Candidate gene analysis distinguishes regulatory mechanisms for phenological transitions in Indian and Snowball cauliflower

ABSTRACT The present study analysed expression of 13 candidate genes in relation to different developmental transitions (vegetative–curding–flowering) in Indian and snowball cauliflwer using quantitative real-time PCR (qRT-PCR). Expression levels of FRUITFULL-c (BoFUL-c), FLOWERING LOCUS T (BoFT), SUPPRESSOR OF OVEREXPRESSION OF CONSTANS1 (SOC1), and LEAFY(BoFLY) increased from seedling to full curd stages in all varieties. VERNALIZATION2 (BoVRN2) and APETALA2 (BoAP2) genes showed a contrasting trend in Pusa Meghna (Indian type) and Pusa Snowball Kt-25 (Snowball type). High expression of BoVRN3, BoVIN2, and BoAP2 in Pusa Snowball Kt-25 fitted well with its vernalisation requirement. The increasing expression of BoFT, SOC1, and BoVIN3 while decreasing trend in BoLFY, REPRODUCTIVE MERISTEM (BoREM1), CAULIFLOWER (BoCAL), and BoFUL-d during seedling–curd initiation–full curd stages in Pusa Shukti (mid-late) matches with Pusa Snowball Kt-25, highlighting their genetic closeness. Hierarchical clustering of these 13 candidate genes grouped all the genes into two major clusters: one had the CAULIFLOWER CURD EXPRESSION 1 (CCE1), BoLFY, BoREM1, BoFUL-c, BoFUL-d, LEAFY Homologous (BoFH), and BoCAL genes while the rest of the genes constituted a second cluster. A significant interaction effect was observed in four candidate varieties and two growing situations (i.e. normal and lower temperature range) for curding-related phenological traits. The information will be helpful in understanding the curding behaviour.


