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

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.

Cloning of a COBL gene determining brittleness in diploid wheat using a MapRseq approach.

Plant tissue brittleness is related to cellular structure and lodging. MED0031 is a mutant identified previously from ethyl methane sulfonate treatment of diploid wheat accession TA2726, showing brittleness in both stem and leaf. In microscopic and histological observations, the mutant was found to have less large vascular bundles per unit area, a thinner sclerenchyma cell wall, and a broader parenchyma, compared with the wild type. The mutated gene, TmBr1, was mapped to a 0.056 cM interval on chromosome 5Am. This gene was cloned using a MapRseq approach that searched the candidate gene through combination of the prior target gene mapping information with SNP calling and discovery of differentially expressed genes from RNA_seq data of the wild type and a BC3F2 bulk showing the mutant phenotype. TmBr1 encodes a COBL protein and a nonsense mutation within the region coding for the conserved COBRA domain caused premature translation termination. Introduction of TmBr1 to Arabidopsis AtCOBL4 mutant rescued the phenotype, demonstrating their functional conservation. Apart from the effect on cellulose content, the TmBr1 mutation might modulate synthesis of noncellulosic polysaccharide pectin as well. Application of the MapRseq approach to isolation of genes present in recombination cold spots and complicated genomes was discussed.