, Volume 250, Issue 4, pp 1073–1088 | Cite as

Two-factor ANOVA of SSH and RNA-seq analysis reveal development-associated Pi-starvation genes in oilseed rape

  • Zhong-Wei Zhang
  • Ling-Yang Feng
  • Jian-Hui Wang
  • Yu-Fan Fu
  • Xin Cai
  • Chang-Quan Wang
  • Jun-Bo Du
  • Ming Yuan
  • Yang-Er Chen
  • Pei-Zhou Xu
  • Ting Lan
  • Guang-Deng Chen
  • Lin-Tao Wu
  • Yun Li
  • Jin-Yao HuEmail author
  • Shu YuanEmail author
Original Article


Main conclusion

The 5-leaf-stage rape seedlings were more insensitive to Pi starvation than that of the 3-leaf-stage plants, which may be attributed to the higher expression levels of ethylene signaling and sugar-metabolism genes in more mature seedlings.


Traditional suppression subtractive hybridization (SSH) and RNA-Seq usually screen out thousands of differentially expressed genes. However, identification of the most important regulators has not been performed to date. Here, we employed two methods, namely, a two-round SSH and two-factor transcriptome analysis derived from the two-factor ANOVA that is commonly used in the statistics, to identify development-associated inorganic phosphate (Pi) starvation-induced genes in Brassica napus. Several of these genes are related to ethylene signaling (such as EIN3, ACO3, ACS8, ERF1A, and ERF2) or sugar metabolism (such as ACC2, GH3, LHCB1.4, XTH4, and SUS2). Although sucrose and ethylene may counteract each other at the biosynthetic level, they may also work synergistically on Pi-starvation-induced gene expression (such as PT1, PT2, RNS1, ACP5, AT4, and IPS1) and root acid phosphatase activation. Furthermore, three new transcription factors that are responsive to Pi starvation were identified: the zinc-finger MYND domain-containing protein 15 (MYND), a Magonashi family protein (MAGO), and a B-box zinc-finger family salt-tolerance protein. This study indicates that the two methods are highly efficient for functional gene screening in non-model organisms.


Ethylene signaling Pi starvation RNA-Seq Sugar metabolism Suppression subtractive hybridization (SSH) 



Differentially expressed gene


Pi-starvation-induced gene


Suppression subtractive hybridization



This work was supported by the National Natural Science Foundation of China (31560402 and 31770322). We thank LetPub ( for its linguistic assistance during the preparation of this manuscript.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

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Supplementary material 1 (XLS 4322 kb)
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Supplementary material 3 (XLS 100 kb)
425_2019_3201_MOESM4_ESM.pdf (4.1 mb)
Supplementary material 4 (PDF 4239 kb)


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Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  1. 1.College of ResourcesSichuan Agricultural UniversityChengduChina
  2. 2.College of AgronomySichuan Agricultural UniversityChengduChina
  3. 3.Horticulture InstituteSichuan Academy of Agricultural SciencesChengduChina
  4. 4.College of Life SciencesSichuan Agricultural UniversitySichuanChina
  5. 5.Rice Research InstituteSichuan Agricultural UniversityChengduChina
  6. 6.Rape Research InstituteGuizhou Academy of Agricultural SciencesGuiyangChina
  7. 7.Rape Research InstituteChengdu Academy of Agriculture and ForestryChengduChina
  8. 8.Research Center for Eco-Environmental EngineeringMianyang Normal UniversityMianyangChina

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