Soil Chemical Analysis

In recent years, molecular markers have been utilized for a variety of applications including examination of genetic relationships between individuals, mapping of useful genes, construction of linkage maps, marker assisted selections and backcrosses, population genetics and phylogenetic studies. Among the available molecular markers, microsatellites or simple sequence repeats (SSRs) which are tandem repeats of one to six nucleotide long DNA motifs, have gained considerable importance in plant genetics and breeding owing to many desirable genetic attributes including hypervariability, multiallelic nature, codominant inheritance, reproducibility, relative abundance, extensive genome coverage including organellar genomes, chromosome specific location and amenability to automation and high throughput genotyping. High degree of allelic variation revealed by microsatellite markers results from variation in number of repeat-motifs at a locus caused by replication slippage and/or unequal crossing-over during meiosis. In spite of limited understanding of the functions of the SSR motifs within the plant genes, SSRs are being widely utilized in plant genome analysis. Microsatellites can be developed directly from genomic DNA libraries or from libraries enriched for specific microsatellites. Alternatively, microsatellites can also be found by searching public databases such as GenBank and EMBL or through cross-species transferability. At present, EST databases are an important source of candidate genes, as these can generate markers directly associated with a trait of interest and may be transferable in close relative genera. A large number of SSR based techniques have been developed and a quantum of literature has accumulated regarding the applicability of SSRs in plant genetics and genomics. In this review we discuss the recent developments (last 4–5 years) made in plant genetics using SSR markers.

Microsatellite markers: an overview of the recent progress in plants

The origin of plant cell and tissue culture can be found in a treatise published during the mid-18th century, entitled La Physique des Arbes, that describes the formation of callus tissue following the for mation of a ring of cortex from elm trees. Over the next two centuries, the discovery of plant growth hormones, in particular auxins and cytokinins, and detailed analyses on the nutritional requirements of plants, led to the formulation of media that could maintain actively dividing cultures derived from gymnosperms, and both dicotyledon ous and monocotyledonous angiosperms. However, much of the prog ress and technological development in the in vitro propagation of plant cells, tissues, and organs has occurred during the last 25 years. Recently, plant tissue culture techniques have been used as basic tools in the rapidly expanding field of plant biotechnology for the development and clonal propagation of new and/or improved plant varieties. Plant tissue culture is used for the micropropagation of commercially valuable cultivars that include ornamentals, oil palm, Glycyrrhiza, Pyrethrum, pine, Eucalyptus, sugar cane, and potatoes. Cultured plant tissue is also used for the selection of cells and, ul timately, the regeneration of plants that are tolerant to physical stresses such as pathogens, drought, and temperature extremes, and to chemical stress agents such as salinity, herbicides, proteins, and pyrethrins. In addition, new plants have been produced by the fusion of protoplasts prepared from cultured cells of different species in cluding sunflower and french bean, tomato and potato, and various cultivars of Datura. Finally, bacterial vectors and various mechanical methods have been used to introduce foreign genes into cultured plant tissues. Genetic transformation can result in profound changes in the phenotype and/or biochemical profile of the regenerated trans genic plants that are not characteristic of the wild type. An impressive variety of technologies in tissue culture, genetic manipulation, and molecular biology have been developed for nu merous plant species. Many of these techniques, sometimes referred to as plant biotechnology, have been extensively summarized and compiled in a well-balanced collection of easy-to-follow protocols presented in three separate, but complimentary, volumes. Plant Cell, Tissue and Organ Culture consists of 22 chapters (with 86 figures) and 5 appendices. The chapters cover critical aspects of (a) the es sential requirements for the operation of a plant tissue culture lab oratory; (b) the basic procedures of micropropagation, regeneration, and somatic embryogenesis; (c) some specific applications of organ culture systems such as embryo rescue and culture, and anther and microspore culture for haploid and double haploid production; (d) elementary transformation technology; and (e) useful microtechnique and analytical protocols specifically adapted to cultured tissues and cells. The appendices provide a convenient summary of media for mulations and commercial suppliers for the materials described in the text.

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Plant Cell, Tissue and Organ Culture

The use of homologous recombination to precisely modify plant genomes has been challenging, due to the lack of efficient methods for delivering DNA repair templates to plant cells. Even with the advent of sequence-specific nucleases, which stimulate homologous recombination at predefined genomic sites by creating targeted DNA double-strand breaks, there are only a handful of studies that report precise editing of endogenous genes in crop plants. More efficient methods are needed to modify plant genomes through homologous recombination, ideally without randomly integrating foreign DNA. Here, we use geminivirus replicons to create heritable modifications to the tomato genome at frequencies tenfold higher than traditional methods of DNA delivery (i.e., Agrobacterium). A strong promoter was inserted upstream of a gene controlling anthocyanin biosynthesis, resulting in overexpression and ectopic accumulation of pigments in tomato tissues. More than two-thirds of the insertions were precise, and had no unanticipated sequence modifications. Both TALENs and CRISPR/Cas9 achieved gene targeting at similar efficiencies. Further, the targeted modification was transmitted to progeny in a Mendelian fashion. Even though donor molecules were replicated in the vectors, no evidence was found of persistent extra-chromosomal replicons or off-target integration of T-DNA or replicon sequences. High-frequency, precise modification of the tomato genome was achieved using geminivirus replicons, suggesting that these vectors can overcome the efficiency barrier that has made gene targeting in plants challenging. This work provides a foundation for efficient genome editing of crop genomes without the random integration of foreign DNA.

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High-frequency, precise modification of the tomato genome

At the end of the class, students will be able to answer the following questions: • What is organic agriculture? • Where do I go to get certification in organic agriculture? • How can I get more information about organic agriculture? What is organic? • The term " organic " is not synonymous to the terms " natural " or " eco-friendly. " • The label " natural " on foodstuff does not guarantee complete adherence to organic practices as defined by a law. • Produce food of high quality in sufficient quantity • Maintain biological diversity within the farming system • Maintain long-term soil fertility • Rely on renewable resources in locally organized agricultural systems • Minimize pollution and protect the environment What is allowed in organic crop production? • New varieties of crops and agricultural technologies • Crop rotations, cover crops and natural-based products that maintain soil fertility • Biological, cultural and physical methods to limit pest expansion and increase population of beneficial insects

英国有机农业（Organic farming）的新举措

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.

Genetic relationships within and between 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.

Polymorphic Microsatellite Markers Transferable Across Capsicum Species

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.

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

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.

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

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

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Ayse Gul Ince

Papers

Genetic relationships within and between Capsicum species.

Polymorphic Microsatellite Markers Transferable Across Capsicum Species

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

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

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