Ribosome binding of phasiRNA precursors accelerates the 24-nt phasiRNA burst in meiotic maize anthers.

Reproductive phasiRNAs (phased, small interfering RNAs), produced from numerous PHAS loci, are essential for plant anther development. PHAS transcripts are enriched on endoplasmic reticulum-bound ribosomes in maize (Zea mays), but the impact of ribosome binding on phasiRNA biogenesis remains elusive. Through ribosome profiling of maize anthers at 10 developmental stages, we demonstrated that 24-PHAS transcripts are bound by ribosomes, with patterns corresponding to the timing and abundance of 24-PHAS transcripts. Ribosome binding to 24-PHAS transcripts is conserved among different maize inbred lines, with ribosomes enriched upstream of miR2275 target sites. We detected short open reading frames (sORFs) in the ribosome-binding regions of some 24-PHAS transcripts and observed a 3-nt periodicity in most sORFs, but mass spectrometry failed to detect peptides corresponding to the sORFs. Deletion of the entire ribosome-binding region of 24PHAS_NO296 locus eliminated ribosome binding and decreased 24-nt phasiRNA production, without affecting 24PHAS_NO296 transcript levels. In contrast, disrupting only the sORFs in 24PHAS_NO296 did not substantially affect the generation of 24-nt phasiRNAs. A newly formed sORF in these mutants may have re-directed ribosome binding to its transcripts. Overall, these findings demonstrate that sORFs facilitate ribosome binding to 24-PHAS transcripts, thereby promoting phasiRNA biogenesis in meiotic anthers.

Genetic basis of maize stalk strength decoded via linkage and association mapping.

Stalk lodging is a severe problem that limits maize production worldwide, although little attention has been given to its genetic basis. Here we measured rind penetrometer resistance (RPR), an effective index for stalk lodging, in a multi-parent population of 1948 recombinant inbred lines (RILs) and an association population of 508 inbred lines (AMP508). Linkage and association mapping identified 53 and 29 single quantitative trait loci (QTLs) and 50 and 19 pairs of epistatic interactions for RPR in the multi-parent population and AMP508 population, respectively. Phenotypic variation explained by all identified epistatic QTLs (up to ~5%) was much less than that explained by all single additive QTLs (up to ~33% in the multi-parent population and ~ 60% in the AMP508 population). Among all detected QTLs, only eight single QTLs explained >10% of phenotypic variation in single RIL populations. Alleles that increased RPR were enriched in tropical/subtropical (TST) groups from the AMP508 population. Based on genome-wide association studies in both populations, we identified 137 candidate genes affecting RPR, which were assigned to multiple biological processes, such as the biosynthesis of cell wall components. Sixty-six candidate genes were cross-validated by multiple methods or populations. Most importantly, 23 candidate genes were upregulated or downregulated in high-RPR lines relative to low-RPR lines, supporting the associations between candidate genes and RPR. These findings reveal the complex nature of the genetic basis underlying RPR and provide loci or candidate genes for developing elite varieties that are resistant to stalk lodging via molecular breeding.

Exploring key developmental phases and phase-specific genes across the entirety of anther development in maize.

Anther development from stamen primordium to pollen dispersal is complex and essential to sexual reproduction. How this highly dynamic and complex developmental process is controlled genetically is not well understood, especially for genes involved in specific key developmental phases. Here we generated RNA sequencing libraries spanning 10 key stages across the entirety of anther development in maize (Zea mays). Global transcriptome analyses revealed distinct phases of cell division and expansion, meiosis, pollen maturation, and mature pollen, for which we detected 50, 245, 42, and 414 phase-specific marker genes, respectively. Phase-specific transcription factor genes were significantly enriched in the phase of meiosis. The phase-specific expression of these marker genes was highly conserved among the maize lines Chang7-2 and W23, indicating they might have important roles in anther development. We explored a desiccation-related protein gene, ZmDRP1, which was exclusively expressed in the tapetum from the tetrad to the uninucleate microspore stage, by generating knockout mutants. Notably, mutants in ZmDRP1 were completely male-sterile, with abnormal Ubisch bodies and defective pollen exine. Our work provides a glimpse into the gene expression dynamics and a valuable resource for exploring the roles of key phase-specific genes that regulate anther development.

