# How can I tell if a certain marker is useful to date a phylogeny?

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If I have a phylogenetic tree for a genus, how can I tell if a certain protein would be a good phylogenetic marker to use if one wanted to use a molecular clock to date the age of some speciation event in that a genus? How can I look for it on a tree?

Speed of Sequence Divergence

You need to use a sequence which diverged only little among most closely related lineages in your tree and diverge quite a lot among most distantly related lineages. In other words, you need a sequence that diverge at a rate that is convenient for your needs.

If you investigate a young tree (a tree which MRCA of all lineages lived recently) you need a sequence that diverge quickly through time. If you investigate an "old tree" (a tree which MRCA of all lineages lived a long time ago) you need a sequence that diverge slowly through time.

Selection

Selection is affecting how fast two sequences diverge. But because selection is specific to the environment experienced by the individuals in each lineages, it can be more difficult to use sequences under selection as a molecular clock (exception of highly conserved selection which are under purifying selection used for very large trees). So we use neutral sequences.

Rate at which mutations fix

Let \$u\$ be the mutation rate for the sequence of interest and \$N\$ be the population size. The number of new mutations per year is equal to \$2Nu\$ (assuming a diploid population). Now, knowing that the sequence is not under selection, the probability of reaching a single mutation to fix (=to reach a frequency of 1 in the population) is \$frac{1}{2N}\$ (see this post for a very introduction to why the probability of fixation is inversely proportional to \$N\$). As a consequence, the rate at which mutations fix is \$2 N u frac{1}{2N}=u\$. In other words, the rate of mutation fixation is the mutation rate (for neutral sequences).

Rate at which mutations fix and sequences divergence

The term, sequence divergence is not clearly define. Typically, one would mean "average number of pairwise differences". Assuming that each new mutation that fixes occurred at a new locus (infinite allele model), then the number of pairwise differences between two lineages is the addition of the number of mutations that fixed in each lineage, that is \$2u\$. Always under the infinite allele model, the probability of the average number of pairwise differences to equal k is given by a Poisson distribution.

Wrapping up

how can I tell if a certain protein would be a good phylogenetic marker

The sequence

1. has to be neutral (it makes things easier)
2. has to have a mutation rate of the right order of magnitude (it improves accuracy of the estimate and therefore improves statistical power)

## Identifying genetic markers for a range of phylogenetic utility–From species to family level

Affiliations Evolutionary Biology & Ecology, Institute for Biology, University of Freiburg, Freiburg (Brsg.), Germany, Australian National Inset Collection, CSIRO National Research Collections Australia, Canberra, Australian Capital Territory, Australia

Roles Data curation, Writing – review & editing

Affiliations The University of Queensland, School of Biological Sciences, Brisbane, Queensland, Australia, Department of Botany, National Museum of Natural History, Smithsonian Institution, Washington, D.C., United States of America

Roles Data curation, Formal analysis, Writing – review & editing

Affiliation The University of Queensland, School of Biological Sciences, Brisbane, Queensland, Australia

Roles Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Supervision, Visualization, Writing – review & editing

Affiliations Division of Ecology and Evolution, Research School of Biology, The Australian National University, Canberra, Australian Capital Territory, Australia, School of Forest Resources and Environmental Science, Michigan Technological University, Houghton, Michigan, United States of America

## New molecular markers for fungal phylogenetics: Two genes for species-level systematics in the Sordariomycetes (Ascomycota)

Although significant progress has been made resolving deep branches of the fungal tree of life, many fungal systematists are interested in species-level questions to both define species and assess fungal biodiversity. Fungal genome sequences are a useful resource to systematic biologists for developing new phylogenetic markers that better represent the whole genome. Here we report primers for two newly identified single-copy protein-coding genes, FG1093 and MS204, for use with ascomycetes. Although fungi were the focus of this study, this methodological approach could be easily applied to marker development for studies of other organisms. The tests used here to assess phylogenetic informativeness are computationally rapid, require only rudimentary datasets to evaluate existing or newly developed markers, and can be applied to other non-model organisms to assist in experimental design of phylogenetic studies. Phylogenetic utility of the markers was tested in two genera, Gnomoniopsis and Ophiognomonia (Gnomoniaceae, Diaporthales). The phylogenetic performance of β-tubulin, ITS, and tef-1α was compared with FG1093 and MS204. Phylogenies inferred from FG1093 and MS204 were largely in agreement with β-tubulin, ITS, and tef-1α although some topological conflict was observed. Resolution and support for branches differed based on the combination of markers used for each genus. Based on two independent tests of phylogenetic performance, FG1093 and MS204 were determined to be equal to or better than β-tubulin, ITS, and tef-1α in resolving species relationships. Differences were found in site-specific rate of evolution in all five markers. In addition, isolates from 15 orders and 22 families of Ascomycota were screened using primers for FG1093 and MS204 to demonstrate primer utility across a wide diversity of ascomycetes. The primer sets for the newly identified genes FG1093 and MS204 and methods used to develop them are useful additions to the ascomycete systematists’ toolbox.

### Highlights

► The markers FG1093 and MS204 are useful for species-level phylogenetics/systematics in Ascomycota. ► Differences in the site specific rate of substitution were determined for β-tubulin/FG1093/ITS/MS204/tef-1α. ► Phylogenetic performance tests show that FG1093/MS204 independently were equal or better markers than β-tubulin/ITS/tef-1α. ► The newly identified genes FG1093/MS204 and methods used to develop them should be useful for systematic biologists.

## Clonal inheritance?

It is widely considered that mtDNA is maternally transmitted, and therefore clonal, in animals ( Birky 2001 ). Paternal mtDNA is eliminated before (as in crayfish, Moses 1961 ), during (as in Ascidia, Ursprung & Schabtach 1965 ) or after (as in mouse, Sutovsky et al. 1999 ) fertilization (see Xu et al. 2005 for review, and White et al. 2008 for exceptions). The transmission of mtDNA in the female germ line, furthermore, is characterized by a strong bottleneck, which reduces the within-individual diversity ( Shoubridge & Wai 2007 ). Because of these two effects (absence of paternal leakage and germ-line bottleneck), distinct mtDNA lineages can co-occur within a zygote, a condition which prevents effective recombination. The lack of genetic exchange has been considered as a useful feature, as it implies that the within-species history of mtDNA can be appropriately represented by a unique tree, which traces back the origins and geographic movements of maternal lineages ( Avise et al. 1987 ). The whole field of phylogeography, therefore, heavily relies on the assumption of clonal mtDNA inheritance.

