Clpf encodes pentatricopeptide repeat protein (PPR5) and regulates pink flesh color in watermelon (Citrullus lanatus L.).

KEY MESSAGE: The gene controlling pink flesh in watermelon was finely mapped to a 55.26-kb region on chromosome 6. The prime candidate gene, Cla97C06G122120 (ClPPR5), was identified through forward genetics. Carotenoids offer numerous health benefits; while, they cannot be synthesized by the human body. Watermelon stands out as one of the richest sources of carotenoids. In this study, genetic generations derived from parental lines W15-059 (red flesh) and JQ13-3 (pink flesh) revealed the presence of the recessive gene Clpf responsible for the pink flesh (pf) trait in watermelon. Comparative analysis of pigment components and microstructure indicated that the disparity in flesh color between the parental lines primarily stemmed from variations in lycopene content, as well as differences in chromoplast number and size. Subsequent bulk segregant analysis (BSA-seq) and genetic mapping successfully narrowed down the Clpf locus to a 55.26-kb region on chromosome 6, harboring two candidate genes. Through sequence comparison and gene expression analysis, Cla97C06G122120 (annotated as a pentatricopeptide repeat, PPR) was predicted as the prime candidate gene related to pink flesh trait. To further investigate the role of the PPR gene, its homologous gene in tomato was silenced using a virus-induced system. The resulting silenced fruit lines displayed diminished carotenoid accumulation compared with the wild-type, indicating the potential regulatory function of the PPR gene in pigment accumulation. This study significantly contributes to our understanding of the forward genetics underlying watermelon flesh traits, particularly in relation to carotenoid accumulation. The findings lay essential groundwork for elucidating mechanisms governing pigment synthesis and deposition in watermelon flesh, thereby providing valuable insights for future breeding strategies aimed at enhancing fruit quality and nutritional value.

Nucleotide variation in the phytoene synthase (ClPsy1) gene contributes to golden flesh in watermelon (Citrullus lanatus L.).

KEY MESSAGE: A gene controlling golden flesh trait in watermelon was discovered and fine mapped to a 39.08 Kb region on chromosome 1 through a forward genetic strategy, and Cla97C01G008760 (annotated as phytoene synthase protein, ClPsy1 ) was recognized as the most likely candidate gene. Vitamin A deficiency is a worldwide public nutrition problem, and ß-carotene is the precursor for vitamin A synthesis. Watermelon with golden flesh (gf, which occurs due to an accumulated abundance of ß-carotene) is an important germplasm resource. In this study, a genetic analysis of segregated gf gene populations indicated that gf was controlled by a single recessive gene. BSA-seq (Bulked segregation analysis) and an initial linkage analysis placed the gf locus in a 290-Kb region on watermelon chromosome 1. Further fine mapping in a large population including over 1000 F2 plants narrowed this region to 39.08 Kb harboring two genes, Cla97C01G008760 and Cla97C01G008770, which encode phytoene synthase (ClPsy1) and GATA zinc finger domain-containing protein, respectively. Gene sequence alignment and expression analysis between parental lines revealed Cla97C01G008760 as the best possible candidate gene for the gf trait. Nonsynonymous SNP mutations in the first exon of ClPsy1 between parental lines co-segregated with the gf trait only among individuals in the genetic population and were not related to flesh color in natural watermelon panels. Promoter sequence analysis of 26 watermelon accessions revealed two SNPs in the cis-acting element sequences corresponding to MYB and MYC2 transcription factors. RNA-seq data and qRT-PCR verification showed that two MYBs exhibited expression trends similar to that of ClPsy1 in the parental lines and may regulate the ClPsy1 expression. Further research findings indicate that the gf trait is determined not only by ClPsy1 but also by ClLCYB, ClCRTISO and ClNCED7, which play important roles in watermelon ß-carotene accumulation.

Identification and mapping of CpPM10.1, a major gene involved in powdery mildew (race 2 France of Podosphaera xanthii) resistance in zucchini (Cucurbita pepo L.).

