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Comparative transcript profiling of gene expression between self-incompatible and self-compatible mandarins by suppression subtractive hybridization and cDNA microarray

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Abstract

Self-incompatibility (SI) is an important trait of Citrus plants that is exploited by farmers to produce seedless fruit. However, the molecular mechanism of SI in Citrus is not well understood. Wuzishatangju (Citrus reticulata Blanco) (SI) is an excellent seedless cultivar selected from a seedy Shatangju cultivar (self-compatible, SC) through spontaneous bud mutation. The two cultivars are therefore excellent materials for studying the mechanisms of SI and/or SC in Citrus. In this study, an integrative strategy combining eight suppression subtractive hybridization libraries with cDNA microarray was used to study the molecular mechanisms that differ between Wuzishatangju and Shatangju (control) mandarins. A custom microarray screen resulted in a total of 1,830 up- or down-regulated clones (false discovery rate <0.05 and a fold change \({ \geqq }\)2) obtained from 9,810 positive clones. The expression of genes involved in embryonic development, ubiquitination pathway, Ca2+-signaling pathway, gibberellins, and auxin was significantly up-regulated in SI Wuzishatangju compared with SC Shatangju mandarin. The microarray analysis suggested that the ubiquitin-mediated proteolysis pathway might be involved in the SI reaction of Wuzishatangju. Additionally, our research highlighted some main genes (mitogen-activated protein kinase, SI S1 family protein, ubiquitin-conjugating factor E4-like, auxin transporter protein 1, and gibberellin receptor) that participate in the SI reaction of Wuzishatangju and could be beneficial for understanding the evolution of SI systems and for breeding seedless citrus fruits in the future.

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Acknowledgments

This work was supported by National Natural Science Foundation of China (No. 31000899 and 31471858), Research Fund for the Doctoral Program of Higher Education of China (No. 20104404120015 and 20114404110018), Guangdong Province Science Foundation of China (No. S2013020013084, S2013010011950 and 06025843), Foundation for Higher Education Discipline and Specialty Construction of Guangdong Provincial Department of Education (No. 2013KJCX0031), Science and Technology Planning Project of Guangzhou (2010r1-C771), the Open Foundation of State Key Laboratory for Conservation and Utilization of Subtropical Agro-bioresources, South China Agricultural University (No. KSL-CUSAb-2012-09), Key Laboratory of Innovation and Utilization for Germplasm Resources in Horticultural Crops in Southern China of Guangdong Higher Education Institutes, South China Agricultural University (No. KBL11008) and “211” Construction Fund for Key Subjects of College of Horticulture, South China Agricultural University.

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Authors and Affiliations

  1. State Key Laboratory for Conservation and Utilization of Subtropical Agro-Bioresources/Key Laboratory of Biology and Genetic Improvement of Horticultural Crops (Ministry of Agriculture), College of Horticulture, South China Agricultural University, Guangzhou, 510642, Guangdong, People’s Republic of China

    Hongxia Miao, Zixing Ye, Guibing Hu & Yonghua Qin

  2. Institute of Tropical Bioscience and Biotechnology, Chinese Academy of Tropical Agricultural Sciences/Key Laboratory of Tropical Crop Bioscience and Biotechnology, Ministry of Agriculture, Haikou, 571101, Hainan, People’s Republic of China

    Hongxia Miao

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  1. Hongxia Miao
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  2. Zixing Ye
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Correspondence to Guibing Hu or Yonghua Qin.

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Supplementary material 1 (DOC 49 kb)

Supplement S2 Superposition of double-color fluorescent marker. A: Superposition; B: Repeat A experiment (TIFF 1193 kb)

11032_2015_204_MOESM3_ESM.tif

Supplement S3 Agarose gel electrophoresis of total RNA. 1: Pistils of Wuzishatangju; 2: Pistils of Shatangju; 3: Pollen of Wuzishatangju; 4: Pollen of Shatangju; 5: 72 h styles after self-pollination of Wuzishatangju; 6: 72 h styles after cross-pollination of Wuzishatangju × Shatangju; 7, 9, 11, 13, 15, 17: Pistils in differential stages (0, 4, 24, 48, 72, and 96 h) after self-pollination of Wuzishatangju; 8, 10, 12, 14, 16, 18: Pistils in differential stages (0, 4, 24, 48, 72, and 96 h) after cross-pollination of Wuzishatangju. (TIFF 326 kb)

11032_2015_204_MOESM4_ESM.tif

Supplement S4 TreeView representation of ESTs from eight SSH libraries. WY: Forward SSH library of Wuzishatangju pistils; YW: Reverse SSH library of Wuzishatangju pistils; H: Forward SSH library of Wuzishatangju pollen; F: Reverse SSH library of Wuzishatangju pollen; T1: Forward SSH library of 72 h styles after self-pollination of Wuzishatangju; T2: Reverse SSH library of 72 h styles after cross-pollination of Wuzishatangju × Shatangju; 0 h1, 24 h5, 48 h7, 72 h9, 96 h11: Forward SSH library of pistils in differential stages (0, 24, 48, 72, and 96 h) after self-pollination of Wuzishatangju; 0 h2, 24 h6, 48 h8, 72 h10, 96 h12: Reverse SSH library of pistils in differential stages (0, 4, 24, 48, 72, and 96 h) after cross-pollination of Wuzishatangju. (TIFF 1730 kb)

11032_2015_204_MOESM5_ESM.tif

Supplement S5 Scatter plots for the eight SSH libraries. WY: Forward SSH library of Wuzishatangju pistils; YW: Reverse SSH library of Wuzishatangju pistils; H: Forward SSH library of Wuzishatangju pollen; F: Reverse SSH library of Wuzishatangju pollen; T1: Forward SSH library of 72 h styles after self-pollination of Wuzishatangju; T2: Reverse SSH library of 72 h styles after cross-pollination of Wuzishatangju × Shatangju; 72 h9: Forward SSH library of pistils in 72 h after self-pollination of Wuzishatangju; 72 h10: Reverse SSH library of pistils in 72 h after cross-pollination of Wuzishatangju × Shatangju. (TIFF 1780 kb)

Supplement S6 The gene expression ratios based on cDNA microarray from the eight SSH libraries. (XLS 5583 kb)

11032_2015_204_MOESM7_ESM.tif

Supplement S7 Up- and down- regulated clones of eight SSH libraries. A (0, 24, 48, 72, and 96 h): Pistils in different stages (0, 24, 48, 72, and 96 h) after self-pollination of Wuzishatangju mandarin. B (0, 24, 48, 72, and 96 h): Pistils in differential stages (0, 24, 48, 72, and 96 h) after cross-pollination of Wuzishatangju × Shatangju mandarin. (TIFF 3079 kb)

Supplement S8 Ubiquitin-mediated proteolysis signaling pathway (TIFF 1355 kb)

11032_2015_204_MOESM9_ESM.jpg

Supplement S9 A tentative model regarding the main genes and/or pathways involved in the SI reaction of Wuzishatangju mandarin ubiquitin-mediated proteolysis signaling pathway (JPEG 165 kb)

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Miao, H., Ye, Z., Hu, G. et al. Comparative transcript profiling of gene expression between self-incompatible and self-compatible mandarins by suppression subtractive hybridization and cDNA microarray. Mol Breeding 35, 47 (2015). https://doi.org/10.1007/s11032-015-0204-x

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