The regulatory landscape of early maize inflorescence development.

BACKGROUND: The functional genome of agronomically important plant species remains largely unexplored, yet presents a virtually untapped resource for targeted crop improvement. Functional elements of regulatory DNA revealed through profiles of chromatin accessibility can be harnessed for fine-tuning gene expression to optimal phenotypes in specific environments. RESULT: Here, we investigate the non-coding regulatory space in the maize (Zea mays) genome during early reproductive development of pollen- and grain-bearing inflorescences. Using an assay for differential sensitivity of chromatin to micrococcal nuclease (MNase) digestion, we profile accessible chromatin and nucleosome occupancy in these largely undifferentiated tissues and classify at least 1.6% of the genome as accessible, with the majority of MNase hypersensitive sites marking proximal promoters, but also 3' ends of maize genes. This approach maps regulatory elements to footprint-level resolution. Integration of complementary transcriptome profiles and transcription factor occupancy data are used to annotate regulatory factors, such as combinatorial transcription factor binding motifs and long non-coding RNAs, that potentially contribute to organogenesis, including tissue-specific regulation between male and female inflorescence structures. Finally, genome-wide association studies for inflorescence architecture traits based solely on functional regions delineated by MNase hypersensitivity reveals new SNP-trait associations in known regulators of inflorescence development as well as new candidates. CONCLUSIONS: These analyses provide a comprehensive look into the cis-regulatory landscape during inflorescence differentiation in a major cereal crop, which ultimately shapes architecture and influences yield potential.

Structural Characterization of ABCB1, the Gene Underlying the d2 Dwarf Phenotype in Pearl Millet, Cenchrus Americanus (L.) Morrone.

Pearl millet is an important food crop in arid and semi-arid regions of South Asia and sub-Saharan Africa and is grown in Australia and the United States as a summer fodder crop. The d2 dwarf germplasm has been widely used in the last half-century to develop high-performing pearl millet hybrids. We previously mapped the d2 phenotype to a 1.6 cM region in linkage group (LG) 4 and identified the ABCB1 gene as a candidate underlying the trait. Here, we report the sequence, structure and expression of ABCB1 in tall (D2D2) and d2 dwarf (d2d2) germplasm. The ABCB1 allele in d2 dwarfs differs from that in tall inbreds by the presence of two different high copy transposable elements, one in the coding region and the second located 664 bp upstream of the ATG start codon. These transposons were present in all d2 dwarfs tested that were reported to be of independent origin and absent in the analyzed wild-type tall germplasm. We also compared the expression profile of this gene in different organs of multiple tall and d2 dwarf inbreds, including the near-isogenic inbreds at the d2 locus, Tift 23B (D2D2) and Tift 23DB (d2d2). Heterologous transformation of the tall (Ca_ABCB1) and the d2 dwarf (Ca_abcb1) pearl millet alleles in the Arabidopsis double mutant abcb1abcb19 showed that the pearl millet D2 but not the d2 allele complements the Arabidopsis abcb1 mutation. Our studies also show the importance of the COOH-terminal 22 amino acids of the ABCB1 protein in either protein function or stability.

Parallel loss of introns in the ABCB1 gene in angiosperms.

BACKGROUND: The presence of non-coding introns is a characteristic feature of most eukaryotic genes. While the size of the introns, number of introns per gene and the number of intron-containing genes can vary greatly between sequenced eukaryotic genomes, the structure of a gene with reference to intron presence and positions is typically conserved in closely related species. Unexpectedly, the ABCB1 (ATP-Binding Cassette Subfamily B Member 1) gene which encodes a P-glycoprotein and underlies dwarfing traits in maize (br2), sorghum (dw3) and pearl millet (d2) displayed considerable variation in intron composition. RESULTS: An analysis of the ABCB1 gene structure in 80 angiosperms revealed that the number of introns ranged from one to nine. All introns in ABCB1 underwent either a one-time loss (single loss in one lineage/species) or multiple independent losses (parallel loss in two or more lineages/species) with the majority of losses occurring within the grass family. In contrast, the structure of the closest homolog to ABCB1, ABCB19, remained constant in the majority of angiosperms analyzed. Using known phylogenetic relationships within the grasses, we determined the ancestral branch-points where the losses occurred. Intron 7, the longest intron, was lost in only a single species, Mimulus guttatus, following duplication of ABCB1. Semiquantitative PCR showed that the M. guttatus ABCB1 gene copy without intron 7 had significantly lower transcript levels than the gene copy with intron 7. We further demonstrated that intron 7 carried two motifs that were highly conserved across the monocot-dicot divide. CONCLUSIONS: The ABCB1 gene structure is highly dynamic, while the structure of ABCB19 remained largely conserved through evolution. Precise removal of introns, preferential removal of smaller introns and presence of at least 2 bp of microhomology flanking most introns indicated that intron loss may have predominantly occurred through non-homologous end-joining (NHEJ) repair of double strand breaks. Lack of microhomology in the exon upstream of lost phase I introns was likely due to release of the selective constraint on the penultimate base (3rd base in codon) of the terminal codon by the splicing machinery. In addition to size, the presence of regulatory motifs will make introns recalcitrant to loss.

Fine-mapping and identification of a candidate gene underlying the d2 dwarfing phenotype in pearl millet, Cenchrus americanus (L.) Morrone.

Pearl millet is one of the most important subsistence crops grown in India and sub-Saharan Africa. In many cereal crops, reduced height is a key trait for enhancing yield, and dwarf mutants have been extensively used in breeding to reduce yield loss due to lodging under intense management. In pearl millet, the recessive d2 dwarfing gene has been deployed widely in commercial germplasm grown in India, the United States, and Australia. Despite its importance, very little research has gone into determining the identity of the d2 gene. We used comparative information, genetic mapping in two F2 populations representing a total of some 1500 progeny, and haplotype analysis of three tall and three dwarf inbred lines to delineate the d2 region by two genetic markers that, in sorghum, define a region of 410 kb with 40 annotated genes. One of the sorghum genes annotated within this region is ABCB1, which encodes a P-glycoprotein involved in auxin transport. This gene had previously been shown to underlie the economically important dw3 dwarf mutation in sorghum. The cosegregation of ABCB1 with the d2 phenotype, its differential expression in the tall inbred ICMP 451 and the dwarf inbred Tift 23DB, and the similar phenotype of stacked lower internodes in the sorghum dw3 and pearl millet d2 mutants suggest that ABCB1 is a likely candidate for d2.