Comparison of ISSR,IRAP and REMAP markers for assessing genetic diversity in different species of <Emphasis Type="Italic">Brassica</Emphasis> sp.

Molecular markers provide facilities in order to study genetic diversity and relationship among genotypes. In this study, genetic diversity among 35 genotype of Brassica sp. (belonging B. napus, B. juncea, B. rapa, B. nigra) were determined using 13 ISSR, 3 IRAP markers and 18 REMAP (primer combinations of ISSR and retrotransposon primer). The percentage of polymorphism for ISSR, IRAP and REMAP was 96.38, 94 and 96%, respectively. By comparison between markers, ISSRs indicated the highest expected heterozygosity (He) and Shannon’s information index (I) with value of 0.34 and 0.51, respectively, while REMAP marker had by far the highest number of polymorphic bands (340) and marker index (7.1) among all fragments scored over all markers. In pattern of clustering based on Bayesian methods, K = 8 was resulted for combined data clustering that was more organized clustering for genotypes compared to others. This research suggests the combined data of ISSR, IRAP and REMAP markers are most reliable than each solely marker whilst have been clustered genotypes in their taxonomic classification of Brassica without any mixture. Principle coordinate analysis (PCoA) separated 35 genotypes in four groups which all of genotypes were clustered correctly based on their taxonomic classification. The findings of this study provide the valuable insight into the Brassica species relationships in terms of similarity among genotypes which can be helpful in breeding programs, and also demonstrate that retrotransposon markers are legible for genetic diversity and next genetic analysis in Brassica genus.     

Genetic diversity and population structure assessment of Chinese <Emphasis Type="Italic">Senna obtusifolia</Emphasis> L. by molecular markers and morphological traits of seed

Senna obtusifolia L. is an important medicinal plant in Asia. This study was the first report on the genetic diversity and population structure of S. obtusifolia which were collected from 47 geographic populations widespread in China. Inter-Simple Sequence Repeat (ISSR) and Start Codon Target Polymorphism (SCoT) combined with seeds morphological traits were used to investigate the relationship of 47 populations. 11 ISSR primers yielded 98 polymorphic bands with 81.67% polymorphism. 24 SCoT primers yielded 267 polymorphic bands with 89.59% polymorphism. The number of allele (Na), the number of effective allele (Ne), Nei’s diversity index (H), and Shannon’s information index (I) reflected a high level of genetic diversity of S. obtusifolia species. The greatest genetic distance (G _D) existed between Southwest and Northwest (0.4022_ISSR/0.5019_SCoT), while the Eastern and Northern showed the least genetic distance (0.1751_ISSR/0.2186_SCoT). The genetic differentiation (Gst) was 0.4875_ISSR/0.4434_SCoT, and the gene flow (Nm) was 0.5256_ISSR/0.6275_SCoT, which indicated that gene exchange among four regions was limited. 47 samples were divided into four clusters mainly according to their geographic distribution through clustering and structure analysis. The analysis on the combined data of ISSR and SCoT showed more reliable and superior results than single analysis of ISSR and SCoT. This study explored the effectiveness of ISSR and SCoT markers to evaluate the genetic diversity and population structure of S. obtusifolia and provided useful information for S. obtusifolia germplasm research and breeding program.     

