Transformation of Teosinte (Zea mays ssp. parviglumis) via Biolistic Bombardment of Seedling-Derived Callus Tissues

Modern maize exhibits a significantly different phenotype than its wild progenitor teosinte despite many genetic similarities. Of the many subspecies of Zea mays identified as teosinte, Zea mays ssp. parviglumis is the most closely related to domesticated maize. Understanding teosinte genes and their regulations can provide great insights into the maize domestication process and facilitate breeding for future crop improvement. However, a protocol of genetic transformation, which is essential for gene functional analyses, is not available in teosinte. In this study, we report the establishment of a robust callus induction and regeneration protocol using whorl segments of seedlings germinated from mature seeds of Zea parviglumis. We also report, for the first time, the production of fertile, transgenic teosinte plants using the particle bombardment. Using herbicide resistance genes such as mutant acetolactate synthase (Als) or bialaphos resistance (bar) as selectable markers, we achieved an average transformation frequency of 4.17% (percentage of independent transgenic events in total bombarded explants that produced callus). Expression of visual marker genes of red fluorescent protein tdTomato and β-glucuronidase (gus) could be detected in bombarded callus culture and in T1 and T2 progeny plants. The protocol established in this work provides a major enabling technology for research toward the understanding of this important plant in crop domestication.

N-cycling microbiome recruitment differences between modern and wild Zea mays

Rewilding modern agricultural cultivars by reintroducing beneficial ancestral traits is a proposed approach to improve sustainability of modern agricultural systems. In this study, we compared recruitment of the rhizosphere microbiome among modern inbred maize and wild teosinte to assess whether potentially beneficial plant microbiome traits have been lost through maize domestication and modern breeding. To do this, we surveyed the bacterial and fungal communities along with nitrogen cycling functional groups in the rhizosphere of 6 modern domesticated maize genotypes and ancestral wild teosinte genotypes, while controlling for environmental conditions and starting soil inoculum. Using a combination of high-throughput sequencing and quantitative PCR, we found that the rhizosphere microbiomes of modern inbred and wild teosinte differed substantially in taxonomic composition, species richness, and abundance of N-cycling functional genes. Furthermore, the modern vs wild designation explained 27% of the variation in the prokaryotic microbiome, 62% of the variation in N-cycling gene richness, and 66% of N-cycling gene abundance. Surprisingly, we found that modern inbred genotypes hosted microbial communities with higher taxonomic and functional gene diversity within their microbiomes compared to ancestral genotypes. These results imply that modern maize and wild maize differ in their interaction with N-cycling microorganisms in the rhizosphere and that genetic variation exists within Zea to potentially ‘rewild’ microbiome-associated traits (i.e., exudation, root phenotypes, etc.).

Combining QTL-seq and linkage mapping to uncover the genetic basis of single vs. paired spikelets in the advanced populations of two-ranked maize×teosinte

Background Teosinte ear bears single spikelet, whereas maize ear bears paired spikelets, doubling the number of grains in each cupulate during maize domestication. In the past 20 years, genetic analysis of single vs. paired spikelets (PEDS) has been stagnant. A better understanding of genetic basis of PEDS could help fine mapping of quantitative trait loci (QTL) and cloning of genes. Results In this study, the advanced mapping populations (BC3F2 and BC4F2) of maize × teosinte were developed by phenotypic recurrent selection. Four genomic regions associated with PEDS were detected using QTL-seq, located on 194.64–299.52 Mb, 0–162.80 Mb, 12.82–97.17 Mb, and 125.06–157.01 Mb of chromosomes 1, 3, 6, and 8, respectively. Five QTL for PEDS were identified in the regions of QTL-seq using traditional QTL mapping. Each QTL explained 1.12–38.05% of the phenotypic variance (PVE); notably, QTL qPEDS3.1 with the average PVE of 35.29% was identified in all tests. Moreover, 14 epistatic QTL were detected, with the total PVE of 47.57–66.81% in each test. The QTL qPEDS3.1 overlapped with, or was close to, one locus of 7 epistatic QTL. Near-isogenic lines (NILs) of QTL qPEDS1.1, qPEDS3.1, qPEDS6.1, and qPEDS8.1 were constructed. All individuals of NIL-qPEDS6.1(MT1) and NIL-qPEDS8.1(MT1) showed paired spikelets (PEDS = 0), but the flowering time was 7 days shorter in the NIL-qPEDS8.1(MT1). The ratio of plants with PEDS > 0 was low (1/18 to 3/18) in the NIL-qPEDS1.1(MT1) and NIL-qPEDS3.1(MT1), maybe due to the epistatic effect. Conclusion Our results suggested that major QTL, minor QTL, epistasis and photoperiod were associated with the variation of PEDS, which help us better understand the genetic basis of PEDS and provide a genetic resource for fine mapping of QTL.

