Genetic regulation of aleurone cell fate in Zea mays

The outermost layer of the endosperm is a specific cell type called the aleurone which is one of the grain quality determining factors because of its high content in lipid, minerals and high quality proteins compared to starchy endosperm. The aleurone layer is also an attractive system to study cell fate determination because of the simplicity and plasticity of aleurone cell fate. Here we report the identification of naked endosperm (nkd) genes which are involved in aleurone differentiation in maize. The nkd mutant shows defects in aleurone cell identity and has approximately 3 outer cell layers instead of the single in WT. However these outer cells do not contain dense granular cytoplasm typical of normal aleurone and have sporadic expression of a Vp1 promoter GUS transgene, which is an aleurone identity marker. The nkd mutant phenotype shows 15:1 segregation ratio in F2 populations suggesting two recessive genes are involved in this phenotype. We performed map-based cloning and found two homologous genes in syntenic regions. The INDETERMINATE1 domain containing transcription factors ZmIDDveg9 and ZmIDD9 correspond to the nkd1 and nkd2 mutant genes on chromosomes 2 and 10, respectively. Independent Ds transposon insertion alleles of nkd1 and nkd2, nkd1-Ds and nkd2-Ds respectively, failed to complement the original nkd mutant. A Nkd2-RNAi line, in which both of nkd genes were knocked down, also showed the nkd mutant phenotype. The nkd transcripts were most abundant in developing kernels around 11 to16 days after pollination. The NKD proteins have putative nuclear localization signals as other IDD genes and GFP fusion proteins showed nuclear localization. The mutant phenotype and gene expression pattern suggest NKD functions in aleurone cell fate acquisition and differentiation

High-throughput linkage analysis of Mutator insertion sites in maize

Insertional mutagenesis is a cornerstone of functional genomics. High-copy transposable element systems such as Mutator (Mu) in maize (Zea mays) afford the advantage of high forward mutation rates but pose a challenge for identifying the particular element responsible for a given mutation. Several large mutant collections have been generated in Mu-active genetic stocks, but current methods limit the ability to rapidly identify the causal Mu insertions. Here we present a method to rapidly assay Mu insertions that are genetically linked to a mutation of interest. The method combines elements of MuTAIL (thermal asymmetrically interlaced) and amplification of insertion mutagenized sites (AIMS) protocols and is applicable to the analysis of single mutants or to high-throughput analyses of mutant collections. Briefly, genomic DNA is digested with a restriction enzyme and adapters are ligated. Polymerase chain reaction is performed with TAIL cycling parameters, using a fluorescently labeled Mu primer, which results in the preferential amplification and labeling of Mu-containing genomic fragments. Products from a segregating line are analyzed on a capillary sequencer. To recover a fragment of interest, PCR products are cloned and sequenced. Sequences with lengths matching the size of a band that co-segregates with the mutant phenotype represent candidate linked insertion sites, which are then confirmed by PCR. We demonstrate the utility of the method by identifying Mu insertion sites linked to seed-lethal mutations with a preliminary success rate of nearly 50%

Revealing biomass heterosis in the allodiploid xBrassicoraphanus, a hybrid between Brassica rapa and Raphanus sativus, through integrated transcriptome and metabolites analysis

Background Heterosis is biologically important but the molecular basis of the phenomenon is poorly understood. We characterized intergeneric hybrids between B. rapa cv. Chiifu and R. sativus cv. WK10039 as an extreme example of heterosis. Taking advantage of clear heterosis phenotypes and the genetic distance between parents, we performed transcriptome and metabolite analysis to decipher the molecular basis of heterosis. Results The heterosis was expressed as fresh weight in the field and as inflorescence stem length in the glass house. Flowering time, distributed as a normal segregating population, ranged from the early flowering of one parent to the late flowering of the other, in contrast to the homogeneous flowering time in a typical F1 population, indicating unstable allelic interactions. The transcriptome and metabolome both indicated that sugar metabolism was altered, suggesting that the change in metabolism was linked to the heterosis. Because alleles were not shared between the hybridized genomes, classic models only partly explain this heterosis, indicating that other mechanisms are involved. Conclusion The differential expression of genes for primary and secondary metabolism, along with the altered metabolite profiles, suggests that heterosis could involve a change in balance between primary and secondary metabolism.This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Ministry of Science and ICT (2016R1D1A1B03930431 and 2013R1A1A2058687 to GY) and by NextGeneration BioGreen 21 Program (PJ013262 to HJH) Rural Development Administration (RDA), Korea. The funding agencies were not involved in the experimental design, collection and interpretation of data, and in preparation of the manuscript

