Genic and nongenic contributions to natural variation of quantitative traits in maize.

The complex genomes of many economically important crops present tremendous challenges to understand the genetic control of many quantitative traits with great importance in crop production, adaptation, and evolution. Advances in genomic technology need to be integrated with strategic genetic design and novel perspectives to break new ground. Complementary to individual-gene-targeted research, which remains challenging, a global assessment of the genomic distribution of trait-associated SNPs (TASs) discovered from genome scans of quantitative traits can provide insights into the genetic architecture and contribute to the design of future studies. Here we report the first systematic tabulation of the relative contribution of different genomic regions to quantitative trait variation in maize. We found that TASs were enriched in the nongenic regions, particularly within a 5-kb window upstream of genes, which highlights the importance of polymorphisms regulating gene expression in shaping the natural variation. Consistent with these findings, TASs collectively explained 44%-59% of the total phenotypic variation across maize quantitative traits, and on average, 79% of the explained variation could be attributed to TASs located in genes or within 5 kb upstream of genes, which together comprise only 13% of the genome. Our findings suggest that efficient, cost-effective genome-wide association studies (GWAS) in species with complex genomes can focus on genic and promoter regions.

Ontogeny of the maize shoot apical meristem.

The maize (Zea mays) shoot apical meristem (SAM) arises early in embryogenesis and functions during stem cell maintenance and organogenesis to generate all the aboveground organs of the plant. Despite its integral role in maize shoot development, little is known about the molecular mechanisms of SAM initiation. Laser microdissection of apical domains from developing maize embryos and seedlings was combined with RNA sequencing for transcriptomic analyses of SAM ontogeny. Molecular markers of key events during maize embryogenesis are described, and comprehensive transcriptional data from six stages in maize shoot development are generated. Transcriptomic profiling before and after SAM initiation indicates that organogenesis precedes stem cell maintenance in maize; analyses of the first three lateral organs elaborated from maize embryos provides insight into their homology and to the identity of the single maize cotyledon. Compared with the newly initiated SAM, the mature SAM is enriched for transcripts that function in transcriptional regulation, hormonal signaling, and transport. Comparisons of shoot meristems initiating juvenile leaves, adult leaves, and husk leaves illustrate differences in phase-specific (juvenile versus adult) and meristem-specific (SAM versus lateral meristem) transcript accumulation during maize shoot development. This study provides insight into the molecular genetics of SAM initiation and function in maize.

Developmental and cell type characterization of bundle sheath and mesophyll chloroplast transcript abundance in maize.

The C4 grass Zea mays separates light and light-independent photosynthetic processes into two leaf cell types: bundle sheath (BS) and mesophyll (M). When mature, BS and M cells have anatomically and biochemically distinct chloroplasts that must cooperate to complete the process of photosynthesis. This report compares changes in transcript abundance between young and mature maize BS and M chloroplasts from specific segments of the leaf developmental gradient. Representative transcripts encoding components of Photosystem I, Photosystem II, Cytochrome b (6) f, thylakoidal NADH dehydrogenase; and the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase as well as nine nuclear-coded transcripts encoding chloroplast proteins were measured using quantitative RT-PCR. In addition, 887 nuclear genes encoding plastid-localized proteins, as well as 64 chloroplast and 34 mitochondrial genes were assayed utilizing a cDNA microarray. In 9 out of the 18 chloroplast-encoded genes and 84 genes from the 985 element microarray revealed greater than twofold transcript abundance differences between developmental stages and/or cell types. Patterns for transcripts associated with operons and gene clusters suggest differing regulatory mechanisms for particular polycistronic stretches. In summary, this report provides evidence that cell type-specific transcript abundance varies more in the young developing chloroplast, and differences plateau or subside as chloroplasts mature.

A next-generation marker genotyping platform (AmpSeq) in heterozygous crops: a case study for marker-assisted selection in grapevine.

Marker-assisted selection (MAS) is often employed in crop breeding programs to accelerate and enhance cultivar development, via selection during the juvenile phase and parental selection prior to crossing. Next-generation sequencing and its derivative technologies have been used for genome-wide molecular marker discovery. To bridge the gap between marker development and MAS implementation, this study developed a novel practical strategy with a semi-automated pipeline that incorporates trait-associated single nucleotide polymorphism marker discovery, low-cost genotyping through amplicon sequencing (AmpSeq) and decision making. The results document the development of a MAS package derived from genotyping-by-sequencing using three traits (flower sex, disease resistance and acylated anthocyanins) in grapevine breeding. The vast majority of sequence reads (⩾99%) were from the targeted regions. Across 380 individuals and up to 31 amplicons sequenced in each lane of MiSeq data, most amplicons (83 to 87%) had <10% missing data, and read depth had a median of 220-244×. Several strengths of the AmpSeq platform that make this approach of broad interest in diverse crop species include accuracy, flexibility, speed, high-throughput, low-cost and easily automated analysis.

Discolored1 (DSC1) is an ADP-Ribosylation Factor-GTPase Activating Protein Required to Maintain Differentiation of Maize Kernel Structures.

The embryo and endosperm are the products of double fertilization and comprise the clonally distinct products of angiosperm seed development. Recessive mutations in the maize gene discolored1 (dsc1) condition inviable seed that are defective in both embryo and endosperm development. Here, detailed phenotypic analyses illustrate that discolored mutant kernels are able to establish, but fail to maintain, differentiated embryo, and endosperm structures. Development of the discolored mutant embryo and endosperm is normal albeit delayed, prior to the abortion and subsequent degeneration of all differentiated kernel structures. Using a genomic fragment that was previously isolated by transposon tagging, the full length dsc1 transcript is identified and shown to encode an ADP-ribosylation factor-GTPase activating protein (ARF-GAP) that co-localizes with the trans-Golgi network/early endosomes and the plasma membrane during transient expression assays in N. benthamiana leaves. DSC1 function during endomembrane trafficking and the maintenance of maize kernel differentiation is discussed.

Nuclear, chloroplast, and mitochondrial transcript abundance along a maize leaf developmental gradient.

In maize, the chloroplast chromosome encodes 104 genes whose roles are primarily in photosynthesis and gene expression. The 2,000-3,000 nuclear gene products that localize to plastids are required both to encode and regulate plastid gene expression as well as to underpin each aspect of plastid physiology and development. We used a new "three-genome" maize biogenesis cDNA microarray to track abundance changes in nuclear, chloroplast and mitochondrial transcripts in stage 2 semi-emerged leaf blades of one month-old maize plants. We report the detection and quantification of 433 nuclear, 62 chloroplast, and 27 mitochondrial transcripts, with the majority of the nuclear transcripts predicted or known to encode plastid proteins. The data were analyzed as ratios of expression of individual transcripts in the green tip (mature chloroplasts) versus the yellow base of the leaf (etioplasts). According to the microarray data at least 51 plastid genes and 121 nuclear genes are expressed at least two-fold higher in the tip of the leaf. Almost all (25) mitochondrial and 177 nuclear transcripts were expressed at least 2-fold higher in the leaf base. Independent quantification of a subset of each transcript population by RNA gel blot analysis and/or quantitative real time RT-PCR concurred with the transcript ratios determined by the array. Ontological distribution of the transcripts suggests that photosynthesis-related RNAs were most highly abundant in the leaf tip and that energy use genes were most highly expressed in the base. Transcripts whose products are used in plastid translation constituted the largest single ontological group with relatively equal numbers of genes in the three expression categories, defined as higher in tip, higher in base, or equally expressed in tip and base.