Tillers are vegetative branches that develop from axillary buds located in the leaf axils at the base of many grasses. Genetic manipulation of tillering is a major objective in breeding for improved cereal yields and competition with weeds. Despite this, very little is known about the molecular genetic bases of tiller development in important Triticeae crops such as barley (Hordeum vulgare) and wheat (Triticum aestivum). Recessive mutations at the barley Uniculme4 (Cul4) locus cause reduced tillering, deregulation of the number of axillary buds in an axil, and alterations in leaf proximal-distal patterning. We isolated the Cul4 gene by positional cloning and showed that it encodes a BROAD-COMPLEX, TRAMTRACK, BRIC-À-BRAC-ankyrin protein closely related to Arabidopsis (Arabidopsis thaliana) BLADE-ON-PETIOLE1 (BOP1) and BOP2. Morphological, histological, and in situ RNA expression analyses indicate that Cul4 acts at axil and leaf boundary regions to control axillary bud differentiation as well as the development of the ligule, which separates the distal blade and proximal sheath of the leaf. As, to our knowledge, the first functionally characterized BOP gene in monocots, Cul4 suggests the partial conservation of BOP gene function between dicots and monocots, while phylogenetic analyses highlight distinct evolutionary patterns in the two lineages.
SummaryEtched1 (et1) is a pleiotropic, recessive mutation of maize that causes ®ssured and cracked mature kernels and virescent seedlings. Microscopic examinations of the et1 phenotype revealed an aberrant plastid development in mutant kernels and mutant leaves. Here, we report on the cloning of the et1 gene by transposon tagging, the localization of the gene product in chloroplasts, and its putative function in the plastid transcriptional apparatus. Several alleles of Mutator (Mu)-induced et1 mutants, the et1-reference (et1-R) mutant, and Et1 wild-type were cloned and analyzed at the molecular level. Northern analyses with wildtype plants revealed that Et1 transcripts are present in kernels, leaves, and other types of tissue, and no Et1 expression could be detected in the et1 mutants analyzed. The ET1 protein is imported by chloroplasts and has been immunologically detected in transcriptionally active chromosome (TAC) fractions derived from chloroplasts. Accordingly, the relative transcriptional activity of TAC fractions was signi®cantly reduced in chloroplasts of et1-R plants. ET1 is the ®rst zinc ribbon (ZR) protein shown to be targeted to plastids. With regard to its localization and its striking structural similarity to the eukaryotic transcription elongation factor TFIIS, it is feasible that ET1 functions in plastid transcription elongation by reactivation of arrested RNA polymerases.Keywords: etched1, plastid nucleoids, transcriptional active chromosome, transposon tagging, plastid transcription, TFIIS. IntroductionThe analysis of genes involved in the development of the maize endosperm is of particular interest in order to understand the structural and regulatory features of seed growth. In maize, many mutants and a number of genes affecting tissue and organ development have been isolated. Numerous mutants have been described, which exhibit abnormal endosperm development (for review: Coe et al., 1988). Among these, a few are pleiotropic and thus of particular interest because they affect the development of different tissue types. Etched1 (et1) is one such mutation, which affects the development of kernels as well as of seedlings.Etched1 is a recessive mutation that was ®rst identi®ed and described by Stadler (1940). The mutant reference allele (et1-reference (et1-R)) was isolated from the progeny of a population of maize plants pollinated with X-ray irradiated pollen. Kernels homozygous for the et1-R allele are ®ssured because of depressions and crevices on the endosperm surface (Figure 1a±c). Prior biochemical and structural analyses of the et1-R kernels revealed that starch synthesis is reduced and starchless endosperm cells are present around the cracks and scars in the kernels (Figure 1; Sangeetha and Reddy, 1991). The et1 kernel phenotype becomes visible approximately 15 days after pollination (DAP). The degree of etching differs among the kernels on an ear and can vary from a weak to a very severe phenotype. This phenotypic variation is apparently not correlated with any speci®c genetic background ...
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.
Microsatellite loci were isolated from the allotetraploid aquatic plant Hymenocallis coronaria. A repeat‐enriched genomic library was constructed and primer pairs designed, resulting in six polymorphic loci. A total of 230 individuals were genotyped, and allelic richness per locus ranged from three to 11, while observed heterozygosity ranged from 0.017 to 0.570. Some amplified products were excised from agarose gel and sequenced to confirm primer specificity and mutation model. These are the first microsatellite markers developed for any member of this genus, and cross‐amplification was successful with the only other member of the genus tested and with a member of the related genus Zephyranthes.
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