Grafting based DNA methylation alteration of snoRNAs in upland cotton (Gossypium L.).

The effects of grafting in response to various biotic and abiotic stressors have been studied, however, the methylation status of small nucleolar RNA (snoRNA) genes in heterograft and homograft cotton needs investigation. This study was undertaken to determine grafting effects on DNA methylation of snoRNA genes in Upland cotton. Rootstocks used were Pima 3-79 (Gossypium barbadense acc. Pima 3-79) and Texas Marker-1 (G. hirsutum acc. TM-1), representing two different species with different fiber properties, adaptations, and morphologies. The methylation ratio and differently methylated cytosines (DMCs) of 10935 snoRNA genes in mature seeds of heterograft and homograft cotton samples were studied using the whole genome bisulfite sequencing method. Seedling vigor and seed weight were studied to investigate phenotype alterations that might be associated with altered methylation levels among grafts. Statistically significant DMC differences among gene elements of snoRNA genes and between homograft and heterograft cotton samples were identified in the absence of DNA sequence alterations. DNA methylation alterations of snoRNA genes associated with seedling vigor and 100 seed weight. The majority of snoRNA genes showed higher numbers of mCG + mCHG-DMCs with increased methylation levels in heterograft, while there were higher numbers of mCG + mCHG-DMCs with decreased methylation levels in homograft. Since snoRNAs regulate essential genes for plant growth and development and plant adaptation to different habitats or extreme environments, their altered methylation levels should be related with plant physiology.

A DNA Extraction Method for Nondestructive Testing and Evaluation of Cotton Seeds (Gossypium L.).

Kernels of cotton provide lint and linter for textiles, oil and protein for food and feed. Cotton seed is formed following fertilization between an ovule and a pollen grain. The seed coat is maternal in origin, whereas the embryo and attached cotyledonary leaves are hybrids of parental lines. The extraction of genomic DNA from an ungerminated whole, a portion or mixed seeds are prerequisite in genetic and genomic studies of cotton. As far as our knowledge, there is only one method of nondescriptive DNA extraction from ungerminated cotton seeds without affecting the seed germination capability, but it has technical difficulties and requires special equipment. Furthermore, the amount of DNA extracted using the published method is low and, therefore, it is only suitable for routine marker assisted selection studies. In this study, a DNA extraction protocol referred to as the CTAB-LiCl was developed for single whole cotton seed, a portion of cotton seed and bulked cotton seeds. This protocol uses a combination of CTAB and LiCl to lyse cells and deplete RNAs simultaneously. The CTAB-LiCl DNA extraction method was evaluated in ninety-six individuals of six different cotton cultivars along with two genetic standards of cotton, TM-1 (G. hirsutum L.), Pima 3-79 (G. barbadense L.), and several other plant species of different plant genera. Results revealed that this method produced high quality and amounts of DNA as confirmed by spectrophotometry, agarose gel, restriction enzyme digestion, polymerase chain reaction, and library production for next generation sequencing studies of whole genome bisulfite sequencing. It does not require the use of liquid nitrogen, RNase, proteinase K, or beta-mercaptoethanol and can be completed in approximately 2 h. Small tissues of the chalaza ends of ungerminated cotton seeds could be used to obtain high quality and quantity of DNA ranging from 14 to 28 µg without affecting the seeds' germination ability, allowing marker-assisted selection before planting and flowering.

Tissue and/or developmental stage specific methylation of nrDNA in Capsicum annuum.

The nuclear ribosomal DNA (nrDNA) sequences are often used for phylogenetic analysis among organisms. Because DNA cytosine methylation and nucleolar dominancy are two common epigenetic mechanisms of nrDNA, we hypothesized that internal transcribed spacer 1 (ITS1), 5.8S rRNA and ITS2 of nrDNA sequences could be used as epigenetic biomarkers. Thus, this research was undertaken to study level and pattern of site-specific cytosine methylation of ITS1, 5.8S and ITS2 in nine tissues and/or developmental stage of pepper Capsicum annuum L. cultivar Demre Sivrisi. Tissues studied consisted of young and old roots at 30 and 90 days after sowing (das), mature dry seeds and seeds at 26 days of post anthesis (dpa), flowering buds at 1 day before flowering, pericarps at 3, 15 and 65 dpa. Levels and patterns of DNA cytosine methylation were identified at single base resolution using bisulfite conversion sequencing. Results of this study revealed that DNA cytosine level and pattern of ITS1, 5.8S and ITS2 were different in most tissues and/or developmental stages studied. In addition, methylation levels of CG, CHG and CHH contexts were also significantly different among the regions. Based on the findings of this study, it was concluded that high level of methylation of nrDNA sequences was relatively higher as observed in transposable element and promoter. On the other hand, its tissue-specific gene expression was effective as that of gene body and promoter methylation. Overall findings revealed that methylation levels of nrDNA could be used as biomarkers for tissue identification or age estimation in plants.

