Carpel number (CN) is an important trait affecting the fruit size and shape of melon, which plays a crucial role in determining the overall appearance and market value. A unique non-synonymous single nucleotide polymorphism (SNP) in CmCLAVATA3 (CmCLV3) is responsible for the variation of CN in C. melo ssp. agrestis (hereafter agrestis), but it has been unclear in C. melo ssp. melo (hereafter melo). In this study, one major locus controlling the polymorphism of 5-CN (multi-CN) and 3-CN (normal-CN) in melo was identified using bulked segregant analysis (BSA-seq). This locus was then fine-mapped to an interval of 1.8 Mb on chromosome 12 using a segregating population containing 1451 progeny. CmCLV3 is still present in the candidate region. A new allele of CmCLV3, which contains five other nucleotide polymorphisms, including a non-synonymous SNP in coding sequence (CDS), except the SNP reported in agrestis, was identified in melo. A cis-trans test confirmed that the candidate gene, CmCLV3, contributes to the variation of CNs in melo. The qRT-PCR results indicate that there is no significant difference in the expression level of CmCLV3 in the apical stem between the multi-CN plants and the normal-CN plants. Overall, this study provides a genetic resource for melon fruit development research and molecular breeding. Additionally, it suggests that melo has undergone similar genetic selection but evolved into an independent allele.

The genus Allium belongs to the botanical family Amaryllidaceae and includes economically important crops such as onion, garlic, bunching onion, and leek, used as vegetables, spices, and traditional medicines. The large sizes of Allium genomes hamper the genetic dissection of agronomically important traits and molecular breeding. With the growing accumulation of genomic, resequencing, transcriptome, and phenotypic data, the demand for an integrative Allium database is increasing. Here we present a user-friendly database, AlliumDB (https://allium.qau.edu.cn), as a functional genomics hub integrating public and in-house data. The database contains all currently available nuclear and organelle genomes for Allium species, with genes comprehensively annotated based on Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses, orthology, gene families, protein families (Pfam), and non-coding RNA families (Rfam). Transcriptome and variation profiles are integrated into dynamic visualization tools. We took phenotypic photographs and generated trait records for hundreds of Allium germplasms collected worldwide, which are included in the database. We incorporated JBrowse for the visualization of gene structures, RNA sequencing data, and variation data. Analysis tools such as the basic local alignment search tool (BLAST), sequence fetch, enrichment, and motif analyses are available to explore potential gene functions. This database incorporates comprehensive Allium genotypic and phenotypic datasets. As the community assembles new genomes and generates resequencing data for Allium germplasms, the database will be improved and continuously updated with these multi-omics data and comparative genomic studies. We expect the AlliumDB database to become a key resource for the study of Allium crops.

KEY MESSAGE:The mutated LsTT2 and Ls2OGD genes are responsible for white seeds and yellow seeds in lettuce, respectively. Three LsCHS genes are involved in the biosynthesis of flavonoid in seed coats. Lettuce seeds have several different colors, including black, yellow, and white. The genetic mechanisms underlying color variations of lettuce seeds remain unknown. We used genome-wide association studies (GWAS) and map-based cloning approaches to clone genes controlling the color of lettuce seeds. LsTT2, which encodes an R2R3-MYB transcription factor and is homologous to the TT2 gene in Arabidopsis, was shown to be the causal gene for the variation of black and white seeds in lettuce. A point mutation leads to the lack of stop codon in the LsTT2 transcript, resulting in white seeds. Knockout of the LsTT2 gene converted black seeds to white seeds. The locus controlling yellow seeds was mapped to Chromosome 2. Knockout of two 2-oxoglutarate-dependent dioxygenases (2OGD) genes from the candidate region converted black seeds to yellow seeds, suggesting that these two 2OGD proteins catalyze the conversion of yellow metabolites to black metabolites. We also showed that three LsCHS genes from the candidate region are associated with flavonoid biosynthesis in seeds. Knockout mutants of the three LsCHS genes decreased color intensity. This study provides new insights into the regulation of flavonoid biosynthesis in plants.

Lettuce (Lactuca sativa) is one of the most popular vegetables worldwide, and diverse leaf shapes, including wavy leaves, are important commercial traits. In this study, we examined the genetics of wavy leaves using an F2 segregating population, and identified a major QTL controlling wavy leaves. The candidate region contained LsKN1, which has previously been shown to be indispensable for leafy heads in lettuce. Complementation tests and knockout experiments verified the function of LsKN1 in producing wavy leaves. The LsKN1∇ allele, which has the insertion of a transposon and has previously been shown to control leafy heads, promoted wavy leaves in our population. Transposition of the CACTA transposon from LsKN1 compromised its function for wavy leaves. High expression of LsKN1 up-regulated several key genes associated with cytokinin (CK) to increase the content in the leaves, whereas it down-regulated the expression of genes in the gibberellin (GA) biosynthesis pathway to decrease the content. Application of CK to leaves enhanced the wavy phenotype, while application of GA dramatically flattened the leaves. We conclude that the changes in CK and GA contents that result from high expression of LsKN1 switch determinate cells to indeterminate, and consequently leads to the development of wavy leaves.