Genetic regulation of aleurone cell fate in Zea mays

The outermost layer of the endosperm is a specific cell type called the aleurone which is one of the grain quality determining factors because of its high content in lipid, minerals and high quality proteins compared to starchy endosperm. The aleurone layer is also an attractive system to study cell fate determination because of the simplicity and plasticity of aleurone cell fate. Here we report the identification of naked endosperm (nkd) genes which are involved in aleurone differentiation in maize. The nkd mutant shows defects in aleurone cell identity and has approximately 3 outer cell layers instead of the single in WT. However these outer cells do not contain dense granular cytoplasm typical of normal aleurone and have sporadic expression of a Vp1 promoter GUS transgene, which is an aleurone identity marker. The nkd mutant phenotype shows 15:1 segregation ratio in F2 populations suggesting two recessive genes are involved in this phenotype. We performed map-based cloning and found two homologous genes in syntenic regions. The INDETERMINATE1 domain containing transcription factors ZmIDDveg9 and ZmIDD9 correspond to the nkd1 and nkd2 mutant genes on chromosomes 2 and 10, respectively. Independent Ds transposon insertion alleles of nkd1 and nkd2, nkd1-Ds and nkd2-Ds respectively, failed to complement the original nkd mutant. A Nkd2-RNAi line, in which both of nkd genes were knocked down, also showed the nkd mutant phenotype. The nkd transcripts were most abundant in developing kernels around 11 to16 days after pollination. The NKD proteins have putative nuclear localization signals as other IDD genes and GFP fusion proteins showed nuclear localization. The mutant phenotype and gene expression pattern suggest NKD functions in aleurone cell fate acquisition and differentiation.</p

High-throughput linkage analysis of Mutator insertion sites in maize

Insertional mutagenesis is a cornerstone of functional genomics. High-copy transposable element systems such as Mutator (Mu) in maize (Zea mays) afford the advantage of high forward mutation rates but pose a challenge for identifying the particular element responsible for a given mutation. Several large mutant collections have been generated in Mu-active genetic stocks, but current methods limit the ability to rapidly identify the causal Mu insertions. Here we present a method to rapidly assay Mu insertions that are genetically linked to a mutation of interest. The method combines elements of MuTAIL (thermal asymmetrically interlaced) and amplification of insertion mutagenized sites (AIMS) protocols and is applicable to the analysis of single mutants or to high-throughput analyses of mutant collections. Briefly, genomic DNA is digested with a restriction enzyme and adapters are ligated. Polymerase chain reaction is performed with TAIL cycling parameters, using a fluorescently labeled Mu primer, which results in the preferential amplification and labeling of Mu-containing genomic fragments. Products from a segregating line are analyzed on a capillary sequencer. To recover a fragment of interest, PCR products are cloned and sequenced. Sequences with lengths matching the size of a band that co-segregates with the mutant phenotype represent candidate linked insertion sites, which are then confirmed by PCR. We demonstrate the utility of the method by identifying Mu insertion sites linked to seed-lethal mutations with a preliminary success rate of nearly 50%.This article is from The Plant Journal 58 (2009): 883–892, doi:10.1111/j.1365-313X.2009.03821.x.</p

Pan-chloroplast genomes for accession-specific marker development in Hibiscus syriacus

Abstract Hibiscus syriacus L. is a renowned ornamental plant. We constructed 95 chloroplast genomes of H. syriacus L. cultivars using a short-read sequencing platform (Illumina) and a long-read sequencing platform (Oxford Nanopore Technology). The following genome assembly, we delineate quadripartite structures encompassing large single-copy, small single-copy, and inverted repeat (IRa and IRb) regions, from 160,231 bp to 161,041 bp. Our comprehensive analyses confirmed the presence of 79 protein-coding genes, 30 tRNA genes, and 4 rRNA genes in the pan-chloroplast genome, consistent with prior research on the H. syriacus chloroplast genome. Subsequent pangenome analysis unveiled widespread genome sequence conservation alongside unique cultivar-specific variant patterns consisting of 193 single-nucleotide polymorphisms and 61 insertions or deletions. The region containing intra-species variant patterns, as identified in this study, has the potential to develop accession-specific molecular markers, enhancing precision in cultivar classification. These findings are anticipated to drive advancements in breeding strategies, augment biodiversity, and unlock the agricultural potential inherent in H. syriacus

The thick aleurone1 Mutant Defines a Negative Regulation of Maize Aleurone Cell Fate That Functions Downstream of defective kernel1

