seven fertile panicle-bearing plants were
mutant progenies were isolated to validate candidate
confirmed for their flowering behavior and selected for
gene-derived allele-specific SNP markers.
spikelet fertility through the M
­ 3 to M
­ 5 generations. The
present study used a photoperiod insensitive mutant
(PPISM), JKOJM-250-22-17-120, which has the highest Bulk segregant analysis (BSA) for the identification
spikelet fertility (88.47%). of linked SSR markers
From the F ­ 2 population of ‘PPIS mutant × Kalijeera’, 10
Test of genome integrity in isolated mutant compared photoperiod-insensitive ­F2 plants and 10 photoperiod-
to available PI rice sensitive ­F2 plants were chosen for genomic DNA isola-
Gamma irradiation results in a variety of random DNA tion, and photoperiod-insensitive bulk (PIS bulk) and
damage events, from modest deletions of 1–16 bp to photoperiod-sensitive bulk (PS bulk) plants were sub-
massive deletions of 9.4–129.7 kb and inversions of sequently prepared by pooling equal molar concentra-
1–3208.5 kb [17, 18]. These processes result in various tions of gDNA from each plant. Initially, 402 SSR and
changes, from point mutations to chromosome aber- InDel markers (Supplementary Table S1) were screened
rations, which ultimately influence the morphological, for polymorphisms between the PPIS mutant and Kon
physiological, and anatomical characteristics of the vari- Joha and Kalijeera strains. Only polymorphic markers
ety or cultivar being improved [19]. Furthermore, Fu were used for bulk segregant analysis [24]. Based on the
et al. [20] suggested that true induced mutants could be BSA results, chromosome 6-specific SSR markers were
Reddy et al. Biological Research (2024) 57:94 Page 4 of 13

synthesized from the genomic interval of 5.0 to 13.2 Mbp Cloning of the Hd1 gene and sequencing
for fine mapping (Supplementary Table S2). The specific primer pairs described in Table 1 amplified

the Q5® High-Fidelity DNA Polymerase (New England

the Hd1 gene from parent and mutant genomic DNA;
Whole‑genome resequencing of parents and mutants
Total DNA was isolated from Kon Joha and PPIS mutants Biolabs, USA) yielded five overlapping fragments. PCR
following the standard CTAB method, and the quality was performed for 35 cycles (30 s at 94 °C, 30 s at 55 °C,
and quantity of the samples were ascertained by resolv- and 30 s at 72 °C), followed by a final amplification for

separated using a 1.5% agarose gel at 75 V. The Wizard®

ing on a 1.2% agarose gel and with a Nanodrop1000 10 min at 72 °C. PCR amplification products were size-
spectrophotometer (Thermo Fisher Scientific, Waltham,
MA, USA), respectively. The DNA samples were then SV Gel and PCR Clean-Up System (Promega, Madi-
sequenced at 30X coverage on the Illumina HiSeq2500 son, USA) was used to purify the PCR products from
platform (Nucleome Informatics Pvt. Ltd., Hyderabad, the gel. The DNA fragment was treated with Taq DNA
India). After filtering the clean reads, the Kon Joha polymerase in excess dATP to obtain dA overhangs at
sequences were aligned to the reference genome Oryza ­3/end. The amplified product was then purified using a
sativa ssp. Japonica cv. Nipponbare (MSU7). The align- High Pure PCR Cleanup Micro Kit (Roche, USA) and
ments are reported in bam (binary alignment and map- ligated into the pTZ57R vector (Thermo Scientific,
ping) format using BWA with the default parameters. USA) according to the manufacturer’s instructions for
InDels were marked if they were ≤ 50 bases in size. The transforming Escherichia coli DH5α cells. Transformed
consensus sequence for wild-type ’Kon Joha’ was gener- colonies containing the cloned fragment were identified
ated using SAMtools [25]. Later, the filtered PPIS mutant following standard blue‒white screening and colony