The mechanisms underlying leafy heads in vegetables are poorly understood. Here, we cloned a quantitative trait locus (QTL) controlling leafy heads in lettuce (Lactuca sativa). The QTL encodes a transcription factor, SAWTOOTH 1 (LsSAW1), which has a BEL1-like homeodomain and is a homolog of Arabidopsis thaliana. A 1-bp deletion in Lssaw1 contributes to the development of leafy heads. Laser-capture microdissection and RNA-sequencing showed that LsSAW1 regulates leaf dorsiventrality and loss-of-function of Lssaw1 downregulates the expression of many adaxial genes but upregulates abaxial genes. LsSAW1 binds to the promoter region of the adaxial gene ASYMMETRIC LEAVES 1 (LsAS1) to upregulate its expression. Overexpression of LsAS1 compromised the effects of Lssaw1 on heading. LsSAW1 also binds to the promoter region of the abaxial gene YABBY 1 (LsYAB1), but downregulates its expression. Overexpression of LsYAB1 led to bending leaves in LsSAW1 genotypes. LsSAW1 directly interacts with KNOTTED 1 (LsKN1), which is necessary for leafy heads in lettuce. RNA-seq data showed that LsSAW1 and LsKN1 exert antagonistic effects on the expression of thousands of genes. LsSAW1 compromises the ability of LsKN1 to repress LsAS1. Our results suggest that downregulation or loss-of-function of adaxial genes and upregulation of abaxial genes allow for the development of leafy heads.

SUMMARYLettuce (Lactuca sativa) is one of the most important vegetable crops in the world. As a leafy vegetable, the polymorphism of lettuce leaves from dark to pale green is an important trait. However, the genetic and molecular mechanisms underlying such variations remain poorly understood. In this study, one major locus controlling the polymorphism of dark‐ and pale‐green leaves in lettuce was identified using genome‐wide association studies (GWAS). This locus was then fine mapped to an interval of 5375 bp on chromosome 4 using a segregating population containing 2480 progeny. Only one gene, homologous to the GLK genes in Arabidopsis and other plants, is present in the candidate region. A complementation test confirmed that the candidate gene, LsGLK, contributes to the variation of dark‐ and pale‐green leaves. Sequence analysis showed that a CACTA transposon of 7434 bp was inserted 10 bp downstream of the stop codon of LsGLK, followed by a duplication of a 1826‐bp fragment covering exons 3–6 of the LsGLK gene. The transposon insertion did not change the expression level of the LsGLK gene. However, because of alternative splicing, only 6% of the transcripts produced from the transposon insertion were wild‐type transcripts, which led to the production of pale‐green leaves. An evolutionary analysis revealed that the insertion of the CACTA transposon occurred in cultivated lettuce and might have been selected in particular cultivars to satisfy the diverse demands of consumers. In this study, we demonstrated that a transposon insertion near a gene may affect its splicing and consequently generate phenotypic variations.

We identified the loss of BoFLC gene as the cause of non-vernalization requirement in B. oleracea. Our developed codominant marker of BoFLC gene can be used for breeding program of B. oleracea crops. Many species of the Brassicaceae family, including some Brassica crops, require vernalization to avoid pre-winter flowering. Vernalization is an unfavorable trait for Chinese kale (Brassica oleracea var. chinensis Lei), a stem vegetable, and therefore it has been lost during its domestication/breeding process. To reveal the genetics of vernalization variation, we constructed an F2 population through crossing a Chinese kale (a non-vernalization crop) with a kale (a vernalization crop). Using bulked segregant analysis (BSA) and RNA-seq, we identified one major quantitative trait locus (QTL) controlling vernalization and fine-mapped it to a region spanning 80 kb. Synteny analysis and PCR-based sequencing results revealed that compared to that of the kale parent, the candidate region of the Chinese kale parent lost a 9,325-bp fragment containing FLC homolog (BoFLC). In addition to the BoFLC gene, there are four other FLC homologs in the genome of B. oleracea, including Bo3g005470, Bo3g024250, Bo9g173370, and Bo9g173400. The qPCR analysis showed that the BoFLC had the highest expression among the five members of the FLC family. Considering the low expression levels of the four paralogs of BoFLC, we speculate that its paralogs cannot compensate the function of the lost BoFLC, therefore the presence/absence (PA) polymorphism of BoFLC determines the vernalization variation. Based on the PA polymorphism of BoFLC, we designed a codominant marker for the vernalization trait, which can be used for breeding programs of B. oleracea crops.

Significance Heading, which facilitates harvest and contributes to taste and shelf life, is an important trait in some vegetable crops. However, litter is known about its genetic and molecular mechanism. Here, we report the genetic cloning of a key regulator ( LsKN1 ) of heading in lettuce, which is homologous to the knotted 1 ( KN1 ) gene in maize. The LsKN1 gene is upregulated by the insertion of a CACTA-like transposon. Its enhanced expression suppressed the LsAS1 gene and consequently altered leaf polarity and led to a heading phenotype.

Different horticultural types of lettuce exhibit tremendous morphological variation. However, the molecular basis for domestication and divergence among the different horticultural types of lettuce remains unknown. Here, we report the RNA sequencing of 240 lettuce accessions sampled from the major horticultural types and wild relatives, generating 1.1 million single-nucleotide polymorphisms (SNPs). Demographic modeling indicates that there was a single domestication event for lettuce. We identify a list of regions as putative selective sweeps that occurred during domestication and divergence, respectively. Genome-wide association studies (GWAS) identify 5311 expression quantitative trait loci (eQTL) regulating the expression of 4105 genes, including nine eQTLs regulating genes associated with flavonoid biosynthesis. GWAS for leaf color detects six candidate loci responsible for the variation of anthocyanins in lettuce leaves. Our study provides a comprehensive understanding of the domestication and the accumulation of anthocyanins in lettuce, and will facilitate the breeding of cultivars with improved nutritional value.