Chapter 26 Use of Organelle Markers to Study Genetic Diversity in Soybean Lidia Skuza, Ewa Filip and Izabela Szućko Additional information is available at the end of the chapter http://dx.doi.org/10.5772/52028 1. Introduction Soybean is the most important crop provider of proteins and oil used in animal nutrition and for human consumption. Plant breeders continue to release improved cultivars with en‐ hanced yield, disease resistance, and quality traits. It is also the most planted genetically modified crop. The narrow genetic base of current soybean cultivars may lack sufficient al‐ lelic diversity to counteract vulnerability to shifts in environmental variables. An investiga‐ tion of genetic relatedness at a broad level may provide important information about the historical relationship among different genotypes. Such types of study are possible thanks to different markers application, based on variation of organelle DNA (mtDNA or cpDNA). 2. Mitochondrial genome 2.1. Genomes as markers Typically, all sufficiently variable DNA regions can be used in genetic studies of popula‐ tions and in interspecific studies. Because of in seed plants chloroplasts and mitochondria are mainly inherited uniparentally, organelle genomes are often used because they carry more information than nuclear markers, which are inherited biparentally. The main benefit is that there is only one allele per cell and per organism, and, consequently, no recombina‐ tion between two alleles can occur. With different dispersal distances, genomes inherited bi‐ parentally, maternally and paternally, also reveal significant differences in their genetic variability among populations. In particular, maternally inherited markers show diversity within a population much better [1]. © 2013 Skuza et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 554 A Comprehensive Survey of International Soybean Research - Genetics, Physiology, Agronomy and Nitrogen Relationships In gymnosperms the situation is somewhat different. Here, chloroplasts are inherited main‐ ly paternally and are therefore transmitted through pollen and seeds, whereas mitochondria are largely inherited maternally and are therefore transmitted only by seeds [2]. Since pollen is distributed at far greater distances than seeds [3], mitochondrial markers show a greater population diversity than chloroplast markers and therefore serve as important tools in con‐ ducting genetic studies of gymnosperms [4]. Mitochondrial markers are also sometimes used in conjunction with cpDNA markers [5]. Mitochondrial regions used in interspecific studies of plants, mainly gymnosperms, in‐ clude, for example, introns of the NADH dehydrogenase gene nad1 [4, 5, 6], the nad7 in‐ tron 1 [7], the nad5 intron 4 [3] and an internally transcribed spacer (ITS) of mitochondrial ribosomal DNA [8, 9]. In addition to the aforementioned organelle markers, microsatellite markers [10, 11] and simple sequence repeats (SSR) are often used in population biology, and sometimes also in phylogeographic studies. Microsatellites are much less common in plants than in animals [12]. However, they are present in both the nuclear genome and the organelle genome. Mi‐ crosatellites may reveal a high variability, which may be useful in genetic studies of popula‐ tions, whereas other sequences or methods such as fingerprinting do not detect mutations sufficiently [9,10,13]. Inherited only uniparentally, organelle markers have a certain quality in phylogeographic analyses. Since they are haploid, the effective population size should be reduced after the analysis using these markers as compared to those in which nuclear mark‐ ers are used [1, 14]. Smaller effective populations sizes should bring about faster turnover rates for newly evolving genotypes, resulting in a clearer picture of past migration history than those obtained using nuclear markers [15-17]. Initially, it was mainly in phylogeographic studies of animal species that mitochondrial markers were used [18]. These studies have provided some interesting data on the begin‐ nings and the evolutionary history of human population [19]. In contrast to studies of ani‐ mals, using mitochondrial markers in studies of plants, especially angiosperms, is limited [20]. Presently, cpDNA markers are most commonly used in phylogeographic studies of an‐ giosperms, whereas mitochondrial markers are prevalent in studies of gymnosperms. 