A 4 bp InDel in the Promoter of Wheat Gene TaAFP-B Affecting Seed Dormancy Confirmed in Transgenic Rice.

Background: Wheat (Triticum aestivum L.) ABA insensitive five (ABI5) binding protein gene (TaAFP) is a homologue of the ABI5 binding protein (AFP) gene in Arabidopsis thaliana. It is well documented that AtAFP is a negative regulator of ABA signaling that regulates embryo germination and seed dormancy. TaABI5 was earlier shown to be expressed specifically in seed and its transcript accumulated during wheat grain maturation and acquisition of dormancy. It plays an important role in seed dormancy. In a previous study, we identified two allelic variants TaAFP-B1a and TaAFP-B1b of TaAFP on chromosome arm 2BS in common wheat, designated as, respectively. Sequence analysis revealed a 4 bp insertion in the promoter of TaAFP-B1a compared with TaAFP-B1b that affected mRNA transcription level, mRNA stability, GUS and tdTomatoER translation level, and GUS activity determining seed dormancy. Results: The transcription and translation levels of TaAFP-B were significantly reduced in TaAFP-Ba and TaAFP-Ba-GFP transgenic plants compared with TaAFP-Bb and TaAFP-Bb-GFP. The average GI (germination index) values of TaAFP-Ba and TaAFP-Ba-GFP were significantly lower than those of TaAFP-Bb and TaAFP-Bb-GFP in T1 and T2 transgenic rice seeds, whereas mature TaAFP-Ba and TaAFP-Ba-GFP transgenic seeds exhibited increased ABA sensitivity and content of endogenous ABA compared with TaAFP-Bb and TaAFP-Bb-GFP. Conclusion: The 4 bp insertion in the promoter of TaAFP-Ba decreased transcript abundance and translation level in transgenic rice. This insertion increased sensitivity to ABA and content of endogenous ABA in mature seeds, leading to a higher seed dormancy and pre-harvest sprouting tolerance in transgenic rice.

Identification of vernalization responsive genes in the winter wheat cultivar Jing841 by transcriptome sequencing.

This study aimed to identify vernalization responsive genes in the winter wheat cultivar Jing841 by comparing the transcriptome data with that of a spring wheat cultivar Liaochun10. For each cultivar, seedlings before and after the vernalization treatment were sequenced by Solexa/Illumina sequencing. Genes differentially expressed after and before vernalization were identified as differentially expressed genes (DEGs) using false discovery rate (FDR) ≤ 0.001 and |log2 (fold change)|>1 as cutoffs. The Jing841-specific DEGs were screened and subjected to functional annotation using gene ontology (GO) database. Vernalization responsive genes among the specific genes were selected for validation by quantitative reverse transcription polymerase chain reaction (qRT-PCR) and the expression change over the time was investigated for the top 11 genes with the most significant expression differences. A total of 138,062 unigenes were obtained. Overall, 636 DEGs were identified as vernalization responsive genes including some known genes such as VRN-1 and COR14a, and some unknown contigs. The qRT-PCR validated changes in the expression of 18 DEGs that were detected by RNA-seq. Among them, 11 genes displayed four different types of expression patterns over time during the 30-day-long vernalization treatment. Genes or contigs such as VRN-A1, COR14a, IRIP, unigene1806 and Cl18953. Contig2 probably have critical roles in vernalization.

Virus-induced gene silencing-based functional verification of six genes associated with vernalization in wheat.

Vernalization requirement is an important characteristic in crop breeding. Wheat is a widely grown crop in the world that possesses enormous economic significance. To better understand the gene networks in vernalization process, we performed a high-throughput RNA sequencing analysis comparing the transcriptomes of spring and winter wheat cultivars, with and without vernalization (unpublished data). In this study, we selected six unigenes (CL14010, CL12788, CL176, Unigene 16777, CL8746 and Unigene10196) from our transcriptome analysis based on their expression differences to further characterize their function. Transient silencing of the six unigenes individually were achieved through virus-induced gene silencing (VIGS) using BSMV vector. The period from germination to spike differentiation were recorded and compared between plants underwent VIGS silencing and the control. Our result showed that VIGS of the six unigenes significantly shortened the period from seedling to double ridge (DR) stage. Resulting in SD period ranging from 59.8 ± 0.60 to 65.8 ± 0.48 days, compared to 85.0 ± 0.73 days in the control. The results indicated that these six unigenes function as suppressors in vernalization process and silence or down-regulation of these genes promoted flower development in wheat. Further characterization of these six unigenes and their function in vernalization and flowering control is needed.