Cardamine hirsuta. We observed strong genetic differentiation among several ancestry groups and broader distribution of Iberian relict strains in European C. hirsuta compared to Arabidopsis. We found synchronization between vegetative and reproductive development and a pervasive role for heterochronic pathways in shaping C. hirsuta natural variation. A single, fast-cycling ChFRIGIDA allele evolved adaptively allowing range expansion from glacial refugia, unlike Arabidopsis where multiple FRIGIDA haplotypes were involved. The Azores islands, where Arabidopsis is scarce, are a hotspot for C. hirsuta diversity. We identified a quantitative trait locus (QTL) in the heterochronic SPL9 transcription factor as a determinant of an Azorean morphotype. This QTL shows evidence for positive selection, and its distribution mirrors a climate gradient that broadly shaped the Azorean flora. Overall, we establish a framework to explore how the interplay of adaptation, demography, and development shaped diversity patterns of 2 related plant species.We study natural DNA polymorphisms and associated phenotypes in the Arabidopsis relative  This study explores how the interplay of adaptation, demography and development shaped diversity patterns in Cardamine hirsuta, a close relative of Arabidopsis, and shows that a QTL in the heterochronic SPL9 gene underlies a distinctive morphotype discovered in the Azores islands. Arabidopsis thaliana have yielded fundamental insights into how genotypes are translated into phenotypes during development of seed plants [A. thaliana [A. thaliana’s demographic history and population structure and allowed investigations of the effects of demography on trait diversity [Cardamine hirsuta, a close relative of A. thaliana, offers excellent opportunities to understand diversity in a comparative fashion because it shares many attributes that make a good model organism, while also showing differences in key traits including floral morphology, seed dispersal, and leaf shape [C. hirsuta leaf into distinct leaflets versus the simple leaf of A. thaliana and the explosive seed dispersal of C. hirsuta versus the passive dispersal in A. thaliana [Comparative analysis of related taxa offers the opportunity to investigate how the interplay of adaptation, historical contingency (the outcome of past events that are often random), and developmental constraints shaped phenotypic and genetic diversity –4. In dod plants ,7. Naturthaliana –15. At tiversity –19. Cardaf shape . These athaliana –24.C. hirsuta also shows natural variation for many traits, which provides an untapped resource for comparative studies of diversity at the population level [A. thaliana has been flowering time, a key life history trait that marks the onset of reproduction and contributes to local adaptation [C. hirsuta provided evidence for trait integration between flowering time and complex leaf morphology [FLOWERING LOCUS C (FLC) acts heterochronically by accelerating the rate of transition from simpler juvenile leaves to adult leaf forms bearing more leaflets. This synchronization of flowering time and leaf development resulted in more leaflets being produced in anticipation of reproduction, which may support resource allocation to the next generation in the form of seeds [C. hirsuta. Notably, many traits are under age-dependent control in seed plants, including stress responses and nutritional status [C. hirsuta, and the ecological factors driving its maintenance remain unknown. Addressing this question and studying the degree of conservation versus divergence of pathways shaping natural trait variation in C. hirsuta and A. thaliana requires a comprehensive survey of genetic and phenotypic diversity in C. hirsuta.on level . A majoraptation –29. Overaptation ,31. Suchaptation –36. A qurphology . Specifiof seeds . Thus, gl status –40, thusl status ,42. HoweC. hirsuta. We find that C. hirsuta from the Iberian Peninsula and the Macaronesian Islands retain considerable genetic diversity, thus resembling relict populations of A. thaliana. However, C. hirsuta relict-like strains extend more than A. thaliana relicts into mainland Europe and therefore have been more able to establish outside glacial refugia. We then use our strain panel to study genetic control of leaflet number and flowering time. We find evidence for their correlated control by the flowering time loci FRIGIDA and FLC as well as a component of the trehalose biosynthesis pathway. Furthermore, polymorphisms at FRIGIDA display a distinct signature of positive selection, providing a striking example of parallel adaptive evolution with A. thaliana, where multiple loss-of-function alleles drive flowering time adaptation in northern latitudes [A. thaliana, a single loss-of-function haplotype dominates variation in C. hirsuta, highlighting how the balance between stochastic and deterministic forces drove parallel trait evolution in these 2 species. Finally, we find abundant diverse C. hirsuta populations in the Azorean islands where seasonality is reduced compared to continental Europe. In exploring the genetic basis for the Azores colonization, we found a QTL cluster that affects age-dependent variation in leaf form without pronounced effect on flowering time and includes a derived allele of the heterochronically acting transcription factor SQUAMOSA PROMOTER BINDING-LIKE 9 (SPL9), which we validate in transgenic assays. This allele shows a polarized East–West geographic distribution in the Azores that correlates with environmental differences, particularly water availability. The same genomic region shows evidence for positive selection at and around the SPL9 locus, indicating that modulation of SPL9-dependent variation in shoot development contributed to adaptation of C. hirsuta in its ecological niche of the Western Azores where plants experience year-round conditions permissive for growth, punctuated by dry summer spells. Our study establishes C. hirsuta as a valuable model for comparative studies at the population level in plants and shows that heterochronic pathways had a major contribution to natural variation in this species under diverse ecological conditions.Here, we use genome-wide single nucleotide polymorphism (SNP) data to analyze the demographic history of European and Macaronesian populations of atitudes . HoweverC. hirsuta and compare it with A. thaliana. Given that the 2 species share similar life cycles, we reasoned that if their distribution and colonization history was shaped by similar ecological forces, then this similarity might be preserved in common patterns of extant genetic diversity in the 2 taxa. Conversely, large differences in the evolutionary trajectories of the 2 species might have left imprints in the distribution of genetic diversity patterns.We set out to understand the demographic history of C. hirsuta strains and investigated patterns of genetic structure based on 5,336,586 biallelic high-quality SNPs. The geographic distribution of our panel is centered on Western Europe but also includes a small number of strains from the United States of America, New Zealand, Australia, and Japan  in which nonadmixed individuals from the 3 populations represented well-differentiated genetic groups, while admixed strains were mainly observed between BAL and NCE  have been identified based on the examination of distributions of pairwise genetic distances (PGDs) between individual strains [A. thaliana. Unlike A. thaliana, for which this distribution has a single major mode around 0.475% , PGDs iand 0.8% . The centhaliana , and it nterpart . This obC. hirsuta and A. thaliana by calculating nucleotide diversity, Tajima’s D, and FST values in the genetic groups described for both species  is similar to the one observed in A. thaliana strains belonging to the Iberian group (relicts and nonrelicts). The lower diversity in NCE is consistent with the effects of recent demographic processes accompanying the colonization of northern latitudes by Southern European lineages after the last glacial maximum [C. hirsuta and A. thaliana  and potentially result from more pronounced population size bottlenecks  is 45% larger than the highest genetic differentiation observed between A. thaliana relict and nonrelict populations in the 1001 Genomes Project  and a more recent split between NCE and BAL . Also, ancestral and present population size estimates largely agreed with the estimates from relate and MSMC2, and all methods identified a reduction in size for NCE following its divergence. Overall, our observations indicate the presence of a relict group in Iberia, thus highlighting striking parallelism in spatial population structure with A. thaliana despite the estimated divergence time of the 2 species being over 25 million years ago [To understand the nature of the evolutionary processes that shaped patterns of variation in e relate  and MSMCe [MSMC2 –50 . FLC influences flowering time and leaf development in both A. thaliana and C. hirsuta [A. thaliana [FLC and TPPI and 6 of them inside their open reading frames or promoter regions. Multilocus GWAS showed that SNPs in both genes had independent effects, indicating that both contribute to phenotypic diversity in our strain panel , a well-studied gene controlling flowering time through the activation of FLC expression [FRI thus emerged as a candidate for determining natural variation in flowering time and leaflet number in C. hirsuta. We found 3 independent polymorphisms predicted to truncate the FRI protein. C. hirsuta Oxford (Ox) lines constitutively expressing the coding sequence (CDS) of any truncated FRI allele driven by the Ubiquitin10-promoter (UBQ10) did not show increased rosette leaf number (RLN), a proxy for flowering time, while lines expressing a nontruncated CDS (FRIfunc) increased RLN by 20%, confirming the functional significance of the truncations (FRIstop) occurred at high frequency and could therefore be responsible for the association on chromosome 8. This allele is specific to NCE only and was present in 45 out of 57 nonadmixed strains , ranging from 4.7 × 10−10 to 6.6 × 10−9 within and flanking the pericentromeric region. We hypothesized that the reduced r in the pericentromeric region could explain why the selective sweep extends further in the proximal pericentromeric area of the FRI locus, in strains with the FRIstop allele. We tested this hypothesis by simulations and found that the sweep profile is consistent with a hard sweep of a single strongly selected beneficial mutation that occurred after the divergence time between lineages of NCE and BAL , while FRIstop was found in 98.2% of the European C. hirsuta strains with loss-of-function alleles (111 out of 113 strains).In the candidate genes  for FRI . Strains for FRI . Further for FRI  negative for FRI . Considethaliana ,43,62–66pFinder2 , which uion Figs  and S2E.ion Figs . Notablymosome 8  . We reasoned that if heterochronic processes have a central role in shoot morphological variation in this species, they might also underlie variation between strains that do not differ in flowering time. This would be similar to the situation in snapdragon, where genetic control of age-dependent leaf shape variation is independent of flowering time [A. thaliana [The ing time , and it thaliana ,74. We athaliana . We hypoC. hirsuta strains from across the Azores archipelago and found that the low leaflet number occurred in approximately 24% of them  that strongly affected leaflet number but not flowering time (LLN4 with 4 heterogeneous inbred families (HIFs) identified a cluster of 3 closely linked QTL for leaflet number located between positions 17.39 Mb and 22.83 Mb, which are referred to as LLN4_1A, LLN4_1B, and LLN4_2 , which is based on the attribute of juvenile plants to not respond to the flowering-inducing stimulus of long photoperiod [LLN4_2 is heterochronic, affecting the timing of the juvenile-to-adult transition and, consequently, leaflet number, largely independent of the flowering transition.To test this hypothesis, we first explored the prevalence of the low-leaflet phenotype. We carried out denser sampling and sequenced 267  of them . ADMIXTUAZ; Figs  and S3C ime Figs  and S3D.ime Figs . We deteime Figs . Five ouing time . Genetic4_2 Figs . In agrenes ILs; , showingtoperiod . We founLLN4_2, we first fine-mapped it to a 49-kb region containing 14 predicted genes. Among these, the C. hirsuta orthologue of the transcription factor encoding gene SQUAMOSA PROMOTER BINDING-LIKE 9  generated by CRISPR-Cas9 genome editing showed reduced leaflet number, similar to the phenotype of the Az1 strain , while the alternative allele of grp2 was prevalent in the East , which is thought to reflect a climatic gradient between warm summers with punctuated precipitations in the West, and extended dry summers in the East, in a context of reduced annual variation in temperature and daylength in the entire archipelago compared to mainland Europe [C. hirsuta revealed potentially adaptive allelic variation in the key heterochronic transcription factor SPL9, which appears as a major driver of morphological differentiation across heterogeneous climatic conditions.To investigate possible drivers of selection at the location . Both grlocation , which wlocation . Haplotythe East . Notablyd Europe –82  and their broader geographic distribution into Western Europe  also influences the pattern of rapid cycling caused by FRI loss-of-function mutations in Central Europe. While in A. thaliana FRI is located near the distal end of the short arm of chromosome 4, in C. hirsuta, it is located in the pericentromeric region of chromosome 8. The extent of genetic hitchhiking in selective sweeps depends on the local recombination rate [FRI might therefore have been restricted in C. hirsuta due to a higher potential genetic load compared to A. thaliana, which limits the capacity to spread through the population. Thus, this case of parallel evolution of FRI based on extensive comparative evaluation of diversity patterns of 2 species along a similar geographic cline illustrates how the complex interplay of demography, natural selection, and genome structure shapes adaptive processes. Natural allelic variation in FRI leading to flowering time variation has also been uncovered in other Brassicaceae such as Brassica napus [Arabidopsis lyrata [C. hirsuta.The study of natural variation in late Fig  largely atitudes ,69. Howeplotypes . Such a plotypes ,87. Alteion rate ,89 and, ion rate . Fixatioca napus  and Arabs lyrata , so onceC. hirsuta germplasm indicates that this island complex harbors considerable C. hirsuta diversity even though previous work had suggested that overall biodiversity in the Azores might be low and very few lineages showed patterns of adaptive radiations there [C. hirsuta established from seeds introduced perhaps by vagrant birds [SPL9 QTL cluster coupled with evidence for positive selection indicates that the phenotypic differentiation we report here may be adaptive in the Western Azores Islands. This observation raises the question of what might be the environmental drivers of this distribution. Examination of climate variables shows that a major difference of the Western Azores relative to the East are the more frequent summer rainfalls in the West, punctuated by dry spells [SPL function and the juvenile state to drought tolerance [SPL9 QTL might help such summer seedling establishment at the face of transient drought. In line with this view, strains harboring the SPL9 QTL cluster were abundant throughout the year and already developmentally advanced in November in the Western Azores .Further information and requests for resources and reagents should be directed to and will be fulfilled by the corresponding author Miltos Tsiantis . Paired-end library preparation and short read sequencing was performed at the Wellcome Trust Centre for Human Genetics  or at the Max-Planck Genome Center  using Illumina HiSeq2000 or HiSeq3000 instruments (Illumina). Read-lengths varied and included 49, 100, 150, and 151 nucleotides across projects . Short read alignment and variant calling was performed using the IMR-DENOM-IRISAS pipeline [The reference sequence of vided in , at httppipeline . Qualitysamtools mpileup version 1.9. IMR-DENOM identified sequence motifs that were particularly diverged and called them as single variants with alleles longer than 1 but not necessarily of equal length. A consequence of recalling the variants by IRISAS was that all differences between both alleles for these motifs were called as SNPs. We therefore masked the regions spanning the variants in question in each strain at the position at which it was called plus the length of the longest allele. To control for uncertainty in called SNPs conferred by sequence ambiguity in the reference genome, we applied Heng Li’s SNPable regions method for all 100-mer subsequences of the reference genome as described here: http://lh3lh3.users.sourceforge.net/snpable.shtml. Any position where the majority of overlapping 100-mers did not map uniquely and without 1 difference was masked in all strains. The masks described above were applied to the variants recalled by IRISAS for all subsequent analyses such that the allele at a SNP was changed to unknown if that position was masked for the strain in question. The number of unmasked sites was considered to be the total number of nucleotides observed for each strain. The SDI files produced by IRISAS were transformed to plink files using IRISAS’ SdiToSnpPlink command. The total number of SNPs was 11,576,732 with a 1.461%, 4.382%, 15.14%, and 12.49% first quantile, median, third quantile, and mean number of missing calls per SNP.Various analyses depended on accurate accounts of polymorphic and nonpolymorphic sites in the resequenced genomes. Several common and strain-specific masks were applied to control for different sources of uncertainty in the genomic data. We masked the resequenced genomes where the read depth was less than 4 reads. Read-depths were extracted for the 8 chromosomes from the bam files containing the reads of each resequenced strain aligned to the reference genome, respectively, using cmdscale and hclust, respectively, to identify and visualize the major groups of distantly related strains. The results of hierarchical clustering were plotted as dendrogram using iTOL [https://itol.embl.de/).PGDs were computed as in  from theing iTOL   leaving 84,065 SNPs. The program ADMIXTURE was then run 75 times for each ancestral population (K = 1) through K = 10, with different random seeds. The individual runs were evaluated for lowest cross-validation (CV) error to determine the best K. The result of the individual replicate run with the lowest CV error was used for further analysis. For the analysis of all 753 strains, including all accessions from the Azores, the same procedure was applied yielding a subset of 421 strains and 24,391 SNPs after filtering . The strains that had been filtered out due to being closely related to retained strains were assigned the same ancestry as the latter.For analysis of population structure with the program DMIXTURE , the setSNPRelate [ADMIXTURE analysis.PCA of genetic variation data was conducted with the R package NPRelate  using alFST values in vcftools version 0.1.16 [C. hirsuta, nonadmixed accessions from the BAL, NCE, and IBE ancestry groups were used. Exons and low-recombining pericentromeric regions , as well as on the VCF file from [C. hirsuta dataset were used: considering only accessions with less than 5% admixture and filtering out exons and pericentromeric regions  were exile from  for the fined by ). SummarpLDdecay  after exMSMC2 [relate [MSMC2, input data were prepared using the masks to determine the total number of observed monomorphic sites between SNPs. Twenty unique datasets were prepared each for the comparisons BAL-IBE, BAL-NCE, and IBE-AZ. Each dataset contained 4 strains per group selected at random from those that showed no evidence of admixture in the ADMIXTURE analysis. The selections of 4 strains per group were ensured to be unique. All SNPs for which at least 2 of the 8 strains in a dataset were polymorphic were included while allowing for missing values. The total number of observed sites between the SNPs was counted as the number of sites for which all 8 strains were unmasked. MSMC2 was then run with the time pattern “1*2+40*1+1*2+1*3” on the within ancestry group strains and across ancestry groups separately. Ne was estimated from within group coalescence rates, and the scaled time segments were converted to generations according to the general MSMC guide (https://github.com/stschiff/msmc/blob/master/guide.md), using a mutation rate per generation, per nucleotide, of either 4 × 10−9 , we used 20 individuals from each of the 3 ancestry groups identified by the software ADMIXTURE that did not show admixture from other groups. Low-recombining pericentromeric as well as exonic regions were filtered out to minimize the effect of direct and indirect negative selection on the site frequency spectrum (SFS). Sites with missing data were excluded, and the number of monomorphic sites  was determined by applying the same quality criterion to polymorphic and monomorphic sites. The unfolded SFS for the BAL, NCE, and IBE ancestry groups was constructed using an in-house python script based on the pyVCF and dadi library [FST, PCA, MSMC2, and relate analyses. Asymmetric migration rates were allowed to vary freely between all 3 pairs of populations and were constant through time. Sizes for each population were constant except for possible bottlenecks (depending on the model) and a final change in size that was allowed to occur concomitantly with the last population split. A detailed description of the models and a table summarizing all parameters including the range used to pick initial parameter values for likelihood optimization are available in fastsimcoal2 was run with the following options “-m -n 100000 -M 0.01 -N 100000 -l 10 -L 40 -q -x–multiSFS”; the maximum likelihood for each model was chosen as the largest likelihood among 50 independent runs. Model choice was conducted based on rAIC values, as described in [−9 [−9 [For the demographic analyses using the software  library ; sites wribed in . Two difd in [−9  and 7·10 [−9 [−9 , and we C. hirsuta strains. The mean flowering time was used for further analysis. Heteroblasty causes the numbers of leaflets on the rosette leaves to be correlated, and we previously described a negative relationship between the rate of