Genetic diversity of Indian jujube cultivars using SCoT,ISSR, and rDNA markers

Genetic variation and relationships among 37 cultivars of Ziziphus mauritiana (Lamk.) native of India were analyzed using start codon targeted (SCoT), inter-simple sequence repeats (ISSR), and ribosomal DNA (rDNA) markers. High level of polymorphism among SCoT (61.6%) and ISSR (61%) primers with higher PIC values ranging from 63.1 to 90.4% of SCoT and 47.3 to 88.8% of ISSR primers was recorded. SCoT and ISSR dendrograms revealed similarity coefficients ranging from 0.80 to 0.92 and 0.79 to 0.96, respectively, and clearly delineated all the cultivars of Z. mauritiana into well-supported distinct clusters. Greater Gst signifies higher amount of differentiation observed over multiple loci among seven Z. mauritiana populations. On the other hand, higher gene flow demonstrating a very high migration rate between Z. mauritiana populations indicated higher rates of transfer of alleles or genes from one population to another. The genetic diversity of population 1 (Rajasthan) was the richest among all the seven populations. The largest genetic distance was measured between Maharashtra and West Bengal and the least between Rajasthan and Punjab cultivars. Most of the genetic diversity exists within population rather than among populations. Substantial variation in the ITS-1 region signifies its phylogenetic utility specifically in assessing genetic diversity in Z. mauritiana. The clustering patterns using three molecular marker systems vis-à-vis place of origin exhibited no consistency in grouping of Z. mauritiana cultivars as cultivars from the same place of origin were genetically cataloged into different SCoT, ISSR, and ITS phylogram clusters indicating wide genetic diversity and distribution across agro-climatic zones validating the robustness of marker systems tested.     

Genetic fidelity assessment of <Emphasis Type="Italic">in vitro</Emphasis>-regenerated plants of <Emphasis Type="Italic">Albizia julibrissin</Emphasis> using SCoT and IRAP fingerprinting

A protocol was established for callus induction and plant regeneration of Albizia julibrissin Durazz., a multipurpose tree. Calli were induced on hypocotyl explants excised from 10- to 14-d-old in vitro seedlings cultured on Murashige and Skoog (MS) medium supplemented with α-naphthaleneacetic acid (NAA) alone or in combination with 6-benzylaminopurine (BA) or 6-furfurylaminopurine (kinetin). The highest frequency of organogenic callus (82.2?±?3.6%) was obtained on MS medium with 10.8 μM NAA and 4.4 μM BA. Calli were then cultured on MS medium with BA or zeatin, singly or in combination, for shoot regeneration. Calli cultured on MS medium with 13.2 μM BA and 4.6 μM zeatin produced the highest frequency of adventitious shoot regeneration (75.3?±?6.3%). Maximum rooting of shoots (73.3?±?5%) was achieved using half-strength MS medium with 4.9 μM indole-3-butyric acid. The genetic fidelity of 12 plants acclimatized to the greenhouse was assessed based on analyses of start codon targeted (SCoT) polymorphism and inter-retrotransposon amplified polymorphism (IRAP). The 14 SCoT and 7 IRAP adapted primers produced 71 and 34 scoreable fragments, of which 33 (46%) and 12 (35%) were polymorphic, respectively. The in vitro-raised plants exhibited 0.129–0.438 genetic distance from the mother plant and 0.000–0.788 distance from one another according to the SCoT and IRAP analyses. Although the culture method described here may not be suitable for clonal propagation of elite genotypes, it can be used for conservation of this plant.     

Development of SCoT-Based SCAR Marker for Rapid Authentication of <Emphasis Type="Italic">Taxus Media</Emphasis>

Taxus media is an important species in the family Taxaceae with high medicinal and commercial value. Overexploitation and illegal trade have led T. media to a severe threat of extinction. In addition, T. media and other Taxus species have similar morphological traits and are easily misidentified, particularly during the seedling stage. The purpose of this study is to develop a species-specific marker for T. media. Through a screening of 36 start codon targeted (SCoT) polymorphism primers, among 15 individuals of 4 Taxus species (T. media, T. chinensis, T. cuspidate and T. fuana), a clear species-specific DNA fragment (amplified by primer SCoT3) for T. media was identified. After isolation and sequencing, a DNA sequence with 530 bp was obtained. Based on this DNA fragment, a primer pair for the sequence-characterized amplified region marker was designed and named MHSF/MHSR. PCR analysis with primer pair MHSF/MHSR revealed a clear amplified band for all individuals of T. media but not for T. chinensis, T. cuspidate and T. fuana. Therefore, this marker can be used as a quick, efficient and reliable tool to identify T. media among other related Taxus species. The results of this study will lay an important foundation for the protection and management of T. media as a natural resource.     

