Together, the genetic and genome–editing data support SlVQ10 as the gene underlying ssm and indicate that it promotes seed size in tomato.
Abstract
Seed size affects seed vigor, seedling establishment, and seed utilization in tomato. Yet the genetic basis of this trait remains poorly defined in tomato itself. Here, we describe a stable small–seed mutant, T31, isolated from an ethyl methanesulfonate (EMS)–mutagenized population of the elite inbred line DL5. Relative to the wild type, T31 showed a 24% reduction in seed width, whereas vegetative growth and major fruit traits were largely unchanged. Throughout this study, ‘seed size’ refers to seed width, which was used as the principal index of seed size because tomato seeds are oblate. Genetic analysis of six populations (P1, P2, F1, F2, BC1, and BC2) indicated that the phenotype is controlled by a single recessive locus, designated ssm. Bulked segregant analysis sequencing (BSA–seq) placed ssm within a 1.77 –Mb interval on chromosome 4. KASP–based fine mapping reduced this interval to 390 kb and identified six EMS–type SNPs. Only one of these SNPs was located in an exon of Solyc04g073950.2, where it caused a Pro337Ser substitution. This gene encodes a VQ motif–containing protein and was designated SlVQ10. To test gene function, we generated CRISPR/Cas9 knockout lines in the DL5 background. Two independent homozygous knockout lines reproduced the small–seed phenotype. Seed size was reduced by 31–33%, and thousand–seed weight decreased by 33–35%. Histological analysis further showed reduced seed–coat cell expansion in the mutants. Together, the genetic and genome–editing data support SlVQ10 as the gene underlying ssm and indicate that it promotes seed size in tomato.
A multiplex CRISPR/Cas12a system is established in eggplant accession MEL3, representing, to the authors' knowledge, the first application of this nuclease for genome editing in eggplant and demonstrating the potential of Cas12a for functional genomics, allele engineering, and precision breeding in eggplant.
Marina Martínez-López, Andrea Solana, Andrea Arrones et al.· bioRxiv· 0 citations
Oilseed rape (Brassica napus L.) is a major oil crop, and both silique and seed size are critical determinants of yield. In Arabidopsis thaliana, mutation of the ABORTED GAMETOPHYTE 1 (AOG1) gene leads to severe defects in gametophyte development and a pronounced reduction in silique length. However, the function of AOG1 homologs in rapeseed remains uncharacterized. In this study, four homologous copies of AOG1 were identified in B. napus: BnaAOG1.A03, BnaAOG1.A10, BnaAOG1.C03, and BnaAOG1.C09. Then a systematic analysis that considered the physiochemical properties, evolution, conserved motifs, gene structure, and cis-regulatory elements of BnaAOG1s family members was conducted. Utilizing the CRISPR/Cas9 system, two of these copies, BnaAOG1.A03 and BnaAOG1.C03, were targeted, and homozygous double mutants were generated. Unlike the Arabidopsis ortholog, the BnaAOG1s were specifically expressed during seed development. Phenotypic evaluation revealed that the double mutants did not exhibit significant changes in silique length, seed number per silique, or thousand-seed weight compared to the wild type. These results indicate that BnaAOG1.A03 and BnaAOG1.C03 are not individually essential for silique and seed development in B. napus. Their functions may be compensated by other homologous copies or have undergone divergence during polyploidization. This investigation provides a valuable case for functional analysis of homologous genes in polyploid crops.
Jiaxu Xiao, Xiaonan Guo, Aoli Liao et al.· Frontiers in Plant Science· 0 citations
Abstract Fruit weight is a key determinant of yield in high-value vegetable crops such as tomato. Despite extensive research, the molecular mechanisms underlying this complex trait remain largely elusive, with only a few genes cloned to date based on quantitative trait loci (QTL). Here, we analyzed 2 populations and reanalyzed 3 previously published populations and identified 945 QTL associated with agro-morphological traits, including both previously reported and unidentified loci. We focused on SlGRF10 (GROWTH-REGULATING FACTOR 10) underlying a fruit weight QTL, fw1.2. Loss of SlGRF10 reduced fruit weight by decreasing cell size, without affecting cell number. Analysis of natural variation in SlGRF10 in over 1,000 tomato accessions revealed that increased single-nucleotide polymorphism diversity in SlGRF10 is associated with lower fruit weight. This suggests that putative impaired activity contributes to reduced fruit weight, while breeding-induced reduction of genetic variation may have promoted increased fruit weight. Transcriptome profiling of SlGRF10 knockout lines 7 and 20 days postanthesis identified several differentially expressed genes involved in cell cycle progression. Our findings not only confirm the role of SlGRF10 in regulating tomato fruit weight but also highlight a set of candidate genes associated with key morpho-physiological traits. With fw11.3, named as CELL SIZE REGULATOR, being the only QTL related to cell size determination in tomato fruits so far, SlGRF10 offers a valuable target for precision breeding and enhances our understanding of fruit weight in tomato and related fruit-bearing species.
