Single-molecule real-time (SMRT) sequencing reveals diverse allelic variations in carotenoid biosynthetic genes in pepper (Capsicum spp.)

First author: Hyo‐Bong Jeong; Affiliations: Seoul National University (国立首尔大学): Seoul, Korea

Corresponding author: Byoung‐Cheorl Kang

The diverse colors of mature pepper (Capsicum spp.) fruit result from the accumulation of different carotenoids. The carotenoid biosynthetic pathway has been well elucidated in Solanaceous (茄科) plants, and analysis of candidate genes involved in this process has revealed variations in carotenoid biosynthetic genes in Capsicum spp. However, the allelic variations revealed by previous studies could not fully explain the variation in fruit color in Capsicum spp. due to technical difficulties in detecting allelic variation in multiple candidate genes in numerous samples. In this study, we uncovered allelic variations in six carotenoid biosynthetic genes, including phytoene synthase 八氢番茄红素合酶 (PSY1, PSY2), lycopene β‐cyclase 番茄红素β-环化酶 (Lcyb), β‐carotene hydroxylase β-胡萝卜素羟化酶 (CrtZ‐2), zeaxanthin epoxidase 玉米黄质环氧化酶 (ZEP), and capsanthin‐capsorubin synthase 辣椒红素/辣椒玉红素合酶 (CCS) genes, in 94 pepper accessions by single‐molecule real‐ ime (SMRT) sequencing. To investigate the relationship between allelic variations in the candidate genes and differences in fruit color, we performed ultra‐performance liquid chromatography (UPLC；超高效液相色谱法) analysis using 43 accessions representing each allelic variation. Different combinations of dysfunctional (功能失调的) mutations in PSY1 and CCS could explain variation in the compositions and levels of carotenoids in the accessions examined in this study. Our results demonstrate that SMRT sequencing technology can be used to rapidly identify allelic variation of target genes in various germplasms. The newly identified allelic variants will be useful for pepper breeding and for further analysis of carotenoid biosynthesis pathways.

成熟的辣椒果实因为积累了不同的类胡萝卜素，具有不同的颜色。茄科中的类胡萝卜素生物合成通路已经研究得很透彻了，对于辣椒中涉及该过程的候选基因的研究揭示了不同程度的变异。然而，由于在不同样本中多个候选基因的等位变异检测技术限制，之前的研究所揭示的等位基因变异并不能完全解释辣椒果实的颜色变异。本文中，作者通过单分子实时测序SMRT技术鉴定了八氢番茄红素合酶PSY1和PSY2、番茄红素β-环化酶Lcyb、β-胡萝卜素羟化酶CrtZ‐2、玉米黄质环氧化酶ZEP以及辣椒红素/辣椒玉红素合酶CCS等6个类胡萝卜素生物合成基因在94株辣椒个体中的等位变异。为了进一步研究候选基因等位变异与果实颜色差异之间的联系，作者利用超高效液相色谱法UPLC对包含所有等位变异类型的43株辣椒进行了鉴定分析。通过PSY1和CCS两个基因的不同组合功能失调突变体，作者解释了本研究中不同辣椒样本之间类胡萝卜素含量水平和组份的差异。本文的研究揭示了SMRT测序技术可以被用来快速鉴定不同种质之间目标基因的等位变异。本文中新鉴定的等位变异将有利于将来辣椒育种研究，并且为进一步的类胡萝卜素生物合成研究提供基础。

doi: https://doi.org/10.1111/pbi.13039

Journal: Plant Biotechnology Journal

First Published: 22 November, 2018