Exploring silique number in Brassica napus L.: Genetic and molecular advances for improving yield

Summary Silique number is a crucial yield‐related trait for the genetic enhancement of rapeseed (Brassica napus L.). The intricate molecular process governing the regulation of silique number involves various factors. Despite advancements in understanding the mechanisms regulating silique number in Arabidopsis (Arabidopsis thaliana) and rice (Oryza sativa), the molecular processes involved in controlling silique number in rapeseed remain largely unexplored. In this review, we identify candidate genes and review the roles of genes and environmental factors in regulating rapeseed silique number. We use genetic regulatory networks for silique number in Arabidopsis and grain number in rice to uncover possible regulatory pathways and molecular mechanisms involved in regulating genes associated with rapeseed silique number. A better understanding of the genetic network regulating silique number in rapeseed will provide a theoretical basis for the genetic improvement of this trait and genetic resources for the molecular breeding of high‐yielding rapeseed.


Introduction
Rapeseed (Brassica napus L., AACC, 2n = 38) is a crucial source of edible oil and biofuel and the third largest oil crop globally after oil palm (Elaeis guineensis) and soybean (Glycine max).Breeders have been improving silique number because it is the trait most highly correlated with yield among the three yield components of rapeseed: silique number, seed number per silique and thousand seed weight ( € Ozer and Oral, 1999).However, silique number is a complex quantitative trait that is susceptible to environmental influences.Hundreds of quantitative trait loci/quantitative trait nucleotides (QTLs/QTNs) for silique number have been identified, but their genetic and molecular bases remain ambiguous (Raboanatahiry et al., 2018;Shi et al., 2015).Silique number (grain number) in crops is mainly controlled by shoot architecture; the shoot apical meristem (SAM) gives rise to leaf primordia, and the axillary meristem (AM) gives rise to vegetative branches or inflorescences (Wang et al., 2018).AMs are established in leaf axils during the vegetative stage and form axillary buds, which subsequently remain dormant or continue to grow and form branches (Bennett and Leyser, 2006).After the transition from the vegetative stage to the reproductive stage, the SAM develops into the inflorescence meristem (IM).The IM then continuously differentiates into the AM and the floral meristem (FM), forming inflorescence branches and floral buds, respectively.These biological processes are influenced by genetics and by environmental conditions such as light, temperature and nutrient status.Therefore, understanding the molecular genetic mechanisms regulating the formation of branches and floral buds is important for the genetic improvement of silique number in rapeseed.
The molecular mechanism controlling the formation of branches and floral buds in plants has been studied extensively (Wang et al., 2018;Zhu and Wagner, 2020).Plant hormone signalling and genetic regulation are largely conserved between monocots and dicots (Teichmann and Muhr, 2015;Wang and Jiao, 2018).In general, auxin and strigolactone (SL) inhibit lateral bud growth, whereas cytokinin directly promotes lateral bud growth (Ongaro and Leyser, 2008).Strigolactone synthesis is conserved in plants.Knockout of MAX1, a strigolactone synthesis gene, results in increased branching or tillering in Arabidopsis, rice and wheat (Cardoso et al., 2014;Sigalas et al., 2023;Yoneyama et al., 2018).The natural auxin indole-3-acetic acid (IAA) promotes the expression of MAX3 and MAX4 genes in the strigolactone synthesis pathway, inhibiting branches or tillers growth (Domagalska and Leyser, 2011;Hayward et al., 2009).Cytokinins can directly promote branches or tillers growth, independent of auxin accumulation (M€ uller et al., 2015).Reduced expression of OsCKX2 causes cytokinin accumulation in inflorescence meristems and increases the spikelet numbers and rice yield (Ashikari et al., 2005).Moreover, numerous genes play crucial roles in the regulation of branching and floral bud formation.For example, BRANCHED1 (BRC1) encodes a TCP transcription factor that promotes bud dormancy but inhibits branch formation by integrating phytohormone and environment signals in most plants (Wang et al., 2019).Lateral repressor (Ls) genes encode members of the VHIID subfamily of plant-specific GRAS (GA INSENSITIVE, REPRESSOR OF GA1-3 SCARECROW) transcription factors that are required for AM initiation in many plant species, such as tomato (Solanum lycopersicum), Arabidopsis (Arabidopsis thaliana) and rice (Greb et al., 2003;Li et al., 2003).Identifying these genetic determinants is helpful for understanding the molecular mechanism regulating silique number and for genetically improving rapeseed.
Here, we summarize the QTLs/QTNs for silique number in rapeseed.We then identify candidate genes for silique number within overlapping QTL/QTN regions.Next, we explore pathways associated with genetic, plant hormonal and environmental stimuli that regulate silique number.By examining gene regulatory networks (GRNs) controlling the formation of branches and floral buds in Arabidopsis and rice, we predict the molecular genetic mechanism underlying silique number in rapeseed.This review summarizes our understanding of the genetic regulation of silique number rapeseed, providing a theoretical basis and potential genetic resources for yield improvement in this important oilseed crop.