Introduction
Cauliflower (Brassica oleracea var.botrytis L.) is a worldwide cultivated vegetable crop which covers nearly 1.38 million ha in area and contributes 24.18 million tonnes to the world's vegetable production (FAOSTAT, 2019).China and India are the leading producers of cauliflower and share almost equally in their joint share of 65% of global production.Originating in the Mediterranean region, cauliflower evolved into distinct ecotypes during the 15 th to 16 th century in Europe, including the Italians or Originals, Cornish, Northerns, Roscoff, Angers, and Erfurts or Snowball.The shift from vegetative to curding to reproductive stages in cauliflower is a temperaturesensitive process.Cauliflower, due to its thermoresponsive characteristics, can be broadly classified into two distinct groups: the European annuals and the Indian or tropical types, each with different temperature optima for transitioning from the vegetative to the curding stage at 10-16°C and 20-25°C, respectively.Interestingly, the Indian type was evolved from the European type, i.e. early and mid-early groups from the Cornish type and the mid-late group from intercrossing of Cornish and other types.However, continuous major and minor mutations have contributed significantly to the evolution of unique attributes in Indian cauliflower for plant habit, tolerance to biotic and abiotic stresses, curding traits, and reproductive behaviour (Swarup & Chatterjee, 1972;Singh & Kalia, 2021).In evolution of tropical types, frequent introgression of genomic regions from European or snowball types could result in desirable phenotypes particularly for curding traits (Dey et al., 2019).This was true for mid-late group genotypes which have genetic materials from both Indian and Snowball groups of cauliflower.Notably, the Indiantype cauliflower has developed an exceptional tolerance to high temperatures ranging from 16 to 27°C, setting it apart from the European summer cauliflower, which is known to thrive at temperatures around 15°C and is reportedly adversely affected when exposed to temperatures above 20°C (Rosen et al., 2018).Reeves et al. (2001) also reported very little apex expansion above 17°C in European winter cauliflower (both `December/January' and `March' groups).The apex expansion is a prerequisite step for curd initiation (CI) in cauliflower.In the Indian or tropical type, the transitions from the vegetative to the reproductive stage through the curding stage occur at higher temperature optima than in the Snowball type.Further, the Snowball type never attains flowering or produces viable seeds in the northern plains of India due to the lack of suitable temperature (Verma & Sharma, 2000).Despite the wealth of knowledge on the evolutionary history of cauliflower, a comprehensive understanding of the underlying molecular changes that have shaped its curding traits is essential.In cauliflower, the 'curd' is the edible part and has economic value.It corresponds to morphological excursions and pre-floral meristems sharing apices of both vegetative and reproductive parts.In Arabidopsis, MADS-box genes have been investigated for their role in developmental processes, namely meristem specification, flowering transition, organ development, and fruit ripening (Smacznaik et al., 2012).CI in cauliflower is a genetically governed trait but temperature plays a key role (Lan & Paterson, 2000;Rakshita et al., 2021).It is a polygenic trait and related to other phenological attributes such as leaf number and plant age (Boss et al., 2004;Matschegewski et al., 2015;Rosen et al., 2018).Various genes were reported to have a role in integration of endogenous and exogenous stimuli to induce curding and flowering at the most favourable conditions (Amasino, 2005;Boss et al., 2004).AP1, CAL, FKF1, GI, CRY2, FE, CO, and FT genes play roles in floral transition in Arabidopsis thaliana (Mouradov et al., 2002;He & Amasino, 2005).Their homologues were identified in cauliflower and broccoli as BoLFY, BoTFL1, BoAP1, and BoCAL (Duclos & Björkman, 2008;Lan & Paterson, 2000).However, these homologues in B. oleracea are not consistent with their Arabidopsis functions (Anthony et al., 1995;Jordan & Anthony, 1994).Thus, information on genes involved in the transition from the vegetative to the curding and further flowering stages is still inconclusive.
Genomic information for prediction of curd induction and flowering in European cauliflower indicated the role of different genes, namely BoAP1-a, BoAP1-c, BoCAL, BoFUL-a, BoFUL-b, BoFUL-c, BoFUL-d, BoLFY, AP2, UFO, BoTFL1, CCE1, and BoREM1 (Duclos & Björkman, 2008;Labate et al., 2006;Lan & Paterson, 2000;Rosen et al., 2018).Further, meristem identity genes (MIGs) were also reported to be involved in curding, transition from inflorescence meristem to floral primordium, and floral organ differentiation (Duclos & Björkman, 2008).The candidate gene approach permits the use of quantitative real-time PCR (qRT-PCR) to understand changes in expression of the target gene(s) at different developmental stages besides simultaneously measuring the expression of several genes having overlapping function (Heid et al., 1996).In the context of tropical cauliflower, a thorough analysis of such genes is crucial to comprehend the genetic changes underlying its historical evolution and to develop genotypes with desired curding behaviour.The present study, therefore, sought to shed light on the molecular genetics of the transition from the vegetative to the curding and reproductive stages in Indian cauliflower, utilising the candidate gene approach.Furthermore, the study aimed to examine the effect of temperature on curding traits by growing the same set of varieties during two distinct growing periods.

Plant material and field evaluation
In the present study, Pusa Meghna, Pusa Sharad, Pusa Shukti, and Pusa Snowball Kt-25 varieties of cauliflower were used as experimental material (Table 1).These varieties form curd at different temperature regimes and are grouped as early (20-27°C), midearly (16-20°C), mid-late (12-16°C), and late or Snowball (10-16°C) maturity groups, respectively.The experimental crop was raised in a split plot design with growing situation as the main plot and varieties as the sub-plot at Research Farm, Division of Vegetable Science, Indian Agricultural Research Institute (IARI), New Delhi in 2017-18 and 2018-19.Five replications were taken for each variety in both situations and both years.A set of these four varieties was sown as per the recommended time for each respective maturity group [i.e. for the normal situation (NS) experiment] whereas delayed sowing (i.e.October sowing) was done for the challenge situation (CS) (Figure 1).Standard crop management practices were followed for raising a healthy trial crop as described by Singh and Sharma (2003).