heteroblastic progression and flowering time [PLINK2 (www.cog-genomics.org/plink/2.0/): (i) a maximum missing call rate of 0.35; (ii) a minimum minor allele count of 10; (iii) (peri)centromeres excluded; and (iv) a squared correlation (r2) less than 0.8. A centered kinship matrix was then calculated using GEMMA [GEMMA with 2,651,721 SNPs that remained after filtering for maximum missing rate of 0.3 and minor allele frequency of 0.04. We also performed GWAS of flowering time using a multilocus mixed model (MLMM) approach [mlmm.gwas version 1.0.6. Based on the results of MLMM, we performed a GWAS with the most significant SNP as a covariate using GEMMA and used the result to produce An experiment was conducted in the greenhouse under long-day conditions in which leaflet numbers on the first 10 rosette leaves, and flowering time were quantified on up to 4 and up to 3 replicates, respectively, for each of 352 ing time . The hetng GEMMA , which iapproach  as impleC. hirsuta, we reconstructed a genetic map from the Ox × Az1 RIL-population from low coverage resequencing data  [As a quality control for the correspondence between the genetic and physical maps of data see . Furtherdata see . The recical map . To do smap-ont) . SecondlFRIGIDA gene of 746 C. hirsuta and 1,212 A. thaliana strains were filtered from SDI files. Genetic variants of A. thaliana strains were called from publicly available resequencing data using the IMR-DENOM-IRISAS pipeline [FRIGIDA alleles were obtained by modifying the reference sequence according to the variants called inside the gene using a custom R script. They were then translated into amino acid sequences in silico using the R package seqinr [A. thaliana, it was possible to recover 29 truncated FRI alleles including the FRIcol and FRILer alleles. The identified alleles of A. thaliana agreed in 757 out of 761 cases for full-length and 239 out of 241 cases for truncated FRI alleles with predictions from Zhang and Jiménez-Gómez [Genetic variants occurring in the pipeline  to ensure seqinr . Truncatez-Gómez , indicatpUBQ10::FRIstop1-3 and pUBQ10::FRIfunc, the ATUBQ10::promoter was amplified with the primers TGCAGTACCCGACGAGTCAGTAATAA and CTCGAGAGTGTTAATCAGAAAAACTCA, thereby attaching the 3′ PstI and 5′ XhoI restriction sites. This fragment was subcloned into pJet 1.2 (Thermo Fisher Scientific) according to the protocol and subsequently cloned as a PstI and XhoI fragment into pBJ36. The open reading frame of FRI was amplified from C. hirsuta Oxford (FRIstop1), C. hirsuta Japan (FRIstop2), C. hirsuta 858B (FRIstop3), and C. hirsuta Azores1 (FRIfunc) using the primers GAATTCATGCAGGAGGAGCAACCCTCAACGGC and GGATCCTTACGTTCTTCCTTTGCGGTCTAGTG, which added 3′ EcoRI and 5′ BamHI restriction sites. The blunt-ended PCR fragments were subcloned into pJet1.2. The FRI alleles were inserted into pBJ36-pUBQ10 using EcoRI/BamHI double digest with subsequent ligation. The pUBQ10::FRI fragments were then cloned into pML-BART using NotI. All plasmids were verified by dideoxy-sequencing and transformed in Agrobacterium tumefaciens strain GV3101. Plants were transformed with A. tumefaciens by floral dip technique [pML-BART, FRIfunc, FRIstop, FRIstop2, and FRIstop3, respectively. We tested for statistical differences between groups using the Dunn test with Bonferroni adjustment for multiple comparisons. Five T2 plants of 20 independent T1 plants of pUBQ10::FRIfunc and pUBQ10::FRIstop, respectively, were sown together with 48 control plants derived from 3 independent transgenics transformed with the pML-BART. After confirming the presence of the transgene via genotyping for the presence of the BASTA resistance gene using the primers GAAGTCCAGCTGCCAGAAAC and TACATCGAGACAAGCACGGT, we scored leaflet number until leaf 7 on at least 33, 37, and 46 plants of FRIfunc, FRIstop, and Control, respectively. We tested for statistical differences between all groups on each leaf separately using the Dunn test with Bonferroni adjustment for multiple comparisons.To obtain echnique . T1 seedPLINK1.9 [FRIfunc) and 45 strains carrying the truncated FRI allele (FRIstop). Pi and Tajima’s D were calculated using VCFtools [PLINK PED files were converted into VCF files keeping only strains with more than 99% of NCE ancestry proportions using PLINK1.9 . The finVCFtools  with a wC. oligosperma as previously described and called SNPs at known polymorphic sites using samtools mpileup. We retained only SNPs that were polymorphic in NCE strains with more than 99% of NCE ancestry proportions. We polarized these SNPs into ancestral and derived alleles according to the outgroup allele and created a site frequency (SF) file using a custom R script. SweepFinder2 [FRIstop and FRIfunc) separately, including monomorphic sites at a step size of 10,000 using the precomputed empirical genome-wide frequency spectrum. To obtain a significance threshold for the composite likelihood ratio, 1,000 neutral simulations of chromosome 8 were performed using msprime version 0.7.3 [fastsimcoal model (−9 recombinations per base pair per generation) and inside (4.39 × 10−10) pericentromeric regions were estimated from the genetic map described in section 3.1 by regressing genetic distances on physical distances, and a mutation rate of 4 × 10−9 mutations per base pair per generation was used. SweeD [We aligned Illumina short reads of the outgroup species pFinder2  was run on 0.7.3  based onal model . Recombid. SweeD  analysisFRIstop on chromosome 8 was consistent with the relatively recent differentiation of NCE. We used SLiM version 3.3 to simulate tree sequences under the Wright–Fisher model [fastsimcoal2 . Recombination rates in the chromosome arms were 9.944·10−8 recombinations·bp−1·generation−1 (rec·bp−1·gen−1), and a reduced rate of 4.945·10−9 rec·bp−1·gen−1 in the pericentromeric region . The seFRIstop was introduced in the position 11,100,000, the approximate position of FRIstop, at or after NCE split from its ancestral population (BAL) 16,030 generations before present. The simulated tree sequences were overlaid with neutral diversity using msprime version 0.4.7 [−9 mutations·bp−1·generation−1. All combinations of the selection coefficients 0.001, 0.01, and 0.1 and appearance of FRIstop at 16,030, 13,030 10,030, 7,030, 4,030, 1,030 before present were simulated 12 times, except for 1 combination 8 times. Simulations in which FRIstop did not achieve the empirical frequency of 79.31% in our C. hirsuta sample were discarded.A selected recessive mutation (h = 0) representing on 0.4.7  with a mVCFtools version 0.1.16 [A sliding window analysis for nucleotide diversity (Pi) was performed on the simulated data using n 0.1.16  with a wThe Az1 strain was collected from the Azorean island of Faial . AfterwaSPL9 in the lab.Seeds or ripe siliques were collected from multiple individual plants in each visited population, but only 1 plant per population was sequenced, with the exception of 12 individuals from a single population collected from Flores in the fall trip. During the same sampling trip, phenological data were collected for the populations visited. Plants were in situ scored for their developmental stage according to 6 categories, from 1 (very young seedlings) to 6 (mature plants shattering seeds). These data were recorded in several representative patches of each population. At the same time, leaf material was harvested and desiccated using silica gel. DNA extracted from this material was later genotyped for the Az1 allele of C. hirsuta strains collected from the Azores were cultivated in a climatic chamber (Controlled Environments) under long-day conditions. Leaflet numbers were counted on the first 6 rosette leaves. Strains with low leaflet numbers were identified as belonging to the first mode of the leaflet number distribution, which included the Az1 strain.Four replicates each of 264 SPL9 locus . The seeds were planted on November 13, 2018. Leaflet numbers of 22 Ox wt strains and 19 introgression lines (IL LLN4_2Az1) that contain a single Az1 introgression in the Ox genetic background flanking the Leaf silhouettes were obtained by scanning the leaves, after gluing them to paper, at 800 dpi and a bit depth of 24 with an Epson Perfection V700 Photo scanner. For Reconstruction [R/qtl [MSTMap [P value < 0.05), were removed. In parallel, we created a genetic map de novo using MSTMap to include markers from unanchored scaffolds using a grouping logarithm of the odds (LOD) of 3, and the Kosambi function. Markers from unanchored scaffolds that could not be mapped to any of the major linkage groups were discarded. In case that adjacent markers of unanchored scaffolds showed a distance larger than 8 cM, these markers were also discarded. The de novo map showed nearly the same order of markers than the physical map, but not entirely. To locate genetically unanchored scaffolds, a marker order was forced according to the physical map of the 8 chromosomes. Markers from unanchored scaffolds were then added to locate them unambiguously based on recombination frequency. As described above, some genetic markers that were originally found on chromosome 8 were transferred to chromosome 7, and the order of markers in the pericentromeric region was inverted. Gaps greater than 5 cM were examined individually to find potential markers of low quality, which would cause an apparent excess of recombination with flanking markers. For that, the length of the genetic map was calculated in the absence and presence of the genetic markers flanking large gaps, and we excluded those markers that caused an increase of 2.5 cM or more. The final map contained 2,720 markers with an average spacing of 0.3 cM, a maximum spacing of 8.9 cM, and a total length of 815.2 cM (see The Az1 strain was propagated through single seed descent (SSD) for 3 generations to reduce the amount of potential heterozygosity before it was crossed to the Ox strain. In total, 192 F2 lines were propagated to the F9 generation by SSD. Genetic markers were obtained by Illumina resequencing at a coverage of 0.5× , and subsequent imputation using truction . One genn [R/qtl  and MSTM [MSTMap . Duplica2 cM see .Phenotyping of the Ox × Az1 recombinant inbred lines: Six replicates of the progeny of the resequenced RILs and 12 replicates of the parental lines were sown on moist soil and stratified for 10 days at 4°C. The plants were grown in the greenhouse under long-day conditions. Leaflet numbers on the first 10 rosette leaves and the total number of rosette leaves were scored.QTL mapping procedures: Multitrait QTL analysis was performed using GENSTAT 20th edition [QMTQTLSCAN procedure using the mean trait values per genotype for leaflet numbers on leaf nodes 2 to 10 and RLN. A simple interval mapping scan was followed by several rounds of composite interval mapping. During the latter, cofactors were added, removed, or their position was refined until no further improvement of the QTL model could be achieved. The final set of cofactors were then used as genetic predictors in the QMTESTIMATE procedure to fit the final QTL model and to estimate the allelic effects and variances explained by those QTL. edition . MultitrR/qtl [SCANTWO interval mapping function at 1 cM resolution using Haley Knott regression [SCANTWO, new QTL were identified through SCANONE