2.2. Plant mitochondrial DNA Mitochondrial genomes of higher plants (208-2000 kbp) are much larger than those of verte‐ brates (16-17 kbp) or fungi (25-80 kbp) [21, 22]. In addition, there are clear differences in size and organization of mitochondrial genomes between different species of plants. Intramolec‐ ular recombination in mitochondria leads to complex reorganizations of genomes, and, in consequence, to alternating arrangement of genes, even in individual plants, and the occur‐ rence of duplications and deletions are common [23]. In addition, the nucleotide substitution rate in plant mitochondria is rather low [24], causing only minor differences within certain loci between individuals or even species. Extensively characterized circular animal mito‐ chondrial genomes are highly conservative within a given species; they do not contain in‐ trons and have a very limited number of intergenic sequences [25]. Plant mitochondrial DNA (mtDNA) contains introns in multiple genes and several additional genes undergoing Use of Organelle Markers to Study Genetic Diversity in Soybean 555 http://dx.doi.org/10.5772/52028 expression when compared to animal mitochondria, but most of the additional sequences in plants are not expressed and they do not seem to be esssentials [26]. The completely se‐ quenced mitochondrial genomes are available for several higher plants, including Arabidop‐ sis thaliana [27] or Marchantia polymorpha [28]. Restriction maps of nearly all plant mitochondrial genomes provide for the occurrence of the master circle with circular subgenomic molecules that arise after recombination among large direct repeats (> 1 kbp) [21, 29-36], which are present in most mitochondrial genomes of higher plants. However, such molecules, whose sizes can be predicted, are very rare or very difficult to observe. It can be explained by the fact that plant mitochondrial genomes are circularly per‐ muted as in the phage T4 [37, 38]. Oldenburg and Bendich reported that mostly linear mole‐ cules in Marchantia mtDNA are circularly permuted with random ends [39]. It shows that plant mtDNA replication occurs similarly to the mechanism of recombination in the T4 [38]. Many reports that have appeared in recent years indicate that mitochondrial genome of yeasts and of higher plants exist mainly as linear and branched DNA molecules with varia‐ ble size which is much smaller than the predicted size of the genomes [39-44]. Using pulsed field gel electrophoresis (PFGE) of in-gel lysed mitochondria from different species revealed that only about 6-12% of the molecules are circular [41, 44]. The observed branched mole‐ cules are very similar to the molecules seen in yeast in the intermediate stages of recombina‐ tion of mtDNA [45] or the phage T4 DNA replication [37, 38]. In all but one known case (Brassica hirta) [46], plant mitochondrial genomes contain repeat recombinations. These sequences, ranging in length from several hundred to several thou‐ sand nucleotides (nt) exist at two different loci in the master circle, yet in four mtDNA se‐ quence configurations [47]. These four configurations correspond to the reciprocal exchange of sequences 5' and 3' surrounding the repeat in the master circle, which suggests that the repeat mediates homologous recombination. Depending on the number and orientation of repeats, the master circle is a more or less complex set of subgenomic molecules [48]. Maternally inherited mutations, which are associated with mitochondria in higher plants, most often occur as a result of intra- and intergenic recombination. This happens in most cases of cytoplasmic male sterility (cms) [41, 49-51], in chm-induced mutation in Arabidopsis [52] and in non-chromosomal stripe mutations in maize [53]. In this way, it is assumed that the recombination activity explains the complexity of the variations detected in the mito‐ chondrial genomes of higher plants. 2.3. Mitochondrial genome of soybean The size of soybean mtDNA has been estimated to be approximately 400 kb [54-56]. Spheri‐ cal molecules have also been observed by electron microscopy [55, 57]. Repeated sequences 9, 23 and 299 bp have been characterized in soybean mitochondria [58, 59]. Also, numerous reorganizations of genome sequences have been characterized among different cultivars of soybean. It has been demonstrated that they occur through homolo‐ gous recombination produced by these repeat sequences [58, 60, 61], or through short ele‐ ments that are part of 4.9kb PstI fragment of soybean mtDNA [62]. The 299 bp repeat 556 A Comprehensive Survey of International Soybean Research - Genetics, Physiology, Agronomy and Nitrogen Relationships sequence has been found in several copies of mtDNA of soybean and in several other higher plants, suggesting that this repeated sequence may represent a hot spot for recombination of mtDNA in many plant species [59, 62]. Previous results suggested that