Modern State of Populations of Endemic <Emphasis Type="Italic">Oxytropis</Emphasis> Species from Baikal Siberia and Their Phylogenetic Relationships Based on Chloroplast DNA Markers

The genetic diversity and population structure of the endemic species of Baikal Siberia Oxytropis triphylla, O. bargusinensis, and O. interposita were studied for the first time on the basis of the nucleotide polymorphism of intergenic spacers psbA–trnH, trnL–trnF, and trnS–trnG of chloroplast DNA. All populations of these species were characterized by a high haplotype (0.762–0.924) and relatively low nucleotide (0.0011–0.0022) diversity. Analysis of the distribution of variability in O. triphylla and O. bargusinensis showed that there was no significant genetic differentiation between populations of each species; the gene flow was 4.43 and 8.91, respectively. The high level of genetic diversity in the studied populations indicates a relatively stable state of these populations. A study of the phylogenetic relationships of closely related species confirms the concept of the origin of O. bargusinensis and O. tompudae as a result of intersectional hybridization of the species of the sections Orobia and Verticillares.     

Comparison of molecular genetic utilities of TD,AFLP, and MSAP among the accessions of <Emphasis Type="Italic">japonica,indica</Emphasis>, and <Emphasis Type="Italic">Tongil</Emphasis> of <Emphasis Type="Italic">Oryza sativa</Emphasis> L.

Rice is one of the most important food crops in the world. Genetic diversity is essential for cultivar improvement programs. We compared genetic diversity derived from insertion–deletion (in–del) or base substitutions by amplified fragment length polymorphism (AFLP), from transposon transposition mutations by transposon display (TD), and from cytosine methylation by methylation-sensitive amplified polymorphism (MSAP) in japonica, indica, and Tongil type varieties of Oryza sativa L. Polymorphic profiles from the three marker systems allowed us to clearly distinguish the three types of varieties. The indica type varieties showed the highest genetic diversity followed by the Tongil and japonica type varieties. Of the three marker systems, TD produced the highest marker indices, and AFLP and MSAP produced similar marker indices. Pair-wise comparisons of the three marker systems showed that the correlation between the two genetic markers systems (AFLP and TD, r = 0.959) was higher than the correlations between the genetic and epigenetic marker systems (AFLP and MSAP, r = 0.52; TD and MSAP, r = 0.505). Both genetic marker systems had similar levels of gene differentiation (G _ST ) and gene flow (N _m ), which differed in the epigenetic marker system. Although the G _ST of the epigenetic marker system was lower than the genetic marker systems, the N _m of the epigenetic marker system was higher than in the genetic marker systems, indicating that epigenetic variations have a greater influence than genetic variations among the O. sativa L. types.     

Retrotransposons in <Emphasis Type="Italic">Betula nana</Emphasis>, and interspecific relationships in the Betuloideae,based on inter-retrotransposon amplified polymorphism (IRAP) markers

The Betulaceae family comprises two subfamilies, Betuloideae and Corylaceae. The subfamily Betuloideae contains two genera, Alnus Mill. and Betula L. Twenty putative long terminal repeat (LTR) retrotransposons were mined from 171 scaffolds containing 5,208,995 bp of dwarf birch (Betula nana) genome sequences. Five retrotransposons were finally selected after filtering the retrotransposon canonical features and nucleotide similarities between left and right LTR sequences. Of the five retroelements, three elements were found to be Ty1/Copia retrotransposons; identity of the other two elements could not be ascertained due to sequence undetermined ‘N’ bases in the sequence database. Inter-retrotranposon amplified polymorphism (IRAP) analysis, based on the LTR sequences of the mined LTR-retrotransposons, produced 179 discernible IRAP bands among the Alnus and Betula genera. Sequence analysis revealed no size homoplasy among the homologous IRAP bands. Phylogenetic and principle coordinate analysis, based on the band sharing among the taxa, showed the species in two different genera were clearly separated. The subgenera in each genus of Alnus and Betula were also distinguishable from the IRAP profiles. In the genus Betula, the species in subgenus Betula showed mixed clustering between species. This is incongruent with the phylogeographical distribution of the species.     