interval mapping at 1 cM resolution. All new QTL were added to the model, and the positions were refined using REFINEQTL. New QTL were added using ADDPAIR and ADDQTL and new interactions using ADDINT. All models were checked using FITQTL, and we retained only QTL above the LOD threshold determined by 10,000 permutations, which corresponds to a LOD of 3.14 for single QTL and 4.17 for epistatic interactions. QTL less than 10 cM apart were considered the same. The search for new QTL was finished when no new QTL could be identified or when the same QTL model occurred twice in the iterative process. The 1.5 LOD interval of each QTL was computed for each QTL in the final QTL model.The multiple QTL model analysis for each trait separately was performed using R/qtl  with a cR/qtl . New QTLgression . When noLLN4_1, we selected recombinant lines in the progeny of HIFLLN4_1 using the markers in HIFs are NILs (sister plants) that segregate only for the region of interest in an otherwise homozygous albeit heterogeneous background. HIFs were selected from the F7 generation of the Ox × Az1 RIL population . These wLLN4_1 and LLN4_2 loci in an Ox genetic background. They were obtained from 2 plants selected from HIF LLN4_1Az1 and HIF LLN4_2Az1, by backcrossing during 4 and 6 generations, respectively, using the C. hirsuta Ox strain as recurrent parent. Marker-assisted selection with the primers in LLN4 region of chromosome 4 (IL LLN4_1Az1+LLN4_2Az1) and only the LLN4_2 region. The line spanning the whole region was used to derive ILs with smaller Az1 introgression fragments for the LLN4_1 locus only.Three ILs were derived carrying Az1 alleles for the Lines IL LLN4_1Az1+LLN4_2Az1 and IL LLN4_1Az1 carried introgression fragments from Az1 chromosome 4 of approximately 9.8 Mb and approximately 6.3 Mb, respectively. Both lines had a proximal recombination event on chromosome 4 between markers located at 13.074 Mb and 13.147 Mb. However, line IL LLN4_1 carried the distal recombination point between markers at 17.504 and 19.361 Mb, whereas the introgression of IL LLN4_1Az1+LLN4_2Az1 spanned until the end of chromosome 4 (22.86 Mb). Line IL LLN4_2Az1 was validated by Illumina whole-genome sequencing . This also allowed determining the Az1 introgression on chromosome 4 to span from position 20,439,000 bp to 20,891,000 bp (approximately 0.5 Mb) and that the total proportion of the genome derived from Az1 in an Ox background is 3.15%. Seeds derived from heterozygous ILs were grown in short-day conditions and genotyped inside the segregating region with genetic markers listed in nls with the formula y ~ A / (1 + B * E^−×). The inflection points were considered a measurement for the juvenile-to-adult shift in days of LD. Confidence intervals (95%) of the inflection points were estimated by 10,000 bootstraps, and the differences between the inflection points were considered significant if the confidence intervals did not overlap.A photoperiod shift experiment was conducted using a HIF that segregated for QTL LLN4_2. Ten plants homozygous for the Ox allele, as well as 10 plants homozygous for the Az1 allele, were shifted from the initial long-day to short-day conditions at 0, 4, 6, 8, 9, 10, 11, 14, 18, 20, and 24 days after sowing. RLN was counted on all plants after they flowered. Statistical analysis was performed in R by fitting a logistic model with RLN as dependent variable and days of long day before shifting as independent variable using the function P values. According to PCR markers, the QTL interval was reduced to 20.570 to 20.619 Mb on chromosome 4  were integrated in the pDE-Cas9 vector optimized for Arabidopsis [C. hirsuta Ox was transformed and transformants (T1) were selected using BASTA. Sequencing of T2 lines revealed two 1 bp insertions of the nucleotides G and C after positions 53 and 179 relative to the start codon, respectively, leading to a frameshift and a premature stop codon resulting in a peptide of 23 amino acids. The T-DNA was segregated out, which was confirmed by PCR and the loss of the BASTA resistance. Homozygous T3 plants were used for genomic complementation experiments. To validate the effect of the spl9 mutation, 10 replicates of the spl9 mutant, IL LLN4_2 homozygous for the Az1 allele, and Ox wild type plants were grown in short-day conditions. For each leaf node, leaflet numbers between the 3 different genotypes were compared using Dunn test.The SPR-Cas9 . A constbidopsis . C. hirspChSPL9::gSPL9 with a genomic clone of SPL9 from Az1 (gSPL9Az1) or Ox (gSPL9Ox), 6 kb genomic fragments containing the open reading frame (ORF), UTRs, and 3-kb upstream sequences were amplified by PCR from the Ox and Az1 strains using the primers TTTGCGGCCGCGAAGTTAACTCGATCTAAATCAAT and CAGCCGCAGCGAGAGACCAGTTGTTATG. The amplified products were cloned in the pGEM-T Easy vector System (Promega). Chimeric constructs with the alleles at the 2 missense SNPs swapped were made from the Az1 and Ox CDS of SPL9. For that, the CDSs were digested with BlpI, and the resulting fragments were religated, to select for those with the alleles interchanged. SPL9mixOx_Az1 has the Ox allele at the first SNP in SPL9 and Az1 at the second SNP and vice versa for the SPL9mixAz1_Ox. All the CDS fragments were cloned into pBJ36 already containing the ChSPL9Ox promoter and the terminator octopine synthase (OCS) by digestion with KpnI–XmaI. The 2 parental versions of pChSPL9::gSPL9, as well as the SPL9mixOx-Az1 and SPL9mixAz1-Ox, were transferred to the binary vector pML-BART using the NotI restriction enzyme. All plasmids were verified by dideoxy-sequencing and transformed in A. tumefaciens strain GV3101. Plants were transformed also with an empty pML-BART as control.To obtain the plasmid SPL9Az1 allele transformed with pML-BART empty, gSPL9Az1, gSPL9Ox, SPL9mixAz1-Ox, SPL9mixOx-Az1, respectively. In the complementation experiment of the spl9 mutant, we propagated 10 independent T1 lines containing a single copy of the transgene for gSPL9Az1 and gSPL9Ox and 3 for the control line. In the T2 generation, 10 replicates of the complemented spl9 mutants and 5 replicates of each control line were grown and phenotyped in long-day conditions. Transgene copy number was estimated in the complemented spl9 mutant and ranged between 0 and 2 copies of the transgene. In the analysis, plants without a transgene were grouped together with the control plants. Ox wild-type plants and IL LLN4_2 plants homozygous for the Az1 were grown and phenotyped along with the other lines. Cumulative leaflet number from the first to the eighth leaf was compared between 92, 42, 23, 50, 26, 19, and 15 plants of control, gSPL9Az1(1), gSPL9Az1(2), gSPL9Ox(1), gSPL9Ox(2), ILLLN4_22Az1, and Ox, respectively. Statistical differences between groups were tested using the Dunn test using the Bonferroni adjustment method of the P values.We obtained and quantified leaflet number of 12, 19, 32, 12, and 20 T1 plants of HIF LLN4_2 homozygous for the DESeq2 [blind argument was set to FALSE. The P value adjustment was performed at a false discovery rate (FDR) of 0.1.For the comparison of the transcriptome of Ox and Az1, seeds were stratified on soil at 4°C for 10 days and then transferred to a growth chamber in short day. Seedlings were cut above the hypocotyl at 7 and 12 days after germination and immediately flash frozen in liquid nitrogen. The lines HIF-LLN4_2 (Rec29) Ox and HIF-LLN4_2 (Rec29) Az1 were grown in long-day conditions. Aboveground tissue of the seedlings was harvested at 12 days after germination. RNA extraction was performed with the Plant Total RNA Kit (Sigma) using the standard protocol. The RNA of 3 replicates of each strain and 2 replicates of each HIF was sequenced in the Max Planck Genome Centre Cologne using Illumina HiSeq3000 and Illumina HiSeq2000, respectively. The Illumina reads were processed as described in , resultiDESeq2 . The couSPL9 amino acid sequences, 21 different sequences including 2 SPL9 sequences from the outgroup species Tarenaya hassleriana were downloaded from https://phytozome-next.jgi.doe.gov/. The evolutionary history was inferred by using the maximum likelihood method and JTT matrix-based model [SPL9 sequences of C. hirsuta Ox and C. hirsuta Az1 amino acid sequences. There was a total of 401 positions in the final dataset. Evolutionary analyses were conducted in MEGA X [For the interspecific comparison of ed model . The tren MEGA X .pcadapt [PLINK1.9 (https://www.cog-genoimcs.org/plink/) to have a minor allele frequency greater than 0.05 and 20% or less missing data per SNP in the 753 global wild strains including the Azores islands. After filtering, 2,247,751 SNPs remained. Initially, a PCA was performed using pcadapt including LD clumping with a threshold of 0.1 to reduce the effect of LD on population structure. The first 9 PCs were above the baseline level of PCs and were retained for the pcadapt analysis for outlier detection. The distribution of P values calculated from the pcadapt analysis was U-shaped, showing an excess of very low and high P values. Since a QQ plot showed an excess of deviating P values, the calculation of the FDR threshold was inappropriate. Alternatively, we classified the 1,000 SNPs with the lowest P values as top outlier SNPs . Then, a PCA was performed with the top 1,000 significant SNPs that were located within the pcadapt peak and that overlapped with the SPL9 QTL cluster . Two groups of highly diverged strains were defined based on the PCA as follows: grp1 with PC1 < 0 and PC2 > 0.07, grp2 with PC1 > 0.11. Visual inspection of the SNPs in the SPL9 haplotype using IGV [pcadapt peak, were performed with pcadapt as described above. LD clumping was not applied either for the PCA of the top1,000 SNPs or the PCA of all SNPs within and outside the pcadapt peak.For the analysis with pcadapt , the SNPsing IGV  allowed n = 103) or grp2 (n = 50), using PLINK1.9 [FST between groups of strains were calculated using VCFtools [n = 1,766 windows) and the pcadapt peak (n = 64 windows) were tested using the Kruskal–Wallis test. For plotting the weighted FST, we kept only windows with more than 700 SNPs to exclude regions with big gaps in the alignment.PLINK ped files were converted into VCF files, keeping only strains belonging to grp1 (PLINK1.9 . NucleotVCFtools  with a whttp://worldclim.org/ using a resolution of 30″ [http://climexp.knmi.nl): PO000008506 , POM00008501 , POM00008512 , POM00008515 , UK000056225 , HRE00105217 , ITM00016310 , and SPE00120566 . For analysis of precipitation, a sliding window was applied on the days of the year with an 11-day window and a stride of 5 days over the time period from 1970 to 2020. In each window, the fraction of days with rain  for the total number of days with observations was calculated. Temperature data were analyzed as mean per day of the year.Climate data were downloaded from n of 30″ . The bion of 30″ . AdditioP values were corrected for multiple testing using either Bonferroni or Benjamini−Hochberg correction. For data preparation and visualization, the R packages data.table, KRIS, Rmisc, packcircles, rnaturalearth, ggplot2  were used [P values < = 0.05 were considered significant. P values ≤0.05, ≤0.01, and ≤0.001 were marked with (*), (**), and (***), respectively.Data were analyzed with R (R Core Development