Firmly enough established from classical genetics, the clonality assumption was little questioned until 1999, when three quasi-simultaneous articles suddenly challenged the dogma in humans. Analysing 29 complete human mitochondrial haplotypes, Eyre-Walker et al. (1999) found an unexpectedly high amount of within-species homoplasy (i.e. phylogenetic conflict between sites), which could apparently not be explained by mutation hotspots – homoplasic sites were not particularly variable across hominid species. They concluded that these many homoplasies were most probably the consequence of recombination events. Using the same data, Awadalla et al. (1999) reported a negative correlation between linkage disequilibrium and physical distance for pairs of sites, again supporting the idea that recombination breaks down allelic associations between distant loci. Hagelberg et al. (1999) , finally, discovered a mitochondrial point mutation shared by distantly related mtDNA haplotypes in a small Melanesian island, but virtually nowhere else in the world. Arguing that two independent occurrences of this rare mutation in the same island were unlikely, the authors concluded that recombination was the most plausible explanation.

These exciting reports in human stimulated the search for instances of mtDNA recombination in various animal species. Systematic surveys of within-species mtDNA data available from public databases revealed significant departure from the clonality assumption in several species, including primates ( Piganeau et al. 2004 Tsaousis et al. 2005 ). The authors, however, carefully noted that part of these bioinformatic-detected instances could correspond to artefacts – e.g. in vitro recombination – and called for experimental corroboration ( Piganeau et al. 2004 ). Population evidence for mtDNA recombination was specifically reported in a mussel ( Ladoukakis & Zouros 2001 ), a butterfly ( Andolfatto et al. 2003 ), scorpions ( Gantenbein et al. 2005 ), a lizard ( Ujvari et al. 2007 ) and fish ( Hoarau et al. 2002 Ciborowski et al. 2007 ), based either on linkage disequilibrium/physical distance analysis, or discovery of obvious recombinants between distant enough parental haplotypes. The mussel case was the least surprising because this species, like several other bivalves ( Breton et al. 2007 ), hosts a paternally transmitted mitochondrial genome, so that all male individuals are heteroplasmic. An instance of paternal leakage followed by recombination, finally, was discovered in a human patient ( Kraytsberg et al. 2004 ).

In flowering plants, while mitochondria, like chloroplast, are thought to be primarily maternally inherited ( Reboud & Zeyl 1994 ), the occurrence of recombination is also debated. In Silene vulgaris, recent recombination events within and among genes, and between mitochondria and chloroplast, have been detected ( Houliston & Olson 2006 ), but not recovered in a subsequent analysis ( Barr et al. 2007 ). On a larger evolutionary scale, phylogenetic conflicts involving mtDNA support the hypothesis of recurrent horizontal transfer of mitochondrial genes during the history of angiosperms ( Bergthorsson et al. 2003 ).

Although some would probably deserve confirmation, these case studies are of great relevance to the field of molecular ecology. They prove that mitochondrial recombination is possible, and call for caution when building and interpreting within-species mtDNA genealogies ( Hey 2000 ). Ironically enough, we now know that the three human studies which motivated this fruitful search for mtDNA recombination were actually questionable. The island-specific pattern ( Hagelberg et al. 1999 ) was resulting from an alignment error ( Hagelberg et al. 2000 ). The decay of linkage disequilibrium with physical distance ( Awadalla et al. 1999 ) was found sensitive to methodological settings, and to the analysed data set ( Ingman et al. 2000 Innan & Nordborg 2002 Piganeau & Eyre-Walker 2004 ). The excess of within-species homoplasy ( Eyre-Walker et al. 1999 ), finally, was most probably created by short-lived, uneasy to detect mutations hotspots ( Galtier et al. 2006 ).

Besides the anecdote, one lesson to be drawn from this interesting debate is that peculiarities of the mitochondrial mutation process can generate recombination-like patterns of sequence variation. For this reason, and because of the lack of power of recombination detection methods ( White & Gemmell 2009 ), the prevalence of mitochondrial recombination across animals is currently difficult to assess. Our view of current literature would tend to suggest that a large fraction of the within-species mtDNA homoplasy is caused by mutation hot-spots ( Innan & Nordborg 2002 Galtier et al. 2006 see below). At any rate, the existence of strong within-species homoplasy is an obvious practical problem when analysing mtDNA population data, whether it is caused by true recombination or mutation-induced convergences.

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## Review on Use of Molecular Markers for Characterizing and Conserving of Plant Genetic Resources

Abstract
Molecular markers have revolutionized and modernized our ability to characterize genetic variation and to rationalize genetic selection, being effective and reliable tools for the analysis of genome architectures and gene polymorphisms in crop plants. The area of plant genomics that has shown the greatest development with respect to the use of molecular marker technology is that of population genetics. All DNA polymorphism assays have proven to be powerful tools for characterizing and investigating Germplasm resources, genetic variation and differentiation of populations, on the basis of gene diversity and gene flow estimates. As a matter of fact, the number of loci for which DNA-based assays have been generated has increased dramatically, the majority using PCR as methodology platform. The information acquired is now being exploited to transfer different traits, including biotic stress resistances and improved quality traits, to important varieties by means of marker-assisted selection (MAS) programs. The most important challenges in the near future are certainly the molecular characterization of Germplasm collections for preserving them from genetic erosion and the identification of phenotypic variants potentially useful for breeding new varieties. Knowing the presence of useful traits, genes and alleles would help in making decisions on the multiplication of plant accessions and the maintenance of seed stocks. There are no doubts that the use of molecular markers for characterization and conservation of genetic resources should be implemented so that potentially useful genes and genotypes can be added to core collections to make them exploitable by breeders.

Keywords: Molecular markers, DNA markers.

