Ayse Gul Ince

Polymorphic Microsatellite Markers Transferable Across Capsicum Species

Pepper (Capsicum annuum L.) is one of the most important crops in the family Solanaceae. However, the number of polymorphic molecular loci detected in this important crop is far behind that of other cultivated plant species. In the present study, a total of 45 microsatellite primer pairs were developed using Capsicum expressed sequence tags databases. Microsatellite primer pairs were tested using several species of Capsicum and several genera in the family Solanaceae including tomato, potato, eggplant, and tobacco. Results indicated that microsatellite primer pairs amplified genomic targets of C. annuum L., Capsicum baccatum L., Capsicum chacoense L., Capsicum chinense L., Capsicum frutescens L., and Capsicum pubescens Ruiz et Pavon, indicating species transferability within Capsicum. Further analyses revealed that amplicons of these primer pairs segregated 1:2:1 or 3:1 Mendelian fashions in 38 F2 individuals of pepper. It was also noted that markers derived from sequences containing dinucleotide repeats were generally more polymorphic at the intraspecific level than sequences containing trinucleotide repeats. All the microsatellite primer pairs developed in this study will be useful for marker-assisted selection and mapping studies in pepper.

Development and utilization of diagnostic DAMD-PCR markers for Capsicum accessions

A polymerase chain reaction (PCR) based approach involving the directed amplification of minisatellite DNA region (DAMD-PCR) was used to identify accession specific DNA markers and study genetic relationships between and within 15 accessions corresponding to 11 species in genus Capsicum. A touch down PCR profile and unique chemical concentration of ingredients resulted in reproducible and reliable DNA amplifications. The number of amplified products varied from 1 to 12 fragments depending on the template DNA and the primers. The DAMD-PCR technique provided a total of 38 accession specific DNA markers (diagnostic DAMD-PCR) which can be utilized in accession identification, preservation and genetic studies of Capsicum germplasm. Based on 1,292 polymorphic and monomorphic DNA markers directed with 22 minisatellite specific primers, accessions were divided into four major groups, three of which corresponded to the three distinct Capsicum complexes. Capsicum chacoense was found to be the most distinct species.

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.

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.

Minisatellites as DNA markers to classify bermudagrasses (Cynodon spp.): confirmation of minisatellite in amplified products

Hyper variable repetitive DNA sequences such as microsatellites, minisatellites, midisatellites and satellites can be of great value in assessing a high level of polymorphism as they are distributed throughout the genomes. Here we used a polymerase chain reaction (PCR) technique, known as the directed amplification of minisatellite-region DNA (DAMD), an inexpensive, PCR-based method to amplify minisatellite DNA regions of 11 Cynodon species. A total of 10 minisatellite core primer sequences derived from other species, including human, were used as primers in DAMD-PCR to assess polymorphisms within 11 Cynodon species. A total of six bands of amplified products were cloned and sequenced. Results indicated that not all the amplified products contained minisatellites. However, the primers used in the DAMD technique offered highly reproducible and polymorphic DNA markers differentiating all the species of bermudagrasses studied. DNA markers are much valuable tool for the plant breeders and taxonomist because they do not have the disadvantages of isozyme and morphological markers, which are usually affected by environmental conditions and different stages of plant development. DNA markers offer a rapid, cost effective, safe and efficient way to test the genetic purity of the varieties, and are a powerful tools in any genome mapping programme to identify DNA markers linked to economically important quantitative traits (Karaca et al. 2002, 2004). There are several DNA-marker techniques for genetic analyses. The restriction fragment length polymorphism (RFLP) technique, is one of the first DNA methods used in a variety of organisms in mapping and genetic diversity studies (Williams and Clair 1993). However, the RFLP technique has several limitations in comparison to various PCRbased marker techniques. The RFLP technique requires large

CAPS-microsatellites: use of CAPS method to convert non-polymorphic microsatellites into useful markers

Microsatellites found in expressed sequence tags (ESTs) have several advantages in comparison to conventional microsatellites developed from genomic DNA. Advantages of EST-microsatellites include better transferability between the species and lower development cost of primer pairs. However, in many cases the amplified products of genomic DNA are larger than their expected sizes based on ESTs and these markers have low level of polymorphism. In the present study we report on a new approach as a modest modification of the cleavage amplified polymorphic sequence (CAPS) called CAPS-microsatellites. Using a total of 20 EST-microsatellite primer pairs on four Capsicum species and two Gossypium species we observed that all 20 monomorphic EST-microsatellites could be converted into polymorphic markers among the species used. Further analyses using a total of 37 F2 individuals obtained from two Capsicum annuum L. lines indicated that CAPS-microsatellite markers usually segregated as co-dominant fashions, discriminating the homozygous from heterozygous genotypes and could be used in genetic analyses of plant species.

A reliable gender diagnostic PCR assay for jojoba (Simmondsia chinensis (Link) Schneider)

Jojoba (Simmondsia chinensis (Link) Schneider) is an obligate cross-pollinated shrub native to the Sonora desert. The most valuable product of the jojoba seeds is the liquid wax or jojoba oil which is marketed extensively in the cosmetic industry. Differing from the most of the cultivated crop species, jojoba has slow-growing habit, difficulties in the determination of sex at the early seedling stages, a male-biased ratio and low seed yield. In jojoba, the development of molecular strategies for the identification of sexes at early stages has been a priority in plantations and breeding programs. Two previous studies reported two candidate male-specific jojoba DNA markers. However, present study indicated that these markers were not useful in jojoba sex identification. A reliable gender diagnostic marker for jojoba is, therefore, needed. In the present study a novel jojoba male-specific touch-down polymerase chain reaction based DNA marker (JMS900) was reported using a total of 120 individual jojoba plants bulked into 16 samples. This sex specific DNA marker may have considerable theoretical and practical applications in the establishment of jojoba plantation and breeding studies.

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.

Early determination of sex in jojoba plant by CAPS assay

Jojoba [Simmondsia chinensis (Link) Schneider] is a dioecious plant grown for its seeds, which are the source of liquid wax or jojoba oil. The sex of jojoba plants cannot be determined with morphological characters until the plants reach reproductive maturity at 3 or more years old. This difficulty of early sex determination imposes severe constraints in breeding studies and in the sex allocation of seedlings in seed orchard establishment, and importantly in a priori mating designs to produce superior jojoba individuals. This study reports three new cleavage-amplified polymorphic sequence (CAPS) assays, which identify male and female individuals distinctly. One of the assays could also identify hermaphrodite jojoba plants existing in nature or obtained using mutagenesis studies.

Differential expression patterns of genes containing microsatellites in Capsicum annuum L.

Microsatellites are important for research in both basic and applied sciences. Understanding how genes containing microsatellites are expressed and regulated in different tissues and developmental stages is a fundamental and challenging question. However, there has been limited study of differential expression of genes containing microsatellites in different plant tissues and developmental stages. We studied microsatellite-containing gene expression profiles in 16 different tissues and at different developmental stages in pepper (Capsicum annuum L.). Results indicated that tissue-specific genes contained more dinucleotide microsatellites, and housekeeping genes contained more trinucleotide microsatellites. Differential expression patterns of microsatellite-containing genes indicated that, in the longer term, development of allele-specific markers for genes controlling agronomic traits will be useful for advancing the science of plant breeding. This is the first report on differential expression of microsatellite-containing genes within tissues and developmental stages in pepper.