Team). Information on the statistical details of the results shown in this manuscript can be found in the respective section of the method details and/or the figure legends. Leaflet number per leaf was compared between different groups  with the nonparametric Wilcoxon-rank test for 2 groups and Kruskal–Wallis test for more than 2 groups. When necessary, ere used –144. DatS1 Fig(A) CV errors in ADMIXTURE analysis with different random seeds. The CV error is plotted against the chosen number of populations (K) for 75 independent analyzes. A lower CV error indicates a better fit to the data. In this analysis, K = 3 was unanimously found to be the best number of populations for the data. (B) PCA of SNP data corroborates ADMIXTURE analysis. The first PC separates IBE (blue) from the others, while the second PC separates BAL (red) and NCE (yellow). Strains with ancestry in only a single ancestry group in the ADMIXTURE analysis (C) Hierarcical clustering (hclust) of the PGDs reveals distant relict-like groups. Hclust was used to identify relict-like strains based on PGD, and distant groups among them. The results of hclust are shown as a dendrogram where branch length is a measure of PGD. The first bifurcation separated the strains into 2 groups with high genetic distance between them, one of which predominantly resembled the relict-like group (colored labels) also discovered in Arabidopsis [ADMIXTURE, this analysis also allowed the identification of groups represented by small numbers of strains, which led to the discovery of 2 distinct relict-like groups of strains with relatively large genetic distance among them. A group of 11 lineages (magenta) from North West Spain, Sweden, the Netherlands, and New Zealand was identified. In ADMIXTURE analysis, these strains were located in either BAL or NCE as admixed lines or they were ungrouped. The large genetic and geographic distance between some of the 11 lineages indicated that they may represent a disparate admixed group, possibly with ancestry of underrepresented groups. (D) The 2 relict-like groups discovered with hclust are responsible for the second major mode in the PGD distribution. The PGD distribution of all strains is shown by the black outline. Colors indicate the contribution of the 2 most distant groups of strains identified by hclust of the PGD matrix, and the remaining pairs of strains are shown in gray. In the legend, the PGD groups are assigned to the ADMIXTURE clusters (E) Relict-like C. hirsuta strains predominantly resemble the Iberian relicts discovered in Arabidopsis. The geographic distribution of the C. hirsuta strains is shown with PGD group membership indicated by corresponding colors . Similar to Iberian relicts in Arabidopsis [C. hirsuta IBE lines (blue) are found at high frequency on the Iberian Peninsula, but also more broadly in Western Europe and on the Macaronesian islands. Map layers were made with Natural Earth and [F) LD decay in comparable ancestry groups of C. hirsuta (C.h.) and A. thaliana (A.t.). The average squared correlation (r2) between pairs of SNPs is plotted against the average physical distance between them in kilobases. For A. thaliana, ancestry groups comparable to C. hirsuta were included: Relicts (C.h. IBE), NWC_Europe , IBC , as well as the Iberian nonrelict population (A.t. Iberia).  Piecewise reconstruction of ancestral effective population sizes (Ne) in the 3 ADMIXTURE groups using MSMC2 (G) and relate (H), and estimates of split times between them considering a mutation rate of 7∙10−9 mutations per base pair, per generation. The top panel shows ancestral changes in Ne within the ADMIXTURE groups plotted against time in years, when considering 1 generation per year. Red, blue, and yellow lines indicate the BAL, IBE, and NCE genetic clusters, respectively. With MSMC2 (G), 20 random sets of 4 strains were analyzed, which are all plotted, while with relate (H), all strains were analyzed jointly, hence a single line. The bottom panels show the RCCRs in BAL vs. NCE (solid lines) and IBE vs. BAL (dashed lines). The split times estimated with fastsimcoal2  rescaled for a mutation rate of 7∙10−9 are indicated by triangles below the x-axes. Light blue shaded areas in the plots show ancient periods of glaciation according to marine isotope stages 2-4, 6, 8, 10, 12, 14, 16, and 18 [I) The best demographic model according to fastsimcoal2 with a mutation rate of 4∙10−9 mutations per generation per base pair, and estimated parameters. The 3 populations identified by ADMIXTURE analysis are shown as columns along the y-axis, which indicates time from present at the bottom to more ancient times at the top. The width of the columns is scaled according to the estimated respective Ne, which is also shown in or above the columns themselves. Split times between the populations are shown by arrows connecting the columns, where all lineages from a population merge with its ancestral population. The times at which this occurs are also marked on the y-axis. Bottlenecks are indicated by temporary constrictions but are not to scale. The time at which a bottleneck period starts when looking backwards in time is indicated on the y-axis. Bidirectional arrows below the figure indicate the corresponding modes of migration in the model. See https://doi.org/10.5281/zenodo.7907435. BAL, Balkan; CV, cross-validation; hclust, hierarchical clustering; IBE, Iberian; LD, linkage disequilibrium; LGM, last glacial maximum; NCE, Northern Central European; PC, principal component; PCA, principal component analysis; PGD, pairwise genetic distance.analysis  are showbidopsis  because clusters  to whichbidopsis , the C. arth and . (F) LD , and 18 , respect, and 18  is likew(TIFF)Click here for additional data file.S2 Fig(A) Variation of flowering time in days after germination and lateral leaflet number of the rosette leaves 2 to 9 (L2-L9) within the 3 ADMIXTURE ancestry groups of C. hirsuta  Alignment of C. hirsuta (CH) FRIGIDA and A. thaliana (AT) FRIGIDA amino acid sequences. The high frequency FRIstop allele in C. hirsuta and the resulting truncated protein are indicated in blue. (C) The presence of the FRIstop allele (black circles) indicated in the PCA shown in FRIstop is not found among the set of 85 and 83 nonadmixed lines from IBE and BAL, respectively, and also only sporadically in admixed lines from those groups. In NCE, the frequency is 45 out of 57 nonadmixed lines after removing closely related strains, where it is primarily found in this group. (D) Transgenic lines harboring a functional FRIGIDA allele exhibit reduced leaflet number in later leaf nodes compared to transgenic lines harboring the FRIstop allele and control plants harboring an empty vector . Black crosses represent the mean ± SEM. (E) Evidence for a selective sweep at the FRIGIDA locus. Genome-wide sliding window analyses of nucleotide diversity , Tajima’s D (middle) are shown as well as the CLR analysis (bottom). The analyses were performed separately in strains with the FRIstop (blue) and the FRIfunc (black) alleles from the NCE population (FRI locus (orange dashed line) displays reduced PI, reduced Tajima’s D, and high CLR, consistent with a selective sweep, exclusively in strains with FRIstop. The horizontal dashed lines in the top and middle panels indicate the genome-wide averages for the respective groups in blue or gray, and in the lower panel, the horizontal dashed line indicates the threshold (α = 0.05) derived from neutral simulations. (F) Graphical representation of allele frequencies on chromosome 8 reveal extended haplotype blocks containing the FRI locus (dashed orange line). Major (black) and minor (white) alleles for all SNPs with a minor allele frequency greater than 5% are shown for the NCE ancestry group. Note the reduced genetic diversity around the FRI locus in FRIstop harboring strains (bottom) as compared to those with FRIfunc. (G) SFS of SNPs in FRIstop harboring strains from the NCE group. The SFS outside of the selective sweep region (H) Physical sizes of swept regions as a function of selection coefficient and duration in simulations indicate that the large haplotype block containing FRIstop in NCE is consistent with recent strong selection. Selection on the FRI locus was simulated in the context of the empirical local recombination rates estimated for chromosome 8 in C. hirsuta for the durations indicated on the x-axis. The physical size of the simulated chromosome was identical to the actual size of chromosome 8 and is indicated by the horizontal dotted line. The median sweep sizes from the simulations are shown by the colored lines and their 95% quantiles by the error bars, where red, blue, and yellow indicate simulated selection coefficients of 0.001, 0.01, and 0.1, respectively. The estimated size of the swept region containing FRI in our C. hirsuta sample is shown by the continuous horizontal line. Frequencies of the various truncated FRI alleles in A. thaliana (AT) and C. hirsuta (CH). Alleles are named as “at” or “ch” followed by the physical position of the polymorphism generating the truncation. Alleles marked with an “x” are found only in strains from outside of Europe. The data underlying the graphs shown in the figure can be found at https://doi.org/10.5281/zenodo.7907435. BAL, Balkan; CLR, composite likelihood ratio; IBE, Iberian; NCE, Northern Central Europe; PCA, principal component analysis; SFS, site frequency spectrum.pulation . Note hop region  is shown(TIFF)Click here for additional data file.S3 Fig(A) Regression of leaflet number on flowering time in IBE strains (blue) and the remaining strains (gray). Points show the cumulative leaflet number of the first 7 rosette leaves of individual strains, and the lines show the fitted linear models. P values and R2 of the regressions are shown in the top right corner for both groups. An asterisk (*) indicates the Azores (Az1) strain showing early flowering time and low leaflet number. (B) Histogram of cumulative leaflet number on the first 6 rosette leaves of C. hirsuta strains collected from the Azores. Strains considered to share a low-leaflet phenotype are indicated by the blue shaded area. (C) CV errors in ADMIXTURE analysis with different random seeds. The CV error is plotted against the chosen number of populations (K) for 75 independent analyzes. A lower CV error indicates a better fit to the data. In this analysis, K = 4 was found to be the best number of populations for the data. (D) Piecewise reconstruction of ancestral effective population sizes (Ne) in the 4 ADMIXTURE groups , IBE vs. BAL (dashed lines), and IBE vs. AZ (dotted line). Light blue shaded areas in the plots show ancient periods of glaciation according to marine isotope stages 2-4, 6, 8, 10, 12, 14, 16, and 18 [E) Multiple QTL model mapping results for different leaflet number traits and RLN in the Ox × Az1 RIL population. Estimated QTL positions are indicated by the solid black symbols. QTL that are involved in a 2-way epistatic interaction share the same open symbol. The boxes around the QTL represent the 1.5 LOD intervals and indicate the direction of the effect  and the percentage of variation explained (color intensity according to the legend). Effects of LLN4_1 and LLN4_2 estimated in ILs carrying Az1 alleles at one or both loci. Points show the leaflet number on leaves of individual replicates plotted against leaf nodes. The vertical bars show the standard error of the mean leaflet numbers. Plants homozygous for Ox (Oxford parent strain) or Az1 alleles (ILs) are shown in yellow or blue, respectively. Significant differences according to Kruskal–Wallis tests are shown with asterisks: *: P ≤ 0.05; **: P ≤ 0.01; ***: P ≤ 0.001. The data underlying the graphs shown in the figure can be found at https://doi.org/10.5281/zenodo.7907435. Az1, Azores1; BAL, Balkan; CV, cross-validation; IBE, Iberian; IL, introgression line; LGM, last glacial maximum; NCE, Northern Central European; Ox, Oxford; QTL, quantitative trait locus; RCCR, relative cross coalescence rate; RIL, recombinant inbreeding line; RLN, rosette leaf number.E groups  using re, and 18 , respect, and 18  is likew(TIFF)Click here for additional data file.S4 Fig(A) Leaflet number progression of C. hirsuta Ox, the Chspl9 mutant, and the introgression line IL-LLN4_2, all in the Ox genetic background. Differences in leaflet numbers between the 3 genotypes were tested with a Dunn test and the P values, adjusted according to the Bonferroni method, are indicated by asterisks: *: P ≤ 0.05; **: P ≤ 0.01; ***: P ≤ 0.001.  