1. INTRODUCTION
Molecular markers have proven to be powerful tools for analyzing germplasm resources and assessing genetic variation within as well as genetic differentiation among populations. In fact, the area of plant genomics that has shown the greatest development with respect to the use of DNA marker technology is that of population genetics. However, both RFLP and PCR-derived markers have also been extensively applied in plant genetics and breeding for mapping Mendelian genes and QTLs. The use of molecular markers for investigating and managing genetic resources should be implemented so that useful information on genes and traits can be added to core collections to make them exploitable by breeders (Barcaccia, 2009).
Conservation of genetic resources entails several activities, many of which may greatly benefit from knowledge generated through applying molecular marker technologies. This is the case for activities related to the acquisition of germplasm (locating and describing the diversity), its conservation (using effective procedures) and evaluation for useful traits. In all, the availability of sound genetic information ensures that decisions made on conservation will be better informed and will result in improved germplasm management. Of the activities related to genetic resources, those involving germplasm evaluation and the addition of value to genetic resources are particularly important as they help identify genes and traits, and thus provide the foundation on which to enhance use of collections. ‘Characterization’ is the description of a character or quality of an individual (Marriem 1991).
The word ‘characterize’ is also a synonym of ‘distinguish’, that is, to mark as separate or different, or to separate into kinds, classes or categories. Thus, characterization of genetic resources refers to the process by which accessions are identified or differentiated. This identification may, in broad terms, refer to any difference in the appearance or make-up of an accession. In the agreed terminology of gene banks and germplasm management, the term ‘characterization’ stands for the description of characters that are usually highly heritable, easily seen by the eye and equally expressed in all environments (IPGRI/CIP. 2003). In genetic terms, characterization refers to the detection of variation as a result of differences in either DNA sequences or specific genes or modifying factors. Standard characterization and evaluation of accessions may be routinely carried out by using different methods, including traditional practices such as the use of descriptor lists of morphological characters. They may also involve evaluation of agronomic performance under various environmental conditions. In contrast, genetic characterization refers to the description of attributes that follow a Mendelian inheritance or that involve specific DNA sequences.
In this context, the application of biochemical assays such as those that detect differences between isozymes or protein profiles, the application of molecular markers and the identification of particular sequences through diverse genomic approaches all qualify as genetic characterization methods. Because of its nature, genetic characterization clearly offers an enhanced power for detecting diversity (including genotypes and genes) that exceeds that of traditional methods. Likewise, genetic characterization with molecular technologies offers greater power of detection than do phenotypic methods (e.g. isozymes). This is because molecular methods reveal differences in genotypes, that is, in the ultimate level of variation embodied by the DNA sequences of an individual and uninfluenced by environment. In contrast, differences revealed by phenotypic approaches are at the level of gene expression (proteins).

2. LITERETURES REVIEW
2.1 Genetic markers in plant breeding:
Genetic markers are the biological features that are determined by allelic forms of genes or genetic loci and can be transmitted from one generation to another, and thus they can be used as experimental probes or tags to keep track of an individual, a tissue, a cell, a nucleus, a chromosome or a gene. Genetic markers used in genetics and plant breeding can be classified into two categories: classical markers and DNA markers (Xu, 2010). Classical markers include morphological markers, cytological markers and biochemical markers. DNA markers have developed into many systems based on different polymorphism-detecting techniques or methods (southern blotting – nuclear acid hybridization, PCR – polymerase chain reaction, and DNA sequencing) (Collard et al., 2005), such as RFLP, AFLP, RAPD, SSR, SNP, etc.

2.1.1. Classical markers
2.1.1.1 Morphological markers
Use of markers as an assisting tool to select the plants with desired traits had started in breeding long time ago. During the early history of plant breeding, the markers used mainly included visible traits, such as leaf shape, flower color, pubescence color, pod color, seed color, seed shape, hilum color, awn type and length, fruit shape, rind (exocarp) color and stripe, flesh color, stem length, etc. These morphological markers generally represent genetic polymorphisms which are easily identified and manipulated. Therefore, they are usually used in construction of linkage maps by classical two- and/or three-point tests. Some of these markers are linked with other agronomic traits and thus can be used as indirect selection criteria in practical breeding. In the green revolution, selection of semi-dwarfism in rice and wheat was one of the critical factors that contributed to the success of high-yielding cultivars. This could be considered as an example for successful use of morphological markers to modern breeding. In wheat breeding, the dwarfism governed by gene Rht10 was introgressed into Taigu nuclear male-sterile wheat by backcrossing, and a tight linkage was generated between Rht10 and the male-sterility gene Ta1. Then the dwarfism was used as the marker for identification and selection of the male-sterile plants in breeding populations (Liu, 1991). This is particularly helpful for implementation of recurrent selection in wheat. However, morphological markers available are limited, and many of these markers are not associated with important economic traits (e.g. yield and quality) and even have undesirable effects on the development and growth of plants.

2.1.1.2 Cytological markers
In cytology, the structural features of chromosomes can be shown by chromosome karyotype and bands. The banding patterns, displayed in color, width, order and position, reveal the difference in distributions of euchromatin and heterochromatin. For instance, Q bands are produced by quinacrine hydrochloride, G bands are produced by Giemsa stain, and R bands are the reversed G bands. These chromosome landmarks are used not only for characterization of normal chromosomes and detection of chromosome mutation, but also widely used in physical mapping and linkage group identification. The physical maps based on morphological and cytological markers lay a foundation for genetic linkage mapping with the aid of molecular techniques. However, direct use of cytological markers has been very limited in genetic mapping and plant breeding.

2.1.1.3 Biochemical/ protein markers
Protein markers may also be categorized into molecular markers though the latter are more referred to DNA markers. Isozymes are alternative forms or structural variants of an enzyme that have different molecular weights and electrophoretic mobility but have the same catalytic activity or function. Isozymes reflect the products of different alleles rather than different genes because the difference in electrophoretic mobility is caused by point mutation as a result of amino acid substitution (Xu, 2010).

2.1.2. DNA markers
DNA markers are defined as a fragment of DNA revealing mutations/variations, which can be used to detect polymorphism between different genotypes or alleles of a gene for a particular sequence of DNA in a population or gene pool. Such fragments are associated with a certain location within the genome and may be detected by means of certain molecular technology. Simply speaking, DNA marker is a small region of DNA sequence showing polymorphism (base deletion, insertion and substitution) between different individuals. There are two basic methods to detect the polymorphism: Southern blotting, a nuclear acid hybridization technique (Southern 1975), and PCR, a polymerase chain reaction technique (Mullis, 1990). Using PCR and/or molecular hybridization followed by electrophoresis (e.g. PAGE – polyacrylamide gel electrophoresis, AGE – agarose gel electrophoresis, CE – capillary electrophoresis), the variation in DNA samples or polymorphism for a specific region of DNA sequence can be identified based on the product features, such as band size and mobility. In addition to Sothern blotting and PCR, more detection systems have been also developed. For instance, several new array chip techniques use DNA hybridization combined with labeled nucleotides, and new sequencing techniques detect polymorphism by sequencing. DNA markers are also called molecular markers in many cases and play a major role in molecular breeding.
Since Botstein et al. (1980) first used DNA restriction fragment length polymorphism (RFLP) in human linkage mapping, substantial progress has been made in development and improvement of molecular techniques that help to easily find markers of interest on a largescale, resulting in extensive and successful uses of DNA markers in human genetics, animal genetics and breeding, plant genetics and breeding, and germplasm characterization and management. Among the techniques that have been extensively used and are particularly promising for application to plant breeding, are the restriction fragment length polymorphism (RFLP), amplified fragment length polymorphism (AFLP), random amplified polymorphic DNA (RAPD), microsatellites or simple sequence repeat (SSR), and single nucleotide polymorphism (SNP). According to a causal similarity of SNPs with some of these marker systems and fundamental difference with several other marker systems, the molecular markers can also be classified into SNPs (due to sequence variation, e.g. RFLP) and non-SNPs (due to length variation, e.g. SSR) (Gupta et al., 2001).