KEY MESSAGE: Powdery mildew resistance in zucchini is controlled by one major dominant locus, CpPM10.1. CpPM10.1 was fine mapped. The expression of candidate gene Cp4.1LG10g02780 in resistant individuals was significantly upregulated after inoculation with the powdery mildew. Powdery mildew (PM) is one of the most destructive fungal diseases, reducing the productivity of Cucurbita crops globally. PM influences the photosynthesis, growth and development of infected zucchini and seriously reduces fruit yield and quality. In the present study, the zucchini inbred line 'X10' had highly stable PM resistance, and the inbred line 'Jin234' was highly susceptible to PM in the seedling stage and adult stages. Genetic analysis revealed that PM resistance in 'X10' is controlled by one major dominant locus. Based on the strategy of QTL-seq combined with linkage analysis and developed molecular markers, the major locus was found to be located in a 382.9-kb candidate region on chromosome 10; therefore, the major locus was named CpPM10.1. Using 1,400 F2 individuals derived from a cross between 'X10' and 'JIN234' and F2:3 offspring of the recombinants, the CpPM10.1 locus was defined in a region of approximately 20.9 kb that contained 5 coding genes. Among them, Cp4.1LG10g02780 contained a conserved domain (RPW8), which controls resistance to a broad range of PM pathogens. Cp4.1LG10g02780 also had nonsynonymous SNPs between the resistant 'X10' and susceptible 'Jin234.' Furthermore, the expression of Cp4.1LG10g02780 was strongly positively involved in PM resistance in the key period of inoculation. Further allelic diversity analysis in zucchini germplasm resources indicated that PM resistance was associated with two SNPs in the Cp4.1LG10g02780 RPW8 domain. This study not only provides highly stable PM resistance gene resources for cucurbit crops but also lays the foundation for the functional analysis of PM resistance and resistance breeding in zucchini.

Identification of Chromosomal Regions and Candidate Genes for Round leaf Locus in Cucumis melo L.

Leaf morphology plays a crucial role in plant classification and provides a significant model for studying plant diversity while directly impacting photosynthetic efficiency. In the case of melons, leaf shape not only influences production and classification but also represents a key genetic trait that requires further exploration. In this study, we utilized forward genetics to pinpoint a recessive locus, dubbed Cmrl (Round leaf), which is responsible for regulating melon leaf shape. Through bulked segregant analysis sequencing and extensive evaluation of a two-year F2 population, we successfully mapped the Cmrl locus to a 537.07 kb region on chromosome 8 of the melon genome. Subsequent genetic fine-mapping efforts, leveraging a larger F2 population encompassing 1322 plants and incorporating F2:3 phenotypic data, further refined the locus to an 80.27 kb interval housing five candidate genes. Promoter analysis and coding sequence cloning confirmed that one of these candidates, MELO3C019152.2 (Cmppr encoding a pentatricopeptide repeat-containing family protein, Cmppr), stands out as a strong candidate gene for the Cmrl locus. Notably, comparisons of Cmrl expressions across various stages of leaf development and different leaf regions suggest a pivotal role of Cmrl in the morphogenesis of melon leaves.

Immune-like sandwich multiple hotspots SERS biosensor for ultrasensitive detection of NDKA biomarker in serum.

Developing the rapid, specific, and sensitive tumor marker NDKA biosensor has become an urgent need in the field of early diagnosis of colorectal cancer (CRC). Surface-enhanced Raman spectroscopy (SERS) with the advantages of high sensitivity, high resolution as well as providing sample fingerprint, enables rapid and sensitive detection of tumor markers. However, many SERS biosensors rely on boosting the quantity of Raman reporter molecules on individual nanoparticle surfaces, which can result in nanoparticle agglomeration, diminishing the stability and sensitivity of NDKA detection. Here, we proposed an immune-like sandwich multiple hotspots SERS biosensor for highly sensitive and stable analysis of NDKA in serum based on molecularly imprinted polymers and NDKA antibody. The SERS biosensor employs an array of gold nanoparticles, which are coated with a biocompatible polydopamine molecularly imprinted polymer as a substrate to specifically capture NDKA. Then the biosensor detects NDKA through Raman signals as a result of the specific binding of NDKA to the SERS nanotag affixed to the capture substrate along with the formation of multiple hotspots. This SERS biosensor not only avoids the aggregation of nanoparticles but also presents a solution to the obstacles encountered in immune strategies for certain proteins lacking multiple antibody or aptamer binding sites. Furthermore, the practical application of the SERS biosensor is validated by the detection of NDKA in serum with the lower limit of detection (LOD) of 0.25 pg/mL, meanwhile can detect NDKA of 10 ng/mL in mixed proteins solution, illustrating high sensitivity and specificity. This immune-like sandwich multiple hotspots biosensor makes it quite useful for the early detection of CRC and also provides new ideas for cancer biomarker sensing strategy in the future.