Morphological observations
The morphological traits were observed from both the NS and CS during 2017-18 and 2018-19.The days to curd initiation (DCI), days to curd harvesting (DCH), and days to bolting (DB) were observed from day of sowing.Curd length (CL) and curd width (CW) were recorded using a metre scale.Gross plant weight (GPW), marketable curd weight (MCW), and net curd weight (NCW) were recorded from five random plants in each plot using a portable electronic chargeable weighing balance.

Tissue sampling and RNA extraction
Leaf samples were collected from healthy plants of all the test varieties at three developmental stages, namely seedling (SL), CI, and full or marketable curd (FC) stage, as per the sampling schedule depicted varietywise in Figure 2A-D.The bolting stage was also observed in the tested varieties.Curd samples at the FC stage were also collected from all four varieties grown in the NS and CS.Further, the expression pattern of genes was validated in eight varieties of different maturity groups, i.e. early: Pusa Meghna, Pusa Ashiwni, Pusa Kartiki, and Pusa Deepali; midearly: Pusa Sharad; mid-late: Pusa Paushja, Pusa Shukti; and Snowball: Pusa Snowball Kt-25.For this, varieties were sown in December and samples were taken only at one time-point (i.e. 45 days after sowing).For this, three biological replicates were collected from each variety in liquid N 2 and stored in a deep freezer (−80°C) until RNA isolation.

Candidate genes and primer designing
Major candidate genes involved in curd proliferation and flower induction (Duclos & Björkman, 2008), reproductive transition from the vegetative stage to CI and initiation of floral primordia (Duclos & Bjorkman, 2015), reproductive development (Ridge et al., 2015), and temperature-related curd induction in cauliflower and broccoli (Matschegewski et al., 2015) were selected for the present study (Table 2).These were synthesised from G Biosciences, Geno Technology, Inc., St. Louis, USA.

RNA extraction and qRT-PCR analysis
Total RNA from the tissue samples was extracted using Tri-Xtract (G Biosciences, Geno Technology, Inc., St. Louis, USA) following the manufacturer's guidelines and quantified with a nano spectrophotometer (Eppendorf Pvt.Ltd).One microlitre of isolated RNA was loaded on a denaturing agarose gel (1.2%) to check its concentration and integrity.Two micrograms of total RNA were reverse transcribed using a Verso cDNA synthesis kit (Thermo Fisher Scientific, Inc.) following the manufacturer's instructions.The cDNA was diluted 10-fold to make 1 µl for the qRT-PCR reaction.Ten microlitres of qRT-PCR reaction mixture were prepared using 5 µl of 2X SYBR Green master mix (Applied Biosystems, Massachusetts, CA, USA), 2 μl nuclease-free water, 1 μl each of forward and reverse primers of the respective gene (100 nM), and 1 μl of template cDNA.The qRT-PCR was performed on the LightCycler® 480 Real-Time PCR (Roche Life Sciences).The Q-PCR programme comprised initial denaturation at 95°C for 120 s, 40 cycles of denaturation at 95°C for 30 s, annealing at 55°C for 60 s, and extension at 72°C for 30 s.For qRT-PCR, two technical replicates were taken for each of three biological replicates from every variety and cq values of both the technical replicates per biological replicate were averaged.In order to select an ideal reference gene, two reference genes, viz.Bo18S (Duclos & Björkman, 2008) and Bo actin (Ridge et al., 2015), were tried by our group.Although both the genes gave stable results across all the samples, the cq values obtained in actin were above 25 and those in the case of 18S were near to 18. Therefore, in all our qPCR studies in cauliflower on expression analysis we use 18S as the reference gene and the ΔCt value of each target gene in the present study was also normalised with the internal control Bo18S.The ΔΔCt value was calculated by taking ΔCt at the SL stage as calibrator.The values were used to plot graphs of relative expression of the genes across varieties.