Molecular Characterization of the Indigenous Stingless Bees (<Emphasis Type="Italic">Tetragonula</Emphasis> spp. Complex) Using ISSR Marker from Southern Peninsular India

India is a country bestowed enormously with stingless bees, but genetic information about them is extremely minimal. This study focused to tap the geographic allocation, genetic variability, and differentiation among Tetragonula species complexes from natural and semi-urban habitats. Genetic analyses were assessed among 36 contrasting genotypes utilizing 20 ISSR primers. The dual combination exquisitely and productively amplified 245 DNA fragments at the loci, of which 240 bands were polymorphic (97.95%). Low to moderate level of genetic differentiation was detected from different estimators (Ht 0.29, G’ _STest 0.16, D _est 0.072, F _ST 0.14, and Nm 2.68). Hierarchical clustering analysis aided to partition the individual genotypes into its respective five species group formed, aided by substantial bootstrap support values, but differing under morphological identification. It also provided valuable insight into the moderate eco-genetic diversity (H 0.39) prevailing from geographically scattered inhabitants. Potential exploitation of hyper-variable ISSR marker turned out fairly as a promising technique for finding valid polymorphisms and infers relevant variations. This baseline information enhances our understanding of the genetic status of the indigenous species from the country.     

A Comparative Analysis of Genetic Variability and Differentiation in <Emphasis Type="Italic">Panax vietnamensis</Emphasis> Ha et Grushv. and <Emphasis Type="Italic">P. ginseng</Emphasis> C.A. Meyer Using ISSR Markers

A comparative analysis of the genetic variability and differentiation of rare medicinal ginseng species, Panax vietnamensis Ha et Grushv. and P. ginseng C.A. Meyer, was carried out using inter-simple sequence repeat markers. It was demonstrated that all the genetic diversity parameters of Vietnamese ginseng were high and considerably exceeded those of P. ginseng. On the contrary, the level of genetic differentiation was higher in true ginseng. It is suggested that the differences in the levels of genetic variability and differentiation of the two ginseng species were influenced by the demographic history, peculiarities of the reproductive system, and human activity.     

Genetic diversity and population structure of <Emphasis Type="Italic">Melia azedarach</Emphasis> in North-Western Plains of India

Key message

Genetic structure among M. azedarach populations was detected and two subpopulations were present among them. A significant ‘isolation by distance’ was found in M. azedarach population in North-Western Plains of India.

Abstract

Melia azedarach is an important forest tree with pharmaceutical, insecticidal, pesticidal, and commercial significance. It is a good reforestation tree because of its fast growth and drought hardy nature. Genetic variation in a species allows itself to adapt, evolve and respond to environmental stress. It provides the basis for survival of a species and critically influences its evolutionary potential. Assessment of genetic diversity is necessary for improvement and conservation of a species. For this, microsatellite markers are of particular interest given the attributes like co-dominance, reproducibility, hyper variability and abundance throughout the genome. In the present study, we analyzed the genetic diversity and population structure of M. azedarach, an ecologically imperative species growing in the North-Western Plains of India. We developed 43 microsatellite markers, of which 20 were subsequently employed for analysis of diversity and population structure among 33 populations encompassing 318 genotypes representing North-Western Plains of India. A moderate level of diversity (Na = 5.1, Ho = 0.506, He = 0.712, I = 1.386) was assessed. The highest value of ΔK estimated using STRUCTURE indicated 2 subpopulations (K = 2). AMOVA exhibited 73 % variation within populations and 12 % variation was found among regions. Significant positive correlation between geographical and genetic distance was found (Rxy = 0.365, P = 0.010). The present study lays a foundation on a better understanding of genetic dynamics of the species and reveals its diversity and population structure in North-Western Plains of India.

     

RAPD-PCR Analysis of Ground Squirrels from the Tobol-Ishim Interfluve: Evidence for Interspecific Hybridization between Ground Squirrel Species <Emphasis Type="Italic">Spermophilus major</Emphasis> and <Emphasis Type="Italic">S. erythrogenys</Emphasis>

