Table_7_QTL and Transcriptomic Analyses Implicate Cuticle Transcription Factor SHINE as a Source of Natural Variation for Epidermal Traits in Cucumber Fruit.docx

The fruit surface is a unique tissue with multiple roles influencing fruit development, post-harvest storage and quality, and consumer acceptability. Serving as the first line of protection against herbivores, pathogens, and abiotic stress, the surface can vary markedly among species, cultivars within species, and developmental stage. In this study we explore developmental changes and natural variation of cucumber (Cucumis sativus L.) fruit surface properties using two cucumber lines which vary greatly for these traits and for which draft genomes and a single nucleotide polymorphism (SNP) array are available: Chinese fresh market type, Chinese Long ‘9930’ (CL9930), and pickling type, ‘Gy14’. Thin-section samples were prepared from the mid-region of fruit harvested at 0, 4, 8, 12, 16, 20, 24 and 30 days post pollination (dpp), stained with Sudan IV and evaluated for cuticle thickness, depth of wax intercalation between epidermal cells, epidermal cell size and shape, and number and size of lipid droplets. ‘Gy14’ is characterized by columnar shaped epidermal cells, a 2–3 fold thicker cuticular layer than CL9930, increased cuticular intercalations between cells and a larger number and larger sized lipid droplets. In both lines maximal deposition of cuticle and increase in epidermal size coincided with exponential fruit growth and was largely completed by approximately 16 dpp. Phenotyping and quantitative trait locus mapping (QTL) of fruit sampled from an F7:F8 Gy14 × CL9930 recombinant inbred line (RIL) population identified QTL regions on chromosomes 1, 4 and 5. Strong QTL for epidermal cell height, cuticle thickness, intercalation depth, and diameter of lipid droplets co-localized on chromosome 1. SSR markers on chromosome 1 were used to screen for recombinants in an extended RIL population to refine the QTL region. Further fine mapping by KASP assay combined with gene expression profiling suggested a small number of candidate genes. Tissue specificity, developmental analysis of expression, allelic diversity and gene function implicate the regulatory factor CsSHINE1/WIN1 as a source of natural variation for cucumber fruit epidermal traits.

果实表皮是一类兼具多重功能的独特组织，可影响果实发育、采后贮藏与果实品质，以及消费者接受度。作为抵御植食性动物、病原菌与非生物胁迫的第一道防线，果实表皮在不同物种、物种内不同栽培品种及不同发育阶段间存在显著差异。本研究选取两类在表皮性状上差异显著，且均已公布草图基因组与单核苷酸多态性（Single Nucleotide Polymorphism, SNP）芯片的黄瓜品系，探究黄瓜（Cucumis sativus L.）果实表皮性状的发育变化与自然变异：二者分别为中国鲜食型黄瓜‘中长9930’（CL9930）与加工腌制型黄瓜‘Gy14’。本研究采集授粉后0、4、8、12、16、20、24及30天（dpp）的果实中部组织制备薄切片，以苏丹IV染色后，对角质层厚度、表皮细胞间蜡质嵌入深度、表皮细胞大小与形态，以及脂滴的数量与尺寸开展观测。‘Gy14’的表皮细胞呈柱状，角质层厚度为CL9930的2~3倍，细胞间角质层嵌入程度更高，且脂滴数量更多、尺寸更大。两类品系中，角质层最大沉积量与表皮细胞尺寸的峰值均与果实指数生长期重合，且基本在授粉后16天左右完成。对由‘Gy14’与‘CL9930’杂交得到的F7至F8代重组自交系（Recombinant Inbred Line, RIL）群体的果实进行表型鉴定与数量性状位点（Quantitative Trait Locus, QTL）定位，在1、4、5号染色体上定位到QTL区域。控制表皮细胞高度、角质层厚度、嵌入深度及脂滴直径的强效QTL均共定位于1号染色体上。研究人员利用1号染色体上的简单序列重复（Simple Sequence Repeat, SSR）标记，对扩大规模的RIL群体进行重组体筛选，以精细定位该QTL区域。通过竞争性等位基因特异性PCR（Kompetitive Allele Specific PCR, KASP）分析开展进一步精细定位，并结合基因表达谱分析，最终筛选出少量候选基因。组织特异性、表达发育模式分析、等位基因多样性及基因功能研究均表明，调控因子CsSHINE1/WIN1是黄瓜果实表皮性状自然变异的来源之一。