Summary: The mechanisms that regulate the vast diversity of plant organ shapes such as the fruit remain to be fully elucidated. TONNEAU1 Recruiting Motif proteins (TRMs) have been implicated in the control of organ shapes in a number of plant species, including tomato. However, the role of many of them is unknown. TRMs interact with Ovate Family Proteins (OFPs) via the M8 domain. However, the in planta function of the TRM‐OFP interaction in regulating shape is unknown. We used CRISPR/Cas9 to generate knockout mutants in TRM proteins from different subclades and in‐frame mutants within the M8 domain to investigate their roles in organ shape and interactions with OFPs. Our findings indicate that TRMs impact organ shape along both the mediolateral and proximo‐distal axes of growth. Mutations in Sltrm3/4 and Sltrm5 act additively to rescue the elongated fruit phenotype of ovate/Slofp20 (o/s) to a round shape. Contrary, mutations in Sltrm19 and Sltrm17/20a result in fruit elongation and further enhance the obovoid phenotype in the o/s mutant. This study supports a combinatorial role of the TRM‐OFP regulon where OFPs and TRMs expressed throughout development have both redundant and opposing roles in regulating organ shape.

Key Message: Six novel fruit weight QTLs were identified in tomato using multiple bi-parental populations developed from ancestral accessions. Beneficial alleles at these loci arose in semi-domesticated subpopulations and were likely left behind. This study paves the way to introgress these alleles into breeding programs. The size and weight of edible organs have been strongly selected during crop domestication. Concurrently, human have also focused on nutritional and cultural characteristics of fruits and vegetables, at times countering selective pressures on beneficial size and weight alleles. Therefore, it is likely that novel improvement alleles for organ weight still segregate in ancestral germplasm. To date, five domestication and diversification genes affecting tomato fruit weight have been identified, yet the genetic basis for increases in weight has not been fully accounted for. We found that fruit weight increased gradually during domestication and diversification, and semi-domesticated subpopulations featured high phenotypic and nucleotide diversity. Columella and septum fruit tissues were proportionally increased, suggesting targeted selection. We developed twenty-one F₂ populations with parents fixed for the known fruit weight genes, corresponding to putative key transitions from wild to fully domesticated tomatoes. These parents also showed differences in fruit weight attributes as well as the developmental timing of size increase. A subset of populations was targeted for QTL-seq, leading to the identification of six uncloned fruit weight QTLs. Three QTLs, located on chromosomes 1, 2 and 3, were subsequently validated by progeny testing. By exploring the segregation of the known fruit weight genes and the identified QTLs, we estimated that most beneficial alleles in the newly identified loci arose in semi-domesticated subpopulations from South America and were not likely transmitted to fully domesticated landraces. Therefore, these alleles could be incorporated into breeding programs using the germplasm and genetic resources identified in this study.

Methyl salicylate imparts a potent flavor and aroma described as medicinal and wintergreen that is undesirable in tomato (Solanum lycopersicum) fruit. Plants control the quantities of methyl salicylate through a variety of biosynthetic pathways, including the methylation of salicylic acid to form methyl salicylate and subsequent glycosylation to prevent methyl salicylate emission. Here, we identified a subclade of tomato methyl esterases, SALICYLIC ACID METHYL ESTERASE1-4, responsible for demethylation of methyl salicylate to form salicylic acid in fruits. This family was identified by proximity to a highly significant methyl salicylate genome-wide association study locus on chromosome 2. Genetic mapping studies in a biparental population confirmed a major methyl salicylate locus on chromosome 2. Fruits from SlMES1 knockout lines emitted significantly (P < 0,05, t test) higher amounts of methyl salicylate than wild-type fruits. Double and triple mutants of SlMES2, SlMES3, and SlMES4 emitted even more methyl salicylate than SlMES1 single knockouts—but not at statistically distinguishable levels—compared to the single mutant. Heterologously expressed SlMES1 and SlMES3 acted on methyl salicylate in vitro, with SlMES1 having a higher affinity for methyl salicylate than SlMES3. The SlMES locus has undergone major rearrangement, as demonstrated by genome structure analysis in the parents of the biparental population. Analysis of accessions that produce high or low levels of methyl salicylate showed that SlMES1 and SlMES3 genes expressed the highest in the low methyl salicylate lines. None of the MES genes were appreciably expressed in the high methyl salicylate-producing lines. We concluded that the SlMES gene family encodes tomato methyl esterases that convert methyl salicylate to salicylic acid in ripe tomato fruit. Their ability to decrease methyl salicylate levels by conversion to salicylic acid is an attractive breeding target to lower the level of a negative contributor to flavor.

Fruit quality traits such as fruit weight, firmness, total soluble solids content, and color strongly influence consumer acceptance, market value, postharv

Developing drought-resistant alfalfa (Medicago sativa L.) that maintains high biomass yield is a key breeding goal to enhance productivity in water-limited areas. In this study, 424 alfalfa breeding families were analyzed to identify molecular markers associated with biomass yield under drought stress and to predict high-merit plants. Biomass yield was measured from 18 harvests from 2020 to 2023 in a field trial with deficit irrigation. A total of 131 significant markers were associated with biomass yield, with 80 markers specifically linked to yield under drought stress; among these, 19 markers were associated with multiple harvests. Finally, genomic best linear unbiased prediction (GBLUP) was employed to obtain predictive accuracies (PAs) and genomic estimated breeding values (GEBVs). Removing low-informative SNPs [SNPs with p-values > 0.05 from the additive Genome-Wide Association (GWAS) model] for GBLUP increased PA by 47.3%. The high number of markers associated with yield under drought stress and the highest PA (0.9) represent a significant achievement in improving yield under drought stress in alfalfa.

Small public breeding programmes have many barriers to adopting technology, particularly creating and using genetic marker panels for genomic-based decisions in selection. Here we report the creation of a DArTag panel of 3,000 loci distributed across the tetraploid genome of blueberry (Vaccinium corymbosum) for use in molecular breeding and genomic prediction. The creation of this marker panel brings cost-effective and rapid genotyping capabilities to public and private breeding programmes. The open access provided by this platform will allow genetic data sets generated on the marker panel to be compared and joined across projects, institutions and countries. This genotyping resource has the power to make routine genotyping a reality for any breeder of blueberry.