Nucleotide Diversity of the Maize ZmCNR13 Gene and Association With Ear Traits

The maize (Zea mays L.) ZmCNR13 gene, encoding a protein of fw2.2-like (FWL) family, has been demonstrated to be involved in cell division, expansion, and differentiation. In the present study, the genomic sequences of the ZmCNR13 locus were re-sequenced in 224 inbred lines, 56 landraces and 30 teosintes, and the nucleotide polymorphism and selection signature were estimated. A total of 501 variants, including 415 SNPs and 86 Indels, were detected. Among them, 51 SNPs and 4 Indels were located in the coding regions. Although neutrality tests revealed that this locus had escaped from artificial selection during the process of maize domestication, the population of inbred lines possesses lower nucleotide diversity and decay of linkage disequilibrium. To estimate the association between sequence variants of ZmCNR13 and maize ear characteristics, a total of ten ear-related traits were obtained from the selected inbred lines. Four variants were found to be significantly associated with six ear-related traits. Among them, SNP2305, a non-synonymous mutation in exon 2, was found to be associated with ear weight, ear grain weight, ear diameter and ear row number, and explained 4.59, 4.61, 4.31, and 8.42% of the phenotypic variations, respectively. These results revealed that natural variations of ZmCNR13 might be involved in ear development and can be used in genetic improvement of maize ear-related traits.

Plasticity and domestication of root anatomy in maize-teosinte derived population

Maize (Zea mays L.) underwent profound changes during domestication in root anatomy for environmental adaptation. However, the genetic mechanism of maize root anatomy domestication and plasticity remains unclear. In this study, a high-resolution mapping was performed for nine root anatomical traits using a maize-teosinte population (mexicana × Mo17) across three environments. Large genetic variations were detected for different root anatomical traits. The cortex area, stele area, aerenchyma area, xylem vessels number and cortical cell number had large variations across three environments, indicating high plasticity. Sixteen quantitative trait loci (QTL) were identified, including seven QTL with QTL × Environment interaction (EIQTL) for high plastic traits and nine QTL without QTL × Environment interaction (SQTL). Most of the root loci were consistent with shoot QTL depicting domestication signals. Combining transcriptome and genome-wide association study (GWAS) revealed that ZmPILS4 serve as a candidate gene underlying a major QTL of xylem traits. The near-isogenic lines (NILs) with lower expression of ZmPILS4 had 18-24% more IAA concentration in the root tip and 8-15% more xylem vessels. Significant domestication signal in promoter region suggested that ZmPILS4 was involved in maize domestication and adaptation. These results divulged the potential genetic basis of root anatomy plasticity and domestication.

Megabase-scale presence-absence variation with Tripsacum origin was under selection during maize domestication and adaptation

Background Structural variants (SVs) significantly drive genome diversity and environmental adaptation for diverse species. Unlike the prevalent small SVs (< kilobase-scale) in higher eukaryotes, large-size SVs rarely exist in the genome, but they function as one of the key evolutionary forces for speciation and adaptation. Results In this study, we discover and characterize several megabase-scale presence-absence variations (PAVs) in the maize genome. Surprisingly, we identify a 3.2 Mb PAV fragment that shows high integrity and is present as complete presence or absence in the natural diversity panel. This PAV is embedded within the nucleolus organizer region (NOR), where the suppressed recombination is found to maintain the PAV against the evolutionary variation. Interestingly, by analyzing the sequence of this PAV, we not only reveal the domestication trace from teosinte to modern maize, but also the footprints of its origin from Tripsacum, shedding light on a previously unknown contribution from Tripsacum to the speciation of Zea species. The functional consequence of the Tripsacum segment migration is also investigated, and environmental fitness conferred by the PAV may explain the whole segment as a selection target during maize domestication and improvement. Conclusions These findings provide a novel perspective that Tripsacum contributes to Zea speciation, and also instantiate a strategy for evolutionary and functional analysis of the “fossil” structure variations during genome evolution and speciation.