tures of positive colonies using the GenElute™ Plasmid

sequences were mapped against the consensus Kon Joha PCR. Plasmid DNA was isolated from overnight cul-
sequence. Genotyping by calling variants was performed
to obtain better support and confidence from BCFtools. Miniprep Kit (Sigma‒Aldrich, USA). The insertion of
These variants are emitted in a cumulative form in a VCF the recombinant plasmid was confirmed through dou-
file (Variant Calling Format), which is then strictly fil- ble digestion with HindIII and EcoRI. Plasmid DNA
tered using BCFtools v1.7, which helps to eliminate false samples were then subjected to Sanger DNA sequenc-
variants created during mapping or variant calling. SNPs ing. The complete assembled sequence of the Hd1 gene
were extracted from the identified variants and then from the parent and mutant Hd1 genes was aligned
annotated using the SnpEff tool [26]. using CLUSTAL W [27].

Table 1 Primer pairs used for cloning and detection of mutations in Hd1
Primer name Sequence (5′-3′) Annealing temp. (oC) Product
Size
(bp)

Primer pairs for cloning of Hd1 genomic fragment

Hd1_FP1 AAA​GCA​AAG​ATG​AAC​AGA​GGTG​ 57.0 700
Hd1_RP1 AGA​GAT​T TT​ACA​GGG​ATC​AATAG​
Hd1_FP2 GAG​ATG​TCC​ATT​GAA​T TG​T TTAG​ 60.4 701
Hd1_RP2 CCT​TGC​T TG​TGG​TAG​TAG​TAG​
Hd1_FP3 GAG​GAC​AAA​CAC​AAT​AGC​T TG​ 57.0 938
Hd1_RP3 AGC​TAG​TAA​TAG​ATG​AAC​TCAC​
Hd1_FP4 ATA​GTG​GTT​ATG​GAG​T TG​TG 62.0 944
Hd1_RP4 GTT​TCA​TAA​CGT​ATT​GTC​T TCTC​
Hd1_FP5 CTG​GAG​CAA​TCA​ATC​TCT​TC 57.0 1095
Hd1_RP5 CTC​ATG​GTT​TAG​TGA​GAA​GATAG​
Allele-specific primer pairs for the nonsynonymous frameshift mutation in Hd1
Hd1_Wild_FP AGG​GAG​GCC​AGG​GTG​C TC​ATG​ 66.5 200
Hd1_common RP CTA​C TG​TCA​GAT​AGA​GCT​GCA​GTG​GAG​AAC​ATC​
Hd1_Mutant_FP GGG​AGG​CCA​GGG​TGC​TCC​GT 66.0 200
Hd1_common RP CTA​C TG​TCA​GAT​AGA​GCT​GCA​GTG​GAG​AAC​ATC​
Reddy et al. Biological Research (2024) 57:94 Page 5 of 13