Interspecific grafting between Gossypium hirsutum, G. barbadense and G. herbaceum lines.

Seedling grafting could provide additional crop improvement strategies for cotton. However, there existed limited studies on interspecific grafting and approaches. Four different grafting approaches were developed and compared between lines representing three of the four cultivated cotton species G. hirsutum, G. barbadense and G. herbaceum. Grafting approaches of this study focused on the cotyledon node and cotyledon leaves retained on scions, rootstocks, without cotyledon node and cotyledon leaves on scions and rootstocks or halved cotyledon node and single cotyledon leaf on scions and rootstocks. Evaluations of the grafting approaches were made by comparing survival and growth rate during the second and fifth weeks after transplantation, respectively. The formation of any lateral shoots at the grafted sites were studied in two of four grafting approaches in the first and the second year during flowering stage. DNA alterations due to grafting were investigated using microsatellite markers. There were no statistically significant differences between grafts and their control in survival rate and locus specific DNA alteration. Growth rate and lateral shoot formation, on the other hand, were different among grafting types and grafts. We concluded that grafting without cotyledon node and cotyledon leaves on rootstocks, and with cotyledon node but without cotyledon leaves on scions were easy to perform and suitable for interspecific cotton grafting. Results suggested that grafting seedlings and allowing time to heal graft wounds prior to spring transplanting or double cropping is suitable for wheat-cotton intercropping to prevent late or early chilling damage associated with seed sowing or conventional transplanting of susceptible seedlings. Furthermore, the rapid and consistent wound healing in seedling grafts along with lateral shoot formation occurring in two of four grafting approaches make them a suitable approach to investigate possible genetic and epigenetic movement between scions and rootstocks, especially across species.

Cross-genera transferable e-microsatellite markers for 12 genera of the Lamiaceae family.

BACKGROUND: The Lamiaceae family contains many high-valued medicinal, aromatic and ornamental plant species. Several members of the genera in this family are under heavy pressure of collection for commercial use. DNA markers such as microsatellites could be used to identify commercially important genotypes and to select high-yielding ones for development of new varieties. RESULTS: A total of 12,432 expressed sequence tags (ESTs) from Salvia fruticosa, S. miltiorrhiza, S. sclarea and Stenogyne rugosa were analyzed. A total of 6216 ESTs were found to be unique according the redundancy test used. Results of this study indicated that the use of redundant ESTs in comparison to non-redundant ESTs was advantageous in terms of higher cross-genera transferability of the markers. A total of 75 EST-microsatellite primer pairs were tested using two different polymerase chain reaction amplification profiles and 52 were found to be cross-genera transferable. Cross-genera transferability of the e-microsatellite primer pairs varied from one species to 12 species tested. It was noted that cross-genera transferability of e-microsatellite primer pairs decreased as the evolutionary distance between the sources and target species increased. CONCLUSION: This study indicated that EST resources from Salvia spp. and Stenogyne rugosa could be successfully used to identify cross-genera transferable e-microsatellite markers for uncharacterized genomes of the genera in the Lamiaceae family. These e-microsatellite markers could allow one to perform comparative analyses of population structure and genomic studies, and facilitate comparative linkage mapping in the genera studied. E-microsatellite primer pairs reported in this manuscript are equivalent to a total of 135 e-microsatellite primer pairs since many e-microsatellite primer pairs show cross-genera transferability.

Genetic relationships within and between Capsicum species.