Genetic regulation of silique number
QTLs/QTNs associated with silique number Silique number is a complex quantitative trait exhibiting a wide range of continuous variation, which is particularly influenced by environmental factors (Xu et al., 2014).More than 100 QTLs/QTNs associated with silique number have been identified from at least 10 linkage mapping populations (Cai et al., 2014;Ding et al., 2012;Qi et al., 2014;Radoev et al., 2008;Shi et al., 2009Shi et al., , 2013;;Wang and Guan, 2010;Yi et al., 2006).Shi et al. (2015) and Raboanatahiry et al. (2018) compared multiple QTLs/QTNs for silique number using B. napus 'Darmor-bzh' as a reference to explore candidate genes regulating yield and its component traits (Chalhoub et al., 2014).Since 2017, 46 additional QTLs/QTNs for silique number have been identified and mapped (Deng et al., 2019;Li et al., 2020b;Lu et al., 2017).Tang et al. (2020) mined four major chromosomal regions containing genes with differential transcript abundance in dpt247 (a recessive mutant with a high density of pods) compared to wild-type B. napus.We refer to these four major chromosomal regions as the major QTLs for silique number.Due to the direct influence of branch number on silique number, QTLs/QTNs for branch number should also be considered.Twenty-seven QTLs/QTNs for branch number have been identified in seven studies since 2015 (He et al., 2017;Li et al., 2016Li et al., , 2020aLi et al., , 2022a;;Liu et al., 2021;Luo et al., 2015;Zheng et al., 2017).In total, 174 QTLs/QTNs for traits related to silique number have been described.We aligned these QTLs with the physical map of the B. napus 'Darmor-bzh' reference genome, revealing that these QTLs/QTNs are found in 19 linkage groups (Figure 1; Table S1).Some of the 174 QTLs/QTNs are located in separate regions, while some are overlapped.Several major regions containing overlapping QTLs/QTNs were detected in three or more studies.Twenty-two regions contain 82 overlapping QTLs/QTNs for siliquerelated traits, which are located on chromosomes A01, A02, A03, A05, A06, A07, A09, C03, C05 and C06 (Figure 2; Table S2).These regions range from 1.0 to 7.1 Mb and are associated with one or more traits related to silique number, such as qSNRT_A03.3for silique number only and qSNRT_A03.2for silique number and branch number.These regions might contain the key genes regulating silique number and its related traits, offering the potential for developing functional markers for marker-assisted breeding.

Identification of candidate genes
There are 213 genes in Arabidopsis and 248 genes in rice related to silique number or grain number, respectively.We used these 461 genes to identify 2091 homologous genes related to silique number in rapeseed (Shi et al., 2009;National Rice Data Center: https://ricedata.cn/;Table S3) using the Browse Data tool at Brassica napus multi-omics information resource (https://yanglab.hzau.edu.cn/BnIR)(Yang et al., 2023) and National Rice Data Center.Of 1831 homologous genes with known chromosome locations to uncover 240 candidate genes within twenty-two regions for traits related to silique number while the remaining 260 orthologues genes could not be used to identify candidate genes since their chromosomal locations were unknown (Figure 2; Tables S2 and S4).These comprised 53 candidate genes for silique number only and 187 candidate genes for multiple traits: 46 for silique number (SN) and branch number (BN); 47 for SN and silique density (SD); 15 for SN and silique number in the main inflorescence (SNMI); 35 for SN and silique number per unit area (SNUA); 11 for SN and silique density of the main inflorescence (SDMI); 24 for SN, BN, and SDMI; and 9 for SN, BN, SNMI and SD.Of the 240 candidates, BnaC06.TB1 (TEOSINTE BRANCHED 1, BnaC06g29550D,), BnaAP1 (APETALA1, BnaA07g24360D, BnaA07g27710D, BnaC06g29980D), BnaC03.TFL1 (TERMINAL FLOWER 1, BnaC03g01440D), BnaBRC1 (BnaA01g26700D, BnaA03g34820D), BnaA02.RAX1 (REGULATOR OF AXILLARY MERISTEMS 1, BnaA02g06100D) and BnaREV (REVOLUTA, BnaA02g06170D, BnaA06g18550D) are orthologs of key genes regulating the formation of branches and floral buds, that is OsTB1, AP1 (OsMADS18), TFL1, BRC1, RAX1 and REV, respectively.Some of these candidate genes have been shown to regulate silique number in rapeseed, such as BnaAP1 (Shah et al., 2018) and BnaTFL1 (Sriboon et al., 2020).This supports the likelihood that these genes control traits related to silique number, making them good candidates for further genetic research in rapeseed.

Genetic control of silique number related traits
Several genes regulating silique number in rapeseed have been identified and characterized through homologous cloning (Figure 3).BnaA02.AP1, encoding a MADS transcription factor, is believed to be a functional homologue of Arabidopsis AP1, encoding a crucial regulator of FM formation (Bl€ umel et al., 2015).Evidence suggests that BnaA01.AP1 is involved in the same biological process as AP1 and may function in a similar manner (Shah et al., 2018).The mutation of an AP1 paralog in rapeseed caused substantial changes in floral morphology as well as traits related to plant architecture, such as branch number (Shah et al., 2018).BnaTFL1 is a member of the phosphatidylethanolamine-binding protein (PEBP) family, which plays an important role in determining FM identity and regulating flowering time in Arabidopsis and soybean (Bradley et al., 1997;Li et al., 2014).There are five copies of BnaTFL1 in rapeseed.CRISPR/Cas9 knockout of different copies of BnaTFL1 resulted in a substantial reduction in the number of siliques on the main inflorescence in all mutants.In addition, the bnaa10.tfl1,bnaa02.tfl1,bnac03.tfl1,bnac03.tfl1/bnac09.tfl1mutants show a considerable reduction in the number of branches (Sriboon et al., 2020).BnaA01.ERF114 is expressed in leaf primordia, the SAM, the leaf margin meristem and reproductive organs; ectopic expression of BnaA01.ERF114 in Arabidopsis resulted in shorter plant height and more branches and siliques per plant (Lyu et al., 2022).
Arabidopsis LAS is a key gene in the regulation of branch number and silique number.Ectopic expression of BnaLAS in Arabidopsis resulted in fewer branches and siliques on the main inflorescences (Yang et al., 2011).Branch number is an important factor affecting the number of siliques in rapeseed.Fine-mapping based on linkage analysis identified some candidate genes that may be involved in regulating branch number in rapeseed, such as BnaC03.BOI, BnaA09.ELP6, BnaA05.URED, BnaA05.ATL9 and BnaA05.ATL38 (He et al., 2017;Li et al., 2020a;Lu et al., 2022;Zhang et al., 2018).Genome-wide association study (GWAS) has also identified candidate genes that may regulate branch number, such as BnaMYB83, BnaSPL5, BnaROP3, BnaLOF2 and BnaCUC3 (Lu et al., 2017;Zheng et al., 2017).The functions and molecular mechanisms of these candidate genes identified by forward genetics require further verification.