Statistical analysis
Analysis of variance (ANOVA) for all eight observed morphological traits was performed using mean values of two years (i.e. 2017-18, 2018-19) in a split plot design using online available SSCNARS software (http://apps.iasri.res.in/sscnars).Here, growing situations were the main plot and varieties were the subplot.Multi Experiment Viewer Mev4.9 software was used for construction of heat maps of differentially expressed genes.Pearson correlation was calculated from distance matrices and cluster analysis was done using a complete linkage clustering pattern.

Morphological observations
The ANOVA for curd phenological stages and curd morphological traits showed significant difference in tested varieties and both growing situations (Table 2).The observations recorded in the present study from the NS and CS are given in Table 3.The maximum difference between mean temperature present during CI in the NS and CS was observed for Pusa Meghna (18.6°C) followed by Pusa Sharad (7.89°C) and Pusa Shukti (4.5°C) while this gap was minimum for Pusa Snowball Kt-25 (0.2°C).In the NS, the varieties behaved according to their normal growth and development pattern.Precocious CI was noticed in all three varieties of Indian cauliflower, namely Pusa Meghna, Pusa Sharad, and Pusa Shukti.However, Pusa Snowball Kt-25, which represented the Snowball or late group of cauliflower, did not show significant difference between the NS and CS for curding traits.The phenological changes from SL to flowering stages in all four varieties of cauliflower are shown in Figure 3.
Under NSs, the CI of the different varieties occurred between 90.8 and 110.6 days after sowing.However, in the challenge condition, CI took place within a range of 64.8-104.6 days.Interestingly, Pusa Snowball Kt-25 exhibited no significant difference in CI time between the two conditions, taking 110.6 days under normal conditions and 104.6 days under challenging conditions.Pusa Meghna was the earliest for CI and curd harvesting in both the normal and challenge conditions.In comparison, Pusa Snowball Kt-25 took 110.6 days for CI in the NS.MCW ranged from 380.0 to 1110.0 g and NCW from 294.0 to 890.0 g, with Pusa Meghna having the minimum values and Pusa Snowball Kt-25 the maximum weights under normal conditions.Curd traits were significantly reduced in the challenge condition in comparison to the NS, particularly in Pusa Meghna and Pusa Sharad.The highest impact of temperature was noticed on NCW and MCW in Pusa Meghna (−194.0%and −131.7%,respectively) followed by Pusa Sharad (−57.7% and −35.6%, respectively) and Pusa Shukti (−23.2% and −26.8%, respectively), while the impact was the least in Pusa Snowball Kt-25  (−15.8% and −21.2%, respectively).Additionally, there were significant differences among the varieties for CL, CW, DB, DCI, DCH, and GPW (Figure 3).

Gene expression analysis
The results of expression analysis of 12 candidate genes are presented in Figure 4A-L.The SL stage of Pusa Meghna, an early group variety, was taken as the reference for interpretation of relative fold changes.
The expression pattern of BoAP2, BoCAL, BoVRN2, SOC1, and BoVIN3 was different in the typical Indian type (Pusa Meghna, Pusa Sharad) from the Snowball type (Pusa Snowball Kt-25).In Pusa Meghna (early group), the transcript abundance of BoFUL-c, BoFT, BoVIN3, and SOC1 was found to increase while that of CCE1, BoAP2, BoCAL, BoREM1, VRN2, and BoFLC2 decreased with the advancement of all three developmental stages, i.e.SL, CI, and FC.BoLFY showed a slight reduction in expression at the CI stage (0.9as against 1.0-fold at the SL stage), but showed highest expression (1.4-fold) at the FC stage.BoFUL-d showed highest expression at the FC (1.2-fold) stage, whereas BoCAL, BoREM, and VRN2 showed highest expression at the SL stage, which was downregulated till the CI stage (Figure 4A-L).
In Pusa Sharad (mid-early group), seven genes showed upregulation and their expression level increased with the developmental stages.These were BoFUL-c, BoFUL-d, BoLFY, BoREM1, BoFLC2, SOC1, and BoFT.Whereas, the BoCAL, BoAP2, CCE1, and BoVIN3 genes were downregulated during the CI and FC stages and VRN2 did not show significant change during the developmental stages.