The maize (Zea mays) aleurone layer occupies the single outermost layer of the endosperm. The defective kernel1 (dek1) gene is a central regulator required for aleurone cell fate specification. dek1 mutants have pleiotropic phenotypes including lack of aleurone cells, aborted embryos, carotenoid deficiency, and a soft, floury endosperm deficient in zeins. Here we describe the thick aleurone1 (thk1) mutant that defines a novel negative function in the regulation of aleurone differentiation. Mutants possess multiple layers of aleurone cells as well as aborted embryos. Clonal sectors of thk1 mutant tissue in otherwise normal endosperm showed localized expression of the phenotype with sharp boundaries, indicating a localized cellular function for the gene. Sectors in leaves showed expanded epidermal cell morphology but the mutant epidermis generally remained in a single cell layer. Double mutant analysis indicated that the thk1 mutant is epistatic to dek1 for several aspects of the pleiotropic dek1 phenotype. dek1 mutant endosperm that was mosaic for thk1 mutant sectors showed localized patches of multilayered aleurone. Localized sectors were surrounded by halos of carotenoid pigments and double mutant kernels had restored zein profiles. In sum, loss of thk1 function restored the ability of dek1 mutant endosperm to accumulate carotenoids and zeins and to differentiate aleurone. Therefore the thk1 mutation defines a negative regulator that functions downstream of dek1 in the signaling system that controls aleurone specification and other aspects of endosperm development. The thk1 mutation was found to be caused by a deletion of approximately 2 megabases.This article is published as Yi, Gibum, Adrienne M. Lauter, M. Paul Scott, and Philip W. Becraft. "The thick aleurone1 mutant defines a negative regulation of maize aleurone cell fate that functions downstream of defective kernel1." Plant physiology 156, no. 4 (2011): 1826-1836, doi: 10.1104/pp.111.177725.</p

The thick aleurone1 Mutant Defines a Negative Regulation of Maize Aleurone Cell Fate That Functions Downstream of defective kernel11[C][W][OA]

The maize (Zea mays) aleurone layer occupies the single outermost layer of the endosperm. The defective kernel1 (dek1) gene is a central regulator required for aleurone cell fate specification. dek1 mutants have pleiotropic phenotypes including lack of aleurone cells, aborted embryos, carotenoid deficiency, and a soft, floury endosperm deficient in zeins. Here we describe the thick aleurone1 (thk1) mutant that defines a novel negative function in the regulation of aleurone differentiation. Mutants possess multiple layers of aleurone cells as well as aborted embryos. Clonal sectors of thk1 mutant tissue in otherwise normal endosperm showed localized expression of the phenotype with sharp boundaries, indicating a localized cellular function for the gene. Sectors in leaves showed expanded epidermal cell morphology but the mutant epidermis generally remained in a single cell layer. Double mutant analysis indicated that the thk1 mutant is epistatic to dek1 for several aspects of the pleiotropic dek1 phenotype. dek1 mutant endosperm that was mosaic for thk1 mutant sectors showed localized patches of multilayered aleurone. Localized sectors were surrounded by halos of carotenoid pigments and double mutant kernels had restored zein profiles. In sum, loss of thk1 function restored the ability of dek1 mutant endosperm to accumulate carotenoids and zeins and to differentiate aleurone. Therefore the thk1 mutation defines a negative regulator that functions downstream of dek1 in the signaling system that controls aleurone specification and other aspects of endosperm development. The thk1 mutation was found to be caused by a deletion of approximately 2 megabases

A genetic characterization of Korean waxy maize (Zea mays L.) landraces having flowering time variation by RNA sequencing

Maize is the second-most produced crop in the Korean peninsula and has been continuously cultivated since the middle of the 16th century, when it was originally introduced from China. Even with this extensive cultivation history, the diversity and properties of Korean landraces have not been investigated at the nucleotide sequence level. We collected 12 landraces with various flowering times and performed RNA-seq in the early vegetative stage. The transcriptomes of 12 Korean landraces have been analyzed for their genetic variations in coding sequence and genetic relationships to other maize germplasm. The Korean landraces showed specific genetic characteristics and were closely related to a Chinese inbred line. Flowering-time related gene profiles pointed to multiple causes for the variation of flowering time within Korean landraces; the profiles revealed significant positive and negative correlations among genes, allowing us to infer possible mechanisms for flowering time variation in maize. Our results demonstrate the value of transcriptome-based genetic and gene expression profiles for information on possible breeding resources, which is particularly needed in Korean waxy landraces.Y