Populations of two ground squirrel species, Spermophilus major and S. erythrogenys, from the interfluvial area of the Tobol and Ishim rivers, where their ranges overlap, have been examined using RAPD-PCR. We have identified 253 loci, which included taxon-specific markers for S. major and S. erythrogenys as well as markers for geographic populations. Estimation of genetic diversity and construction of phylogenetic relationships were performed using software programs POPGENE, TEPGA, and TREECON. In all, based on morphological traits, animals from the Tobol-Ishim interfluve were assigned to the two parental morphotypes and showed similar levels of genetic variability (H, n_a, n_e). However, the total polymorphism level proved to be higher in ground squirrels with the major morphotype (P = 40.32%,P₉₅ = 27.27%) than in animals with the erythrogenys morphotype (P = 32%,P₉₅ = 22.13%). Nevertheless, the number of rare alleles was high in both cases, constituting about 70% of the total number. Interpopulation differentiation was considerably higher in S. major δ = 0.50) than in S. erythrogenys δ = 0.41). The genetic differentiation between local samples from the Tobol-Ishim interfluvial area was lower than that between the parental species. A significant part of the genetic diversity of the species examined and animals from the zone of overlapping ranges was accounted for by intrapopulation variability. Animals from the northern and southern parts of the Tobol-Ishim interfluve were charac-terized by the core traits of S. major and S. erythrogenys, respectively, falling into two distinct clusters in the UPGMA and NJ reconstructions. In addition to three hybrid individuals, identified by the bioacoustic method, three hybrid animals were distinguished using RAPD analysis. These animals earlier were thought to be “pure” species and formed their own clusters in phylogenetic reconstructions. Thus, the RAPD-PCR results directly showed the existence of stable hybridization (20% genetic hybrids) between S. major and S. erythrogenys in the Tobol-Ishim interfluvial area, which is more extensive than inferred previously from morphological and bioacoustic data.     

LTR retrotransposons from the <Emphasis Type="Italic">Citrus x clementina</Emphasis> genome: characterization and application

Long terminal repeat retrotransposons (LTR-RTs) are a large portion of most plant genomes, and can be used as a powerful molecular marker system. The first citrus reference genome (Citrus x clementina) has been publicly available since 2011; however, previous studies in citrus have not utilized the whole genome for LTR-RT marker development. In this study, 3959 full-length LTR-RTs were identified in the C. x clementina genome using structure-based (LTR_FINDER) and homology-based (RepeatMasker) methods. LTR-RTs were first classified by protein domain into Gypsy and Copia superfamilies, and then clustered into 1074 families based on LTR sequence similarity. Three hundred fifty Copia families were grouped into four lineages: Retrofit, Tork, Sire, and Oryco. One hundred seventy-eight Gypsy families were sorted into six lineages: Athila, Tat, Renia, CRM, Galadriel, and Del. Most LTR-RTs (3218 or 81.3%) were anchored to the nine Clementine mandarin linkage groups, accounting for 9.74% of chromosomes currently assembled. Accessions of 25 Rutaceae species were genotyped using 17 inter-retrotransposon amplified polymorphism (IRAP) markers developed from conserved LTR regions. Sequence-specific amplified polymorphism (SSAP) makers were used to distinguish ‘Valencia’ and ‘Pineapple’ sweet oranges (C. x sinensis), and 24 sweet orange clones. LTR-RT markers developed from the Clementine genome can be transferred within the Rutaceae family demonstrating that they are an excellent tool for citrus and Rutaceae genetic analysis.     

Variability of microsatellite loci in <Emphasis Type="Italic">Vincetoxicum</Emphasis> Wolf species in southeastern Ukraine

Genetic diversity of 13 species of the genus Vincetoxicum Wolf found in Ukraine with the use of four of eight nuclear microsatellite markers previously developed for Vincetoxicum atratum from Japan was studied. The number of alleles in studied loci varied in the range from 8 to 25. The expected heterozygosity was 0.690–0.938; the observed heterozygosity varied in the range from 0.205 to 0.806. The total rate of genetic variability of studied species was found to be comparable to the rate of variability of Vincetoxicum atratum from Japan. Microsatellite loci Vinc5, Vinc104, Vinc123, and Vinc124 can be successfully used for estimating the intra- and interspecific polymorphism of the species of genus Vincetoxicum Wolf in Ukraine.     