Table 1. Comparison of most widely used DNA marker systems in plants Adapted from Collard et al. (2005), Semagn et al. (2006a), Xu (2010), and others.

2.2 Genetic Characterization and Its Use in Decision-Making for the Conservation of Crop Germplasm
Characterization, at present is carried out either based on morphological traits or on molecular markers (biochemical and DNA markers). Morphology-based characterization has some limitations in the accurate identification of the accessions, such as limited number of traits to characterize (Rao 2004). The characterization, conservation and exploitation of crop plant germplasm maintained in gene banks propound a number of challenges to the researchers dedicated to the investigation of plant genetic resources. Common problems include the development of strategies for sampling representative individuals in natural and experimental populations, the improvement of tools and technologies for long-term conservation and for high-throughput characterization of large numbers of stored accessions. The knowledge of the genetic diversity present in a gene bank is crucial for developing sustainable conservation strategies and it is also essential for the profitable exploitation of a gene bank by specific breeding programs. As a matter of fact, germplasm characterization of plant accessions deposited in gene banks has been limited and this likely represents a major cause for the limited adoption of conserved accessions in crop breeding programs (Ferreira 2006). Consequently, the genetic characterization of accessions belonging to a given collection and the examination of genetic relationships among them should be strengthened and perpetrated not only for maintaining but also for exploiting crop genetic resources.
Conservation of the genetic resources in the agro-ecosystem in which they have evolved (in situ conservation) is now being more widely considered, as complementary to strategies based on gene banks (ex situ conservation), for limiting genetic erosion and so preserving genetic diversity. If it is true that in situ conservation has been proposed essentially for wild relatives of cultivated plants, it is also true that when considered for major crops this alternative can very often be unfeasible from a socio-economic perspective (Negri et al. 2000 Lucchin et al. 2003). Genomic DNA-based marker assays have revolutionized and modernized our ability to characterize genetic variation and to rationalize genetic selection (Lanteri and Barcaccia 2006). Molecular markers are known as particularly effective and reliable tools for the characterization of genome architectures and the investigation of gene polymorphisms in crop plants.
Besides linkage mapping, gene targeting and assisted breeding, the plant DNA polymorphism assays are powerful tools for characterizing and investigating germplasm resources and genetic relatedness. These techniques include restriction fragment length polymorphism (RFLP) markers and PCR-based molecular markers, such as simple sequence repeat (SSR) or microsatellite markers (Morgante and Olivieri 1993), amplified fragment length polymorphism (AFLP) markers (Vos et al. 1995).

2.3 Genetic Diversity and Similarity Statistics for Characterizing Plant Germplasm at the Population Level
Genetic diversity and similarity measurements are very useful for describing the genetic structure of populations. The genetic structure of natural populations of a crop plant species is strongly influenced by the reproductive system of their individuals and the union types occurring within populations. Breeding schemes that can be adopted as well as variety types that can be constituted depend on the reproductive barriers and mating systems of plants (Barcaccia 2009). Natural populations of species that reproduce by apomixis or that propagate vegetatively are polyclonal, being composed by several genetically distinct clones and usually dominated by a few well-adapted genotypes. Therefore, genetic variation within populations is distributed among clones and most populations are characterized by different levels of differentiation among genotypes.
Landraces of self-pollinated species (e.g., bean, lentil, wheat and barley) are composed of a mixture of pure lines, genetically related but reproductively independent each other. Thus, genetic as well as phenotypic variation is mainly detectable among lines due to the presence within natural populations of fixed genotypes mainly homozygous for different alleles. Spontaneous hybridization is however possible to some extent depending on the species, environmental factors and germplasm stocks. Cultivated varieties of selfing species are usually represented by pure lines obtained by repeated self-pollination of a number of hybrid individuals stemmed from two parental lines chosen for complementary morphological and commercial traits. Maize is one of the most commercially important cross-pollinated species. In many countries, existing landraces are selected by farmers for their own use and eventually sale to neighbors. Traditionally, landraces are developed by mass selection in order to obtain relatively uniform populations characterized by valuable production locally. Synthetics are also produced by intercrossing a number of phenotypically superior plants, selected on the basis of morpho-phenological and commercial traits. More rarely, plants are also evaluated genotypically by means of progeny tests. Compared to landraces, synthetics have a narrower genetic base but are equivalently represented by a heterogeneous mixture of highly heterozygous genotypes sharing a common gene pool. However, newly released varieties are exclusively represented by F1 hybrids developed by private breeders and seed companies using inbred lines belonging to distinct heterotic groups.
Genetic characterization is providing new information to guide and prioritize conservation decisions for crop plants. The most urgently required action is the effective protection of all remaining wild ancestral populations and closely related species of crop plants, most of them now endangered. They are the only remaining sources of putative alleles of economic values that might have been lost during domestication events. It is equally important to ensure that the plant genetic resources selected for conservation include populations from the geographic areas representing the different domestication centres where high estimates of genetic diversity within and differentiation among populations are expected(Barcaccia 2009).

