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.

Carpels in maize undergo programmed cell death in half of the flowers initiated in ears and in all flowers in tassels. The HD-ZIP I transcription factor gene GRASSY TILLERS1 (GT1) is one of only a few genes known to regulate this process. To identify additional regulators of carpel suppression, we performed a gt1 enhancer screen and found a genetic interaction between gt1 and ramosa3 (ra3). RA3 is a classic inflorescence meristem determinacy gene that encodes a trehalose-6-phosphate (T6P) phosphatase (TPP). Dissection of floral development revealed that ra3 single mutants have partially derepressed carpels, whereas gt1;ra3 double mutants have completely derepressed carpels. Surprisingly, gt1 suppresses ra3 inflorescence branching, revealing a role for gt1 in meristem determinacy. Supporting these genetic interactions, GT1 and RA3 proteins colocalize to carpel nuclei in developing flowers. Global expression profiling revealed common genes misregulated in single and double mutant flowers, as well as in derepressed gt1 axillary meristems. Indeed, we found that ra3 enhances gt1 vegetative branching, similar to the roles for the trehalose pathway and GT1 homologs in the eudicots. This functional conservation over ∼160 million years of evolution reveals ancient roles for GT1-like genes and the trehalose pathway in regulating axillary meristem suppression, later recruited to mediate carpel suppression. Our findings expose hidden pleiotropy of classic maize genes and show how an ancient developmental program was redeployed to sculpt floral form.

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.

The abrupt origin and rapid diversification of the flowering plants presents what Darwin called "an abominable mystery". Floral diversification was a key factor in the rise of the flowering plants, but the molecular underpinnings of floral diversity remain mysterious. To understand the molecular biology underlying floral morphological evolution, genetic model systems are essential for rigorously testing gene function and gene interactions. Most model plants are eudicots, while in the monocots genetic models are almost entirely restricted to the grass family. Likely because grass flowers are diminutive and specialized for wind pollination, grasses have not been a major focus in floral evo-devo research. However, while grass flowers do not exhibit any of the raucous morphological diversification characteristic of the orchids, there is abundant floral variation in the family. Here, we discuss grass flower diversity, and review what is known about the developmental genetics of this diversity. In particular, we focus on three aspects of grass flower evolution: (1) the evolution of a novel organ identity-the lodicule; (2) lemma awns and their diversity; and (3) the convergent evolution of sexual differentiation. The combination of morphological diversity in the grass family at large and genetic models spread across the family provides a powerful framework for attaining deep understanding of the molecular genetics of floral evolution.

Widespread, comprehensive sequencing of patient tumors has facilitated the usage of precision medicine (PM) drugs to target specific genomic alterations. Therapeutic clinical trials are necessary to test new PM drugs to advance precision medicine, however, the abundance of patient sequencing data coupled with complex clinical trial eligibility has made it challenging to match patients to PM trials. To facilitate enrollment onto PM trials, we developed MatchMiner, an open-source platform to computationally match genomically profiled cancer patients to PM trials. Here, we describe MatchMiner’s capabilities, outline its deployment at Dana-Farber Cancer Institute (DFCI), and characterize its impact on PM trial enrollment. MatchMiner’s primary goals are to facilitate PM trial options for all patients and accelerate trial enrollment onto PM trials. MatchMiner can help clinicians find trial options for an individual patient or provide trial teams with candidate patients matching their trial’s eligibility criteria. From March 2016 through March 2021, we curated 354 PM trials containing a broad range of genomic and clinical eligibility criteria and MatchMiner facilitated 166 trial consents (MatchMiner consents, MMC) for 159 patients. To quantify MatchMiner’s impact on trial consent, we measured time from genomic sequencing report date to trial consent date for the 166 MMC compared to trial consents not facilitated by MatchMiner (non-MMC). We found MMC consented to trials 55 days (22%) earlier than non-MMC. MatchMiner has enabled our clinicians to match patients to PM trials and accelerated the trial enrollment process.

The systematic deployment of next generation sequencing means patient tumors can be genomically profiled and specific genetic alterations can be targeted with precision medicine (PM) drugs. More therapeutic clinical trials are needed to test new PM drugs to advance precision medicine, however, the availability of comprehensive patient sequencing data coupled with complex clinical trial eligibility has made it challenging to match patients to PM trials. To facilitate enrollment onto PM trials, we developed MatchMiner. MatchMiner is an open-source platform to computationally match genomically profiled cancer patients to PM trials. Here, we describe MatchMiner's capabilities, outline its deployment at Dana-Farber Cancer Institute (DFCI), and characterize its impact on PM trial enrollment. MatchMiner's two primary goals are to (1) facilitate PM trial options for all patients, and (2) accelerate trial enrollment onto PM trials. MatchMiner has 3 main modes of use: (1) patient-centric, where a clinician looks up trial options for an individual patient, (2) trial-centric, where a trial team identifies candidate patients for their trial by setting up a filter, and (3) trial search, where a clinician can find trial options for patients that have external genomic reports. From the time MatchMiner was first deployed at DFCI in March 2016 through March 2021, we curated 354 PM trials containing a broad range of genomic and clinical eligibility criteria and MatchMiner facilitated 166 trial consents (MatchMiner consents, MMC) for 159 patients. To quantify MatchMiner's impact on trial consent, we retrospectively measured time from genomic sequencing report date to trial consent date for the 166 MMC compared to trial consents not facilitated by MatchMiner (non-MMC). We found MMC consented to trials 55 days (22%) earlier than non-MMC. MatchMiner has enabled our clinicians to match patients to PM trials and accelerated the trial enrollment decision making process.

Floral morphology is immensely diverse. One developmental process acting to shape this diversity is growth suppression. For example, grass flowers exhibit extreme diversity in floral sexuality, arising through differential suppression of stamens or carpels. In maize, carpels undergo programmed cell death in half of the flowers initiated in ears and in all flowers in tassels. The HD-ZIP I transcription factor gene GRASSY TILLERS1 (GT1) is one of only a few genes known to regulate this process. To identify additional regulators of carpel suppression, we performed a gt1 enhancer screen, and found a genetic interaction between gt1 and ramosa3 (ra3). RA3 is a classic inflorescence meristem determinacy gene that encodes a trehalose-6-phosphate (T6P) phosphatase (TPP). Dissection of floral development revealed that ra3 single mutants have partially derepressed carpels, whereas gt1; ra3 double mutants have completely derepressed carpels. Surprisingly, gt1 suppresses ra3 inflorescence branching, revealing a role for gt1 in meristem determinacy. Supporting these genetic interactions, GT1 and RA3 proteins colocalize to carpel nuclei in developing flowers. Global expression profiling revealed common genes misregulated in single and double mutant flowers, as well as in derepressed gt1 axillary meristems. Indeed, we found that ra3 enhances gt1 vegetative branching, similar to the roles for the trehalose pathway and GT1 homologs in the eudicots. This functional conservation over ~160 million years of evolution reveals ancient roles for GT1-like genes and the trehalose pathway in regulating axillary meristem suppression, later recruited to mediate carpel suppression. Our findings expose hidden pleiotropy of classic maize genes, and show how an ancient developmental program was redeployed to sculpt floral form.

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