Julia von Steimker, M. Macho, Regina Wendenburg et al.· Plant Physiology· 0 citations
CRISPR-Cas9 has emerged as a powerful tool for targeted genome editing in plants; however, its application in tetraploid potato (Solanum tuberosum ssp. tuberosum) remains challenging due to its vegetative propagation and complex highly heterozygous genome. Availability of whole-genome sequence data for the specific genotype is crucial to ensure complete knockout of all alleles of target genes while minimizing off-target mutations. In this study, using the tetraploid potato cultivar Désirée, we report, a complete CRISPR-Cas9-mediated knockout of the BEL5 gene, encoding a transcription factor, known as one of the key regulators driving tuber formation. We employed Agrobacterium-mediated transformation and demonstrated that repeated de novo regeneration could improve editing efficiency by promoting emergence of new mutations. BEL5 knockout plants exhibited a delayed onset of tuberization under inductive short-day conditions in hydroponics; however, their overall tuber yields were comparable to wild type plants. Based on our results, we propose a regulatory role of BEL5 in the timing of tuber onset but, unexpectedly, its dispensability for tuber development in modern cultivated potato. Besides providing functional insight into the BEL5 role in potato, this study includes a methodological approach for efficient CRISPR-Cas9 gene editing in this vegetatively propagated polyploid crop, along with strategies for detecting mutations in genes that lack clear phenotypic manifestation.
Andrea Zounková, Daniele Chirivì, A. Přibylová et al.· bioRxiv· 0 citations
Plant height is a key agronomic trait closely related to yield performance in many crops. Moderately reducing plant height can enhance lodging resistance and improve the harvest index. We obtained a dwarf mutant
Bndwarf3
in
Brassica napus
by ethyl methanesulfonate (EMS) mutagenesis of our germplasm NJ7982, which displays a reduced height and compact architecture. Genetic analysis using F
1
, F
2
and F
2:3
populations derived from a cross between
Bndwarf3
and ZS11 (Zhongshuang11) revealed that the dwarfism is controlled by a single semi‐dominant nuclear locus designated
BnDF3
. Through Bulked Segregant Analysis (BSA) combined with high‐throughput resequencing, we mapped
BnDF3
to a 3.92‐Mb interval on chromosome C04. Further mapping with insertion/deletion (InDel) markers and simple sequence repeat (SSR) markers narrowed the mapping interval to 403 kb containing 43 predicted genes. Among these genes,
BnaC04G0506100ZS
encoding a glycogen‐synthase‐kinase‐3 (GSK3)‐like kinase can be regarded as candidate gene responsible for dwarfing plant type due to its Thr‐to‐Ile substitution (Thr‐291‐Ile) within the conserved TREE motif of the STKc_GSK3 domain. A CAPS marker designed based on this mutation co‐segregated perfectly with the dwarf phenotype in a large segregating population. Overexpression of the mutant allele
BnDF3
in ZS11 leads to dwarf and compact transgenic plants, whereas overexpression of the wild‐type allele (
Bndf3
) did not alter plant type.
BnDF3
overexpression reduced plant sensitivity to exogenous brassinolide (BL) and partially restored plant growth under GSK3 inhibitor (LiCl) treatments. These results demonstrated that
BnDF3
is a gain‐of‐function allele functioning as the gain‐of‐function BIN2 gene in brassinosteroid (BR) signalling pathway. Our work may provide both a functional marker and a novel genetic resource useful in variety breeding targeted to strong lodging resistance.
Mao Yang, Yifei Guo, Jiayang Guo et al.· Plant Breeding· 0 citations
Brassica napus yellow-seeded genotypes have higher seed oil content and quality than black-seeded types, but the mechanisms controlling seed coat color (SCC) remain unclear, partly due to limited gap-free references. We generated the telomere-to-telomere genome assembly of the black-seeded cultivar Zhongyou 821 using PacBio HiFi, ultralong ONT, and Hi-C sequencing. Using this reference, genome-wide association study (GWAS) of 504 accessions identified BnaWRKY44 as a major SCC candidate gene. CRISPR-Cas9 knockout of BnaWRKY44 lightened SCC, increased oil content and unsaturated fatty acids, and improved oil quality. Haplotype analysis defined the elite BnaWRKY44Hap4 allele and enabled development of a diagnostic CAPS marker for breeding. Transcriptomic, Y1H and dual-LUC assays showed that BnaWRKY44 directly binds and regulates the BnaVPT1 promoter. BnaVPT1 knockout confirmed its roles in SCC lightening and flavonoid biosynthesis. This work provides a T2T rapeseed resource and a BnaWRKY44-BnaVPT1 module for breeding high-oil, yellow-seeded rapeseed.
Haijiang Liu, Yongheng Yuan, Kaijie Ye et al.· Cell Reports· 0 citations