Comparative analyses reveal potential uses of Brachypodium distachyon as a model for cold stress responses in temperate grasses

Background: Little is known about the potential of Brachypodium distachyon as a model for low temperature stress responses in Pooideae. The ice recrystallization inhibition protein (IRIP) genes, fructosyltransferase (FST) genes, and many C-repeat binding factor (CBF) genes are Pooideae specific and important in low temperature responses. Here we used comparative analyses to study conservation and evolution of these gene families in B. distachyon to better understand its potential as a model species for agriculturally important temperate grasses. Results: Brachypodium distachyon contains cold responsive IRIP genes which have evolved through Brachypodium specific gene family expansions. A large cold responsive CBF3 subfamily was identified in B. distachyon, while CBF4 homologs are absent from the genome. No B. distachyon FST gene homologs encode typical core Pooideae FST-motifs and low temperature induced fructan accumulation was dramatically different in B. distachyon compared to core Pooideae species. Conclusions: We conclude that B. distachyon can serve as an interesting model for specific molecular mechanisms involved in low temperature responses in core Pooideae species. However, the evolutionary history of key genes involved in low temperature responses has been different in Brachypodium and core Pooideae species. These differences limit the use of B. distachyon as a model for holistic studies relevant for agricultural core Pooideae species.Keywords: Gene family evolution, Cold climate adaptation, Fructosyltransferase, Brachypodium distachyon, Ice recrystallization inhibition protein, C-repeat binding factor, Gene expressio

Comparative SNP and Haplotype Analysis Reveals a Higher Genetic Diversity and Rapider LD Decay in Tropical than Temperate Germplasm in Maize

Understanding of genetic diversity and linkage disequilibrium (LD) decay in diverse maize germplasm is fundamentally important for maize improvement. A total of 287 tropical and 160 temperate inbred lines were genotyped with 1943 single nucleotide polymorphism (SNP) markers of high quality and compared for genetic diversity and LD decay using the SNPs and their haplotypes developed from genic and intergenic regions. Intronic SNPs revealed a substantial higher variation than exonic SNPs. The big window size haplotypes (3-SNP slide-window covering 2160 kb on average) revealed much higher genetic diversity than the 10 kb-window and gene-window haplotypes. The polymorphic information content values revealed by the haplotypes (0.436–0.566) were generally much higher than individual SNPs (0.247–0.259). Cluster analysis classified the 447 maize lines into two major groups, corresponding to temperate and tropical types. The level of genetic diversity and subpopulation structure were associated with the germplasm origin and post-domestication selection. Compared to temperate lines, the tropical lines had a much higher level of genetic diversity with no significant subpopulation structure identified. Significant variation in LD decay distance (2–100 kb) was found across the genome, chromosomal regions and germplasm groups. The average of LD decay distance (10–100 kb) in the temperate germplasm was two to ten times larger than that in the tropical germplasm (5–10 kb). In conclusion, tropical maize not only host high genetic diversity that can be exploited for future plant breeding, but also show rapid LD decay that provides more opportunity for selection

RNA-seq raw data of maize CMS-C line

RNA-seq raw data of biological replicate 1 of cytoplasmic male sterility line C48-2 spikelets at mononuclear stag

Insight into the maize CMS-associated mitochondrial-nuclear interaction at the DNA methylation level

The mitochondrial-nuclear interaction is essential for cellular homeostasis and proper function. DNA methylation plays important roles in gene expression regulation as well as maintaining genomic stability. Isonuclear-alloplasmic and isoplasmic-allonuclear lines were used as materials in this study. The mitochondrial DNA (mtDNA) and nuclear DNA (nDNA) from maize tassels were used for DNA methylation analysis with high-performance liquid chromatography (HPLC). The results as follow: (1) Different mtDNA methylation levels were detected within the isoplasmic-allonuclear lines, and varied nDNA methylation levels also were detected among the isonuclear-alloplasmic lines. (2) From pollen mother cell stage to binucleate stage, the change pattern of DNA methylation levels varied among CMS-C, CMS-T and CMS-S lines. The DNA methylation level peaked at tetrad stage for CMS-C line, mononuclear stage for CMS-T line and binucleate stage for CMS-S line, respectively. Interestingly, the peaked periods for DNA methylation level just meets their critical abortive periods of CMS-C, CMS-T and CMS-S lines. (3) The change trends of methylation levels both for mtDNA and nDNA show highly consistent for every experimental material. Based on these results, we speculated that the epigenetic status affected by mitochondrial-nuclear interaction may be involved in the regulation of maize CMS.The accepted manuscript in pdf format is listed with the files at the bottom of this page. The presentation of the authors' names and (or) special characters in the title of the manuscript may differ slightly between what is listed on this page and what is listed in the pdf file of the accepted manuscript; that in the pdf file of the accepted manuscript is what was submitted by the author

