The Unstable Restorer-of-Fertility Locus in Pepper (Capsicum Annuum. L) is Delimited to a Genomic Region Containing PPR Genes

The use of cytoplasmic-genic male sterility (CGMS) systems greatly increases the efficiency of hybrid seed production. Although marker development and candidate gene isolation have been performed for the Restorer-of-fertility (Rf) gene in pepper (Capsicum annuum L.), the broad use of CGMS systems has been hampered by the instability of fertility restoration among pepper accessions, especially sweet peppers, due to the widespread presence of the Unstable Restorer-of-fertility (Rfu) locus. Therefore, to investigate the genetic factors controlling unstable fertility restoration in sweet peppers, we developed a segregation population from a cross between a male-sterile line and an Rfu-containing line to examine the inheritance of Rfu. Individuals with unstable restoration vs. sterility segregated at a 3:1 ratio, indicating that a single dominant gene controls unstable fertility restoration. Genetic mapping delimited the Rfu locus to a 479 kb genomic region on chromosome 6 flanked by two markers, which is close to but different from the previously identified Rf-containing region. The Rfu-containing region harbors a pentatricopeptide repeat (PPR) gene, along with 13 other candidate genes. In addition, this region is syntenic to the genomic region containing the largest number of Rf-like PPR genes in tomato. Therefore, the dynamic evolution of PPR genes might be responsible for both the restoration and instability of fertility in pepper. During genetic mapping, we developed various molecular markers, including one that co-segregated with Rfu. These markers showed higher accuracy for genotyping than previously developed markers, pointing to their possible use in marker-assisted breeding of sweet peppers.

Development and validation of CAPS-marker associated with the Rf2 gene in sorghum (Sorghum bicolor (L.) Moench)

Background. The development of heterotic hybrids based on cytoplasmic male sterility (CMS) is the leading strategy in breeding sorghum (Sorghum bicolor (L.) Moench). The trait of pollen fertility restoration in forms with CMS A1 (milo), predominantly used in sorghum breeding, is determined by at least two dominant complementary genes Rf1 and Rf2, and also gene Rf5. The development of accessible molecular markers of sorghum Rf genes is highly relevant for hybrid breeding, since they can significantly accelerate the process of creating female sterile forms (A lines), sterility maintainers (B lines) and pollen fertility restorers (R lines).Material and methods. The studied material included 36 sorghum accessions from the VIR collection, which differed by the ability to restore pollen fertility in forms with A1-type CMS. The nucleotide polymorphism of 935 bp fragments of the PPR genes Sobic.002G057050, Sobic.002G054100, and Sobic.002G054200 located at the chromosome 2 was studied.Results. The fragments obtained with the use of a pair of 2459403fw and 2459403 primers were 935 bp long and included parts of three genes: Sobic.002G057050, Sobic.002G054100, Sobic.002G054200. For identifying the sequence variant Sobic.002G057050-1090 associated with the Rf2 gene, Tru9 I restrictase was chosen, which allows obtaining a 572 bp fragment unique for all the studied R lines. Such a marker was found in 10 sorghum lines from West China and Kyrgyzstan, which are widely used in breeding as fertility restorers. The fragment was found neither in three lines with sterile cytoplasm and their fertile analogues, nor in 7 accessions of kafir sorghum, which lacked functional alleles of Rf genes.Conclusions. It has been demonstrated that the marker can be used for selection and checking purity of R and B/A lines. It is also applicable for verifying hybridity of F1 seeds and analyzing hybrid populations from crosses of R lines 924-4, 928-1, 929-3, 931-1, 933-1/6, 1237-3, 1243-2, 1251, 1150-1, F10BC2 with A lines Nizkorosloe 81s, А-83 and А-10598. It may be suggested that the ability to restore pollen fertility in R lines, which lack the marker CAPS- 572, is determined by another Rf gene. The studied 935 bp fragment of Sobic.002G057050 harbours 22 SNP, therefore the development of CAPS-markers for their identification and differentiation can be promising.

