Rapid identification of fruit length loci in cucumber (Cucumis sativus L.) using next-generation sequencing (NGS)-based QTL analysis

Abstract

The cucumber (Cucumis sativus L.) exhibits extensive variations in fruit size and shape. Fruit length is an important agronomic and domesticated trait controlled by quantitative trait loci (QTLs). Nonetheless, the underlying molecular and genetic mechanisms that determine cucumber fruit length remain unclear. QTL-seq is an efficient strategy for QTL identification that takes advantage of bulked-segregant analysis (BSA) and next-generation sequencing (NGS). In the present study, we conducted QTL mapping and QTL-seq of cucumber fruit length. QTL mapping identified 8 QTLs for immature and mature fruit length. A major-effect QTL fl3.2, which explained a maximum of 38.87% of the phenotypic variation, was detected. A genome-wide comparison of SNP profiles between two DNA bulks identified 6 QTLs for ovary length. QTLs ovl3.1 and ovl3.2 both had major effects on ovary length with a △ (SNP-index) of 0.80 (P < 0.01) and 0.74 (P < 0.01), respectively. Quantitative RT-PCR of fruit size-related homologous genes localized in the consensus QTL FL3.2 was conducted. Four candidate genes exhibited increased expression levels in long fruit genotypes. Our results demonstrated the power of the QTL-seq method in rapid QTL detection and provided reliable QTL regions for fine mapping of fruit length-related loci and for identifying candidate genes.

Figure 1. Fruits of CC3, NC76 and RIL populations.

(a) Ovaries of the maternal CC3 (left) and paternal NC76 (right) on day-of-anthesis; (b) Extreme long and short mature fruits harvested from RIL populations.

Figure 2. Correlation coefficiency among immature fruit length, mature fruit length and ovary length.

(a) Correlation coefficiency between immature length and mature fruit length using the means of F₃ families in spring 2013 and autumn 2014. The X-axis represents immature fruit, and the Y-axis represents mature fruit length; (b) Correlation coefficiency between mature fruit length and ovary length using F₇ RILs in spring 2015. The X-axis refers to ovary length on day-of-anthesis, and the Y-axis refers to mature fruit length.

Figure 3. SNP-index graphs of L-pool, S-pool and Δ (SNP-index) graphs from QTL-seq analysis.

The Δ (SNP-index) plot with statistical confidence intervals under the null hypothesis of no QTL (green, P < 0.01; red, P < 0.05). The X-axis represents the position of seven chromosomes, and the Y-axis represents the SNP-index. The SNP-index was calculated based on a 1 Mb interval with a 10 kb sliding window. a: using CC3 genome sequence as the reference genome; b: using NC76 genome sequence as the reference genome.

Figure 4. The co-localization of six fruit size homologues within chromosomal interval of the consensus QTL FL3.2.

Chromosomal intervals of ovl3.2 (green), fl3.2 (red) and mfl3.2 (blue) are given in Mb.

Figure 5. Distribution of homologous genes for fruit size detected on seven cucumber chromosomes.

Physical locations for fruit size homologues on cucumber chromosomes (Chr. 1–7, on the top). Chromosomal distances (Mb) are on the left, and gene names are on the right. The six homologous genes co-localized within QTL FL3.2 are depicted by green box.

Figure 6. qRT-PCR results of six candidate genes localized in chromosome regions of the consensus QTL FL3.2.

The blue bars represent CC3, and the red bars represent NC76; The X-axis indicates 0, 2, 4, 6 days before anthesis, whereas the Y-axis indicates expression levels. The alphabets (A–D) represent significant levels of gene expression.