Genetic relationships were estimated among 24 accessions belonging to 11 species of Capsicum, using 2,760 RAPD markers based on touch-down polymerase chain reactions (Td-RAPD-PCR). These markers were implemented in analyses of principal coordinates, unweighted pair group mean average, and 2,000 bootstrap replications. The accessions were divided into four groups, corresponding to previously described Capsicum complexes: C. annuum complex (CA), C. baccatum complex (CB), C. pubescens complex (CP), and C. chacoense accessions (CA/B). Their overall mean genetic similarity index was 0.487 +/- 0.082, ranging from 0.88 to 0.32, based on Jaccard's coefficient. The highest genetic variation was observed among the accessions in CP; the accessions in CB had a low level of variation as judged from the standard deviations of the genetic similarity indices. Based on the Td-RAPD-PCR markers, the 24 accessions were divided into four major groups, three of which corresponded to the three distinct Capsicum complexes. Accessions of C. chacoense were found to be equally related to complexes CA, CB, and CP.

Exact tandem repeats analyzer (E-TRA): a new program for DNA sequence mining.

Exact Tandem Repeats Analyzer 1.0 (E-TRA) combines sequence motif searches with keywords such as 'organs', 'tissues', 'cell lines' and 'development stages' for finding simple exact tandem repeats as well as non-simple repeats. E-TRA has several advanced repeat search parameters/options compared to other repeat finder programs as it not only accepts GenBank, FASTA and expressed sequence tags (EST) sequence files, but also does analysis of multiple files with multiple sequences. The minimum and maximum tandem repeat motif lengths that E-TRA finds vary from one to one thousand. Advanced user defined parameters/options let the researchers use different minimum motif repeats search criteria for varying motif lengths simultaneously. One of the most interesting features of genomes is the presence of relatively short tandem repeats (TRs). These repeated DNA sequences are found in both prokaryotes and eukaryotes, distributed almost at random throughout the genome. Some of the tandem repeats play important roles in the regulation of gene expression whereas others do not have any known biological function as yet. Nevertheless, they have proven to be very beneficial in DNA profiling and genetic linkage analysis studies. To demonstrate the use of E-TRA, we used 5,465,605 human EST sequences derived from 18,814,550 GenBank EST sequences. Our results indicated that 12.44% (679,800) of the human EST sequences contained simple and non-simple repeat string patterns varying from one to 126 nucleotides in length. The results also revealed that human organs, tissues, cell lines and different developmental stages differed in number of repeats as well as repeat composition, indicating that the distribution of expressed tandem repeats among tissues or organs are not random, thus differing from the un-transcribed repeats found in genomes.

A software program combining sequence motif searches with keywords for finding repeats containing DNA sequences.

MOTIVATION: One of the most interesting features of genomes (both coding and non-coding regions) is the presence of relatively short tandemly repeated DNA sequences known as tandem repeats (TRs). We developed a new PC-based stand-alone software analysis program, combining sequence motif searches with keywords such as organs, tissues, cell lines or development stages for finding exact, inexact and compound, TRs. Tandem Repeats Analyzer 1.5 (TRA) has several advanced repeat search parameters/options over other repeat finder programs as it does not only accept GenBank, FASTA and expressed sequence tag (EST) sequence files but also does analysis of multifiles with multisequences. Advanced user-defined parameters/options let the researchers use different motif lengths search criteria for varying motif lengths simultaneously. The outputs show statistical results to be evaluated by the user. The discovery of TRs in ESTs could be useful for both gene mapping and association studies and discovering TRs located in coding regions of important genes that are expressed under various conditions of environment, stress, organ, tissue and development stage. RESULTS: In this paper, we demonstrated applications of TRA using 175 899 ESTs sequences for three Arabidopsis spp. downloaded from GenBank. The EST-SSRs/ESTs ratios were found 43.1%, 15.3% and 2.34% in A.lyrata, A.thaliana and A.halleri, respectively. Analysis revealed that organs, tissues and development stages possessed different amounts of repeats and repeat compositions. This indicated that the distribution of TRs among the tissues or organs may not be random differing from the untranscribed repeats found in genomes. AVAILABILITY: The program can be obtained free by anonymous FTP from ftp.akdeniz.edu.tr/Araclar/TRA.