Development of allele‑specific SNP markers and marker segregation confirmed the single recessive gene inherit-
validation ance pattern for this photoperiod insensitivity trait in the
Based on the sequencing of Hd1, a single base deletion PPIS mutant. Plotting the graphs using data on days to
was found in exon 2. Allele-specific SNP markers were flowering of parents and ­F2 individuals of both the PPIS
developed through WebSNAPPER tools (Supplementary mutant/Kon Joha and PPIS mutant/Kalijeera crosses
Table S3) [28]. The first pair of oligonucleotides could dif- resulted in a bimodal distribution curve (Supplementary
ferentiate both the parent and the mutant for the wild- Figs. 1 & 2). Two peaks were observed for the flower-
type allele-specific primer. We synthesized two sets of ing of ­F2 individuals in response to photoperiod: one for
forward primers to identify mutant allele-specific prim- the photoperiod-insensitive group and the other for the
ers; the second oligonucleotide clearly differentiated the photoperiod-sensitive group (Supplementary Figs. 1 & 2).
wild type from the mutant (Supplementary Table S3; These results clearly indicated a monogenic inheritance
Table 1). After confirming the polymorphic reaction in pattern for photoperiod insensitivity.
the wild type (both Kalijeera & Kon Joha) and mutant,
the allele-specific markers were used for marker valida- Genome integrity analysis and genome sequencing
tion in the component wild type and mutant type bulk revealed more point mutations than repeat length/indel
of the ‘mutant × Kalijeera’ ­F2 population. Similarly, variation
SNP-based PCR markers were also used for confirma- Based on genome integrity analysis of the mutant, par-
tion in 10 homozygous wild-type plants and 10 homozy- ent (Kon Joha), Kalijeera and photoperiod-insensitive
gous mutant-type plants of the ‘mutant × Kon Joha’ ­F3 Ahu Jiha rice genotypes with 150 SSR and InDel mark-
population. ers (Supplementary Table S1), none of the markers could
detect any polymorphisms between the parent and PPIS
Structural analysis of wild‑type and mutant Hd1 proteins mutant. In contrast, the PPIS mutant was distinct from
Three-dimensional structures of wild-type and mutant the already available photoperiod-insensitive Ahu Joha
Hd1 proteins were predicted using AlphaFold2 [29]. rice cultivars; this established the novelty of the isolated
AlphaFold2, as implemented in ColabFold, was used for mutant (Fig. 2). Fifty-four descriptors comprising traits
the computational modeling [30]. The modeled structure at the seedling, vegetative, flowering and maturity phases
of only the CCT domain was compared to the crystal were recorded (Supplementary Table S4) for the PPIS
structure of the CCT domain (PDB ID 7C9O). Struc- mutant and Kon Joha. The PPIS mutant was monomor-
tural analysis and illustrations were generated using the phic and similar to Kon Joha in terms of fifty-two traits.
PyMOL molecular graphics system (v.2.6.0a0; Schrod- Polymorphism was evident for the other two traits: the
inger, LLC), an open-source molecular visualization soft- time of heading and maturity (Supplementary Table S4).
ware. The sequence alignment figure was prepared using However, the mutant was polymorphic to Kon Joha for
the ESPript server (https://​espri​pt.​ibcp.​fr; [31]). ­ anicle−1, plant height (cm) and panicle length
filled grain p
(cm) (Supplementary Table S5). These morphological dif-
Results ferences may arise due to variations in the environment
Inheritance of photoperiod insensitivity during flowering in the mutant and parent plants.
Inheritance of the mutant trait in the PPIS mutant Genome sequencing of the parent and mutant revealed
was confirmed in both the F ­ 2 (Fig. 1) and F
­ 3 genera- as many as 3,57,562 nucleotide variations between the
tions of the hybrids ‘PPIS mutant × Kon Joha’ and ‘PPIS Kon Joha and PPIS mutants. Genome sequencing gen-
mutant × Kalijeera’. Of the 514 plants in the F ­ 2 popula- erated 161,333,716 reads (29.5% duplicated data) in Kon
tion of ‘PPIS mutant × Kon Joha’, 381 were photoperiod Joha and 165,632,496 reads (33.7% repeated data) in the
sensitive, and 133 were photoperiod insensitive (χ2 = 0.21; PPIS mutant. The overall GC content was 44.4%. Of the
P = 0.647). Similarly, among the 502 ­ F2 plants of the 3,57,562 nucleotide variants, 23.17%, 72.42% and 4.41%
‘PPIS mutant × Kalijeera’, 364 and 138 plants were pho- were silent, missense and nonsense mutations, respec-
toperiod sensitive and photoperiod insensitive, respec- tively. Among these missense and nonsense mutations,
tively (χ2 = 1.66; P = 0.197). When 250 ­F2 progenies (66 8.68% were in the exon region. The average mutation
photoperiod-insensitive: 184 photoperiod-sensitive) density was 1 in 1046 bases. The Ts/Tv ratio for all the
of ‘PPIS mutant × Kon Joha’ were tested in the ­F3 gen- point mutations was 2.38.
eration, all 66 photoperiod-insensitive mutant proge-
nies were found to be true bred. A total of 122 F ­ 2 plant BSA identified two closely spaced SSRs on chromosome 6
progenies were heterozygous (3:1), and the remaining 62 linked to the mutant trait
plants were subjected to true breeding for photoperiod For quick mapping of the mutant trait in the
sensitivity [χ2 (1:2:1) = 0.272; P = 0.873]. Thus, ­F2 and ­F3 PPIS mutant, it was hybridized with a distant
Reddy et al. Biological Research (2024) 57:94 Page 6 of 13