Genome-wide RNA-seq analyses of entire seedlings. (B) Comparison of C. hirsuta Az1 and C. hirsuta Ox, and (C) the NILs HIF-LLN4_2 (Rec29) with Az1 and Ox alleles at the SPL9 region. Negative log base 10 transformed P values are plotted against fold change of expression and each point is a gene. Red-colored points are significantly differentially expressed, while the black ones are not. The SPL9 gene is indicated in each plot. (D) Phylogeny and homology of SPL9 genes in 16 Brassicaceae. The left panel shows the SPL9 gene tree. The top panel shows the proportion of genes harboring the most common AA. The bottom-middle panel shows the entire SPL9 protein sequence, while the bottom-right panel corresponds to the region around the SPL9 missense SNPE242Q (indicated by asterisk). The data underlying the graphs shown in the figure can be found at https://doi.org/10.5281/zenodo.7907435. AA, amino acid; Az1, Azores1; Chspl9, C. hirsuta loss-of-function allele of SPL9; Ox, Oxford; SPL9, SQUAMOSA PROMOTER BINDING PROTEIN-LIKE 9.(TIFF)Click here for additional data file.S5 Fig(A) Reduced leaflet number in an IL carrying the SPL9Az1 locus in the Ox background (IL LLN4_2Az1) compared to the wild type when grown in a common garden on the island of Faial, Azores. The difference between the means was tested for significance with the Wilcoxon rank-sum test. (B) Genome-wide scan for selection with pcadapt. A Manhattan plot is shown with the results from the analysis of 753 C. hirsuta strains. The negative log base 10 transformed P values for SNPs are plotted against their physical positions on each chromosome. The dashed horizontal line indicates the genome-wide threshold on or above which there are only 1,000 SNPs. The functional missense SNP E242Q of SPL9 underlying QTL LLN4_2 is highlighted by a red circle. Yellow boxes in the lower part of the panel indicate the locations of QTL LLN4_1A, LLN4_1B, and LLLN4_2. (C) PCA of 753 C. hirsuta strains with pcadapt using all SNPs  outside the pcadapt peaks at the SPL9 QTL cluster  and inside . Each point is a strain and colors indicate whether it belongs to the Western Azores group grp1 (blue), Eastern Azores group grp2 (red), or others (gray). (D) An east–west climatic gradient on the Azorean archipelago as indicated by precipitation of the driest quarter (BIO17) and the distribution of strains grouped according to pcadapt analysis . Each independent sampling is represented by a pie chart, indicated by season  and year . Pie charts show the proportions of strains from the different groups in our sample colored according to SPL9 cluster; gray—others). The size of the pie chart is scaled to the number of strains according to the legend in the bottom left. Collection locations are indicated on the map by points colored according to BIO17 as indicated by the legend on the right. The latitudinal and longitudinal gradients for BIO17 across the collection sites on the Azores are also indicated along the upper and right margins of the figure. Map layers were made with Natural Earth and [E) Boxplots showing climatic differences between locations of C. hirsuta strains belonging to grp1 from West Azores (blue) and grp2 from East Azores (red). Only bioclimatic variables that were significantly different between both groups according to a Kruskal–Wallis test are shown. The P values of the test are indicated by asterisks: * 0.01 < P ≤ 0.05, *** P ≤ 0.001. (F) Geographic locations of weather stations from which data were analyzed. Stations were chosen to represent the 4 major groups discovered with ADMIXTURE analysis . Daily mean temperature is plotted against day of the year. Annual changes in temperature on the Azores are much reduced compared to the other locations primarily due to mild temperature in both winter and summer. The data underlying the graphs shown in the figure can be found at https://doi.org/10.5281/zenodo.7907435. AZ, Azores; BAL, Balkan; GBG, genomic background; IBE, Iberian; IL, introgression line; NCE, Northern Central European; Ox, Oxford; PCA, principal component analysis; QTL, quantitative trait locus; SPL9, SQUAMOSA PROMOTER BINDING PROTEIN-LIKE 9.arth and . (E) Box(TIFF)Click here for additional data file.S6 Fig(A) The RF of genetic markers within the Ox × Az1 RIL population reveal 2 regions on chromosome 8 that most likely belong to chromosome 7 instead of previous assignment to chromosome 8 (arrows). In addition, markers of the pericentromeric region  might be inverted.  Genetic (B) and physical (C) maps based on previous (left side) and new assembly (right side) of chromosome 8. Regions colored in red, yellow, and blue depict unchanged segments of the map, whereas the region in gray color corresponds to the inverted segment. A genetic map with inverted order of markers shows reduced genetic length supporting the inverted assembly (B). The breakpoints of the predicted inversion indicated by 1 and 2 have been previously uncovered by consistent breakpoints in Nanopore long reads mapped to the original assembly of chromosome 8 (C).  Images representing Nanopore reads  that span the regions 1 (D) and 2 (E) of the reassembled chromosome 8 and confirm the correctness of the new assembly. The data underlying the graphs shown in the figure can be found at https://doi.org/10.5281/zenodo.7907435. Az1, Azores1; Ox, Oxford; RF, recombination fraction; RIL, Recombinant Inbred Line.(TIFF)Click here for additional data file.S1 Table(CSV)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.S3 Table(XLSX)Click here for additional data file.S4 Table(CSV)Click here for additional data file.S5 Table(CSV)Click here for additional data file.S6 Table(XLSX)Click here for additional data file.S7 Table(CSV)Click here for additional data file.S8 Table(XLSX)Click here for additional data file.S9 Table(XLSX)Click here for additional data file.S10 Table(XLSX)Click here for additional data file.S11 Table(XLSX)Click here for additional data file.S12 Table(XLSX)Click here for additional data file.S13 Table(XLSX)Click here for additional data file.


Wolbachia, Rickettsia, and Cardinium. So, we studied the spatial and temporal distribution of Wolbachia and Rickettsia in Asia II 1 Bemisia tabaci. The results showed that the titers of Wolbachia and Rickettsia in the 3–120 h old eggs changed in a “w” pattern, and the location of Wolbachia and Rickettsia in the egg changed from egg stalk to egg base, and then from the egg base to egg posterior, and finally back to the middle of egg in a Rickettsia and Wolbachia coinfected whitefly host. Our study helps to explain the vertical transmission mechanism of bacterial endosymbionts and the distribution of bacterial endosymbionts in different tissues in the host.Bacterial endosymbionts play important roles in the life history of herbivorous insects, including supplying nutrients allowing exploitation of unbalanced diets, increasing the survivorship and fecundity of hosts, protecting hosts against entomopathogenic fungi and parasitoid wasps, ameliorating the detrimental effects of heat, and broadening the range of suitable food plants. The important function of bacterial endosymbionts is that they can manipulate the reproduction of their hosts. However, all functions of endosymbionts must rely on vertical transmission to spread within the population, such as Wolbachia and Rickettsia are bacterial endosymbionts that can induce a number of reproductive abnormalities in their arthropod hosts. We screened and established the co-infection of Wolbachia and Rickettsia in Bemisia tabaci and compared the spatial and temporal distribution of Wolbachia and Rickettsia in eggs (3–120 h after spawning), nymphs, and adults of B. tabaci by qPCR quantification and fluorescent in situ hybridization (FISH). The results show that the titer of Wolbachia and Rickettsia in the 3–120 h old eggs showed a “w” patterned fluctuation, while the titers of Wolbachia and Rickettsia had a “descending–ascending descending–ascending” change process. The titers of Rickettsia and Wolbachia nymphal and the adult life stages of Asia II1 B. tabaci generally increased with the development of whiteflies. However, the location of Wolbachia and Rickettsia in the egg changed from egg stalk to egg base, and then from egg base to egg posterior, and finally back to the middle of the egg. These results will provide basic information on the quantity and localization of Wolbachia and Rickettsia within different life stages of B. tabaci. These findings help to understand the dynamics of the vertical transmission of symbiotic bacteria. Buchnera in aphids and Carsonella in psyllids [The symbiotic relationship between insects and endosymbionts is widely prevalent . Obligatpsyllids ,4. Additpsyllids ,6,7. Intpsyllids . The verpsyllids .Bemisia tabaci  (Hemiptera: Aleyrodidae) is a complex of cryptic species, among which at least 40 cryptic species are morphologically indistinguishable but biologically different, and has become one of the most economically important crop pests worldwide [B. tabaci species complex shares a long-term and intimate association with only one obligate bacterial symbiont (Portiera) and facultative bacterial endosymbionts  [Wolbachia and Rickettsia are the most studied endosymbionts. Both of them can affect some aspects of host fitness, such as weight, fecundity, longevity, and defense [Wolbachia and Rickettsia are the master manipulators of arthropod reproduction that can induce a number of reproductive abnormalities, such as male killing, feminization, parthenogenesis, and cytoplasmic incompatibility (CI), thus increasing the spread of Wolbachia in host populations [Wolbachia or Rickettsia as a pest control agent requires generating novel Wolbachia or Rickettsia–host interactions by transinfection or microinjection of Wolbachia or Rickettsia strains into different insect pests [Wolbachia or Rickettsia on host fitness and behavior and their potential application as biological control agents [orldwide ,11. The kettsia) . Wolbach defense ,14,15. Fulations ,17. CI hulations ,20. The ct pests . Althougl agents , there iWolbachia and Rickettsia spread within different life stages of B. tabaci was detected under laboratory conditions. We compared the spatial and temporal distribution of Wolbachia and Rickettsia in eggs (3–120 h after egg laying), nymphs, and adults of B. tabaci using qPCR quantification and fluorescent in situ hybridization (FISH). These results will further enhance our understanding of the vertical transmission of symbiotic bacteria in insects. This study will also provide basic dynamic change pattern on the quantity and localization of Wolbachia and Rickettsia within different life stages of B. tabaci and will serve as a reference for the selection of the optimal time for the horizontal transfer of symbiotic bacteria within the eggs of different whiteflies by microinjection techniques, to study the interactions between the same symbiotic bacteria and different whitefly cryptic species or transfection of symbiotic bacteria inducing cytoplasmic incompatibility for pest control by microinjection technique.In this study, the spread of Gossypium hirsutum Linnaeus 1763. (var. Lumianyan no. 32) was used to rear B. tabaci Asia II 1. Cultivate cotton seedlings according to Li et al. [B. tabaci Asia II 1 individuals were collected from the soybean plants, Glycine max (Linn.) Merr in Guangzhou in 2017 and reared on the cotton plants at 26.0 ± 0.5 °C and 70–80% relative humidity under a 14:10 h (L:D) photoperiod in the laboratory at South China Agricultural University. The species and infection rate of symbiotic bacteria in the original population of B. tabaci were determined by PCR detection, and the experimental population line was screened and established based on the original laboratory line using the “single-pair” and PCR detection method  were released into a leaf cage, which was attached onto a clean cotton leaf to allow egg laying to establish the population), and a laboratory line that was coinfected with Rickettsia and Wolbachia (IWR).The cotton plant i et al. . Healthy16S rRNA, 23S rRNA, and wsp genes of endosymbionts . A 3 μL volume of the PCR product was visualized on 1% agarose gel containing GoldView colorant  and then photographed. When bands with the expected size were visible on the gels, the remaining 17 μL of PCR product was sent for sequencing in Sangon Biotech (Shanghai) Co., Ltd. Portiera aleyrodidarum DNA was used as a positive control and ddH2O was used as a negative control to eliminate potential confounding variables and judge the quality of extracted DNA.On the basis of clarifying the type and infection rate of secondary endosymbionts, newly emerged adults were randomly selected in the original population was measured by PCR with special primers of ymbionts . Resultsnd 83.3% . So, theu et al. . Adult iRickettsia and Wolbachia in the IWR line, which included both females and males, was monitored prior to and during the experiments using polymerase chain reaction (PCR) using Rickettsia-specific 16S rRNA and Wolbachia-specific wsp primers as described previously [Rickettsia and Wolbachia was monitored for three generations. The maternal transmission efficiency of Rickettsia and Wolbachia was studied according to the methods of Nguyen [Rickettsia and Wolbachia was recorded in the arrhenotokous parthenogenetic progeny of IWR virgin females and the amphigenetic progeny of IWR virgin females and IWR virgin males. The infection status of Rickettsia and Wolbachia was tested with PCR using Rickettsia-specific 16S rRNA and Wolbachia-specific wsp primers  samples were collected. Newly emerged adults were collected and separated with dactylethrae, and their sex was examined under a microscope. DNA samples were extracted using a TIANamp Genomic DNA kit . Quantitative real-time PCR (qPCR) was used to detect the titer of Rickettsia and Wolbachia in the line of B. tabaci Asia II1 following the protocol of Ghanim and Kontsedalov [2O . The procedure was 5 min activation at 95 °C, then 45 cycles of 20 s at 95 °C, 30 s at 60 °C, and 45 s at 72 °C in a quantitative real-time PCR machine . Melting ramp included 30 s at 50 °C to 98 °C, rising 1 °C at each step, and waiting 5 s after each step. The primers of gltA and coxA genes were used as described previously [WR line quantified the titer of Rickettsia and Wolbachia as one repeat, and the experiment was repeated three times. Three technical replicates were performed for each biological replicate.To collect eggs, nymphs, and newly emerged males and females of Asia II1 for quantitative detection of endosymbionts, 500 females and males of Itsedalov . The qPCeviously . A β-acteviously . Thirty Wolbachia and Rickettsia in the eggs, nymphs, and adults of whiteflies was studied by FISH using the previously described protocol [2O2 in ethanol for 24 h in the dark and incubation in a hybridization buffer  containing 10 pmol/mL of the 6-FAM-labeled Wolbachia 16S rRNA probe and the Cyanine3-labeled Rickettsia 16S rRNA probe as described previously [Eggs, nymphs, and adults of Asia II1 were collected for fluorescence in situ hybridization visualization of endosymbionts. Localizations of protocol . Whitefleviously . After o−ΔΔCt [The Bio-Rad real-time PCR system (USA) and accompanying software (Bio-Rad CFX Manager) were used for qPCR data normalization, and the relative quantities of endosymbionts were calculated using the method of 2−ΔΔCt . StatistRickettsia and Wolbachia in the IWR line of B. tabaci Asia II1, PCR detection was confirmed by the presence of Rickettsia and Wolbachia in adult males and females of B. tabaci Asia II1  maternal transmission of Rickettsia and Wolbachia was observed in the male and female progeny of IWR line  at different time intervals. The titers of endosymbionts Rickettsia and Wolbachia showed a downward trend at 3–9 h and 12–72 h, especially at 3–9 h. The titers of endosymbionts Rickettsia and Wolbachia showed an upward trend at 9–12 h and 72–120 h. Compared with other time intervals, the increase at 9–12 h was significantly higher , whereas the titer of Rickettsia observed in nymphs were significantly higher than the titer of adult females  were infected with two secondary endosymbionts (Rickettsia and Hamiltonella), whereas the populations of B. tabaci MEAM1 from Montenegro also contained a third endosymbiont (Wolbachia). The Guangzhou population of B. tabaci MEAM1 contains Rickettsia, Hamiltonella, and Hemipteriphilus [ulations ,35. In tritschea . The speriphilus . The aboriphilus .Rickettsia and Wolbachia moved from the egg stalk to the egg, and the content of symbiotic bacteria (Rickettsia and Wolbachia) in eggs increased obviously for the first time at 12 h. The fluorescence intensity and the infection area of symbiotic bacteria in the FISH diagram did not reflect the change in Rickettsia and Wolbachia content at 12–24 h. The infected cell area of symbiotic bacteria increased after the symbiotic bacteria are transferred from the stalk position to the egg base, resulting in a decrease in the content of symbiotic bacteria per unit area, which is best proved by the FISH picture of the original symbiotic bacteria Portiera in the 1-day-old whitefly eggs and the localization of Wolbachia in the 1-day-old eggs of citrus psylla [coxA and gltA gene were presented in Rickettsia and Wolbachia can be reasonably explained from the biological point of view  in thrips were higher in male hosts than in female hosts. The localization of Rickettsia and Wolbachia examined in nymphal instars and adults of Asia II1 B. tabaci by FISH revealed the presence of Rickettsia and Wolbachia in a confined pattern limited to the bacteriocytes localized in the abdomen. These results are consistent with Gottlieb et al. [Wolbachia around and inside the bacteriocytes of nymphs and adults; however, in some individuals, the bacterium was also found in the abdomen.The titers of endosymbionts v et al. , who alsunctions . Nguyen unctions  revealedb et al. , who detWolbachia and Rickettsia within different life stages of B. tabaci. These findings will serve as a reference for the interaction between symbiotic bacteria and different tissues of host insects and the horizontal transfer of symbiotics between different hosts for their management through cytoplasmic incompatibility strategy by microinjection technique.In conclusion, these results will improve our understanding of the vertical transmission of symbiotic bacteria in insects and also provide basic information on the quantity and localization of


To screen and identify single nucleotide polymorphisms (SNPs) associated with geese meat quality traits, we performed genome-wide association study (GWAS) in a population of male Sichuan white geese  at 70 days old to screen the SNPs in the goose genome. Additionally, genotypes of the selected SNPs associated with goose meat were detected using the matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOP MS) method. The results revealed 43 SNPs that showed potentially significant associations with these traits. Additionally, we detected 28 annotated genes as potential candidate genes for the five meat quality traits. The enrichment analysis of genes within a 1Mb vicinity of SNPs showed potentially significant enrichment in the protein digestion and absorption pathway, as well as the Glycolipid metabolism pathway. The findings of this study offer novel genetic markers and candidate genes that can be used for marker-assisted selection of geese, laying the groundwork for understanding the genetic basis of goose meat quality traits and establishing a foundation for comprehending their genetic basis.