A Rapid Pipeline for Pollen- and Anther-Specific Gene Discovery Based on Transcriptome Profiling Analysis of Maize Tissues

Abstract

Recently, crop breeders have widely adopted a new biotechnology-based process, termed Seed Production Technology (SPT), to produce hybrid varieties. The SPT does not produce nuclear male-sterile lines, and instead utilizes transgenic SPT maintainer lines to pollinate male-sterile plants for propagation of nuclear-recessive male-sterile lines. A late-stage pollen-specific promoter is an essential component of the pollen-inactivating cassette used by the SPT maintainers. While a number of plant pollen-specific promoters have been reported so far, their usefulness in SPT has remained limited. To increase the repertoire of pollen-specific promoters for the maize community, we conducted a comprehensive comparative analysis of transcriptome profiles of mature pollen and mature anthers against other tissue types. We found that maize pollen has much less expressed genes (>1 FPKM) than other tissue types, but the pollen grain has a large set of distinct genes, called pollen-specific genes, which are exclusively or much higher (100 folds) expressed in pollen than other tissue types. Utilizing transcript abundance and correlation coefficient analysis, 1215 mature pollen-specific (MPS) genes and 1009 mature anther-specific (MAS) genes were identified in B73 transcriptome. These two gene sets had similar GO term and KEGG pathway enrichment patterns, indicating that their members share similar functions in the maize reproductive process. Of the genes, 623 were shared between the two sets, called mature anther- and pollen-specific (MAPS) genes, which represent the late-stage pollen-specific genes of the maize genome. Functional annotation analysis of MAPS showed that 447 MAPS genes (71.7% of MAPS) belonged to genes encoding pollen allergen protein. Their 2-kb promoters were analyzed for cis-element enrichment and six well-known pollen-specific cis-elements (AGAAA, TCCACCA, TGTGGTT, [TA]AAAG, AAATGA, and TTTCT) were found highly enriched in the promoters of MAPS. Interestingly, JA-responsive cis-element GCC box (GCCGCC) and ABA-responsive cis-element-coupling element1 (ABRE-CE1, CCACC) were also found enriched in the MAPS promoters, indicating that JA and ABA signaling likely regulate pollen-specific MAPS expression. This study describes a robust and straightforward pipeline to discover pollen-specific promotes from publicly available data while providing maize breeders and the maize industry a number of late-stage (mature) pollen-specific promoters for use in SPT for hybrid breeding and seed production.

Rapid Identification of Pollen- and Anther-Specific Genes in Response to High-Temperature Stress Based on Transcriptome Profiling Analysis in Cotton

“…In order to evaluate the transcript abundance in each of the sequenced tissues, we chose genes whose TPM (transcripts per kilobase of exon model per million mapped reads) value was higher than 1.00 as expressed genes (EGs), and we divided the EG into four groups: low expressed genes (LEGs, 1–10 TPM), medium expressed genes (MEGs, 10–30 TPM), high expressed genes (HEGs, 30–300 TPM), and very high expressed genes (VHEGs, ≥300 TPM) [ 39 , 40 ]. Of the 70,199 genes reported or predicted in the Gossypium cotton TM-1 reference genome, we found that pollen grains only have 12,005 EGs occupying 17.1% of known cotton genes ( Table 1 ).…”

“…Pollen-specific genes were termed preferentially expressed in pollen or not expressed in other tissues except pollen. Thus, the criterion was used for screened PSGs as reported: (1) a TPM of at least 1 in mature pollen, and (2) expression at least 100-fold higher in pollen than in other vegetative or reproductive tissues [ 40 ]. In total, 1111 PSGs were identified ( Table S2 ).…”

“…Unique characteristics of the pollen transcriptome were identified in Arabidopsis: the number of expressed genes in pollen is approximately one-third of the number of other organs on average [ 49 ]. Transcriptome profiling based on RNA-seq revealed that mature pollen grains possess 9242 EGs, which is less than 30% of known maize genes, whereas other reproductive tissues and vegetative tissues express approximately 80% of maize genes, with 24,175 to 27,659 EGs [ 40 ]. The transcripts of the top 300 EGs and top 1000 EGs in pollen occupied 75.5% and 91.7% of the pollen transcriptome, while this percentage in other tissues was only were 28.2–47.2% and 43.4–69.2% [ 40 ].…”

“…Transcriptome profiling based on RNA-seq revealed that mature pollen grains possess 9242 EGs, which is less than 30% of known maize genes, whereas other reproductive tissues and vegetative tissues express approximately 80% of maize genes, with 24,175 to 27,659 EGs [ 40 ]. The transcripts of the top 300 EGs and top 1000 EGs in pollen occupied 75.5% and 91.7% of the pollen transcriptome, while this percentage in other tissues was only were 28.2–47.2% and 43.4–69.2% [ 40 ]. In maize, microarray-based transcriptome profiling revealed that mature haploid pollen expresses approximately 10,000 transcripts, half of the less than 20,000 transcripts expressed in post-meiotic anthers [ 46 ].…”

“…Deveshwar et al (2011) identified 1000 genes specifically expressed in rice anther stages and the proportions of anther-specific genes in the four stages of anther development, pre-meiotic, meiotic, anthers at single-celled and tri-nucleate pollen, were 0.3%, 0.5%, 2.0%, and 4.4%, respectively, which suggested that a late stage of anther includes more anther-specific genes and that the number of anther-specific genes increases with the development of anther [ 47 ]. Shi et al (2021) identified 1215 mature pollen-specific genes using a more stringent 100-fold criterion, and 1009 mature anther-specific genes were identified in the late stage of anther development using the same criterion [ 40 ]. Moreover, similar GO terms were found in mature pollen-specific genes and mature anther-specific genes, and 623 shared genes (mature anther- and pollen-specific genes) in mature anther-specific genes and mature pollen-specific genes were also identified [ 40 ].…”