BoFUL-c, BoFUL-d, CCE1, BoCAL, BoLFY, REM1, and
BoFLC2 had the highest expression level at the CI stage.BoFT had the highest expression level at the FC stage (50.2-fold) and lowest at the CI (0.3-fold) stage.
Comparison of expression of 13 different genes in curd samples at the FC stage from the normal and challenge conditions is presented in Figure 5.In the challenge condition, the expression level of BoFT, BFUL-d, SOC1, BoAP2, and BoVRN2 was higher in all three varieties than their expression levels in the NS.Further, BoCAL, REM1, and BoLFY expression was prominently high in Pusa Sharad and Pusa Shukti in the CS.However, the expression was also high in Pusa Snowball Kt-25 but at an insignificant level.A marginal increment was noticed in all three varieties for expression of CCE1.BoFLC-2 showed the reverse trend and expression was decreased in the challenge condition in comparison to the NS in all three varieties.Hierarchical clustering analysis of the genes at similar growth stages grouped these genes into two major clusters (Figure 6).CCE1, BoLFY, BoREM1, BoFUL-c, BoFUL-d, BoFH, and BoCAL formed one cluster while AP2, BoVRN2, BoVIN3, and SOC1 formed a sub-cluster of a second cluster.
The experiment on validation of the expression patterns of 12 genes was performed using eight varieties and results are presented in Figure 7.
The expression pattern of BoFT was high in three varieties of the early group.BoVIN3, CCE, BoFLC2, and AP2 were expressed at a high level only in Pusa Kartiki (early group) and BoFUL-c in Pusa Meghna.Overall, expression of eight genes, namely AP2, BoREM1,BoVRN2,BoFLC2,BoCAL,BoLFY,and BoFH, was found to be the highest in the early group varieties, i.e.Pusa Ashwani and Pusa Kartiki, and the mid-early group variety Pusa Sharad which had attained the CI stage.The transcript abundance of these genes was low in Pusa Deepali (of the early group but which showed   more delayed bolting than other early group varieties) and Pusa Meghna which attained the bolting stage.In mid-late group genotypes, Pusa Paushja showed higher expression than another variety Pusa Shukti of the same group for AP2, BoREM1,BoVRN2,BoCAL,BoFLC2,BoFT,BoFLY,CCE,BoVIN3, and BoFH.None of the genes showed high expression in Pusa Shukti (midlate group) and Pusa Snowball Kt-25 (late or Snowball) at 45 days after sowing because both these varieties were in the vegetative stage.