Phytohormonal control of silique number
Multiple signals, including auxin and cytokinin, play important roles in regulating the formation of both branches and floral buds (Figure 3).Auxin is biosynthesized in the apices and transported in a polar manner towards the base of the plant to repress the growth of axillary buds, whereas cytokinins promote this process (Ongaro and Leyser, 2008;Shimizu-Sato et al., 2009; Wang  et al., 2006).The location and timing of axillary bud initiation depend on auxin concentrations for the polar transport of PIN proteins (Benkov a et al., 2003;Heisler et al., 2005;Reinhardt et al., 2000Reinhardt et al., , 2003;;Vernoux et al., 2010).BnaA03.IAA7 encodes an Aux/IAA protein that is involved in improving plant architecture; its mutation increased the number of siliques per plant and improved yield in rapeseed (Li et al., 2019).BnaA01.KAT2, a potassium channel protein, down-regulates the expression of ARR3/4/6/7/9 (encoding negative regulators of cytokinin signalling) and IAA1/2 (key genes in the auxin signalling pathway) (Yuan et al., 2022).Overexpressing BnaA01.KAT2 increased the length of the main inflorescence and silique number in rapeseed.
Cytokinin oxidase/dehydrogenases (CKXs) play key roles in the irreversible degradation of cytokinin, thereby regulating plant growth and development (Figure 3).Twenty-three BnaCKX genes have been identified genome-wide in rapeseed.Transcriptome analysis showed that most BnaCKX genes are highly expressed in axillary buds, flowers or siliques at the reproductive growth stage, indicating that these genes are involved in regulating axillary bud, flower, and silique growth and development (Liu et al., 2018).Moreover, four BnaCKX3 genes and two BnaCKX5 genes are highly expressed in reproductive organs, and sextuple bnackx3b-nackx5 mutants have increased cytokinin concentrations in reproductive tissues, resulting in a larger and more active IM, as well increased silique number, compared with the wild type (Schwarz et al., 2020).Overexpressing BnaA09.CKX2 increased thousand seed weight while decreasing silique number per plant in rapeseed (Yan et al., 2023).These findings suggest that cytokinin status can be modulated via mutagenesis of specific CKX genes to improve the yield of dicot crops such as rapeseed.
A third class of plant hormones affecting branch number is SLs, which play a negative role in bud outgrowth; this function is highly conserved in both monocots and dicots (Gomez-Roldan et al., 2008;Umehara et al., 2008) (Figure 3).MAX1 encodes a cytochrome P450 monooxygenase (CYP711A1) involved in SL biosynthesis (Lazar and Goodman, 2006).Arabidopsis max1 mutants display reduced stature, increased branching and rounder rosette leaves.By contrast, overexpressing MAX1 repressed bud outgrowth at the stem base in Arabidopsis (Abe et al., 2014;Booker et al., 2005;Lazar and Goodman, 2006).Rapeseed BnaMAX1 genes have redundant functions resembling those of Arabidopsis MAX1, regulating plant height and axillary bud outgrowth.Simultaneous knockout of all four BnaMAX1 alleles resulted in a semidwarf phenotype with increased branching and more siliques, contributing to improved yield per plant (Zheng et al., 2020).SL is perceived by Arabidopsis AtD14 and rice DWARF14 (OsD14), leading to the degradation of SL response regulators mediated by the F-box protein MAX2 in Arabidopsis and its homologue D3 in rice (Chevalier et al., 2014;Jiang et al., 2013;Zhou et al., 2013).The d14 mutant exhibits increased shoot branching with reduced plant height, similar to the SL-deficient mutants d10 and max4 in Arabidopsis and rice, respectively (Arite et al., 2007;Bainbridge et al., 2005).Knockout of BnaD14 in rapeseed also resulted in a semidwarf phenotype with increased branch number, contributing to increased yield (Stanic et al., 2021).
Brassinosteroids (BRs) are also involved in regulating branch number and FM formation (Fang et al., 2020;Li and He, 2020;Xu et al., 2020).AtDWF4 encodes a 22a hydroxylase that functions in a rate-limiting step in the BR biosynthetic pathway.Ectopic and organ-specific expression of AtDWF4 led to greater inflorescence height, branch number, number of seeds per plant and seed weight in Arabidopsis, tobacco (Nicotiana tabacum) and rice (Choe et al., 2001;Liu et al., 2007;Wu et al., 2008).These observations suggest that the role of AtDWF4 is conserved in different plants (Vriet et al., 2013).Ectopic expression of AtDWF4 also resulted in more branches and siliques on the main inflorescences in rapeseed (Sahni et al., 2016).BRs and auxin are essential regulators of plant architecture.The rapeseed ed1 mutant shows reduced plant height, branch number and silique number per plant.ED1 is a homologue of AtIAA7.BnaARF8 interacts directly with ED1 and BnaBZR1, indicating that ED1 interacts with BR signalling via BnaARF8 and BnaBZR1 to regulate plant architecture in rapeseed (Zheng et al., 2019).
Gibberellins (GAs) are required for the normal growth of almost all plant organs via the promotion of cell division and cell elongation.In Arabidopsis, GAs activate the expression of the key genes SOC1 and LFY, leading to FM formation (Blazquez et al., 1998;Bonhomme et al., 2000;Moon et al., 2003).
Endogenous GA concentrations in rapeseed are related to floral bud initiation, and external application of GA biosynthesis inhibitors inhibits flower development (Rood et al., 1989).