2.4 Using molecular characterization to make informed decisions on the conservation of crop genetic resources
Information about the genetic make-up of accessions helps decision making for conservation activities, which range from collecting and managing through identifying genes to adding value to genetic resources. Well-informed sampling strategies for germplasm material destined for ex situ conservation and designation of priority sites (i.e. identifying specific areas with desirable genetic diversity) for in situ conservation are both crucial for successful conservation efforts. In turn, defining strategies is dependent on knowledge of location, distribution and extent of genetic diversity.
Molecular characterization, by itself or in conjunction with other data (phenotypic traits or geo-referenced data), provides reliable information for assessing, among other factors, the amount of genetic diversity (Perera et al.,2000), the structure of diversity in samples and populations (shim et al.,2000, Figliuolo et al 2004), rates of genetic divergence among populations (Maestri et al.,2002) and the distribution of diversity in populations found in different locations (Maestri et al ., 2004, Perera et al.,2000).
A recent study on the genetic diversity of cultivated Capsicum species in Guatemalan home gardens compared the diversity present in an array of home gardens in the Department of Alta Verapaz with a countrywide representative sample of 40 accessions conserved ex situ in the national collection (Guzmán et al., 2005). The results showed that home gardens of Alta Verapaz (H = 0.251) contained as much diversity as the entire national ex situ collection (H = 0.281). These results thus suggest that, (1) home gardens are indeed an extremely important resource for in situ conservation of Capsicum germplasm in Guatemala, and as such they should not be neglected (2) if further collecting activities were to be undertaken, special emphasis should be given to collecting in Alta Verapaz and (3) additional collecting in Alta Verapaz alone could disclose novel genetic diversity that is absent from the national collection. Conservation of clonally propagated crops demands more complex and expensive procedures. If these crops are maintained on-farm, their existence is endangered by several factors, one of which being the introduction of alternative improved varieties. Conservation efforts need then to be based on solid knowledge of clonal diversity. This was the case for Abyssinian banana or ensete (Ensete ventricosum (Welw.) Cheesman) from Ethiopia, which was analysed with AFLP markers (Negash, A. et al 2002) Of the 146 clones from five different regions, only 4.8% of the total genetic variation was found between regions, whereas 95.2% was found within regions. The results led to a reduced number of clones for conservation and indicated the existence of a common practice of exchange of local types between regions, which, in its turn, emphasized the need to collect further in different farming systems.
A study on taro (Colocasia esculenta (L.) Schott) genetic diversity in the Pacific, using SSR markers, showed that many of the accessions from countries of the Pacific region were identical to those of Papua New Guinea. This indicates that originally the cultivars may have been introduced throughout the region from Papua New Guinea (Mace, E.S. et al 2005) and that collection of taro genetic diversity could focus on Papua New Guinea alone. Molecular characterization also helps determine the breeding behaviour of species, individual reproductive success and the existence of gene flow, that is, the movement of alleles within and between populations of the same or related species, and its consequences (Papa, R. & Gepts P. 2003 Papa, R. & Gepts P. 2003). Molecular data improve or even allow the elucidation of phylogeny, and provide the basic knowledge for understanding taxonomy, domestication and evolution (Nwakanma, D. C., et al 2003).
As a result, information from molecular markers or DNA sequences offers a good basis for better conservation approaches. Management of germplasm established in a collection (usually a field, seed or in vitro gene bank) comprises several activities. Usually, such activities seek to ensure the identity of the individually stored and maintained samples, to ensure the safeguarding of genetic integrity and genetic diversity and to have the material available for distribution to users. These tasks are primarily a responsibility of gene bank managers and curators, and involve the control of accessions on arrival at the facilities, as well as their continuous safeguarding for the future through regeneration and multiplication. For all these routine activities, information about the genetic constitution of samples or accessions is critical and provides possibly the most important means of measuring the quality of the work being performed. Börner et al. (2000) analysed bulk seed of wheat accessions to test their genetic integrity after 24 cycles of regeneration and after more than 50 years of storage at room temperature in a gene bank. They found neither contamination nor incorrect manipulation effects such as mechanical mixtures, but did identify one case of genetic drift in one accession.
However, in the same gene bank, a study examined the genetic constitution of rye accessions that underwent frequent regeneration. Results showed that (1) a significant number of alleles present in the original sample was lacking in the newly regenerated material, and (2) new alleles in the new material were not present in the first regeneration sample (Chebotar, S., et al 2003). Thus, the use of molecular markers can quickly help check whether changes in alleles or allele frequencies are taking place. Molecular information has been used to weigh the need for decreasing the size of germplasm collections, which otherwise would add costs to the long-term conservation of germplasm. For instance, Dean et al. (1999) used microsatellite markers to analyse the genetic diversity and structure of 19 sorghum accessions known as ‘Orange’ in the USDA’s national sorghum collection. They found two redundant groups (involving five entries) among the 19 accessions evaluated. They also found that much of the total genetic variation was partitioned among accessions. As a result, the authors concluded that the number of accessions held by the US National Plant Germplasm System (NPGS) could be significantly reduced without risking the overall amount of genetic variation contained in these holdings.
Markers were also helpful in examining genetic identities and relationships of Malus accessions (Hokanson, S.C., et al 1998.). Eight primer pairs unambiguously differentiated 52 of 66 genotypes in a study that calculated the probability of any two genotypes being similar at all loci analysed as being about 1 in 1,000 million. The results not only discriminated among the genotypes, but were also shown to be useful for designing strategies for the collection and in situ conservation of wild Malus species. Selected molecular technologies render cost-effective and comprehensive genotypic profiles of accessions (‘fingerprints’) that may be used to establish the identity of the material under study. Simultaneously, these technologies can detect contaminants (and, in the case of material mixtures, contamination with introgressed genes from other accessions or commercial varieties), as well as the presence of redundant materials (or ‘duplicates’) (McGregor, et al 2002).
Moreover, molecular data provide the baseline for monitoring natural changes in the genetic structure of the accession (Chwedorzewska,et al., 2002)or those occurring as a result of human intervention (e.g. seed regeneration or sampling for replanting in the field). Whatever the case, analysis of molecular information allows the design of strategies for either purging the consequences of inappropriate procedures or amending them to prevent future inconveniences (de Vicente, M.C. 2002). A small number of potential duplicates were identified in a core collection of cassava (Manihot esculenta Crantz) when isozyme and AFLP profiles were compared (Chavarriaga-Aguirre, P., et al 1999). The core collection had been assembled with information from traditional markers, which proved to be highly effective for selecting unique genotypes. Molecular data were used for efficiently verifying the previous work on the collection and ensure minimum repetition. Thus, gene bank managers can easily realize the potential value of using molecular methods to support and possibly modify or improve a gene bank’s operations.
A special and increasingly important role of genetic characterization is that of identifying useful genes in germplasm, that is, of maximizing conservation efforts. Because the major justification for the existence of germplasm collections is use of the conserved accessions, it is important to identify those valuable genes that can help develop varieties that will be able to meet the challenges of current and future agriculture.
Characterization has benefited from several approaches resulting from advances in molecular genetics such as genetic and QTL mapping, and gene tagging (Yamada, T.,et al 2004 , Kelly, J.D.,et al 2003). Research in this field has led to the acknowledgement of the value of wild relatives, in which modern techniques have discovered useful variation that could contribute to varietal improvement (Xiao, J., et al 1996, de Vicente, M.C. et al 1993). Knowledge of molecular information in major crops and species and of the synteny of genomes, especially conservation of gene order, has also opened up prospects for identifying important genes or variants in other crop types, particularly those that receive little attention from formal research.
Until now in India, identification and classification of Hibiscus have mainly been based on morphology and according to (Wachira F, Tanaka J and Takeda Y, 2001) even if these descriptors are useful, they show limited levels of inter and intra-varietal polymorphism and hence, may not account for all the diversity in the species. Since it is difficult to identify cultivar based entirely on these morphological features, several kinds of methods which can be used to measure levels and patterns of it is important to find an effective method to accurately identify the varieties to meet research needs. The novelty of this project lies in the use of different molecular markers with increasing order of specificity to study genetic diversity which will help in development of new cultivars of Hibiscus varieties with superior properties to meet changing agronomic requirements. polymorphism and hence, may not account for all the diversity in the species. Since it is difficult to identify cultivars based entirely on these morphological features, several kinds of methods which can be used to measure levels and patterns of it is important to find an effective method to accurately identify the varieties to meet research needs.
Modern molecular techniques have been developed in order to meet the demands of the horticulture industry genetic variation, which range from morphological characterization to various DNA-based markers such as restriction fragment length polymorphism (RFLP), randomly amplified polymorphic DNA (RAPD), amplified fragment length polymorphism (AFLP) and simple sequence repeats (SSR) (Crawford, D. J.2000, Newton, A. C., et al 2002, Martinez, L.,et al 2003, Fontaine, C., et al 2004, Murtaza, N.2006, Ferdousi Begum.,2013). Identification and characterization of germplasm is essential for the conservation and utilization of plant genetic resources (Suvakanta-Barik., et al 2006). Characterization of plant with the use of molecular markers is an ideal way to conserve plant genetic resources. Molecular characterization helps to determine the breeding behaviour of species, individual reproductive success and the existence of gene flow, the movement of alleles within and between populations of the same or related species, and its consequences (Papa R and Gepts P.2003).
Molecular data improves the elucidation of phylogeny, and provide the basic knowledge for understanding taxonomy, domestication and evolution of plants (Nwakanma D C., et al 2003). Random amplified polymorphic DNA (RAPD) technique has been widely used in many plant species for varieties analysis, population studies and genetic linkage mapping (Williams J. G. K., et al 1990, Yu K., et al 1993, Rout G. R., et al 2003). Optimization of the RAPD analysis depends on selection of primers. Although, the RAPD method uses arbitrary primer sequences, many of these primers must be screened in order to select primers that provide useful amplification products. By contrast, single-locus markers are usually characterized by co-dominance (i.e. both alleles identified in heterozygous individuals) and thus are more flexible and supply more robust and comparable data (Karp, 2002).
An appropriate use of molecular markers techniques requires to clearly define the issues addressed, what type of information will be needed (on genetic diversity) and to know what the different techniques can offer not only in terms of genetic information but also resource requirements, reproducibility, adaptability for automation. Furthermore, it is of pivotal importance to consider how the information will be gathered and the way in which the data will be scored and analysed. For accurate and unbiased estimates of genetic diversity adequate attention has to be devoted to:
a. sampling strategies,
b. utilization of various data sets on the basis of the understanding of their strengths and constraints,
c. choice of genetic similarity estimates or distance measures, clustering procedures and other multivariate methods in analyses of data and
d. objective determination of genetic relationships (Mohammadi and Prasanna, 2003).
For all these reasons, choosing the most appropriate technique may be difficult and often a combination of techniques is needed to gather the information one is interested in. Up to now most conservation efforts have focused on agriculturally important crops and about one third of all ex situ accessions in gene bank represents just five species: i.e. wheat (Triticum sp.), barley, rice, maize and beans (Phaseolus spp). The relative over-representation of five species does not necessarily mean that their genetic diversity has been fully covered (Graner et al. 2003) but, on the other hand, there is significant lack of knowledge about the diversity and geographic distribution of less utilized crops as well as their wild relatives (Hammer et al. 2003). Genetic studies in selected crops have demonstrated that widespread and localised alleles occurring in the entire collection are usually contained in the core subset, with only rare localized alleles excluded (van Hintum et al. 2000). Findings suggest that, although a high variability can be found among plants, most of their genotypes belong to the same landrace locally called ‘Nostrano di Storo’ (Barcaccia et al., 2003).