Identification of Maize <i>Rf4</i>-Restorer Lines and Development of a CAPS Marker for <i>Rf4</i>

Rf4 is one of the dominant restorer genes for maize C-type cytoplasmic male sterility (CMS-C), which has significant value in hybrid maize seed production. However, the highly complex fertility restoration mechanism of CMS-C makes it difficult to screen Rf4-restorer lines, and insufficient Rf4-restorer lines limit its use in current agricultural production. To search for Rf4-restorer lines, in this study, the genotypes of eighteen inbred maize lines at the Rf4 locus were analyzed based on the male fertility investigation of hybrid F1, the genetic analysis of F2 populations, molecular marker mapping, allelic tests, and Rf4 genomic sequence analysis. Our results indicated that of the eighteen maize inbred lines, ten were able to completely rescue CMS-C line CHuangzaosi (CHZS) male sterility. A genetic analysis showed that DAN598, PHT77, 78551S, and LH212Ht only contained one dominant restorer gene each, and the molecular-marker mapping indicated that their restorer genes were located at the short arm of chromosome 8. The allelic testing of the fertility of the restorer (Rf) demonstrated that the restorer gene of twelve inbred lines, including DAN598, PHT77, 78551S, and LH212Ht, was allelic to one restorer gene of A619. Furthermore, the genomic sequence alignment of Rf4 revealed that there were two different amino acids in the coding sequence between the A619 (Rf4Rf4) restorer lines and four CMS-C lines (rf4rf4). For the crucial S1596 site variation (TTT/TAC), DAN598, PHT77, 78551S, and LH212Ht shared the same bases (TTT) with A619 and encoded phenylalanine, while the four CMS-C sterile lines had the TAC and encoded tyrosine. Our results revealed that these tester lines, DAN598, PHT77, 78551S, and LH212Ht, were the Rf4-restorer lines. Additionally, derived from the sequence variants of Rf4, 39 possible Rf4-restorer lines from 129 inbred maize lines were detected. Furthermore, we developed a Cleaved Amplified Polymorphism Sequences (CAPS) marker based on the S1596 variations. The PCR amplification product of S1596 (TAC) was digested by the TatI endonuclease into two bands with sizes of ~260 bp and ~100 bp. In comparison, when S1596 was TTT, the PCR product could not be digested. In conclusion, in this study, we identified various Rf4-restorer lines for maize CMS-C and developed a molecular marker for Rf4. The reported results will contribute to the popularization and application of Rf4 in hybrid maize-seed production

Comparative transcriptome analysis of isonuclear-alloplasmic lines unmask key transcription factor genes and metabolic pathways involved in sterility of maize CMS-C

Although C-type cytoplasmic male sterility (CMS-C) is one of the most attractive tools for maize hybrid seed production, the detailed regulation network of the male sterility remains unclear. In order to identify the CMS-C sterility associated genes and/or pathways, the comparison of the transcriptomes between the CMS-C line C48-2 and its isonuclear-alloplasmic maintainer line N48-2 at pollen mother cell stage (PS), an early development stage of microspore, and mononuclear stage (MS), an abortive stage of microspore, were analyzed. 2,069 differentially expressed genes (DEGs) between the two stages were detected and thought to be essential for the spikelet development of N48-2. 453 of the 2,069 DEGs were differentially expressed at MS stage between the two lines and thought to be participated in the process or the causes of microspore abortion. Among the 453 DEGs, 385 (84.99%) genes were down-regulated and only 68 (15.01%) genes were up-regulated in C48-2 at MS stage. The dramatic decreased expression of the four DEGs encoding MYB transcription factors and the DEGs involved in “polyamine metabolic process”, “Cutin, suberine and wax biosynthesis”, “Fatty acid elongation”, “Biosynthesis of unsaturated fatty acids” and “Proline metabolism” might play an important role in the sterility of C48-2. This study will point out some directions for detailed molecular analysis and better understanding of sterility of CMS-C in maize

Cloning, molecular evolution and functional characterization of ZmbHLH16, the maize ortholog of OsTIP2 (OsbHLH142)