Control of silique number by environmental stimuli
In rapeseed, silique number depends on the initiation and outgrowth of axillary buds and floral bud formation.Several environmental factors affect silique number in rapeseed, including plant population density and the availability of nitrogen, sugars and boron.
Rapeseed plants show high adaptability to changing plant density, which is an important factor affecting yield and yield components (Diepenbrock, 2000;R o_ zyło and Pałys, 2014).SNUA (silique number per unit area) is the most important factor determining yield.Silique number per plant and branch number increase under low planting density, thus compensating for the decreased population size, while the opposite occurs at a higher planting density (Ma et al., 2014;Zheng et al., 2022).Increasing planting density can increase the competition of individual plants for nutrients and light by altering root morphology and canopy structure (Li et al., 2017;Ma et al., 2014;Rondanini et al., 2017).Planting density can affect photosynthesis, carbon metabolism and dry matter accumulation in rapeseed (Kuai et al., 2022).Planting density also affects rosette leaf diameter, which can increase the efficient capture of radiation at flowering, as well as floral branching, which can increase silique number per area (Rondanini et al., 2017).Therefore, increasing planting Nitrogen (N) is one of the most important mineral elements for plant growth and development and a key factor in improving crop yield (Figure 3).Compared with other crops, rapeseed requires higher N fertilization for production (Rathke et al., 2005).N fertilizer can significantly affect rapeseed yield by altering branch number and silique number per plant (Islam and Evans, 1994).Therefore, breeding rapeseed with high N efficiency is of great strategic importance for ensuring the security of grain and oil and the sustainable development of the rapeseed industry (Zhan et al., 2023a).An effective way to reduce N fertilizer use is to cultivate N-efficient cultivars identified by characterizing genotypic variation in rapeseed under contrasting N supply (Balint et al., 2008;Kessel et al., 2012;Sve cnjak and Rengel, 2006).The N transporter genes BnaAMT1.1, BnaNRT1.1,BnaNRT2.2,BnaNRT2.5, BnaNRT2.6 and BnaNRT2.7 are expressed at markedly higher levels in the roots of N-efficient genotypes compared to N-inefficient genotypes, which may help explain why Nefficient materials have more siliques and higher yields than Ninefficient materials (Wang et al., 2014(Wang et al., , 2015)).In recent years, several N-responsive genes regulating yield and yield component traits have been identified in rapeseed.The WRKY transcription factor gene BnaA09.WRKY47 is responsive to N deficiency in rapeseed.Overexpressing BnaA09.WRKY47 improved N reutilization in older leaves by regulating the expression of BnaC07.SGR1, BnaA09.AAP1 and BnaA02.NRT1.7,resulting in greater silique number per plant and increased yield under low-N conditions (Cui et al., 2023).Interactions between N and auxin regulate tillering and panicle branching in rice (Luo et al., 2020).N deficiency in rapeseed induces the expression of the auxin biosynthesis gene BnaAUX1 and the polar auxin transporter gene BnaPIN1, suggesting that N might regulate silique number and branch number via auxin biosynthesis, transport and signalling pathways (Yang et al., 2022).
Sugars may play a role in activating bud outgrowth, while the growing stem apex may suppress branch outgrowth by acting as a sugar sink (Barbier et al., 2015;Kebrom, 2017).Association analysis using a diverse panel of 55 rapeseed lines identified single-nucleotide polymorphisms in the promoter and coding sequences of the sucrose transporter gene BnaA07.SUT1 that were significantly associated with branch number and silique number per plant (Li et al., 2011) (Figure 3).Elevated activities of cytosolic fructose-1,6-bisphosphatase (cyFBPase) and sedoheptulose-1,7-bisphosphatase (SBPase) are associated with higher yields in plants.Ectopic expression of the rapeseed cyFBPase and SBPase genes in tobacco resulted in greater growth and biomass and a greater number of flowers compared to wildtype plants (Li et al., 2022b).Therefore, increasing the expression of cyFBPase and SBPase genes may offer an opportunity for improving yield in rapeseed.
Boron (B) is an essential micronutrient for the growth of broad bean (Vicia faba), barley (Hordeum vulgare), miscellaneous plants (Warington, 1923), rapeseed (Chen et al., 2018) and watermelon (Citrullus lanatus) (Shireen et al., 2020) (Figure 3).Symptoms of B deficiency include inhibited apical shoot growth, repressed root elongation, curved leaves, dried-up floral buds and abortion of fertile pollen, which directly lead to decreases in plant productivity and reduce agricultural production and quality (Durbak et al., 2014;Lordkaew et al., 2011;Quiroga et al., 2020;Yang et al., 2013;Yuan et al., 2017).The uptake and translocation of B in Arabidopsis occur via boric acid influx channel proteins and efflux transporters (Takano et al., 2002(Takano et al., , 2006(Takano et al., , 2010)).Expressing the B transporter gene BnaC04.BOR1.1c in the Arabidopsis bor1-1 mutant led to wild-type growth and rescued the bor1-1 mutant phenotype.Knockdown of BnaC04.BOR1.1c in rapeseed increased branch number and floral organ number in the main inflorescence, but flower development was abnormal, resulting in low silique number and yield (Zhang et al., 2017b).BnaA03.-NIP5.1 was identified as a candidate gene for efficient B uptake in rapeseed by QTL fine-mapping.Transgenic lines with increased BnaA03.NIP5.1 expression exhibit improved tolerance to low B levels at both the seedling and mature stages.The BnaA03.NIP5.1 Q haplotype in the natural population confers high BnaA03.NIP5.1 expression and tolerance to low B. Field tests using a natural population and near-isogenic lines confirmed that varieties carrying the BnaA03.NIP5.1 Q allele have significantly higher silique number and yield per plant (He et al., 2021a).BnaA02.NIP5.1 and BnaA02.NIP6.1aalso positively regulate B uptake and transport (He et al., 2021b).Knockdown of BnaA02.-NIP5.1 or BnaA02.NIP6.1a in rapeseed resulted in lower B accumulation under B deficiency, leading to fewer siliques (Song et al., 2021).The application of B fertilizer also improves N uptake and N use efficiency, which increases branch number and silique number in rapeseed (Wang et al., 2022).

