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Physiological characterization, transcriptomic profiling, and microsatellite marker mining of Lycium ruthenicum

黑果枸杞生理指标测定、 转录组分析以及分子标记开发研究

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Abstract

Lycium ruthenicum is a perennial shrub species that has attracted considerable interest in recent years owing to its nutritional value and ability to thrive in a harsh environment. However, only extremely limited transcriptomic and genomic data related to this species can be found in public databases, thereby limiting breeding research and molecular function analysis. In this study, we characterized the physiological and biochemical responses to saline-alkaline mixed stress by measuring photochemical efficiency, chlorophyll content, and protective enzyme activity. We performed global transcriptomic profiling analysis using the Illumina platform. After optimizing the assembly, a total of 68 063 unique transcript sequences with an average length of 877 bp were obtained. Among these sequences, 4096 unigenes were upregulated and 4381 unigenes were down-regulated after saline-alkaline mixed treatment. The most abundant transcripts and over-represented items were assigned to gene ontology (GO) terms or Kyoto Encyclopedia of Genes and the Genomes (KEGG) categories for overall unigenes, and differentially expressed unigenes were analyzed in detail. Based on this set of RNA-sequencing data, a total of 9216 perfect potential simple sequence repeats (SSRs) were identified within 7940 unigenes with a frequency of 1/6.48 kb. A total of 77 primer pairs were synthesized and examined in wet-laboratory experiments, of which 68 loci (88.3%) were successfully amplified with specific products. Eleven pairs of polymorphic primers were verified in 225 individuals from nine populations. The inbreeding coefficient and the polymorphism information content value ranged from 0.011 to 0.179 and from 0.1112 to 0.6750, respectively. The observed and expected heterozygosities ranged from 0.064 to 0.840 and from 0.115 to 0.726, respectively. Nine populations were clustered into three groups based on a genetic diversity study using these novel markers. Our data will be useful for functional genomic investigations of L. ruthenicum and could be used as a basis for further research on the genetic diversity, genetic differentiation, and gene flow of L. ruthenicum and other closely related species.

概要

目 的

以高耐盐碱多年生沙漠经济灌木黑果枸杞为研究材料, 对其在盐碱胁迫处理下的生理指标进行测定, 确定转录组测试的时间。 通过转录组分析挖掘潜在抗逆基因, 并挖掘全转录组水平的分子标记。 旨在为黑果枸杞的优良基因资源利用、 野生品种保护和新品种培育提供理论依据和实践指导。

创新点

首次对黑果枸杞进行盐碱胁迫下的生理指标变化和全转录组水平的基因表达变化进行分析, 并基于转录组进行大规模简单重复序列标记开发和验证, 并将所获取的分子标记应用到9 个野生群体进行遗传多样性分析。

方 法

采用双通道 PAM-100 荧光仪研究盐碱胁迫对黑果枸杞P700 (PS I) 和叶绿素荧光 (PS II) 的影响; 通过盐碱胁迫下丙二醛 (MDA) 含量、 超氧化物歧化酶 (SOD) 和过氧化物酶 (POD) 活性变化选定转录组测序 (RNA-seq) 取样时间; 采用 Illumina 高通量测序平台进行转录组从头测序; 选取 20 个基因采用荧光定量聚合酶链式反应 (PCR) 法进行基因表达分析; 基于转录组序列组装结果进行简单重复序列扫描; 采用聚丙烯酰胺凝聚和毛细管电泳法鉴定引物多态性, 选取其中 11 对多态性引物应用于遗传多样性分析。

结 论

通过对对照以及混合盐碱处理的黑果枸杞无菌苗进行生理和生化测试, 结果选定处理 6 小时为取样点。 RNA-seq 结果共获得 68 063 个 unigene, 平均长度为 877 bp, 其中 4096 个基因在混合盐碱处理下表现为上调, 4381 个表现为下调。 随机选取 24 个基因进行荧光定量表达分析, 结果显示, 荧光定量表达结果与 RNA-seq 结果呈显著正相关。 基于转录组测试数据, 在 7940 个基因中挖掘出 9216 个简单重复序列标记, 对其中 77 个进行检测, 显示有 68 个位点清晰存在, 选取其中 11 个多态性位点对来自西北四个省份或自治区的 9 个野生种质资源进行遗传多样性分析, 结果显示分析可靠。

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Acknowledgements

We sincerely thank Dr. Qiang-zhang DU of Beijing Forestry University, China for providing valuable suggestions for this study.

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Correspondence to Jin-huan Chen.

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Project supported by the Fundamental Research Funds for the Central Universities (No. 2016ZCQ05), the Forestry Industry Research Special Funds for Public Welfare Projects (No. 201504101), and the Ningxia Goji Special Funds of Establishment and Application of Technical System of Molecular Breeding for Wolfberry, China

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Chen, Jh., Zhang, Dz., Zhang, C. et al. Physiological characterization, transcriptomic profiling, and microsatellite marker mining of Lycium ruthenicum. J. Zhejiang Univ. Sci. B 18, 1002–1021 (2017). https://doi.org/10.1631/jzus.B1700135

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