The transcription factor ZmbHLH16, the maize ortholog of OsTIP2 (OsbHLH142), was isolated in the present study. Tissue expression analysis showed that ZmbHLH16 is preferentially expressed in male reproductive organs. Subcellular location analysis of ZmbHLH16 via rice protoplast indicated that it is located in the nucleus. Through nucleotide variation analysis, 36 polymorphic sites in ZmbHLH16, including 23 single nucleotide polymorphisms and 13 InDels, were detected among 78 maize inbred lines. Neutrality tests and linkage disequilibrium analysis showed that ZmbHLH16 experienced no significant evolutionary pressure. Yeast one-hybrid experiment showed that the first 80 residues in the N-terminus of ZmbHLH16 had transactivation activity, whereas the full length did not. Genome-wide coexpression analysis showed that 395 genes were coexpressed with ZmbHLH16. Among these genes, the transcription factor ZmbHLH51 had similar expression pattern and identical subcellular localization to those of ZmbHLH16. Subsequently, the interaction between ZmbHLH51 and ZmbHLH16 was verified by yeast two-hybrid experiment. Through yeast two-hybrid analysis of series truncated ZmbHLH16 fragments, we found not only the typical bHLH domain [175-221 amino acids (a.a.)], but also that the 81-160 a.a. and 241-365 a.a. of ZmbHLH16 could interact with ZmbHLH51. All these results lay the foundation for further understanding the functions of ZmbHLH16

Preparation and Characterization of Magnetic Metal–Organic Frameworks Functionalized by Ionic Liquid as Supports for Immobilization of Pancreatic Lipase

Enzymes are difficult to recycle, which limits their large-scale industrial applications. In this work, an ionic liquid-modified magnetic metal–organic framework composite, IL-Fe3O4@UiO-66-NH2, was prepared and used as a support for enzyme immobilization. The properties of the support were characterized with X-ray powder diffraction (XRD), Fourier-transform infrared (FTIR) spectra, transmission electron microscopy (TEM), scanning electronic microscopy (SEM), and so on. The catalytic performance of the immobilized enzyme was also investigated in the hydrolysis reaction of glyceryl triacetate. Compared with soluble porcine pancreatic lipase (PPL), immobilized lipase (PPL-IL-Fe3O4@UiO-66-NH2) had greater catalytic activity under reaction conditions. It also showed better thermal stability and anti-denaturant properties. The specific activity of PPL-IL-Fe3O4@UiO-66-NH2 was 2.3 times higher than that of soluble PPL. After 10 repeated catalytic cycles, the residual activity of PPL-IL-Fe3O4@UiO-66-NH2 reached 74.4%, which was higher than that of PPL-Fe3O4@UiO-66-NH2 (62.3%). In addition, kinetic parameter tests revealed that PPL-IL-Fe3O4@UiO-66-NH2 had a stronger affinity to the substrate and, thus, exhibited higher catalytic efficiency. The results demonstrated that Fe3O4@UiO-66-NH2 modified by ionic liquids has great potential for immobilized enzymes

A preliminary identification of Rf*-A619, a novel restorer gene for CMS-C in maize (Zea mays L.)

C-type cytoplasmic male sterility (CMS-C) is widely utilized for hybrid maize seed production. However, genetic mechanisms underlying the fertility restoration are very complicated. At present, there is a divergence on the number of fertility restorer genes in maize inbred line A619 for CMS-C. To further elucidate the restoring mechanism of A619, we used genetic analysis and molecular markers to confirm the restorer genes of maize inbred line A619 for C-type male sterile line C48-2 in this study. Firstly, the fertility segregations of (C48-2 × A619)F2 populations were investigated under three environments during 2013–2015. The segregation ratio of fertile and sterile plants in the F2 population fit to 15:1 via chi-square test and this result suggested that there are two dominant restorer genes in A619 for CMS-C, i.e., Rf4 and a novel gene named Rf*-A619. Next, based on the sequence differences between Rf4 and its recessive allelic rf4, a novel dominant marker F2/R2 was developed and validated to genotyping Rf4 in the F2 population. Through genotypic analysis, we found that there were a certain amount of fertile individuals without Rf4 which accounted for 3/16 in the F2 population via chi-square test at the 0.05 level. These results provided another proof to sustain that the inbred line A619 contains one additional restorer gene for CMS-C fertility restoration except Rf4. At last, we used one SSR marker which is tightly linked with the dominant restorer gene Rf5 to analyze those fertile plants without Rf4 in the F2 population. The PCR amplification results showed that Rf*-A619 is not allelic to Rf5 but a novel restorer gene for CMS-C. These results not only provide a basis for the mapping and characterization of a novel restorer gene but also give a new insight into the mechanism of CMS-C fertility restoration