Discussion
Two different growing situations, namely (i) normal sowing time (sowing the crop in such a time that it is exposed to the desired range of temperature during the curding phase) and (ii) CS (sowing the crop in such a time that it encounters a lower range of temperature during the curding phase), were taken for the present study.Both situations had a wide difference for temperature during curding for early (Pusa Meghna), mid-early (Pusa Sharad), and mid-late (Pusa Shukti) varieties.The difference in mean temperature during CI between the NS and CS for Pusa Meghna, Pusa Sharad, Pusa Shukti, and Pusa Snowball Kt-25 was 18.6°C, 7.89°C, 4.5°C, and 0.2°C, respectively.The ascending order of varieties for DCI was Pusa Meghna<Pusa Sharad<Pusa Shukti<Pusa Snowball Kt-25 in both the NS and CS.Cauliflower is thermo-sensitive for CI as well as for flowering (Singh & Kalia, 2021;Matschegewski et al., 2015).Reeves et al. (2001) also highlighted the significance of the lower limit of mean temperature in apex expansion followed by CI in winter cauliflower (i.e.4°C for 'March' and 8°C for `December/January' groups).
Quantitative trait loci (QTLs) play a key role in determining the curding traits of cauliflower.Rakshita et al. (2021) identified 12 QTLs associated with 8 curdingrelated traits in Indian cauliflower, whereas Matschegewski et al. (2015) found 18 QTLs related to temperature-regulated curding traits in cauliflower.Rosen et al. (2018) also reported the significance of leaf appearance rate (LAR) in CI and identified 10 QTLs for LAR.The early group variety Pusa Meghna is known for its heat tolerance mechanism, allowing for CI to occur at higher temperatures.Sharma et al. (1997) attributed this to the occurrence of the desired temperature range (20-27°C for Pusa Meghna and 16-20°C for Pusa Sharad) during the early plant growth stage in challenging growing situations, although the initial gestation period was observed in Pusa Meghna.In NSs, CI curd initiation and maturity occurred in the month of September (27-30°C), which was also present during the month of October (the sowing time in challenging growing situations).Pusa Meghna's small curd size, observed in both normal and challenging growing situations, can be attributed to the small frame of its plants and the limited time for curd expansion when compared to other varieties.These factors are strongly correlated with curd size and weight.The increasing trend of curd size and weight was as follows: Pusa Meghna<Pusa Sharad<Pusa Shukti<Pusa Snowball Kt-25.The findings agree with reports of Singh et al. (2014) for a strong correlation between curd weight and plant frame (r 2 = 0.9168).Lin et al. (2019) also highlighted the role of temperature in CI and curd size in cauliflower while studying two different maturity group cultivars of cauliflower, namely H-37 (early maturity) and H-80 (mid-late maturity).They reported that the BoFLC2 expression was significantly downregulated in 'H-37' compared to 'H-80' after CI and advanced growth.Rakshita et al. (2021) also reported a similar trend for curding traits in early, mid-early, mid-late, and late group varieties of cauliflower.Matschegewski et al. (2015) performed transcriptional profiling of BoFLC and BoVRN2 and reported increased expression levels of BoVRN2 in fastercurding genotypes.The inconsistent function of both genes suggested the existence of facultative and/or BoVRN2/BoFLC-independent mechanisms in temperature-regulated floral transition in cauliflower, as reported by previous studies.Duclos and Björkman (2008) explained the role of BoCAL,CCE1,BoREM1,and AP2 genes in curd proliferation and flower induction.Moreover, BoFT and BoVIN3 genes have a role in reproductive development, as reported by Ridge et al. (2015), whereas BoLFY and SOC1 are involved in reproductive transition (Duclos & Bjorkman, 2015).Our study focused on the expression of these genes in four cauliflower varieties, namely Pusa Meghna, Pusa Sharad, Pusa Shukti, and Pusa Snowball Kt-25, belonging to early, mid-early, mid-late, and late maturity groups, respectively.Gene expression analysis was conducted at four different growth stages, including the SL stage, CI stage, and fully mature curd (FC) stage, revealing a stage-specific expression pattern of these genes.
Studies on natural variation as well as mutants in Arabidopsis led to the identification of several flowering genes.It has been proposed that in the vernalisation pathway, FLOWERING LOCUS C (FLC) and VERNALIZATION2 (VRN2) genes play a pivotal role in the regulation of the transition from the vegetative to the generative phase (Schmitz & Amasino, 2007).FLC encodes a MADS-box protein that prevents floral transition by directly repressing floral integrators including FLOWERINGTIME (FT) (Alexandre & Henning, 2008;Deng et al., 2011;Michaels & Amasino, 1999).In response to vernalisationinductive temperature, expression of VRN2), together with VERNALIZATION 1 (VRN1) and VERNALIZATION INSENSITIVE3 (VIN3), is induced, which mediates repression of FLC and thus releases flowering genes FT (flowering locus T), FD, and SOC1 (SUPPRESSOR OF OVEREXPRESSION OF CONSTANS1) from FLC suppression (He & Amasino, 2005;Jung & Muller, 2009).This in turn, results in subsequent activation of meristem identity genes, e.g.LFY and AP2 that promote floral