(1) Background: Goose meat is highly valued for its economic significance and vast market potential due to its desirable qualities, including a rich nutritional profile, tender texture, relatively low-fat content, and high levels of beneficial unsaturated fatty acids. However, there is an urgent need to improve goose breeding by identifying molecular markers associated with meat quality. (2) Methods: We evaluated meat quality traits, such as meat color, shear force (SF), cooking loss rate (CLR), and crude fat content (CFC), in a population of 215 male Sichuan white geese at 70 days of age. A GWAS was performed to identify potential molecular markers associated with goose meat quality. Furthermore, the selected SNPs linked to meat quality traits were genotyped using the MALDI-TOP MS method. (3) Results: A dataset of 2601.19 Gb of WGS data was obtained from 215 individuals, with an average sequencing depth of 10.89×. The GWAS revealed the identification of 43 potentially significant SNP markers associated with meat quality traits in the Sichuan white goose population. Additionally, 28 genes were identified as important candidate genes for meat quality. The gene enrichment analysis indicated a substantial enrichment of genes within a 1Mb vicinity of SNPs in both the protein digestion and absorption pathway and the Glycerolipid metabolism pathway. (4) Conclusion: This study provides valuable insights into the genetic and molecular mechanisms underlying goose meat quality traits, offering crucial references for molecular breeding in this field. Goose meat is a nutrient-rich meat that is considered a healthy food due to its high protein, low fat, and low cholesterol content, which is regarded as one of the ideal meat products and an important source of meat-based foods by the World Health Organization . In 2021GJA1 may serve as a functional gene in the development of breast muscle in chickens [SHH, LMBR1, IGF1R, and SLC16A, which might be involved in controlling abdominal fat content. In the case of Pekin ducks, GWAS of body size and carcass traits revealed a specific mutation site located within the anti-lipogenesis gene NR2F2 in high sebaceous Pekin ducks [Genetic factors play a crucial role in determining meat quality traits alongside external factors. Genome-wide association studies (GWAS) have been utilized to investigate the relationship between phenotype and genotype in populations, yielding significant findings in the identification of genetic variations associated with meat quality traits in livestock and poultry ,16,17,18chickens . Anotherin ducks ,23.The utilization of whole-genome resequencing successfully detected single nucleotide polymorphisms (SNPs) associated with meat quality traits in the genome of Sichuan geese. Subsequent to that, a GWAS was conducted to precisely identify specific genomic regions and candidate genes associated with these traits. Moreover, we examined the frequency of SNPs associated with meat quality traits. The comprehensive findings of this study lay a solid foundation for future investigations into goose meat traits and make significant contributions to the advancement of genetic selection efforts in this field.The male Sichuan white geese, selected as experimental animals from a shared incubation batch, were reared at the AnFu Waterfowl Breeding Base in Chongqing City, China . The geese were subjected to standardized conditions of diet, temperature, lighting, and unrestricted access to water during their rearing period. When the geese reached 70 days of age, a total of 215 healthy individuals were randomly chosen as experimental subjects. Blood samples were collected from the geese’s wings using vacuum tubes containing the anticoagulant EDTA, and these samples were subsequently stored at −20 °C to prepare them for future experiments. To measure the three meat color parameters  in three fixed points of 215 goose right pectoral muscle samples after 2 h of slaughter, an automatic colorimeter and a portable pH meter were used after calibration. The muscle samples were standardized for thickness and weight (M0) and then bathed in an 80 °C-thermostat water bath . Once the center temperature of the samples reached 70 °C, they were incubated for a duration of 30 min. Subsequently, the samples were removed from the heat source and allowed to cool down to room temperature. The sample was then weighed (M1), and the cooking loss rate (CLR) was calculated as CLR = (M0 − M1)/M0 × 100%.R) was dried at 105 °C for 2 h. The crude fat content (CFC) was then calculated as CFC = (1 − MR) × Mp/100 × 100%.The cooled breast muscle was fixed perpendicular to the muscle fiber into long strips with a 1 cm × 1 cm cross-sectional area, and the strips were sheared with a tenderness meter (CLM-3), repeated 5 times to obtain the average shearing force (SF) of these samples. Fresh meat samples of 100g were freeze dried into powder, and the powder was weighed (Mp). The moisture content of freeze dried (MCFD) was calculated as MCFD = (100 − Mp)/100 × 100%. The fat was extracted by petroleum benzene from 1g of the freeze-dried powder, and the remaining material  was performed using the Illumina HiSeq X Ten platform. Following the generation of WGS data, we performed quality control procedures using the NGS QC Toolkit software , which iP = 0.05/N or 1/N. Here, P represents the Bonferroni-corrected p-value for significant or potential associations across the entire genome, while N denotes the number of independent SNPs obtained from the population structure analysis. In this particular study, the determined thresholds for significant and potentially significant associations were 4.94 × 10−9  and 1.0 × 10−7 , respectively. The Manhattan plots and quantile-quantile (Q-Q) plots were created using the “gap” package  [https://cran.r-project.org/web/packages/qqman/) [We utilized the GEMMA software  to condues/gap/)  and “qqm/qqman/)  in the R/qqman/) .To determine the genotypes of the SNPs significantly associated with meat quality traits in the Sichuan white goose population using GWAS, we designed amplification and extension primers  to amplihttps://metascape.org/, accessed on 12 December 2019).In this study, the BEDTools software  was usedA fast and efficient fattening method is commonly used for raising meat geese. Generally, geese can be marketed after 70 days, and the meat quality of 70-day-old geese holds significant economic value in production practice. The statistical data of meat quality traits for the breast muscles of 70-day-old male Sichuan white geese are presented in We obtained 2608.44 Gb of WGS data from 215 male Sichuan white geese, with an average depth coverage of 10.89×. After filtering, we obtained 2601.19 Gb of high-quality sequencing data, with an average mapping rate of 97.88% (ranging from 97.72% to 98.10%) when aligned to the reference genome sequence of geese. The average Q20 and Q30 values were 96.77% and 91.90%, respectively. The average GC content was calculated to be 43.63%. The subsequent analysis focused on 10,072,006 SNPs identified in the goose genome (version ASM1303099v1) .The PCA analysis indicated that there was no significant population stratification . Hence, We detected the 11 SNPs genotypes and compared the meat quality traits of different genotypes identified by GWAS , includip value = 1.80 × 10−3), human papillomavirus infection , ECM-receptor interaction , protein digestion and absorption , and Glycerolipid metabolism   .The Sichuan white goose is a dual-purpose breed of Chinese poultry known for producing both meat and eggs. It is a valuable genetic resource and contributes significantly to the poultry industry, providing substantial economic and nutritional benefits in China. The quality of meat in the poultry industry is a crucial factor that affects consumer acceptance and, ultimately, the economic success of the industry. Poultry breeders use selective breeding and genetic improvement programs to enhance meat quality traits, aiming to produce birds that meet consumer preferences and demand. In this study, we investigated the meat quality traits of 70-day-old male Sichuan white geese. Using a GWAS, we screened and identified a total of 43 SNPs associated with CFC, CLR, meat color, and SF traits in a population of 215 Chinese local geese (Sichuan white goose).The sample size is a critical factor in research design that can affect the validity and reliability of research findings . A largeTMEM65) and mothers against decapentaplegic homolog 6 (SMAD6) genes. TMEM65 is a mitochondrial inner-membrane protein that plays a crucial role in oxidative stress response, oxygen consumption, and citrate synthase activity [TMEM65 may be involved in fat oxidation metabolism, indirectly affecting CFC levels. Fatty acid metabolism has a significant impact on the flavor and production of intramuscular fat, which, in turn, affects meat tenderness, water retention, and flavor presentation. Therefore, the identification of genetic markers associated with CFC, such as the identified SNP within the TMEM65 in this study, can have important implications for the selection and breeding of animals with desirable meat quality traits.The crude fat content (CFC) of meat is a critical indicator of its quality, and it has been shown to be associated with several other quality traits. In this study, we investigated the genetic factors underlying CFC in geese and identified two potentially significant SNPs located in the transmembrane protein 65 , one of the identified candidate genes, plays a crucial role in several cellular processes. Its main function is the production of glutathione, which serves to protect cells from oxidative damage and aids in amino acid transport. Additionally, GSS is involved in the detoxification of foreign compounds. The presence of GSS as a candidate gene suggests its significance in safeguarding cells and maintaining cellular homeostasis. Further exploration of GSS’s role in these processes would provide valuable insights into its contribution to cellular health and the potential impact on various biological functions [RRAGC) and solute carrier organic anion transporter family member 5A1 (SLCO5A1) play crucial roles in the cellular response to amino acid availability. RRAGC, along with three other Rag proteins, plays a crucial role in the activation of the mTORC1 pathway, which is sensitive to amino acid levels [GSS, RRAGC, and SLCO5A1 may be responsible for the color presentation of the carcass by oxidative metabolism.Meat color  reflects changes in muscle physiology, biochemistry, and microbiology and is an important index for evaluating meat appearance. Meat color can impact the functional characteristics and quality of further processed meat products. Our study  identifiunctions . Ras-reld levels ,42, whicd levels ,44,45. Ad levels . Then, tBMF), Nuclear envelope integral membrane protein 2 (NEMP2), and mitochondrial NAD-dependent malic enzyme (NAD-ME) that were potentially significantly associated with SF in goose. The correlations were further validated by the results of MALDI-TOF MS. In this study, we confirmed significant associations between the genotypes of SNP (chr2:36812553) in BMF and two SNPs  in NAD-ME with the shear force (SF) for geese meat. BMF, a member of the Bcl2 protein family, is a sensor for intracellular damage that can trigger cell apoptosis and potentially mediate muscle fiber atrophy [NAD-ME participates in various metabolic pathways and provides reducing power NADPH for the synthesis of various cellular components [BMF may directly impact muscle fiber structure, while NAD-ME may affect oxidoreductase activity and intramuscular fat generation in muscle, ultimately affecting SF.The shearing force (SF) is a critical parameter for assessing meat tenderness, which is influenced by various factors such as muscle fiber characteristics , connective tissue composition , myogenic fibrin hydrolase properties, and intramuscular fat content. In this study, we identified seven SNPs located in Bcl-2-modifying factor  as the reference, which resulted in improved genome assembly and annotation compared to previous studies . FurtherMetabolic pathways have comprehensive effects on meat quality traits , involviIn this study, we performed GWAS with goose meat quality traits and identified 43 SNPs that demonstrated potentially significant associations with these traits. Additionally, we used the MALDI-TOP MS method to genotype the selected SNPs associated with the traits in the same population. Our findings revealed 28 annotated genes that show promise as potential candidate genes linked to the five investigated meat quality traits. These genes represent novel genetic markers and can be implemented in marker-assisted selection programs for geese. Importantly, our study contributes to a comprehensive understanding of the genetic mechanisms underlying meat quality traits in geese, establishing a solid foundation for future research in this field.