Fig. 1 Hybridity confirmation in ­F1 individuals of the cross PPISM/Kalijeera: a intermediate husk color of ­F2 seeds; b RM480 amplified both the bands
of mutant (M) and Kalijeera (KJ) in ­F1; c sensitive Kalijeera (left), PPISM (right) and segregating ­F2 of their cross (center); d sensitive Kon Joha (left),
PPISM (right) and segregating ­F2 of their cross (center)

parent (Kalijeera) to develop a segregating F

­ 2 popula-
tion. Screening of 402 SSR and InDel markers revealed
57 (15.17%) polymorphic markers between the mutant
and Kalijeera strains. The BSA of these polymorphic
markers in the photoperiod-sensitive bulk (PS bulk)
and photoperiod-insensitive bulk (PIS bulk) samples
revealed a close association between RM527 and the
mutant trait on chromosome 6. Moreover, other SSR
markers (RM204, RM225, RM586, RM587, RM588,
and RM589) on chromosome 6 were not associated
with the mutant trait. The RM527 marker lies between
9,862,291 and 9,862,523 bp on chromosome 6 of rice.
Other markers remained at the proximal end of chro-
mosome 6 (beyond 5.0 Mbp). We then synthesized 40
Fig. 2 Dendrogram depicting the genetic relationship of the PPIS
additional SSR markers between 5.0 and 13.2 Mbp on
mutant with the parent and other Ahu Joha genotypes
Reddy et al. Biological Research (2024) 57:94 Page 7 of 13

chromosome 6 (Supplementary Table S2). By screen- The genotyping assay in these individual 10 PIS plants
ing for polymorphisms between Kalijeera and the revealed no crossing over for RM527 and RM19725,
PPIS mutant, five additional SSR markers (RM3794, whereas all the other polymorphic SSR markers on
RM19592, RM19725, RM5850 and RM19902) were chromosome 6 showed recombination and proved their
found to be polymorphic (Supplementary Table S2). incomplete linkage with a photoperiod-insensitive gene
Furthermore, BSA revealed a close association between in the PPIS mutant (Supplementary Fig. 3).
RM19725 (8,134,068 to 8,134,111 bp in chromosome
6) and the mutant trait. Thus, BSA delineated a marker A single‑base deletion in Hd1 between RM19725
interval (RM19725—RM527) on chromosome 6 that and RM527
is tightly associated with photoperiod insensitivity Several flowering-related genes were located on rice
in the PPIS mutant (Figs. 3 & 4). To confirm this, we chromosome 6. The Hd17 (OsELF3), Hd3a, and RFT1
genotyped all 10 individual mutant-type plants (con- genes are between 2.23 Mbp and 3.0 Mbp long. Another
stituting the PIS bulk) with polymorphic SSR mark- three genes, Os06g40080/Se5, Os06g41090/OsFTIP and
ers (RM589, RM225, RM3794, RM19592, RM19725, Os06g45640/OsNF-YC4, were present at 23.85 Mbp to
RM527, RM5850 and RM19902) on chromosome 6. 27.64 Mbp. Heading date 1 (Hd1) included 9,336,359

Fig. 3 Capillary gel electrophoresis of the RM527 marker in the BSA experiment and bulk F2 individuals of ‘Mutant × Kalijeera’

Fig. 4 Agarose gel electrophoresis of the RM19725 marker in the BSA experiment and bulk individuals of ­F2 of ‘Mutant × Kalijeera’
Reddy et al. Biological Research (2024) 57:94 Page 8 of 13