3. CONCLUSION
In conclusion, the most important challenges in the near future are certainly collections for preserving crops from genetic erosion, the molecular characterization of germplasm, and the identification of useful variation in germplasm, potentially useful for breeding new varieties. Knowing the presence of useful traits, genes and alleles would help in making decisions on the multiplication of accessions and the maintenance of seed stocks for responding to an expected higher demand of materials. Such information may also help in making decisions on heterogeneous accessions, where only some genotypes may possess useful alleles. Thus, the gene bank curator may have to decide to maintain the original material as it is and separate a subpopulation carrying the desirable alleles and give it new accession numbers and management protocols. This will facilitate germplasm use and add value to the collections.

In bacteria and archaea, there is a single ITS, located between the 16S and 23S rRNA genes. Conversely, there are two ITSs in eukaryotes: ITS1 is located between 18S and 5.8S rRNA genes, while ITS2 is between 5.8S and 28S (in opisthokonts, or 25S in plants) rRNA genes. ITS1 corresponds to the ITS in bacteria and archaea, while ITS2 originated as an insertion that interrupted the ancestral 23S rRNA gene. [1] [2]

In bacteria and archaea, the ITS occurs in one to several copies, as do the flanking 16S and 23S genes. When there are multiple copies, these do not occur adjacent to one another. Rather, they occur in discrete locations in the circular chromosome. It is not uncommon in bacteria to carry tRNA genes in the ITS. [3] [4]

In eukaryotes, genes encoding ribosomal RNA and spacers occur in tandem repeats that are thousands of copies long, each separated by regions of non-transcribed DNA termed intergenic spacer (IGS) or non-transcribed spacer (NTS).

