Bulked-Segregant Analysis Coupled to Whole Genome Sequencing (BSA-Seq) for Rapid Gene Cloning in Maize

Forward genetics remains a powerful method for revealing the genes underpinning organismal form and function, and for revealing how these genes are tied together in gene networks. In maize, forward genetics has been tremendously successful, but the size and complexity of the maize genome made identifying mutant genes an often arduous process with traditional methods. The next generation sequencing revolution has allowed for the gene cloning process to be significantly accelerated in many organisms, even when genomes are large and complex. Here, we describe a bulked-segregant analysis sequencing (BSA-Seq) protocol for cloning mutant genes in maize. Our simple strategy can be used to quickly identify a mapping interval and candidate single nucleotide polymorphisms (SNPs) from whole genome sequencing of pooled F2 individuals. We employed this strategy to identify narrow odd dwarf as an enhancer of teosinte branched1, and to identify a new allele of defective kernel1. Our method provides a quick, simple way to clone genes in maize

Boundary domain genes were recruited to suppress bract growth and promote branching in maize

Grass inflorescence development is diverse and complex and involves sophisticated but poorly understood interactions of genes regulating branch determinacy and leaf growth. Here, we use a combination of transcript profiling and genetic and phylogenetic analyses to investigate tasselsheath1 (tsh1) and tsh4, two maize genes that simultaneously suppress inflorescence leaf growth and promote branching. We identify a regulatory network of inflorescence leaf suppression that involves the phase change gene tsh4 upstream of tsh1 and the ligule identity gene liguleless2 (lg2). We also find that a series of duplications in the tsh1 gene lineage facilitated its shift from boundary domain in nongrasses to suppressed inflorescence leaves of grasses. Collectively, these results suggest that the boundary domain genes tsh1 and lg2 were recruited to inflorescence leaves where they suppress growth and regulate a nonautonomous signaling center that promotes inflorescence branching, an important component of yield in cereal grasses

B and C class MADS-box genes and the developmental genetics of maize flower development

The ABC model of flower development describes how a flower is patterned and the genes necessary for floral organ identity. However, it is not clear that the ABC model can be generally applied to the flowering plants, as it was based solely on genetic studies from the core eudicot species Arabidopsis and Antirrhinum. This dissertation describes an examination of maize orthologs of B and C class genes, and compares their function with B and C class genes of Arabidopsis to understand the degree to which the ABC model is conserved. B class genes from maize were found to rescue Arabidopsis B class mutants, and the maize B class proteins were shown to bind DNA as an obligate heterodimer as has been demonstrated in Arabidopsis. These findings indicate conservation in biochemical function of the maize and Arabidopsis B class proteins. Furthermore, these findings support the conclusion that the lodicule, a grass specific organ of uncertain homology, represents a modified petal. A comparative expression approach was used to further verify the relationship of lodicules to the organs of non-grass flowers. B class genes were shown to be expressed in a whorl of foliar organs outside the stamens in Streptochaeta, a basal grass that diverged before the evolution of lodicules, and in the petals of the outgroup species Joinvillea and Chondropetalum strongly supporting the interpretation that lodicules are modified petals, and further supporting conservation of B class function between Arabidopsis and maize. Zag1 and Zmm2 are duplicate pair of C class genes from maize that are hypothesized to have partitioned the C class function of establishing stamen and carpel identity. Rescue of the Arabidopsis C class mutant ag with the two maize genes confirms that their protein products have subfunctionalized, with ZAG1 better able to promote carpel identity, and ZMM2 better able to promote stamen identity. A more recent duplicate of Zmm2 was isolated, Zmm23, as were mutant alleles of zmm2 and zmm23. While the zmm2 zmm23 double mutant had no phenotype, the zag1 zmm2 zmm23 showed a considerable enhancement of the previously described zag1 phenotype substantiating a C class function for Zmm2 and Zmm2

Protein change in plant evolution: tracing one thread connecting molecular and phenotypic diversity