Genome-Wide Identification of U-To-C RNA Editing Events for Nuclear Genes in Arabidopsis thaliana

Cytosine-to-Uridine (C-to-U) RNA editing involves the deamination phenomenon, which is observed in animal nucleus and plant organelles; however, it has been considered the U-to-C is confined to the organelles of limited non-angiosperm plant species. Although previous RNA-seq-based analysis implied U-to-C RNA editing events in plant nuclear genes, it has not been broadly accepted due to inadequate confirmatory analyses. Here we examined the U-to-C RNA editing in Arabidopsis tissues at different developmental stages of growth. In this study, the high-throughput RNA sequencing (RNA-seq) of 12-day-old and 20-day-old Arabidopsis seedlings was performed, which enabled transcriptome-wide identification of RNA editing sites to analyze differentially expressed genes (DEGs) and nucleotide base conversions. The results showed that DEGs were expressed to higher levels in 12-day-old seedlings than in 20-day-old seedlings. Additionally, pentatricopeptide repeat (PPR) genes were also expressed at higher levels, as indicated by the log2FC values. RNA-seq analysis of 12-day- and 20-day-old Arabidopsis seedlings revealed candidates of U-to-C RNA editing events. Sanger sequencing of both DNA and cDNA for all candidate nucleotide conversions confirmed the seven U-to-C RNA editing sites. This work clearly demonstrated presence of U-to-C RNA editing for nuclear genes in Arabidopsis, which provides the basis to study the mechanism as well as the functions of the unique post-transcriptional modification.

1RS.1BL molecular resolution provides novel contributions to wheat improvement

SUMMARYWheat-rye 1RS.1BL translocation has a significant impact on wheat yield and hence food production globally. However, the genomic basis of its contributions to wheat improvement is undetermined. Here, we generated a high-quality assembly of 1RS.1BL translocation comprising 748,715,293 bp with 4,996 predicted protein-coding genes. We found the size of 1RS is larger than 1BS with the active centromere domains shifted to the 1RS side instead of the 1BL side in Aikang58 (AK58). The gene alignment showed excellent synteny with 1BS from wheat and genes from 1RS were expressed well in wheat especially for 1RS where expression was higher than that of 1BS for the grain-20DPA stage associated with greater grain weight and negative flour quality attributes. A formin-like-domain protein FH14 (TraesAK58CH1B01G010700) was important in regulating cell division. Two PPR genes were most likely the genes for the multi fertility restoration locus Rf multi. Our data not only provide the high-resolution structure and gene complement for the 1RS.1BL translocation, but also defined targets for enhancing grain yield, biotic and abiotic stress, and fertility restoration in wheat.

Mitochondrial Pentatricopeptide Repeat Protein, EMB2794, Plays a Pivotal Role in NADH Dehydrogenase Subunit nad2 mRNA Maturation in Arabidopsis thaliana

The Arabidopsis genome encodes >450 proteins containing the pentatricopeptide repeat (PPR) motif. The PPR proteins are classified into two groups, termed as P and P Long-Short (PLS) classes. Typically, the PLS subclass proteins are mainly involved in the RNA editing of mitochondrial and chloroplast transcripts, whereas most of the analyzed P subclass proteins have been mainly implicated in RNA metabolism, such as 5′ or 3′ transcript stabilization and processing, splicing and translation. Mutations of PPR genes often result in embryogenesis and altered seedling developmental defect phenotypes, but only a limited number of ppr mutants have been characterized in detail. In this report, we show that null mutations in the EMB2794 gene result in embryo arrest, due to altered splicing of nad2 transcripts in the Arabidopsis mitochondria. In angiosperms, nad2 has five exons that are transcribed individually from two mitochondrial DNA regions. Biochemical and in vivo analyses further indicate that recombinant or transgenic EMB2794 proteins bind to the nad2 pre-mRNAs in vitro as well as in vivo, suggesting a role for this protein in trans-splicing of nad2 intron 2 and possibly in the stability of the second pre-mRNA of nad2. Homozygous emb2794 lines, showing embryo-defective phenotypes, can be partially rescued by the addition of sucrose to the growth medium. Mitochondria of rescued homozygous mutant plants contain only traces of respiratory complex I, which lack the NADH-dehydrogenase activity.

Dynamic response of RNA editing to temperature in grape by RNA deep sequencing

RNA editing is a post-transcriptional process of modifying genetic information on RNA molecules, which provides cells an additional level of gene expression regulation. Unlike mammals, in land plants, RNA editing converts C-to-U residues in organelles. However, its potential roles in response to different stressors (heat, salt, and so on) remains unclear. Grape is one of the most popular and economically important fruits in the world, and its production, like other crops, must deal with abiotic and biotic stresses, which cause reductions in yield and fruit quality. In our study, we tested the influence of the environmental factor temperature on RNA editing process in the whole mRNA from grape organelle. In total, we identified 122 and 627 RNA editing sites in chloroplast and mitochondria respectively with the average editing efficiency nearly ~ 60%. The analyses revealed that number of non-synonymous editing sites were higher than that of synonymous editing sites, and the amino acid substitution type tends to be hydrophobic. Additionally, the overall editing level decreased with the temperature rises, especially for several gene transcripts in chloroplast and mitochondria (matK, ndhB, etc.). We also found that the expression level of most PPR genes decreased with the temperature rises, which may contribute to the decline of RNA editing efficiency at high temperature. Our findings suggested that the RNA editing events were very sensitive to heat stress; the changes of amino acid in RNA editing genes may contribute to the stress adaption for grape.