Each eukaryotic ribosomal cluster contains the 5' external transcribed spacer (5' ETS), the 18S rRNA gene, the ITS1, the 5.8S rRNA gene, the ITS2, the 26S or 28S rRNA gene, and finally the 3' ETS. [5]

During rRNA maturation, ETS and ITS pieces are excised. As non-functional by-products of this maturation, they are rapidly degraded. [6]

Sequence comparison of the eukaryotic ITS regions is widely used in taxonomy and molecular phylogeny because of several favorable properties: [7]

• It is routinely amplified thanks to its small size associated to the availability of highly conserved flanking sequences.
• It is easy to detect even from small quantities of DNA due to the high copy number of the rRNA clusters.
• It undergoes rapid concerted evolution via unequal crossing-over and gene conversion. This promotes intra-genomic homogeneity of the repeat units, although high-throughput sequencing showed the occurrence of frequent variations within plant species. [8]
• It has a high degree of variation even between closely related species. This can be explained by the relatively low evolutionary pressure acting on such non-coding spacer sequences.

For example, ITS markers have proven especially useful for elucidating phylogenetic relationships among the following taxa.

Taxonomic group Taxonomic level Year Authors with references
Asteraceae: Compositae Species (congeneric) 1992 Baldwin et al. [9]
Viscaceae: Arceuthobium Species (congeneric) 1994 Nickrent et al. [10]
Poaceae: Zea Species (congeneric) 1996 Buckler & Holtsford [11]
Leguminosae: Medicago Species (congeneric) 1998 Bena et al. [5]
Orchidaceae: Diseae Genera (within tribes) 1999 Douzery et al. [12]
Odonata: Calopteryx Species (congeneric) 2001 Weekers et al. [13]
Yeasts of clinical importance Genera 2001 Chen et al. [14]
Poaceae: Saccharinae Genera (within tribes) 2002 Hodkinson et al. [15]
Plantaginaceae: Plantago Species (congeneric) 2002 Rønsted et al. [16]
Jungermanniopsida: Herbertus Species (congeneric) 2004 Feldberg et al. [17]
Pinaceae: Tsuga Species (congeneric) 2008 Havill et al. [18]
Chrysomelidae: Altica Genera (congeneric) 2009 Ruhl et al. [19]
Symbiodinium Clade 2009 Stat et al. [20]
Brassicaceae Tribes (within a family) 2010 Warwick et al. [21]
Ericaceae: Erica Species (congeneric) 2011 Pirie et al. [22]
Diptera: Bactrocera Species (congeneric) 2014 Boykin et al. [23]
Scrophulariaceae: Scrophularia Species (congeneric) 2014 Scheunert & Heubl [24]
Potamogetonaceae: Potamogeton Species (congeneric) 2016 Yang et al. [25]

ITS2 is known to be more conserved than ITS1 is. All ITS2 sequences share a common core of secondary structure, [26] while ITS1 structures are only conserved in much smaller taxonomic units. Regardless of the scope of conservation, structure-assisted comparison can provide higher resolution and robustness. [27]

## Molecular Markers

### Peptide Fingerprinting

The peptides can be used as molecular markers specially the polypeptides (proteins) and among them the enzymes ( Allison, 1954 ). That is possible because the peptides are indeed the product of the expression of the genes, which may show mutations (polymorphisms) between the individuals of the same species. In the case of the enzymes, these variants are known as isozymes or isoenzymes. They may have different enzymatic activities, and thus can be detected with biochemical assays. In short, the biological samples are homogenized to obtain the protein extracts, which are subsequently segregated by Agarose Gel Electrophoresis (AGE) or – if more resolution is needed – PolyAcrylamide Gel Electrophoresis (PAGE). Then the isozymes can be revealed by means of a specific biochemical reaction in the presence of a staining cocktail ( Figure 1 ).

Figure 1 . Peptide fingerprinting. The protein extracts from the samples are subjected to agarose gel electrophoresis, with further staining by the isozyme activity. The example on the left shows a dominant marker with a null allele for descriptive purposes the one on the right shows a codominant marker (the heterozygote is explained because the enzyme is a dimer).

The peptide molecular markers can be codominant, and thus in such cases the heterozygote can be differentiated from each of the two homozygotes. Proteomics brings a new level of resolution to the peptide fingerprinting, including peptide microarrays as previously indicated, albeit being more expensive. Nevertheless, the increase of information on the protein databases coupled with a reduction of price can bring this powerful technology to the forefront of peptide fingerprinting for routine use.

## Contents

In genetics, a molecular marker (identified as genetic marker) is a fragment of DNA that is associated with a certain location within the genome. Molecular markers are used in molecular biology and biotechnology to identify a particular sequence of DNA in a pool of unknown DNA.

### Types of genetic markers Edit

There are many types of genetic markers, each with particular limitations and strengths. Within genetic markers there are three different categories: "First Generation Markers", "Second Generation Markers", and "New Generation Markers". [5] These types of markers may also identify dominance and co-dominance within the genome. [6] Identifying dominance and co-dominance with a marker may help identify heterozygotes from homozygotes within the organism. Co-dominant markers are more beneficial because they identify more than one allele thus enabling someone to follow a particular trait through mapping techniques. These markers allow for the amplification of particular sequence within the genome for comparison and analysis.

Molecular markers are effective because they identify an abundance of genetic linkage between identifiable locations within a chromosome and are able to be repeated for verification. They can identify small changes within the mapping population enabling distinction between a mapping species, allowing for segregation of traits and identity. They identify particular locations on a chromosome, allowing for physical maps to be created. Lastly they can identify how many alleles an organism has for a particular trait (bi allelic or poly allelic). [7]

List of Markers Acronym
Restriction Fragment Length Polymorphism RFLP
Random Amplified Polymorphic DNA RAPD
Amplified Fragment Length Polymorphism AFLP
Variable Number Tandem Repeat VNTR
Oligonucleotide Polymorphism OP
Single Nucleotide Polymorphism SNP
Allele Specific Associated Primers ASAP
Inverse Sequence-tagged Repeats ISTR
Inter-retrotransposon Amplified Polymorphism IRAP