Characterization of a major QTL and Genome-Wide Epistatic Interactions for the Transformation of Single Spikelet in Teosinte Ears into Paired Spikelets in Maize Ears During Maize Domestication

Maize ear carries paired spikelets, whereas the ear of its wild ancestor, teosinte, bears single spikelets. However, little is known about the genetic basis of the processes of transformation of single spikelets in teosinte ear to paired spikelets in maize ear. In this study, a two-ranked, paired-spikelets primitive maize and a two-ranked, single-spikelet teosinte were utilized to develop an F2 population, and QTL mapping for single vs. paired spikelets (PEDS) was performed. Two QTL (qPEDS1.1 and qPEDS3.1) for PEDS located on chromosomes 1L and 3S were identified in the 162 F2 plants using the inclusive composite interval mapping of additive (ICIM-ADD) module, explaining 1.93% and 23.79% of the phenotypic variance, respectively. Out of the 409 F2 plants, 43 plants with PEDS = 0% and 43 plants with PEDS > 20% were selected for selective genotyping; the QTL (qPEDS3.1) accounting for 64.01% of the phenotypic variance for PEDS was also detected. Moreover, the QTL (qPEDS3.1) was validated in three environments, which explained 31.05%, 38.94% and 23.16% of the phenotypic variance, respectively. In addition, 50 epistatic QTLs were detected in 162 F2 plants using the two-locus epistatic QTL (ICIM-EPI) module; they were distributed on all 10 chromosomes and explained 94.40% of the total phenotypic variance. The results contribute to a better understanding of the genetic basis of domestication of paired spikelets and provide a genetic resource for future map-based cloning; in addition, the systematic dissection of epistatic interactions underlies a theoretical framework for overcoming epistatic effects on QTL fine mapping.

Wild Progenitor and Landraces Led Genetic Gain in the Modern-Day Maize (Zea mays L.)

Maize (Zea mays ssp. mays) originated from Mexico and Central America and grew worldwide for food, feed and industrial products components. It possesses ten chromosomes with a genome size of 2.3 gigabases. Teosinte (Z. mays ssp. parviglumis) is the probable progenitor of the modern-day maize. The maize domestication favored standing gain of function and regulatory variations acquired the convergent phenotypes. The genomic loci teosinte branched 1 (tb1) and teosinte glume architecture 1 (tga1) played a central role in transforming teosinte to modern-day maize. Under domestication and crop improvement, only 2% (~1200) genes were undergone selection, out of ~60000 genes. Around ~98% of the genes have not experienced selection; there is enormous variation present in the diverse inbred lines that can be potentially utilized to identify QTLs and crop improvement through plant breeding. The genomic resources of wild relatives and landraces harbor the unexplored genes/alleles for biotic/abiotic tolerance, productivity and nutritional quality. The human-made evolution led to the transformation of wild relatives/landraces to the modern-day maize. This chapter summarized the maize’s wild relatives/landraces and the genetic gain over time in biotic/abiotic, productivity, and nutritional quality traits.

Population genomics of the maize pathogen Ustilago maydis: demographic history and role of virulence clusters in adaptation

The tight interaction between pathogens and their hosts results in reciprocal selective forces that impact the genetic diversity of the interacting species. The footprints of this selection differ between pathosystems because of distinct life-history traits, demographic histories, or genome architectures. Here, we studied the genome-wide patterns of genetic diversity of 22 isolates of the causative agent of the corn smut disease, Ustilago maydis, originating from five locations in Mexico, the presumed center of origin of this species. In this species, many genes encoding secreted effector proteins reside in so-called virulence clusters in the genome, an arrangement that is so far not found in other filamentous plant pathogens. Using a combination of population genomic statistical analyses, we assessed the geographical, historical and genome-wide variation of genetic diversity in this fungal pathogen.We report evidence of two partially admixed subpopulations that are only loosely associated with geographic origin. Using the multiple sequentially Markov coalescent model, we inferred the demographic history of the two pathogen subpopulations over the last 0.5 million years. We show that both populations experienced a recent strong bottleneck starting around 10,000 years ago, coinciding with the assumed time of maize domestication. While the genome average genetic diversity is low compared to other fungal pathogens, we estimated that the rate of non-synonymous adaptive substitutions is three times higher in genes located within virulence clusters compared to non-clustered genes, including non-clustered effector genes. These results highlight the role that these singular genomic regions play in the evolution of this pathogen.Significance statementThe maize pathogen Ustilago maydis is a model species to study fungal cell biology and biotrophic host-pathogen interactions. Population genetic studies of this species, however, were so far restricted to using a few molecular markers, and genome-wide comparisons involved species that diverged more than 20 million years ago. Here, we sequenced the genomes of 22 Mexican U. maydis isolates to study the recent evolutionary history of this species. We identified two co-existing populations that went through a recent bottleneck and whose divergence date overlaps with the time of maize domestication. Contrasting the patterns of genetic diversity in different categories of genes, we further showed that effector genes in virulence clusters display a high rate of adaptive mutations, highlighting the importance of these effector arrangements for the adaptation of U. maydis to its host.