induction (Jung & Muller, 2009;Sung & Amasino, 2004).In our study we observed a similar phenomenon because when we tried to see the difference in gene expression at CI and the FC stage in Pusa Snowball Kt-25 (a variety with a specific vernalisation requirement), lower expression of BoVIN3 (10.5-fold) and BoVRN2 (2.3-fold) was observed at the CI stage (though the expression in both genes was higher than at the SL stage) as compared to the FC stage and this was associated with higher expression of BoFLC2 (2.9-fold) at the CI stage which in turn repressed the expression of BoFT and SOC1 (0.3-and 4.6-fold, respectively) at this stage.On the onset of vernalisation-inducing conditions during January, expression of BoVIN3 and BoVRN2 (38.9-and 5.2-fold, respectively) was induced, therefore their expression was observed to be higher at the FC stage, which repressed BoFLC2 (lowering its expression at the FC stage to 2.3 fold), thereby releasing BoFT and SOC2 which therefore expressed at a higher level at the FC stage (50.2-and5.6-fold, respectively).As regards expression of meristem identity genes, LEAFY (BoLFY) and APETALA2 (BoAP2), studied in the present investigation, the expression of BoAP2 was lower (4.4-fold) at the CI stage and higher at the FC stage (6.3-fold), which is in line with the observations of Sung and Amasino (2004) as well as Jung and Muller (2009).However, BoLFY did not show compliance with these reports as expression of BoLFY was at 2.0-fold at the CI stage and 1.3-fold at the FC stage.In general, our observations confirmed the earlier reports of Schmitz and Amasino (2007), He and Amasino (2005), and Jung and Muller (2009).However, in three other varieties of cauliflower, namely Pusa Meghna, Pusa Sharad, and Pusa Shukti, which do not have a vernalisation requirement, the expression of all these genes was low at the CI stage and high at the FC stage.However, the expression remained the same at both stages in the case of BoVRN2 in Pusa Shukti, BoFLC2 in Pusa Meghna, BoAP2 in Pusa Sharad, and BoLFY in Pusa Shukti.Kempin et al. (1995) investigated the molecular basis of cauliflower phenotype (curd) in Arabidopsis and reported that this morphotype was due to the lack of a functional CAULIFLOWER (CAL) gene product.We also observed lower expression of BoCAL during CI and curd development in Pusa Meghna and Pusa Sharad.Late group cultivar Pusa Snowball Kt-25 showed its slightly higher expression at the CI stage but downregulation in the FC stage.Interestingly, the expression of BoCAL was observed to be higher in Pusa Shukti at both the CI and FC stages than the SL stage but the trend matched with Pusa Sharad (Indian type) and Pusa Snowball Kt-25 (snowball type).This supports the earlier theory of intermediate genetic constitution of the mid-late group (i.e.Pusa Shukti) which was hypothesised to be evolved by intentional crossing attempts between Indian-type and Snowball-type cauliflower genotypes (Swarup & Chatterjee, 1972).
Further, earlier studies have reported that the curdspecific gene CCE1 is involved in maintaining the developmental arrest (Palmer et al., 2001) while BoREM1 breaks the arrest to allow continued reproductive development (Franco-Zorrilla et al., 1999, Duclos & Bjorkman, 2008).We observed that expression of CCE1 was high at the CI stage whereas it declined at the FC stage in all the varieties under study except Pusa Sharad where the reverse was true.In the case of BoREM, Pusa Snowball Kt-25 and Pusa Shukti showed a similar behaviour with high expression at the curd induction stage and low at the FC stage, whereas Pusa Meghna and Pusa Sharad exhibited contrasting behaviour to this.
In Arabidopsis, FUL has a redundant role with AP1, CAL, and LFY in promoting flowering time and floral primordium initiation.It is also involved in cauline leaf morphology and carpel/fruit development (Gu et al., 1998;Ferrandiz et al., 2000;Duclos & Bjorkman, 2008).We observed that the vernalisationrequiring variety Pusa Snowball Kt-25 had a different expression pattern for both these BoFUL paralogues from the varieties insensitive to vernalisation.In the case of BoFUL-c the expression in Pusa Snowball Kt-25 was higher at the CI stage and lower at the FC stage while the rest of the varieties showed an opposite profile.In the case of BoFUL-d Pusa Shukti also showed behaviour similar to Pusa Snowball Kt-25 as both had shown higher expression at the CI stage than in the FC stage, contrary to Pusa Meghna and Pusa Sharad.These observations appear to be in line with that of Duclos and Bjorkman (2008) in the case of F 1 hybrid cauliflower cv.Green Harmony.
The differential expression pattern of the genes in Pusa Kartiki from the other three early group varieties can be explained with its curding behaviour (Figure 7).It has better curding plasticity and forms full-size curds even at delayed planting.Close agreement in early and mid-early group varieties for expression of the genes is attributed to their true tropical nature and common line of evolution (i.e. from European materials) (Rakshita et al., 2021).Further, the common expression profile of genes in Pusa Shukti (mid-late group) and Pusa Snowball Kt-25 (late or Snowball) was supportive of the theory of evolution of mid-late group cauliflower from crossing of Indian-or tropical-type and Snowball cauliflower.