Control of axillary and floral bud formation
The molecular mechanisms underlying plant branching and floral bud formation have been studied extensively in many species.AM initiation and development are regulated by transcriptional regulators that are largely conserved between dicots (Arabidopsis/tomato) and monocots (rice), with homologous genes having the same or similar functions in different species.Therefore, the regulatory networks for branching and floral bud formation in other plants, such as Arabidopsis, tomato and rice, provide a reference for the regulation of these processes in rapeseed.Systematically collating information on the genetic regulatory networks of branching and floral bud formation in Arabidopsis and rice characterized in previous studies and reviews (Cao et al., 2015;Cao and Jiao, 2020;Chongloi et al., 2019;Fang et al., 2020;Han et al., 2014Han et al., , 2020;;Li et al., 2020cLi et al., , 2023b;;Luo et al., 2020;Mutasa-G€ ottgens and Hedden, 2009;Su et al., 2020;Wang et al., 2018Wang et al., , 2020;;Wils and Kaufmann, 2017;Zhan et al., 2023b;Zhang et al., 2022;Zhu and Wagner, 2020) allows us to shed light on the GRNs of key genes regulating silique number in rapeseed based on their homologous genes in Arabidopsis and rice (Figure 4; Table S4).
BnaLAS encodes a member of the VHIID subfamily of plantspecific GRAS transcription factors required for AM initiation in many plants (Greb et al., 2003), such Arabidopsis (LAS ), rice (OsMOC1), tomato (Ls) and wheat (Triticum aestivum; TaMOC1) (Greb et al., 2003;Li et al., 2003;Schumacher et al., 1999;Zhang et al., 2015).The Arabidopsis las mutant cannot form lateral shoots during vegetative development but forms lateral buds during the reproductive phase.During the vegetative stage, AMs initiate at a distance from the SAM and require LAS function; after the floral transition, AMs initiate close to the apex of the IM and do not require LAS (Greb et al., 2003).The function of CUC2 and CUC3 overlaps with that of LAS, and LAS expression is reduced in the cuc2 mutant (Hibara et al., 2006;Raman et al., 2008).Celltype-specific gene expression analysis and genome-wide yeastone-hybrid studies indicated that CUC2 is a positive regulator of LAS expression (Tian et al., 2014).SPL9 and SPL15, belonging to the plant-specific SPL transcription factor family, are regulators that directly inhibit the expression of LAS to inhibit the initiation of AMs, thus linking the ageing pathway with AM initiation (Tian et al., 2014).DELLA proteins interact with SPL9 and attenuate the activity of SPL9 in repressing LAS, subsequently promoting the initiation of axillary buds (Yu et al., 2012).LAS modulates the expression of the gene encoding the GA deactivation enzyme GA2ox4 to form a low-GA cell niche in the leaf axil region, which is required for axillary bud formation (Zhang et al., 2020).In addition, during vegetative development, LAS activity is required for the expression of REVOLUTA (REV; encoding a HD-ZIPIII transcription factor) and AUXIN RESISTANT1 (AXR1; encoding an E1 ligase in the RUB1 pathway) in the developing AM (Greb et al., 2003).OsMOC1, encoding a GRAS family nuclear protein, is Genetic basis on rapeseed silique number 1903 mainly expressed in axillary buds and plays a role in axillary bud initiation and outward growth in rice (Li et al., 2003).OsMOC3, a homologue of Arabidopsis WUS, is involved in the initial development of the AM (Tanaka et al., 2015).OsMOC1 interacts with OsMOC3 and transcriptionally enhances the expression OsFON1, which regulates FM size and the number of all floral organs (Shao et al., 2019;Suzaki et al., 2004).
After the floral transition, the SAM is transformed into an IM.The IM either produces FMs from its flanks or remains indeterminate, iterating the pattern of inflorescence branches (Teo et al., 2014).TFL1 is a key gene that affects flowering and regulates plant architecture throughout the lifecycle of plants (Conti and Bradley, 2007;Ratcliffe et al., 1998).The regulation of inflorescence architecture by TFL1 is conserved among plants, including Arabidopsis (Ratcliffe et al., 1998), rice (Nakagawa et al., 2002), maize (Zea mays) (Danilevskaya et al., 2010), soybean (Tian et al., 2010) and pea (Pisum sativum) (Foucher et al., 2003).Not only does TFL1 expression determine persistent axillary bud growth but low TFL1 expression results in the conversion of IMs to FMs (P erilleux et al., 2019;Teo et al., 2014).Flowering and the conversion of shoot meristems to FMs occur early in tfl1 mutants, whereas overexpression of TFL1 led to greater inflorescence production and delayed flowering (Ratcliffe et al., 1998;Shannon and Meeks-Wagner, 1991).TFL1 antagonizes the floral-fate-inducing factors LFY and AP1 (Denay et al., 2017;Wagner, 2017).FM identity genes such as LFY and AP1 are ectopically expressed in the IM and bind to the TFL1 regulatory region, and AP1 contributes to the dissociation of the gene loop involving the 3 0 distal region and transcription start site at the TFL1 locus, which is associated with TFL1 transcription (Kaufmann et al., 2010;Liu et al., 2013;Moyroud et al., 2011;Winter et al., 2011).Up-regulation of LFY and AP1 is delayed in plants overexpressing TFL1 during the floral transition (Bowman et al., 1993;Bradley et al., 1997;Prusinkiewicz et al., 2007;Ratcliffe et al., 1998).TFL1 is recruited to LFY chromatin and regulates LFY expression with the help of the bZIP transcription factor FD (Goretti et al., 2020;Zhu et al., 2020).TFL1 negatively regulates FD-dependent transcription of its target genes to finetune flowering and IM development (Hanano and Goto, 2011).The expression of TFL1 is inhibited by SOC1, SVP, AGL24 and SEP4 in the emerging FM (Liu et al., 2013).
OsTFL1 represses flowering and controls inflorescence architecture in rice.The molecular mechanism of the phase transition and inflorescence architecture is conserved between grass and dicot species (Nakagawa et al., 2002).All four RCN genes (rice TFL1-like genes) are predominantly expressed in the vasculature, and RCN proteins are transported to the shoot apex, where they antagonize florigen activity and regulate inflorescence development.The antagonistic activity of RCN against Hd3a, a rice homologue of FT, depends on its 14-3-3 binding activity (Kaneko-Suzuki et al., 2018).OsMADS34 promotes the transition from the vegetative stage to the reproductive stage and IM development (Kobayashi et al., 2012), as well as spikelet meristem formation (Kobayashi et al., 2010).The osmads34 mutant exhibits altered inflorescence morphology, with an increase in the primary branch number and a decrease in the secondary branch number and seedsetting rate (Gao et al., 2010;Zhang et al., 2016).The repression of RCN4 by OsMADS34 primarily functions to counterbalance secondary branch meristem identity and promote the transition to spikelet meristem identity (Zhu et al., 2022).
AP1 and LFY are key genes in the transition of an IM into an FM.AP1 is a key target of LFY, a MADS box transcription factor that functions in the primordium to determine the fate of the IM for flowering (Bowman et al., 1993;Wagner et al., 1999).The establishment of FM identity and subsequent flower development are largely dependent on the activity of the transcription factors LFY and AP1 (Grandi et al., 2012;Pastore et al., 2011;Saddic et al., 2006;Wils and Kaufmann, 2017).LFY is expressed before the formation of the first floral primordium, and AP1 is subsequently up-regulated in the young floral primordium, which determines the fate of flower formation (Bl azquez et al., 1997;Hempel et al., 1997;Mandel et al., 1992;Yamaguchi et al., 2009).LFY, through a series of coherent and logical feed-forward loops, directly or indirectly up-regulates AP1 (Pastore et al., 2011;Wagner et al., 1999;Yamaguchi et al., 2014).During FM development, AP1 down-regulates several floral repressor genes of the AP2 family of transcription factors such as SNZ, TOE1 and TOE3 (Kaufmann et al., 2010) and multiple florals promoting transcription factor genes such as AGL24, SOC1, FD, FUL, SVP and TFL1 to promote the continuous emergence of floral primordia (Hanano and Goto, 2011;Kaufmann et al., 2010;Liljegren et al., 1999;Wigge et al., 2005).In the above regulatory pathways, AP1 induces LFY expression through a positive feedback loop to establish the identity of FM, ensuring its expression for downstream floral organ speciation and development while maintaining its determinism (Grandi et al., 2012).The repression of SVP and AGL24 by AP1 relieves the negative regulation of SEP3 (Pos e et al., 2012), leading to the formation of AP1/SEP3 heterodimers and inducing the expression of AP3 and PI, thereby initiating the development of floral organs (Gregis et al., 2008;Wils and Kaufmann, 2017).
SLs are key phytohormones that regulate the growth of lateral meristems in plants.The role of MAX1 in regulating SL biosynthesis is functionally conserved in rice, Arabidopsis, wheat, maize and rapeseed (Al-Babili and Bouwmeester, 2015;Booker et al., 2005;Cardoso et al., 2014;Sigalas et al., 2023;Yoneyama et al., 2018;Zheng et al., 2020).MAX1 converts carlactone (CL) to carlactonoic acid (CLA) (Abe et al., 2014).A SABATH methyltransferase then catalyses the conversion of CLA to Me-CLA in Arabidopsis (Wakabayashi et al., 2021).Me-CLA is a non-canonical SL that interacts with AtD14, a receptor protein in the SL signalling pathway, indicating that it is biologically active in suppressing shoot branching (Abe et al., 2014).SL induces changes in the structure of AtD14 protein and is then hydrolysed into CLIM (covalently linked intermediate molecule), promoting an interaction between AtD14 and AtD3 (Yao et al., 2018).AtD14 may also play a role in SL signal transduction pathways.The mutation of OsD14 increases tiller number and decreases plant height in rice (Arite et al., 2009;Liu et al., 2009), and the transcription of OsD14 is repressed by transcription factors such as OsMADS57 and OsCCA1.OsMADS57 interacts with OsTB1, reducing the inhibition of OsD14 transcription by OsMADS57 (Guo et al., 2013).OsCCA1 positively regulates the expression of OsTB1, OsD14 and IPA1, thus repressing tiller-bud outgrowth (Wang et al., 2020).OsD14 is associated with OsD3 in a GR24-dependent manner, suppressing shoot branching in rice (Zhao et al., 2014).