to 9,338,643 bp. Hd1 lies perfectly between the identi- band in seven wild-type plants in the F ­ 2 population of
fied markers RM19725 and RM527 on chromosome 6. ‘PPIS mutant × Kalijeera’ was due to their heterozygous
After cloning, we amplified Hd1 from Kon Joha and PPIS nature, as revealed by the amplification of the RM19725
mutants and sequenced four gene fragments. After align- marker (Fig. 4). The heterozygosity shown through the
ing the complete sequences of Kon Joha (OR113693) and allele-specific SNP marker was well matched with the
the PPIS mutant (OR113694), a single-base deletion (G to RM19725 amplification due to the close physical prox-
−) was identified in exon 2 of Hd1 (Fig. 5). imity with Hd1. The use of allele-specific SNP primer
pairs further confirmed the heterozygosity and homozy-
Validating the relationship between mutant traits gosity of wild-type F­ 2 plants of ‘mutant × Kalijeera’. Of
and candidate gene mutations in two segregating the 10 plants, 7 were heterozygous wild type, and 3 were
populations homozygous. Thus, the segregation ratio in the wild-
From the above pattern of mutations in Hd1, we could type pool (10 plants) for heterozygous and homozygous
visualize the nonsynonymous nature of single-base plants maintained genotypic segregation at 2:1 (χ2 = 0.13,
deletions in exon 2. The single-base deletion leads to a P value = 0.937). For the next level of validation, we used
frameshift mutation phenomenon and a possible increase true breeding (deduced from ­F3 segregation), wild-type
in nonfunctional proteins. Using the Web-SNAPPPER (10 plants), and mutant-type (10 plants) plants from
tool, two allele-specific forward primers were designed ‘PPIS mutant × Kon Joha’. Due to the homozygosity
for the parent (wild type) and mutant (PPIS mutant) of the plants, all the wild-type plants amplified bands
strains, along with a standard reverse primer. The ampli- with parental-allele-specific primer pairs but no bands
fication of both of these allele-specific primer pairs was with mutant-allele-specific primer pairs (Supplemen-
first confirmed in Kon Joha and PPIS mutants (Supple- tary Fig. 4). The bands of all 10 mutant-type plants were
mentary Fig. 4 & 5). The wild-type allele-specific primer amplified with mutant allele-specific primer pairs, but no
pairs amplified a 200 bp band in Kon Joha but not in the bands were amplified with parental allele-specific prim-
PPIS mutant. Mutant-allele-specific primer pairs ampli- ers (Supplementary Fig. 5).
fied the band in mutants but did not amplify the band in
Kon Joha. For validation, two different populations were A frameshift in exon 2 of Hd1 leads to drastic changes
used. Initially, individual gDNA of bulk components in the C‑terminal region of the Hd1 protein
of the BSA study in ­F2 of ‘PPIS mutant × Kalijeera (dis- A single-base deletion in exon 2 of Hd1 leads to a
tant parent)’ was used. The bands of all 10-individual frameshift mutation at the C-terminus of the Hd1 pro-
mutant-type plants were amplified with mutant-allele- tein. The C-terminus of Hd1 contains a CCT {Constant
specific primer pairs, but no amplicons were amplified (CO), CO-LIKE (COL) and TIMING OF CAB EXPRES-
with parental-allele-specific primer pairs (Supplemen- SION1 (TOC1)} domain. Due to this frameshift, the
tary Fig. 6 & 7). The bands of seven out of the 10 wild- mutant Hd1 protein has an altered primary sequence at
type plants were amplified with mutant allele-specific the C-terminus, which includes the CCT domain (Sup-
primer pairs, and the 200 bp amplicons of all 10 plants plementary Fig. 8). To gain insight into the structural
were amplified with parental allele-specific primer pairs changes caused by frameshift mutations, three-dimen-
(Supplementary Fig. 6 & 7). The amplification of the sional models of wild-type and mutant Hd1 proteins were

a.

b.
WT
Mutant

Fig. 5 ClustalW sequence alignment of parental and mutant Hd1 surrounding the mutation site. a It represents the genomic sequences,
where at 2962 position, a single base deletion (G to −) occurred. b It represents corresponding triplets match of the ORF
Reddy et al. Biological Research (2024) 57:94 Page 9 of 13

constructed using AlphaFold2, and only the CCT domain [34, 35] and is controlled by multiple quantitative trait
was used for structural analysis (Supplementary Fig. 9). loci (QTLs). Various genetic mapping approaches have
The modeled structure of the wild-type CCT domain was mapped a large number of heading date QTLs [9, 36–50].
compared to the crystal structure of the CCT domain. Hd1 is an Arabidopsis CONSTANS (CO) ortholog in
The very high structural similarity shows that AlphaFold rice that delays and promotes heading under long-day
can correctly predict the structure of the CCT domain of and short-day conditions, respectively [8, 51, 52]. This
the Hd1 protein (Supplementary Fig. 9). The wild-type gene regulates panicle development and affects yield