Genome sizes and phylogenetic relationships suggest recent divergence of closely related species of the <Emphasis Type="Italic">Limonium vulgare</Emphasis> complex (Plumbaginaceae)

Limonium vulgare and related species form a complex group, but until now cytological and genetic studies have been based on single species and specific geographical areas. We investigated genome size, karyological and genetic diversity in samples from Western Mediterranean and evaluated the phylogenetic relationships among the species of this complex. Genome size was assessed using flow cytometry on samples from natural populations of L. vulgare, L. maritimum and L. narbonense. Chromosome counts were conducted in plants obtained from seeds collected in the field. The internal transcribed spacer ITS1 of the nuclear rDNAs was used to assess ITS polymorphisms as well as the phylogenetic relationships within the L. vulgare complex. Our analyses showed that all species were tetraploid, with the chromosome number of L. maritimum being presented here for the first time. Significant differences were observed in genome size, with L. narbonense having lower genome sizes than the other two species, and possible aneuploids being detected. Ten new ITS sequences from L. vulgare, L. narbonense and L. maritimum were provided. Most species’ populations showed unique ribotypes, and L. narbonense has the highest ribotype diversity. One of the L. maritimum populations presented a closer genetic relationship with L. vulgare, whereas the other two seemed to be more related with L. narbonense. Phylogenetic analyses confirmed that L. vulgare and L. narbonense form a monophyletic group, sister to the remaining Limonium species. Our results put into evidence that the studied species may represent a relatively early stage of divergence.     

Morphometric and ISSR-Analysis of Local Populations of <Emphasis Type="Italic">Geranium molle</Emphasis> L. from the Southern Coast of the Caspian Sea

Geranium molle is known as Dovefoot Geranium or Awnless Geranium. Dovefoot Geranium is a low-growing herb with pink flowers and sharply toothed leaves. Dovefoot Geranium is native to Eurasia and has been introduced to many habitats of the world. This species is very similar to G. robertianum but its palmate-like leaves and bilobed petals show differences. This plant is considered to be anodyne, astringent and vulnerary. We have no information on its population genetic structure, genetic diversity, and morphological variability in Iran. Therefore, due to the importance of these plant species, we performed a combination of morphological and molecular data for this species. For this study, we used 132 randomly collected plants from 18 geographical populations in 4 provinces. Genetic diversity parameters were determined in these populations. STRUCTURE analysis and K-Means clustering identified 14 gene pools in the country and revealed isolation by distance among the studied populations. The Mantel test showed correlation between genetic and geographical distance. AMOVA revealed a significant genetic difference among populations and showed that 40% of total genetic variation was due to within-population diversity. The consensus tree of both molecular and morphological data identified divergent populations. These data may be used in future breeding and conservation of this important medicinal plant in the country.     

Assessment of genetic diversity in <Emphasis Type="Italic">Mucuna</Emphasis> species of India using randomly amplified polymorphic DNA and inter simple sequence repeat markers

Genus Mucuna which is native to China and Eastern India comprises of perennial climbing legume with long slender branches, trifoliate leaves and bear green or brown pod covered with soft or rigid hairs that cause intense irritation. The plants of this genus are agronomically and economically important and commercially cultivated in India, China and other regions of the world. The high degrees of taxonomical confusions exist in Mucuna species that make authentic identification and classification difficult. In the present study, the genetic diversity among the 59 accessions of six species and three varieties of M. pruriens has been assessed using DNA fingerprinting based molecular markers techniques namely randomly amplified polymorphic DNA (RAPD), inter simple sequence repeats (ISSR) and combined dataset of RAPD and ISSR. Also, genetic relationship among two endemic species of Mucuna namely M. imbricata and M. macrocarpa and two varieties namely IIHR hybrid (MHR) and Dhanwantari (MD) with other species under study was investigated by using cluster analysis and principal coordinate analysis. The cluster analysis of RAPD, ISSR and combined dataset of RAPD and ISSR clearly demonstrated the existence of high interspecific variation than intra-specific variation in genus Mucuna. The utility and efficacy of RAPD and ISSR for the study of intra species and interspecies genetic diversity was evident from AMOVA and PCoA analysis. This study demonstrates the genetic diversity in Mucuna species and indicates that these markers could be successfully used to assess genetic variation among the accessions of Mucuna species.