GRNs of key genes for silique number
Transcriptional regulation is important for plant growth and development; the downstream target genes of transcription factors can be identified through ChIP-seq (Schmidt et al., 2009).We therefore compiled a list of the downstream target genes of the key transcription factors WUS (Ma et al., 2019), AP1 (Goslin ª 2024 The Authors.Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd., 22, 1897-1912et al., 2017;Kaufmann et al., 2010;Pajoro et al., 2014;Winter et al., 2015), LFY (Goslin et al., 2017;Jin et al., 2021;Moyroud et al., 2011;Sayou et al., 2016;Winter et al., 2015;Zhu et al., 2020), STM (Li et al., 2018), FLC (Deng et al., 2011;Mateos et al., 2017), REV (Brandt et al., 2012), SVP (Gregis et al., 2013;Mateos et al., 2015Mateos et al., , 2017;;Tao et al., 2012) and SOC1 (Immink et al., 2012;Tao et al., 2012) identified by ChIP-seq (Table S5).The 13 793 target genes included 146 candidate genes for silique number.We used 54 transcription factor genes identified from among the 146 candidate genes as well as the eight key transcription factors to construct a GRN (Figure 5; Table S5).This analysis revealed several regulatory pathways, such as the direct binding of LFY to the promoter of AP1 to regulate its expression (Bowman et al., 1993;Wagner et al., 1999).However, a large number of downstream genes that may regulate axillary bud initiation and elongation and control floral bud formation remain unclear.The GRN reveals some important genes that control silique number.For example, TEM1 (AT1G25560) was enriched in the ChIP-seq data for the eight key transcription factors.The potential regulatory relationships revealed by Arabidopsis ChIP-seq data provide a reference for studying the genetic regulatory mechanism of silique number in rapeseed.