Proteins change over the course of evolutionary time. New protein-coding genes and gene families emerge and diversify, ultimately affecting an organism’s phenotype and interactions with its environment. Here we survey the range of structural protein change observed in plants and review the role these changes have had in the evolution of plant form and function. Verified examples tying evolutionary change in protein structure to phenotypic change remain scarce. We will review the existing examples, as well as draw from investigations into domestication, and quantitative trait locus (QTL) cloning studies searching for the molecular underpinnings of natural variation. The evolutionary significance of many cloned QTL has not been assessed, but all the examples identified so far have begun to reveal the extent of protein structural diversity tolerated in natural systems. This molecular (and phenotypic) diversity could come to represent part of natural selection’s source material in the adaptive evolution of novel traits. Protein structure and function can change in many distinct ways, but the changes we identified in studies of natural diversity and protein evolution were predicted to fall primarily into one of six categories: altered active and binding sites; hypomorphic and hypermorphic alleles; altered protein-protein interactions; altered domain content; altered protein stability; and altered activity as an activator or repressor. Variability was also observed in the evolutionary scale at which particular changes were observed. Some changes were detected at both micro- and macroevolutionary timescales, while others were observed primarily at deep or shallow phylogenetic levels. This variation might be used to determine the trajectory of future investigations in structural molecular evolution

Conservation of B class gene expression in the second whorl of a basal grass and outgroups links the origin of lodicules and petals

Studies of flower development in core eudicot species have established a central role for B class MADS-box genes in specifying petal and stamen identities. Similarly in maize and rice, B class genes are essential for lodicule and stamen specification, suggesting homology of petals and lodicules and conservation of B class gene activity across angiosperms. However, lodicules are grass-specific organs with a morphology distinct from petals, thus their true homology to eudicot and nongrass monocot floral organs has been a topic of debate. To understand the relationship of lodicules to the sterile floral organs of nongrass monocots we have isolated and observed the expression of B class genes from a basal grass Streptochaeta that diverged before the evolution of lodicules, as well as the outgroups Joinvillea and Elegia, which have a typical monocot floral plan. Our results support a conserved role for B function genes across the angiosperms and provide additional evidence linking the evolution of lodicules and second whorl tepal/petals of monocots. The expression data and morphological analysis suggest that the function of B class genes should be broadly interpreted as required for differentiation of a distinct second floral whorl as opposed to specifying petal identity per se

A Conserved Mechanism of Bract Suppression in the Grass Family[W][OA]

Bract suppression in maize, rice, and barley is regulated by a conserved genetic mechanism. Interestingly, the orthologous gene in Arabidopsis has no role in bract suppression, suggesting distinct bract suppression mechanisms have evolved in these two lineages

Finemapping Genotype and Phenotype

Genotype and phenotype data from 45 RC-NILs and 2 NI-RILs used in fine-mapping etb1.

Data from: A gene for genetic background in Zea mays: fine-mapping enhancer of teosinte branched1.2 to a YABBY class transcription factor

The effects of an allelic substitution at a gene often depend critically on genetic background, i.e., the genotypes at other genes in the genome. During the domestication of maize from its wild ancestor (teosinte), an allelic substitution at teosinte branched (tb1) caused changes in both plant and ear architecture. The effects of tb1 on phenotype were shown to depend on multiple background loci, including one called enhancer of tb1.2 (etb1.2). We mapped etb1.2 to a YABBY class transcription factor (ZmYAB2.1) and showed that the maize alleles of ZmYAB2.1 are either expressed at a lower level than teosinte alleles or disrupted by insertions in the sequences. tb1 and etb1.2 interact epistatically to control the length of internodes within the maize ear, which affects how densely the kernels are packed on the ear. The interaction effect is also observed at the level of gene expression, with tb1 acting as a repressor of ZmYAB2.1 expression. Curiously, ZmYAB2.1 was previously identified as a candidate gene for another domestication trait in maize, nonshattering ears. Consistent with this proposed role, ZmYAB2.1 is expressed in a narrow band of cells in immature ears that appears to represent a vestigial abscission (shattering) zone. Expression in this band of cells may also underlie the effect on internode elongation. The identification of ZmYAB2.1 as a background factor interacting with tb1 is a first step toward a gene-level understanding of how tb1 and the background within which it works evolved in concert during maize domestication