Genomic markers as mentioned, have particular strengths and weakness, so, consideration and knowledge of the markers is necessary before use. For instance, a RAPD marker is dominant (identifying only one band of distinction) and it may be sensitive to reproducible results. This is typically due to the conditions in which it was produced. RAPD's are used also under the assumption that two samples share a same locus when a sample is produced. [6] Different markers may also require different amounts of DNA. RAPD's may only need 0.02ug of DNA while an RFLP marker may require 10ug of DNA extracted from it to produce identifiable results. [8] currently, SNP markers have turned out to be a potential tool in breeding programs in several crops. [9]

Molecular mapping aids in identifying the location of particular markers within the genome. There are two types of maps that may be created for analysis of genetic material. First, is a physical map, that helps identify the location of where you are on a chromosome as well as which chromosome you are on. Secondly there is a linkage map that identifies how particular genes are linked to other genes on a chromosome. This linkage map may identify distances from other genes using (cM) centiMorgans as a unit of measurement. Co-dominant markers can be used in mapping, to identify particular locations within a genome and can represent differences in phenotype. [10] Linkage of markers can help identify particular polymorphisms within the genome. These polymorphisms indicate slight changes within the genome that may present nucleotide substitutions or rearrangement of sequence. [11] When developing a map it is beneficial to identify several polymorphic distinctions between two species as well as identify similar sequence between two species.

When using molecular markers to study the genetics of a particular crop, it must be remembered that markers have restrictions. It should first be assessed what the genetic variability is within the organism being studied. Analyze how identifiable particular genomic sequence, near or in candidate genes. Maps can be created to determine distances between genes and differentiation between species. [12]

Genetic markers can aid in the development of new novel traits that can be put into mass production. These novel traits can be identified using molecular markers and maps. Particular traits such as color, may be controlled by just a few genes. Qualitative traits (requires less than 2 genes) such as color, can be identified using MAS (marker assisted selection). Once a desired marker is found, it is able to be followed within different filial generations. An identifiable marker may help follow particular traits of interest when crossing between different genus or species, with the hopes of transferring particular traits to offspring.

One example of using molecular markers in identifying a particular trait within a plant is, Fusarium head blight in wheat. Fusarium head blight can be a devastating disease in cereal crops but certain varieties or offspring or varieties may be resistant to the disease. This resistance is inferred by a particular gene that can be followed using MAS (Marker Assisted Selection) and QTL (Quantitative Trait Loci). [13] QTL's identify particular variants within phenotypes or traits and typically identify where the GOI (Gene of interest) is located. Once the cross has been made, sampling of offspring may be taken and evaluated to determine which offspring inherited the traits and which offspring did not. This type of selection is becoming more beneficial to breeders and farmers because it is reducing the amount of pesticides, fungicides and insecticides. [13] Another way to insert a GOI is through mechanical or bacterial transmission. This is more difficult but may save time and money.

### Applications of markers in cereal breeding Edit

1. Assessing variability of genetic differences and characteristics within a species.
2. Identification and fingerprinting of genotypes.
3. Estimating distances between species and offspring.
4. Identifying location of QTL's.
5. Identification of DNA sequence from useful candidate genes [13]

### Application Edit

It has 5 applications in fisheries and aquaculture:

1. Species Identification
2. Genetic variation and population structure study in natural populations
3. Comparison between wild and hatchery populations
4. Assessment of demographic bottleneck in natural population
5. markers assisted breeding

Biochemical markers are generally the protein marker. These are based on the change in the sequence of amino acids in a protein molecule. The most important protein marker is alloenzyme. alloenzymes are variant forms of an enzyme that are coded by different alleles at the same locus and this alloenzymes differs from species to species. So for detecting the variation alloenzymes are used. These markers are type-i markers.

## Introduction

As the global architecture of human Y-chromosome phylogeny has become increasingly well-defined, researchers have found a powerful tool that helps explain a great deal of human population history that was previously inaccessible [1]–[3]. For eastern and southeastern Asia, the Y-chromosome haplogroup O-M175 is particularly important, as it is the most prevalent Y-chromosome lineage in these regions and comprises around 75% of the male populations in mainland China [4]–[7] and roughly 87% in Southeast Asia [8]–[13]. To date, studies have shown three major sub-lineages under O-M175: O1a-M119, O2a-M95 and O3-M122 [14]. The extant phylogenies of O3-M122 and O1a-M119 have been adequately resolved with many SNP markers, and subsequently studied in many Asian populations [4], [10], [14]. However, O2a-M95, which comprises some 58% of the male populations in Southeast Asia [8]–[13], [15], the phylogeny still lacks resolution, with only two characterized sub-branches (O2a1*-M95 and O2a1a-M88) [16], greatly limiting the genetic and historical inferences that can be made from this key Y chromosome lineage in Asia and the Pacific.

The importance of O2a-M95, aside from its genetic prevalence, is its predominance among populations of the Austro-Asiatic language family, the eighth largest family in the world in terms of population size (104 millions) [17]. In Southeast Asia, Austro-Asiatic is the first language of many ethnic groups in Cambodia, Vietnam, Laos, Thailand, Burma and Malaysia, and serves as the main official language in Cambodia and Vietnam. More importantly, a recent genome-wide survey of sequence variations in extensive Asian populations found that the Austro-Asiatic speaking populations are located at the basal position of the phylogenetic tree covering all major Asian populations, suggesting that they may represent one of the most ancient populations in Southeast Asia [18]. We recently demonstrated that the Austro-Asiatic speaking populations from Cambodia harbor many ancient polymorphisms in their mitochondrial genomes, consistent with the proposed ancientness [19]. The postulated southern origin and northward migration of East Asian populations then places mainland Southeast Asia (MSEA) and southern China as the potential cradle of modern human settlement during their initial dispersal into eastern Asia [4], [5], [15], [18], [20]. Though a variety of data supports this position, this theory needs greater evidence to more accurately trace the history of early human migration into Asia. As the major Y chromosome lineage in Austro-Asiatic populations, improving the phylogenetic resolution of O2a-M95 would greatly improve our understanding of early human migrations in Asia and the Pacific.

In this study, we aimed to improve the resolution of the O2a-M95 lineage by analyzing the newly discovered SNP markers among Austro-Asiatic speaking populations. After genotyping of 10 novel Y chromosome SNPs in 22 Austro-Asiatic populations from Cambodia, Thailand and southwestern China, we were able to markedly improve the resolution of O2a-M95 and establish 5 new sub-branches, providing a more detailed within-lineage structure for this key Y chromosome lineage.