Summary and future perspectives
Identification of genes and loci for the genetic improvement of silique number Silique number is a complex quantitative trait in rapeseed.Hundreds of QTLs regulating silique number have been identified; however, few genes responsible for controlling silique number have been cloned.This may be due to the low heritability of rapeseed silique number and the lack of omics evidence.It is still challenging to identify the key genes regulating silique number in rapeseed by forward genetics.The release of several reference genomes, such as Darmor v4.1 and v10 (Chalhoub et al., 2014;Rousseau-Gueutin et al., 2020) andZS11.v0, v10 andv2 (Chen et al., 2021;Song et al., 2020;Sun et al., 2017), and the application of SNP chips (Edwards et al., 2013;Li et al., 2023a;Xiao et al., 2021) in rapeseed provide opportunities for discovering key genes controlling silique number in rapeseed.In addition, several QTL mapping methods have been developed for identifying candidate genes associated with important agronomic traits in rapeseed, such as natural-population-based GWAS (He et al., 2017;Khan et al., 2021), transcriptome-wide association analysis (TWAS) (Tan et al., 2022;Tang et al., 2021) and parental-population-based linkage analysis methods such as bulked segregant RNA sequencing (BSR) (Fu et al., 2019) and bulked segregant analysis (BSA) (Wang et al., 2016;Yu et al., 2023;Zhao et al., 2020).Databases built upon these data, such as A Gene Expression Database for Brassica Crops (BrassicaEDB, https://brassica.biodb.org/)(Chao et al., 2020), qPCR Primer Database (qPrimerDB, https://qprimerdb.biodb.org/) (Lu et al., 2018), Brassicaceae Database (BRAD, http://www.brassicadb.cn/#/)(Chen et al., 2022) and Brassica napus multi-omics information resource (BnIR, https://yanglab.(Yang et al., 2023), provide important data Genetic basis on rapeseed silique number 1905 resources and analysis platforms for genetic breeding research in rapeseed.Advances in QTL mapping methods and multiomics data analysis will help overcome the difficulties in cloning key genes regulating silique number in rapeseed.
Dissection of the molecular mechanism underlying silique number to enable its genetic improvement Silique number in rapeseed is mainly determined by the initiation and elongation of axillary buds and the formation of floral buds.Axillary bud initiation and elongation and floral bud formation are very complex biological processes, but some progress has been made in understanding their mechanisms in Arabidopsis and rice.Homologous cloning-based approaches in rapeseed have identified several genes that regulate silique number, such as BnaMAX1, BnaLAS, BnaTFL1 and BnaAP1.However, these genes are not sufficient for the genetic improvement of silique number.
As rapeseed is an allotetraploid, genetic regulation is more complex in rapeseed than in Arabidopsis and rice, with not only simple functional redundancy but also possible subfunctionalization between homologous genes (Babula-Skowro nska et al., 2015;Schiessl, 2020;Schiessl et al., 2019).RNA interference (RNAi) and CRISPR-based editing are important tools for studying gene function.RNAi provides an opportunity to develop novel traits in transgenic plants and shows high specificity, high efficiency and reliability for studying gene function (Fishilevich et al., 2016;Ramon et al., 2014).CRISPR-based editing can target a single gene as well as multiple orthologous genes, such as the five BnaTFL1 gene copies; CRISPR/Cas9 gene editing technology has been used to generate different mutants such as bnac03.tfl1a,bnaa02.tfl1and bnac03.tfl1/bnac09.tfl1(Sriboon et al., 2020).More importantly, mutants generated by CRISPR editing can be used directly in crop production or as pre-breeding materials (Hussain et al., 2018;Zhang et al., 2017a).Therefore, CRISPR-based editing is a powerful tool for analysing key genes related to silique number in rapeseed.