Genome-Wide Analysis of the DYW Subgroup PPR Gene Family and Identification of GmPPR4 Responses to Drought Stress

Pentatricopeptide-repeat (PPR) proteins were identified as a type of nucleus coding protein that is composed of multiple tandem repeats. It has been reported that PPR genes play an important role in RNA editing, plant growth and development, and abiotic stresses in plants. However, the functions of PPR proteins remain largely unknown in soybean. In this study, 179 DYW subgroup PPR genes were identified in soybean genome (Glycine max Wm82.a2.v1). Chromosomal location analysis indicated that DYW subgroup PPR genes were mapped to all 20 chromosomes. Phylogenetic relationship analysis revealed that DYW subgroup PPR genes were categorized into three distinct Clusters (I to III). Gene structure analysis showed that most PPR genes were featured by a lack of intron. Gene duplication analysis demonstrated 30 PPR genes (15 pairs; ~35.7%) were segmentally duplicated among Cluster I PPR genes. Furthermore, we validated the mRNA expression of three genes that were highly up-regulated in soybean drought- and salt-induced transcriptome database and found that the expression levels of GmPPR4 were induced under salt and drought stresses. Under drought stress condition, GmPPR4-overexpressing (GmPPR4-OE) plants showed delayed leaf rolling; higher content of proline (Pro); and lower contents of H2O2, O2− and malondialdehyde (MDA) compared with the empty vector (EV)-control plants. GmPPR4-OE plants exhibited increased transcripts of several drought-inducible genes compared with EV-control plants. Our results provided a comprehensive analysis of the DYW subgroup PPR genes and an insight for improving the drought tolerance in soybean.

Dynamic response of RNA editing to temperature in Grape by RNA deep-sequencing

RNA editing is a post-transcriptional process of modifying genetic information on RNA molecules, which provides cells an additional level of gene expression regulation. Unlike mammals, in land plants, RNA editing converts C to U residues in organelles. However, its potential role in response to different stressors (heat, salt and so on) remains unclear. Grape is one of the most popular and economically important fruits in the world, and its production, like other crops, must deal with abiotic and biotic stresses, which cause reductions in yield and fruit quality. In our study, we tested the influence of the environmental factor temperature on RNA processing in the whole mRNA from grape organelle. In total, we identified 123 and 628 RNA editing in chloroplast and mitochondria respectively with the average editing extent nearly ~60%. The analyses revealed that number of non-synonymous editing sites were higher than that of synonymous editing sites, and the amino acid substitution type tend to be hydrophobic. Additionally, the overall editing level decreased with the temperature rises, especially several gene transcripts in chloroplast and mitochondria (matK, ndhB etc.). 245 sites were furthermore determined as stress-responsive sites candidates. We also found that the expression level of PPR genes decreased with the temperature rises, which may contribute to the loss of RNA editing at high temperature. Our findings suggest that the RNA editing events are very sensitive to high temperature, the changes of amino acid in these genes may contribute to the stress adaption for grape.

GhYGL1, a pentatricopeptide repeat protein, is required for chloroplast development in cotton

Background The pentatricopeptide repeat (PPR) gene family, which contains multiple 35-amino acid repeats, constitutes one of the largest gene families in plants. Pentatricopeptide repeat genes function in organelles to target specific transcripts and are involved in plant development and growth. However, the function of PPR genes in cotton is still unknown. Results In this study, we characterized a PPR gene (GhYGL1), producing a yellow green leaf phenotype, that is required for cotton plastid development. The GhYGL1 gene has a DYW domain in C-terminal and is highly express in leaves, localized to the chloroplast fractions. GhYGL1 share high amino acid-sequence homology with AtECB2. In ecb2-mutated Arabidopsis, overexpression of GhYGL1 rescued the seedling lethal phenotype and restored the editing of accD and ndhF transcripts. Silencing of GhYGL1 led to the reduction of chlorophyll and phenotypically yellow-green leaves in cotton. Compared with wild type, GhYGL1-silenced cotton showed significant deformations of thylakoid structures. Furthermore, the expression levels of plastid-encoded polymerase- (PEP) and nuclear-encoded polymerase- (NEP) dependent genes were significantly decreased in GhYGL1-silenced cotton. Conclusions Our data indicate that GhYGL1 not only controls the editing of accD and ndhF genes, but also controls the expression of NEP- and PEP-dependent genes to regulate the development of thylakoids, and therefore regulates leaf variegation in cotton.