Conclusion
Our study on the expression analysis of candidate genes involved in developmental transition has uncovered a fascinatingly intricate genetic system underlying the curding phenomenon in Indian cauliflower.Our findings revealed that BoFT was consistently upregulated from the SL stage to the fully mature curd stage in all four varieties studied.Interestingly, the expression patterns of BoFUL-d and BoAP2 in Pusa Meghna and Pusa Sharad, the typical Indian cauliflowers, were in stark contrast to those observed in Pusa Snowball Kt-25 and Pusa Shukti.The latter two exhibited similar expression patterns for BoFULd, CCE1, and BoFLY.BoVRN2 and BoFUL-c were highly expressed exclusively in Pusa Snowball Kt-25 whereas in the other varieties, their expression levels were similar to those observed at the SL stage.Pusa Meghna and Pusa Snowball Kt-25 had similar patterns for BoREM1 and BoFT.Intriguingly, the BoAP2, BoVIN2, BoVIN3, and SOC1 genes could differentiate between Snowball and Indian types, as these genes were highly expressed in Pusa Snowball Kt-25 only.These findings also substantiated to some extent the evolutionary pattern of the mid-late group from Indian and Snowball types, as revealed by the expression pattern of the curding genes at different phenological stages.Our study is the first to report in detail on the role of developmental genes in the vegetativecurding-reproductive transition in Indian cauliflower, and it has shed light on the complexities of this generalised mechanism.

Figure 3 .
Figure 3. Relative change in curding traits in cauliflower varieties grown in the CS over the normal growing situation (with standard error bars).DB, date of bolting, DCH-days to curd harvesting, DCI-days to curd initiation, GPW-gross 194 plant weight, MCW-marketable curd weight and NCW-net curd weight.

Figure 5 .
Figure 5. Relative expression levels of genes in curd portion at FC phase in cauliflower.NS-Natural sowing; CS-challenge sowing.

Table 1 .
Leaf sample collection schedule in the study for gene expression analysis in 2019 (same period was maintained in 2020).

Table 2 .
Primers of different flowering-related genes used for study of gene expression in cauliflower varieties.

Table 3 .
Pooled interaction of morphological observations from cauliflower varieties and growing situations (in the NS and CS).