ɑ-helices (ɑ1 and ɑ2) and 2 loops (Fig. 6 and Supplemen-

Hd1 protein has a typical CCT domain containing two [53]. Adapting temperate japonica rice cultivars in tropi-
cal regions results in the loss of function of Hd1 alleles
tary Fig. 9). However, the mutant Hd1 protein showed [54]. Hd1 was found to be a possible target of artificial
changes in secondary structural elements, as illustrated selection during domestication to diversify the head-
in Fig. 6. ing dates of rice cultivars [55]. The present study reports
the isolation of a photoperiod-insensitive mutant with
Discussion a loss-of-function mutation in Hd1. This particular
Mutations are essential to evolution. Even deleterious mutant was isolated from a gamma ray-induced muta-
mutations can cause evolutionary changes, especially genized population of Kon Joha rice, a landrace grown
in small populations, by removing individuals carrying for its short aromatic grain in Assam, India. After veri-
adaptive alleles at other genes [32]. During domestica- fying its true breeding nature in successive mutant gen-
tion, loss-of-function mutations resulting in seed dor- erations, the mutant was found to be genetically similar
mancy and shattering of wild rice have become favorable to its parent but remains highly dissimilar to the exist-
for human consumption and usage. Following domes- ing photoperiod-insensitive Ahu Joha rice genotypes
tication in the tropics and subtropics, Oryza sativa cul- (Fig. 2). The distinctness of the isolated mutant from
tivars gradually spread to temperate and cool regions other Ahu Joha genotypes prompted us to further char-
due to the loss of photoperiod sensitivity [33]. Naturally, acterize the mutant at the genetic and molecular levels.
rice flowers are under SD conditions. Heading date is a In the inheritance study, two mapping populations were
critical trait for rice diversification and domestication raised for phenotypic and genotypic segregation in the

Fig. 6 Comparison of the wild-type and mutant HD1-CCT domains. A Comparison of the primary sequence and secondary structural elements.
B Predicted secondary structure elements of the wild-type HD1-CCT domain (yellow). C Predicted secondary structure elements of the mutant
HD1-CCT domain (magenta). D Superposition of the wild-type (yellow) and mutant (magenta) HD1-CCT domains
Reddy et al. Biological Research (2024) 57:94 Page 10 of 13

­F2 and F ­ 3 generations, respectively. The segregation pat- (Fig. 6 and Supplementary Fig. 9 & 10). Helix ɑ2 (residues
terns revealed that the induced photoperiod insensitiv- 361–369) and loop2 (residues 370–379) together form
ity in Kon Joha is governed by a single recessive gene. the DNA recognition element (DRE, residues 361–379)
The monogenic inheritance of photoperiod insensitivity of CCT, which anchors into the minor groove of the
was previously reported by Jones et al. [56] and Chand- ’CCACA’ box (Fig. 6 and Supplementary Fig. 9).
raratna [57]. In parallel, screening for polymorphisms of The crystal structure of the H ­ d1CCT​-GHD8/OsNF-

and K348 from ɑ1 and R352, K353, R359 and Y360 from
genome-wide SSR and InDel markers between a mutant YC2 trimer shows that residues R341, R344, Y345, R346,
and a distant parent, Kalijeera, detected almost 14.2% of
the polymorphisms. A BSA approach in the F ­ 2 popula- loop1 regions form hydrogen bonds with different resi-
tion of ‘PPIS mutant × Kalijeera’ detected the association dues of GHD8 and OsNF-YC2 (Supplementary Figs. 11
of the RM527 marker with the photoperiod insensitiv- & 12). Among these residues, R338 and Y345 in the PIE
ity trait. RM527 is present on chromosome 6 of the rice are crucial for the binding of Hd1 to the GHD8/OsNF-
plant. The successful screening of 40 chromosome-6-spe- YC2 dimer, and R338A and Y345A mutations abrogate
cific SSR markers (with 5.0 and 13.2 Mbp genomic inter- the formation of the HD1/GHD8/OsNF-YC2 trimer [10].
vals) revealed five additional polymorphisms. RM19725 These residues are also conserved in the HD1 homolog
was linked to photoperiod insensitivity in BSA. The in Arabidopsis and participate in similar interactions
map interval delimited by RM527 and RM19725 repre- [59]. ­Hd1CCT​ residues involved in the interactions with
sented an approximately 1.73 Mbp interval (8,134,111 GHD8 and OsNF-YC2 are primarily positively charged
to 9,862,291 bp), within which the heading date 1 (Hd1) residues that interact with negatively charged residues
gene is present. Other photoperiod insensitivity genes, from GHD8 and OsNF-YC2 in a zipper-like arrange-
such as Hd17 (OsELF3), Hd3a and RFT (Rice flower- ment (Supplementary Fig. 13). The structure of the HD1/