Genome-Wide Association Analyses Reveal the Importance of Alternative Splicing in Diversifying Gene Function and Regulating Phenotypic Variation in Maize.

Alternative splicing (AS) enhances transcriptome diversity and plays important roles in regulating plant processes. Although widespread natural variation in AS has been observed in plants, how AS is regulated and contribute to phenotypic variation is poorly understood. Here, we report a population-level transcriptome assembly and genome-wide association study to identify splicing quantitative trait loci (sQTLs) in developing maize (Zea mays) kernels from 368 inbred lines. We detected 19,554 unique sQTLs for 6570 genes. Most sQTLs showed small isoform usage changes without involving major isoform switching between genotypes. The sQTL-affected isoforms tend to display distinct protein functions. We demonstrate that nonsense-mediated mRNA decay, microRNA-mediated regulation, and small interfering peptide-mediated peptide interference are frequently involved in sQTL regulation. The natural variation in AS and overall mRNA level appears to be independently regulated with different cis-sequences preferentially used. We identified 214 putative trans-acting splicing regulators, among which ZmGRP1, encoding an hnRNP-like glycine-rich RNA binding protein, regulates the largest trans-cluster. Knockout of ZmGRP1 by CRISPR/Cas9 altered splicing of numerous downstream genes. We found that 739 sQTLs colocalized with previous marker-trait associations, most of which occurred without changes in overall mRNA level. Our findings uncover the importance of AS in diversifying gene function and regulating phenotypic variation.

Natural variations in stearoyl-acp desaturase genes affect the conversion of stearic to oleic acid in maize kernel.

KEY MESSAGE: We identified 11 SAD genes, and mined their natural variations associated with the conservation of stearic to oleic acid, especially ZmSAD1 supported by both the QTL and an expression QTL. Maize oil is generally regarded as a healthy vegetable oil owing to its low abundance of saturated fatty acids. Stearoyl-ACP desaturase (SAD) is a key rate-limiting enzyme for the conservation of stearic (C18:0) to oleic (C18:1) acid. Here, 11 maize SAD genes were identified to have more divergent functions than Arabidopsis SAD genes. The genomic regional associations in a maize panel including 508 inbred lines identified 6 SAD genes significantly associated (P < 0.01) with the C18:0/C18:1 ratio or the level of C18:0 or C18:1, one gene of which co-localized with a quantitative trait locus (QTL) and 5 of which co-localized with an expression QTL. ZmSAD1, supported by both the QTL and an expression QTL, had the largest effect on C18:0/C18:1. One nonsynonymous single-nucleotide polymorphism in exon 3 and one 5-bp insertion/deletion in the 3' untranslated region were further shown to contribute to the natural variation in C18:0/C18:1 according to ZmSAD1-based association mapping. Finally, selection tests of ZmSAD1 in teosinte, regular maize, and high-oil maize indicated that ZmSAD1 was not a selection target during the process of maize domestication and high-oil maize development. These results will guide the manipulation of the ratio between saturated and unsaturated fatty acids in maize.

ZmcrtRB3 encodes a carotenoid hydroxylase that affects the accumulation of α-carotene in maize kernel.

