Identifying differentially expressed genes in pollen from self-incompatible "Wuzishatangju" and self-compatible "Shatangju" mandarins

Abstract

Self-incompatibility (SI) is one of the important factors that can result in seedless fruit in Citrus. However, the molecular mechanism of SI in Citrus is not yet clear. In this study, two suppression subtractive hybridization (SSH) libraries (forward, F and reverse, R) were constructed to isolate differentially expressed genes in pollen from "Wuzishatangju" (SI) and "Shatangju" (self-compatibility, SC) mandarins. Four hundred and sixty-eight differentially expressed cDNA clones from 2077 positive clones were sequenced and identified. Differentially expressed ESTs are possibly involved in the SI reaction of "Wuzishatangju" by regulating pollen development, kinase activity, ubiquitin pathway, pollen-pistil interaction, and calcium ion binding. Twenty five SI candidate genes were obtained, six of which displayed specific expression patterns in various organs and stages after self- and cross-pollination. The expression level of the F-box gene (H304) and S1 (F78) in the pollen of "Wuzishatangju" was 5-fold higher than that in "Shatangju" pollen. The F-box gene, S1, UBE2, UBE3, RNaseHII, and PCP were obviously up-regulated in pistils at 3 d after self-pollination of "Wuzishatangju", approximately 3-, 2-, 10-, 5-, 5-, and 2-fold higher, respectively than that at the same stage after cross-pollination of "Wuzishatangju" × "Shatangju" pistils. The potential involvement of these genes in the pollen SI reaction of "Wuzishatangju" is discussed.

Figure 1

Gene distribution from two suppression subtractive hybridization (SSH) libraries. In the F library, self-incompatibility (SI) “Wuzishatangju” pollen was used as the “tester”, and self-compatibility (SC) “Shatangju” as the “driver”. In the R library, SC “Shatangju” pollen was used as the “tester”, and SI “Wuzishatangju” as the “driver”. The Arabic numerals represent the number of unique sequences at each step of the annotation process. NoBlast, no blast result; NoAnnot, no annotation; Annot, annotation. Data analysis and visualization of results were performed by the Blast2GO software.

Figure 2

Classification of the unique sequence from the SSH according to Gene Ontology criteria. Gene Ontology analysis was carried out on the transcripts isolated from the two libraries. The graph, which combined biological processes, was made based on ontology level 2.

Figure 3

Expression analysis of 10 pollen SI-related genes in different organs of “Wuzishatangju” and “Shatangju” mandarins using SqRT-PCR. CPBP, Calcium-dependent phospholipid binding protein; UBE2, Ubiquitin-conjugating enzyme E2; S1-protein, self-incompatibility S1 family protein; UBE3, Ubiquitin-protein ligase E3; PCP, Pollen coat-like protein; RNase HII, Ribonuclease HII family protein; SDPP, Sexual differentiation process protein; SMP, Seed maturation protein; CWI, Cell wall invertase. Data are based on 0.5 g of random samples collected from each organ.

Figure 4

Expression analysis of 10 pollen SI-related genes in different organs of “Wuzishatangju” and “Shatangju” mandarins using qPCR. CPBP, Calcium-dependent phospholipid binding protein; UBE2, Ubiquitin-conjugating enzyme E2; S1-protein, self-incompatibility S1 family protein; UBE3, Ubiquitin-protein ligase E3; PCP, Pollen coat-like protein; RNase HII, Ribonuclease HII family protein; SDPP, Sexual differentiation process protein; SMP, Seed maturation protein; CWI, Cell wall invertase. The error bars represent SD of three replicates. Data are based on 0.5 g of random samples collected from each organ.

Figure 5

Expression analyses of 10 pollen SI-related genes in different stages of pistils after self-pollination of “Wuzishatangju” and cross-pollination of “Wuzishatangju” × “Shatangju” mandarins using qPCR. “W*W”, self-pollination of “Wuzishatangju” × “Wuzishatangju”; “W*Y”, cross-pollination of “Wuzishatangju” × “Shatangju”. CPBP, Calcium-dependent phospholipid binding protein; UBE2, Ubiquitin-conjugating enzyme E2; S1-protein, self-incompatibility S1 family protein; UBE3, Ubiquitin-protein ligase E3; PCP, Pollen coat-like protein; RNase HII, Ribonuclease HII family protein; SDPP, Sexual differentiation process protein; SMP, Seed maturation protein; CWI, Cell wall invertase. The error bars represent the SD of three replicates. Data are based on 0.5 g of random samples collected from each organ.