that loop 1 acts as a flexible linker between the ɑ1 and

ing locus T), are also present in tandem on the proximal GHD8/OsNF-YC2 trimer-DNA complex also suggested

ɑ2 helices and helps in the proper positioning of the ɑ2

region of chromosome 6 (2,234,119 to 2,942,452 bp).
None of the other SSR markers around these genomic
boundaries of chromosome 6 showed association/link- helix in the minor groove of the ’CCACA’ box. Therefore,
age with photoperiod insensitivity in the isolated mutant both the positively charged residues and the nature of the
(Supplementary Fig. 3; Supplementary Table S2). The secondary structural elements needed to properly orient
other two photoperiod insensitivity genes, Se5/OsHY1 these residues are crucial for binding the CCT domain to
and OsNF-YC4/OsHAP5B, are present at the very distal the GHD8/OsNF-YC2 dimer.
end of chromosome 6, from 23,853,714 to 23,858,061 bp A single-base deletion in exon 2 of Hd1 leads to a
and 27,627,767 to 27,633,058 bp, respectively [58]. frameshift mutation in the middle of the CCT domain,
After confirming the position of the mutant photo- resulting in a mutant Hd1 protein with an altered pro-
period insensitivity gene in the PPIS mutant, we cloned tein sequence at the C-terminal region (Fig. 6). Since the
Hd1 from both the mutant and the parent genomic DNA. mutation site is located in the middle of the PIE (Protein
A single-base deletion in exon 2 of Hd1 results in a dys- interaction element) of the CCT domain, it changes the
functional Hd1 protein. This single-base deletion causes primary sequence and the secondary structure of the lat-
a frameshift mutation in the C-terminus of Hd1 proteins, ter part of the PIE and completely alters the sequence
leading to a mutant Hd1 protein with an altered primary and structure of the DRE (DNA recognition element) of
sequence at the C-terminus (Supplementary Fig. the CCT (Fig. 6 and Supplementary Fig. 9). The mutant
8). The Hd1 protein has a zinc finger B-box domain CCT domain likely folds into a helix and loop structure.
near the N-terminus and a CCT domain (residues 323– However, computational modeling suggested distinct dif-

the mutant CCT domain, the crucial ɑ1 helix is trun-

387) near the C-terminus, followed by a single helix and ferences in the wild-type and mutant CCT domains. In

cated, and the C-terminal part of the ɑ1 helix becomes

a 10-residue tail. The CCT domain at the C-terminus is
crucial for the binding of Hd1 to the GHD8/OsNF-YC2
dimer to form the Hd1-GHD8/OsNF-YC2 trimer, which unstructured.

more prominent helix in continuity with helix ɑ2 (Fig. 6).

specifically targets a CCACA nucleotide sequence within Similarly, loop 1 regions become helical and form a
the Hd3a promoter [10]. In the crystal structure, Shen

ɑ-helices (ɑ1 and ɑ2) and 2 loops (L1 and L2) (Fig. 6 and
et al. [10] reported that the CCT domain folds into 2 At the residue level, in the PIE of the mutant CCT

