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.

Isolation of Metallothionein (MT) Gene from Phytoremediation Agent, Eleusine indica

Belulang grass (Eleusine indica) is a plant in the Poaceae family that is commonly found in the coastal area of Dumai, Riau Province. Eleusine indica is characterized by narrow leaves, concave stems that can reach up to 95 cm high and strong roots. E. indica is known to be very tolerant of its environment, including the environment contaminated with heavy metals. The ability of E. indica as a phytoremediation agent in absorbing heavy metals has been widely known as the role of metallothionein (MT) protein. MT is believed to have a function in the metal metabolism and detoxification process through the metal chelating interaction between the cysteine amino acid residues. This unique function prompted the interest to isolate the MT gene from E. indica. This method involves the isolation of genomic DNA from E. indica followed by the process of amplification of the MT gene using specific primers, namely MTFS and MTRS by polymerase chain reaction (PCR) technology. The success of the MT gene isolation process from E. indica was evidenced by the presence of a single band size of around 172 bp via the visualization process on 1% agarose gel. Furthermore, the results of the PCR product are purified for the purpose of sequencing activity. The results of sequencing analysis of the 172 bp fragment showed 99.31% identical similarity with the complete metallothionein gene from E. indica (DQ082855.1) by using the BLASTN tool, NCBI website.

Topological isolation of developmental regulators in mammalian genomes

Precise control of mammalian gene expression is facilitated through epigenetic mechanisms and nuclear organization. In particular, insulated chromosome structures are important for regulatory control, but the phenotypic consequences of their boundary disruption on developmental processes are complex and remain insufficiently understood. Here, we generated deeply sequenced Hi-C data for human pluripotent stem cells (hPSCs) that allowed us to identify CTCF loop domains that have highly conserved boundary CTCF sites and show a notable enrichment of individual developmental regulators. Importantly, perturbation of such a boundary in hPSCs interfered with proper differentiation through deregulated distal enhancer-promoter activity. Finally, we found that germline variations affecting such boundaries are subject to purifying selection and are underrepresented in the human population. Taken together, our findings highlight the importance of developmental gene isolation through chromosomal folding structures as a mechanism to ensure their proper expression.

Seeing the Forest through the (Phylogenetic) Trees: Functional Characterisation of Grapevine Terpene Synthase (VviTPS) Paralogues and Orthologues

Gene families involved in specialised metabolism play a key role in a myriad of ecophysiological and biochemical functions. The Vitis vinifera sesquiterpene synthases represent the largest subfamily of grapevine terpene synthase (VviTPS) genes and are important volatile metabolites for wine flavour and aroma, as well as ecophysiological interactions. The functional characterisation of VviTPS genes is complicated by a reliance on a single reference genome that greatly underrepresents this large gene family, exacerbated by extensive duplications and paralogy. The recent release of multiple phased diploid grapevine genomes, as well as extensive whole-genome resequencing efforts, provide a wealth of new sequence information that can be utilised to overcome the limitations of the reference genome. A large cluster of sesquiterpene synthases, localised to chromosome 18, was explored by means of comparative sequence analyses using the publicly available grapevine reference genome, three PacBio phased diploid genomes and whole-genome resequencing data from multiple genotypes. Two genes, VviTPS04 and -10, were identified as putative paralogues and/or allelic variants. Subsequent gene isolation from multiple grapevine genotypes and characterisation by means of a heterologous in planta expression and volatile analysis resulted in the identification of genotype-specific structural variations and polymorphisms that impact the gene function. These results present novel insight into how grapevine domestication likely shaped the VviTPS landscape to result in genotype-specific functions.

VPB1 Encoding BELL-like Homeodomain Protein Is Involved in Rice Panicle Architecture

Inflorescence architecture in rice (Oryza sativa) is mainly determined by spikelets and the branch arrangement. Primary branches initiate from inflorescence meristem in a spiral phyllotaxic manner, and further develop into the panicle branches. The branching patterns contribute largely to rice production. In this study, we characterized a rice verticillate primary branch 1(vpb1) mutant, which exhibited a clustered primary branches phenotype. Gene isolation revealed that VPB1 was a allele of RI, that it encoded a BELL-like homeodomain (BLH) protein. VPB1 gene preferentially expressed in the inflorescence and branch meristems. The arrangement of primary branch meristems was disturbed in the vpb1 mutant. Transcriptome analysis further revealed that VPB1 affected the expression of some genes involved in inflorescence meristem identity and hormone signaling pathways. In addition, the differentially expressed gene (DEG) promoter analysis showed that OsBOPs involved in boundary organ initiation were potential target genes of VPB1 protein. Electrophoretic mobility shift assay (EMSA) and dual-luciferase reporter system further verified that VPB1 protein bound to the promoter of OsBOP1 gene. Overall, our findings demonstrate that VPB1 controls inflorescence architecture by regulating the expression of genes involved in meristem maintenance and hormone pathways and by interacting with OsBOP genes.

Fine Mapping and Candidate Gene Analysis of the Up Locus Determining Fruit Orientation in Pepper (Capsicum spp.)

Fruit orientation is an important horticultural and domesticated trait, which is controlled by a single semi-dominant gene (up) in pepper. However, the gene underlying up locus has not yet been identified. In this study, the previously detected major QTL UP12.1 was firstly verified using an intraspecific backcross population (n=225) stem from the cross of BB3 (C. annuum) and its wild relative Chiltepin (C. annuum var. glabriusculum) using BB3 as the recurrent parent. Then, a large BC1F2 population (n=1827) was used for recombinant screening to delimit the up locus into an interval with ~169.51 kb in length. Sequence comparison and expression analysis suggested that Capana12g000958, encoding a developmentally regulated G-protein 2, was the most likely candidate gene for up. The findings of this study will form a basis for gene isolation and reveal of genetic mechanism underlying the fruit orientation domestication in pepper.