α-carotene is one of the important components of pro-vitamin A, which is able to be converted into vitamin A in the human body. One maize (Zea mays L.) ortholog of carotenoid hydroxylases in Arabidopsis thaliana, ZmcrtRB3, was cloned and its role in carotenoid hydrolyzations was addressed. ZmcrtRB3 was mapped in a quantitative trait locus (QTL) cluster for carotenoid-related traits on chromosome 2 (bin 2.03) in a recombinant inbred line (RIL) population derived from By804 and B73. Candidate-gene association analysis identified 18 polymorphic sites in ZmcrtRB3 significantly associated with one or more carotenoid-related traits in 126 diverse yellow maize inbred lines. These results indicate that the enzyme ZmcrtRB3 plays a role in hydrolyzing both α- and ß-carotenes, while polymorphisms in ZmcrtRB3 contributed more variation in α-carotene than that in ß-carotene. Two single nucleotide polymorphisms (SNPs), SNP1343 in 5'untranslated region and SNP2172 in the second intron, consistently had effects on α-carotene content and composition with explained phenotypic variations ranging from 8.7% to 34.8%. There was 1.7- to 3.7-fold change between the inferior and superior haplotype for α-carotene content and composition. Thus, SNP1343 and SNP2172 are potential polymorphic sites to develop functional markers for applying marker-assisted selection in the improvement of pro-vitamin A carotenoids in maize kernels.

Convergent selection of a WD40 protein that enhances grain yield in maize and rice.

A better understanding of the extent of convergent selection among crops could greatly improve breeding programs. We found that the quantitative trait locus KRN2 in maize and its rice ortholog, OsKRN2, experienced convergent selection. These orthologs encode WD40 proteins and interact with a gene of unknown function, DUF1644, to negatively regulate grain number in both crops. Knockout of KRN2 in maize or OsKRN2 in rice increased grain yield by ~10% and ~8%, respectively, with no apparent trade-offs in other agronomic traits. Furthermore, genome-wide scans identified 490 pairs of orthologous genes that underwent convergent selection during maize and rice evolution, and these were enriched for two shared molecular pathways. KRN2, together with other convergently selected genes, provides an excellent target for future crop improvement.

Genome-wide association study dissects the genetic architecture of oil biosynthesis in maize kernels.

Maize kernel oil is a valuable source of nutrition. Here we extensively examine the genetic architecture of maize oil biosynthesis in a genome-wide association study using 1.03 million SNPs characterized in 368 maize inbred lines, including 'high-oil' lines. We identified 74 loci significantly associated with kernel oil concentration and fatty acid composition (P < 1.8 × 10(-6)), which we subsequently examined using expression quantitative trait loci (QTL) mapping, linkage mapping and coexpression analysis. More than half of the identified loci localized in mapped QTL intervals, and one-third of the candidate genes were annotated as enzymes in the oil metabolic pathway. The 26 loci associated with oil concentration could explain up to 83% of the phenotypic variation using a simple additive model. Our results provide insights into the genetic basis of oil biosynthesis in maize kernels and may facilitate marker-based breeding for oil quantity and quality.

Genome-wide association studies identified three independent polymorphisms associated with α-tocopherol content in maize kernels.

Tocopherols are a class of four natural compounds that can provide nutrition and function as antioxidant in both plants and animals. Maize kernels have low α-tocopherol content, the compound with the highest vitamin E activity, thus, raising the risk of vitamin E deficiency in human populations relying on maize as their primary vitamin E source. In this study, two insertion/deletions (InDels) within a gene encoding γ-tocopherol methyltransferase, Zea mays VTE4 (ZmVTE4), and a single nucleotide polymorphism (SNP) located ~85 kb upstream of ZmVTE4 were identified to be significantly associated with α-tocopherol levels in maize kernels by conducting an association study with a panel of ~500 diverse inbred lines. Linkage analysis in three populations that segregated at either one of these three polymorphisms but not at the other two suggested that the three polymorphisms could affect α-tocopherol content independently. Furthermore, we found that haplotypes of the two InDels could explain ∼33% of α-tocopherol variation in the association panel, suggesting ZmVTE4 is a major gene involved in natural phenotypic variation of α-tocopherol. One of the two InDels is located within the promoter region and associates with ZmVTE4 transcript level. This information can not only help in understanding the underlying mechanism of natural tocopherol variations in maize kernels, but also provide valuable markers for marker-assisted breeding of α-tocopherol content in maize kernels, which will then facilitate the improvement of maize as a better source of daily vitamin E nutrition.