Derek W. Barchenger, Joseph I. Said, Franchesca A. Ortega, and Paul W. ... [OPP13-CAPS (Kim, 2005), AFRF8-CAPS (Kim et al., 2006), PR-CAPS (Lee et al.,.
Genome-wide Identification of Chile Pepper Pentatricopeptide Repeat (PPR) Domains Provides Insight into Restoration of Fertility Derek W. Barchenger, Joseph I. Said, Franchesca A. Ortega, and Paul W. Bosland
Introduction
Department of Plant and Environmental Sciences, New Mexico State University, Las Cruces NM 88003.
Results
Pentatricopeptide-repeat (PPR) proteins are a large family of proteins that are common in the plant kingdom, and are important in gene regulation.
Fig. 1. Chromosome maps of the 552 putative C. annuum pentatricopeptide repeat domains identified in this project. The PPR domains were identified using the known Arabidopsis PPRs queried against the chile pepper genome. The number directly to the right of each chromosome is the location of each PPR domain in 10,000 bp. The name given to each PPR domain in this study is to the left of its location. The chromosome number is at the bottom of each map.
Encoded in the nucleus, PPRs facilitate processing, splicing, editing, stability, and translation of RNA, primarily in organelles. Cytoplasmic male sterility (CMS) is the failure to produce functional pollen and is controlled by plant mitochondrial genome. Restoration-of-fertility (Rf) genes act to mask the mitochondrial genes that determine CMS, thus restoring fertility. Many Rf genes are known to encode PPRs proteins, although their mode of action is largely unknown. CMS and Rf are important traits for efficient and less expensive F1 hybrid seed production. Hybrid cultivars are often advantageous for farmers as they can display heterosis, or hybrid vigor, resulting in an increase in the level of quality traits and yield. Hybrid cultivars can be expensive to produce because without a CMS/Rf system in place they require laborious and costly hand emasculation and pollinator exclusion. An effective molecular marker for CMS in chile pepper is currently in use, allowing for faster selection of this trait. Markers for Rf in chile pepper [OPP13-CAPS (Kim, 2005), AFRF8-CAPS (Kim et al., 2006), PR-CAPS (Lee et al., 2008), CRF-SCAR (Gulyas et al., 2006), and PPe1 (Jo et al., 2010)] have limited applicability due to lack of agreement between the marker and the phenotype across all chile pepper cultivars.
Objectives To identify and map the PPR domains in chile pepper, providing a basis for future work with this increasingly important gene family. To characterize the PPR encoding Rf genes in chile pepper in an attempt to explain the lack of phenotype and marker agreement for this important trait.
Materials and Methods The known Arabidopsis PPR domains were queried against the C. annuum genome using MAKER2, creating a GFF file of the PPR domains of chile pepper. BLOSUM (Block Substitution Matrix) was employed to match PPRs to the genome, and a BLAST (Basic Local Alignment Search Tool) score was assigned to each match. Candidate PPR domain sequences were extracted using script written within the BioStrings package in R software (Version 3.1.2).
Nucleotide sequences were translated into protein sequences, and scanned for PPR motifs using ExPASy software. BLAST was used with the protein sequences containing PPR motifs to identify possible function.
References Gulyas, G., K. Pakozdi, J.S. Lee, and Y. Hirata. 2006. Analysis of fertility restoration by using cytoplasmic male-sterile red pepper (Capsicum annuum L.) lines. Breeding Sci. 56:331-334. Jo, Y.D., Y.M. Kim, M.N. Park, J.H. Yoo, M. Park, B.D. Kim, and B.C. Kang. 2010. Development and evaluation of broadly applicable markers for Restorer-of-fertility in pepper. Mol. Breeding 25:187-201. Kim, D.S. 2005. Development of RAPD and AFLP markers linked to fertility restorer (Rf) gene in chili pepper (Capsicum annuum L.). Thesis. Seoul National University. Kim, D.S., D.H. Kim, J.H. Yoo, B.D. Kim. 2006. Cleaved amplified polymorphic sequence and amplified fragment length polymorphism markers linked to the fertility restorer gene in chili pepper (Capsicum annuum L.). Mol. Cells 21:135-140. Lee, J. J.B. Yoon, and H.G. Park. 2008. Linkage analysis between the partial restoration (pr) and the restorer-of-fertility (Rf) loci in pepper cytoplasmic male sterility. Theor. Appl. Genet. 117:383-389.
Table 1. Location and gene length of fourteen PPR genes identified in chile pepper as having similarity to known restorer-of-fertility genes. C. annuum PPRs Chromsome Gene location (bp) Gene length (bp) CaPPR1.20 1 167995867 1128 CaPPR1.21 1 167995224 285 CaPPR6.34 6 174129855 180 CaPPR6.35 6 174130124 213 CaPPR6.38 6 206431066 1731 CaPPR6.39 6 206566726 1062 CaPPR6.40 6 206567906 423 CaPPR6.41 6 206731489 468 CaPPR6.42 6 207392625 648 CaPPR6.43 6 207393499 603 CaPPR6.44 6 211275521 537 CaPPR6.45 6 211280934 273 CaPPR6.46 6 214033838 2259 CaPPR6.48 6 217079895 507 Fig. 2. Dendrogram of the candidate Rf genes identified in this study (those that begin with CaPPR) with Rf and Rflike genes from several species. Zea mays Rf-2 was used as the root of the tree because it does not encode a PPR protein. The dendrogram was made with protein sequences using ClustalW for multiple sequence alignment.
Discussion We successfully identified and mapped 552 PPR domains in the chile pepper genome. These domains could possibly include complete PPR genes, pseudogenes, and gene fragments, which could be the result of transposition, a common phenomenon in chile pepper. Although not displayed here, we found 154 of the PPR genes identified in this study to have high similarity to known PPRs in Arabidopsis. These conserved genes could provide a basis for future work in this area, as similar genes among species could have similar function. Interestingly, we found 14 of the chile pepper PPR domains to have similarity to restorer-offertility (Rf) genes of other species. These 14 Rf gene copies could be the reason a more widely applicable molecular marker for this trait has not yet been found in chile pepper. Most of these possible Rf genes were found to be clustered on chromosome 6, which is similar to the findings of Jo et al. (2010). We also found 2 possible Rf genes on chromosome 1. This is noteworthy, because the Rf genes of Arabidopsis are known to be on chromosome 1.
Outlook The next step is to characterize the 14 possible Rf genes identified in this study in individuals displaying different phenotypes for this trait. We then plan on identifying a set of molecular markers that have the potential to identify Rf individuals among diverse populations of chile peppers.