Supplementary Fig. 9). Helix ɑ1 (residues 335–351) and

domain, the crucial tyrosine residue (Y345) is replaced

as R346 and K348 from ɑ1 and R352, K353, R359 and

with threonine, and all the other essential residues, such
loop1 (residues 352–360) together form a protein-inter-
acting element (PIE, residues 335–360) and interact with Y360 of the loop region, are also altered (Fig. 6). Since
the GHD8/OsNF-YC2 dimer, yielding a functional trimer both positively charged residues and specific secondary
Reddy et al. Biological Research (2024) 57:94 Page 11 of 13

structures are required for the interaction of the CCT Vice-Chancellor and Director of Research (Agri.), Assam Agricultural University,
and Jorhat for their encouragement and support.
domain with the GHD8/OsNF-YC2 dimer, these altera-
tions are likely to cause the loss of this crucial interaction. Author contributions
Therefore, this altered CCT domain of Hd1 is unlikely to Concept and design (D.S., N.S.B., B.K.D., S.M. and A.R.B.) were a result of our
collective brainstorming; mutant development (D.S., D.B. and T.V.S.S.M.) was a
bind to the GHD8/OsNF-YC2 dimer to form a repres- joint effort; experimentation and data collection (B.J.R., S.M.B., S.M., T.V.S.S.M.
sive protein trimer complex. This particular CCT domain and D.B.) were carried out by our team; data analysis (B.J.R., S.M. and S.C.B.) was
is essential for yielding a histone-like structure along a collaborative task; first manuscript draft (B.J.R., S.M. and S.C.B.) was a product
of our shared insights; critical review and editing (D.S.) was done by our
with the DTH8 and NF-Y proteins via interaction of the meticulous reviewer; and all authors approved the final manuscript, signifying
promoter element of the florigen gene containing the our unanimous agreement.
CCACA element [10]. The changes in the primary and
Funding
secondary structures of the CCT domain in the mutant The Board of Research in Nuclear Sciences, Department of Atomic Energy,
Hd1 protein are likely to compromise the interaction Government of India (35/14/17/2016-BRNS/35056 Dated. 04/06/2016) partially
with the CCACA element that leads to relaxed repression funded this research.
of Hd3a and RFT genes under long-day conditions. In Data availability
the absence of the trimeric complex interaction with the All data generated or analyzed during this study are included in this published
CCACA box of the Hd3a promoter, the interaction is also article and its supplementary information files.
likely to be affected, leading to the observed phenotype.
Declarations
Ethics approval and consent to participate
Conclusion Not applicable.
The cultivation of Assam’s aromatic Joha rice, a unique
rice class famous for its aroma, taste, and nutritional Consent for publication
Not applicable.
benefits and high market prices, is decreasing to 5%
of the winter rice area in the state, mainly due to poor Competing interests
productivity, long duration, limited seasonal adapt- The authors have meticulously reviewed their personal and professional affilia-
tions and declare with utmost certainty that they do not have any competing
ability, and high production costs. Short-grain aro- financial or non-financial interests.
matic Joha rice is mostly photoperiod sensitive and least
responsive to applied fertilizers. These fragrant low- or Author details
1
Department of Plant Breeding and Genetics, Assam Agricultural University,
no-input rice cultivars, which are organic by default, have Jorhat, Assam 785013, India. 2 Nuclear Agriculture and Biotechnology Division
evolved to thrive in rainfed shallow lowland rice eco- (NA&BTD), Bhabha Atomic Research Centre (BARC), Trombay, Mumbai 400085,
systems. Here, we report a novel mutant allele of head- India. 3 Protein Crystallography Section, Bio‑Science Group, Bhabha Atomic
Research Centre (BARC), Trombay, Mumbai 400085, India. 4 Homi Bhabha
ing date 1 (Hd1) conferring photoperiod insensitivity National Institute, Training School Complex, Anushaktinagar, Mumbai 400094,
in the famous Joha rice variety of Assam. This mutant India. 5 Department of Agricultural Biotechnology, Assam Agricultural Univer-
allele, when adopted, could lead to significant increases sity, Jorhat, Assam 785013, India.
in yield, reducing production costs and increasing profits Received: 9 May 2024 Accepted: 3 October 2024
for farmers. The deciphered genetic basis of this mutant
allele, which involves a mutation in the Hd1 gene, has
underscored the urgent need for a structured breed-
ing programme in high-value Joha rice, highlighting the
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