Potential application of silique number molecular mechanism in rapeseed breeding
Genetic improvement of silique number is very important in improving rapeseed yield.In this study, twenty-two regions were identified, some of which were associated with one trait only, such as qSNRT_A03.3for silique number, and some regions associated with multiple traits, such as qSNRT_A03.2for silique number and branch number.These regions that regulate multiple traits may have pleiotropic candidate genes or linkage between two genes that regulate silique number and branch number, respectively.The development of molecular markers for twentytwo regions could be helpful in genetic improvement of silique number-related traits.
Haplotype-assisted breeding is an important approach for highyield breeding in rapeseed.At present, a number of silique number candidate genes have been identified in rapeseed, including 240 silique number candidate genes predicted in this study, and some branch number candidate genes were excavated by linkage analysis and association analysis (He et al., 2017;Li et al., 2020a;Lu et al., 2017Lu et al., , 2022;;Zhang et al., 2018;Zheng et al., 2017).The excellent haplotype of these candidate genes will be helpful to silique number improvement and increase yield in rapeseed.
At present, although, our knowledge of the molecular mechanisms regulating traits related to silique number is limited.It is known that gene regulation of silique number could provide some ways for yield improvement.The BnaD14 is strigolactone signalling pathway genes in rapeseed.Mutation bnad14 could notably increase the number of branches in rapeseed, but it has no remarkable effect on increasing yield (Stanic et al., 2021).BnaCKX3 and BnaCKX5 are a group of enzymes that regulate oxidative cleavage to maintain cytokinin homeostasis.Sextuple bnackx3bnackx5 mutants have increased cytokinin concentrations in reproductive tissues, resulting in a larger and more active IM, as well increased silique number (Schwarz et al., 2020).Therefore, simultaneous knockout of BnaD14, BnaCKX3 and BnaCKX5 in rapeseed may increase branch number, increase silique number per branch and greatly improve rapeseed yield.However, breeding rapeseed with ideal architecture requires the dissection of genetic loci associated with traits related to silique number or the cloning of the underlying genes (Liu et al., 2022).The application of multiomics including genome, transcriptome, and epigenome and advanced technologies such as GWAS and CRISPR-based editing in rapeseed will facilitate the identification of key genes controlling silique number.As more regulatory genes are cloned and studied, the genetic networks controlling silique number in rapeseed will be revealed, paving the way for improving rapeseed yield by increasing silique number.

Figure 1
Figure1Alignment map displaying regions of QTLs/QTNs for traits related to silique number in rapeseed.From outside to inside, different coloured blocks in the outermost circle represent different genetic linkage groups, the six inner circles represent six traits related to silique number (BN, SD, SDMI, SN, SNMI and SNUA, respectively), and different geometric shapes within the six inner circles with the same colour as their respective blocks represent QTLs/QTNs associated with specific linkage groups identified in previous studies.

Figure 2
Figure2Regions containing overlapping QTLs/QTNs associated with silique number.Overlapping QTLs/QTNs are named q + SNRT (silique-numberrelated trait) + chromosome + number, and different coloured dots represent different traits related to silique number.Blue to red shading on the chromosomes indicates gene density from low to high.

Figure 3
Figure 3 Summary of genetic, plant hormonal and environmental control of silique number in rapeseed.Black solid and dotted arrows represent direct and indirect positive regulation, respectively.Red inhibition symbols indicate negative regulation.Black and blue gene names represent genes confirmed and predicted to be involved in regulating silique number, respectively.Phytohormones are indicated in purple.

Figure 4
Figure 4 Genetic interactions of major regulatory genes that control SAM activity, shoot branching and FM formation in Arabidopsis and rice.From top to bottom, orange shading indicates FM formation during reproductive growth, and green shading indicates axillary bud elongation, axillary bud initiation and SAM activity during vegetative growth (Arabidopsis on the left and rice on the right).Adjacent solid boxes of different colours indicate gene interactions, with different colours indicating different source references describing the same step.Purple and blue gene names represent homologues of confirmed and candidate genes that regulate silique number in rapeseed.Black arrows indicate positive regulation, red lines indicate inhibition, and solid and dashed lines indicate direct and indirect regulation, respectively.

Figure 5
Figure5Gene regulatory network for silique number in Arabidopsis.The 54 genes in the genetic regulatory network were derived from the intersection of Arabidopsis homologues of transcription factor gene among candidate genes and eight genes (WUS, LFY, FLC, AP1, SOC1, STM, SVP and REV ) using ChIP-seq.Large and small grey dots represent genes identified by ChIP-seq and target genes, respectively.Different coloured lines connecting different genes indicate transcriptional regulation of target genes by different transcription factors.