Molecular diversity and genetic relationships in <Emphasis Type="Italic">Secale</Emphasis>

The objective of this study was to quantify the molecular diversity and to determine the genetic relationships among Secale spp. and among cultivars of Secale cereale using RAPDs, ISSRs and sequence analysis of six exons of ScMATE1 gene. Thirteen ryes (cultivated and wild) were genotyped using 21 RAPD and 16 ISSR primers. A total of 435 markers (242 RAPDs and 193 ISSRs) were obtained, with 293 being polymorphic (146 RAPDs and 147 ISSRs). Two RAPD and nine ISSR primers generated more than 80% of polymorphism. The ISSR markers were more polymorphic and informative than RAPDs. Further, 69% of the ISSR primers selected achieved at least 70% of DNA polymorphism. The study of six exons of the ScMATE1 gene also demonstrated a high genetic variability that subsists in Secale genus. One difference observed in exon 1 sequences from S. vavilovii seems to be correlated with Al sensitivity in this species. The genetic relationships obtained using RAPDs, ISSRs and exons of ScMATE1 gene were similar. S. ancestrale, S. kuprijanovii and S. cereale were grouped in the same cluster and S. segetale was in another cluster. S. vavilovii showed evidences of not being clearly an isolate species and having great intraspecific differences.     

Analysis of DNA polymorphism in a relict Uralian species,large-flowered foxglove (<Emphasis Type="Italic">Digitalis grandiflora</Emphasis> Mill.), using RAPD and ISSR markers

Genetic polymorphism of the Uralian relict plant species, large-flowered foxglove Digitalis grandiflora Mill. (family Scrophulariaceae), was examined using RAPD and ISSR techniques. A total of 149 RAPD and 74ISSR markers were tested. The indices characterizing polymorphism and genetic diversity were calculated. The data obtained pointed to a high level of genetic variation of D. grandiflora (P ₉₅ = 65%). The cenopopulation examined was weakly differentiated with most of genetic diversity accounted by within-population differentiation.     

Estimation of the Genetic Structure of <Emphasis Type="Italic">Helix pomatia</Emphasis> L. Populations (Mollusca,Pulmonata) by Capillary Electrophoresis of ISSR DNA Fragments

Using the capillary electrophoresis of ISSR DNA fragments, the genetic structure of the adventitious populations of Helix pomatia L. (Mollusca, Pulmonata) inhabiting the territory of Eastern Europe was studied. A significant decrease in genetic heterogeneity (H_e = 0.095 ± 0.002) and population differentiation level (F_st = 0.074) was revealed in comparison with similar data obtained using the ISSR method with electrophoretic detection in agarose gel and allozymes in polyacrylamide gel. Similar to other methods, an effect of spatial isolation (R_M =–0.476) was found on the basis of regression analysis between genetic and geographical distances. The effective population size, calculated with the help of M. Slatkin’s model, turned out to be the largest of the previously calculated values (N_e = 41.8), which indicates the high viability of the studied groups of Roman snails in newly developed territories.     

Evaluation of genetic diversity of <Emphasis Type="Italic">Clinacanthus nutans</Emphasis> (Acanthaceaea) using RAPD,ISSR and RAMP markers

Three polymerase chain reaction (PCR) techniques were compared to analyse the genetic diversity of Clinacanthus nutans eight populations in the northern region of Peninsular Malaysia. The PCR techniques were random amplified polymorphic deoxyribonucleic acids (RAPD), inter-simple sequence repeats (ISSR) and random amplified microsatellite polymorphisms (RAMP). Leaf genomic DNA was PCR amplified using 17 RAPD, 8 ISSR and 136 RAMP primers . However, only 10 RAPD primers, 5 ISSR primers and 37 RAMP primers produced reproducible bands. The results were evaluated for polymorphic information content (PIC), marker index (MI) and resolving power (RP). The RAMP marker was the most useful marker compared to RAPD and ISSR markers because it showed the highest average value of PIC (0.25), MI (11.36) and RP (2.86). The genetic diversity showed a high percentage of polymorphism at the species level compared to the population level. Furthermore, analysis of molecular variance revealed that the genetic diversity was higher within populations, as compared to among populations of C. nutans. From the results, the RAMP technique was recommended for the analysis of genetic diversity of C. nutans.     

Genetic diversity of <Emphasis Type="Italic">Opuntia</Emphasis> spp. varieties assessed by classical marker tools (RAPD and ISSR)

Opuntia, commonly named “nopal” in Mexico, is an important crop for its agronomical, economical, ecological and cultural value. Furthermore, it is known for its taxonomic complexity. In this paper, we report the genetic variability of 52 Opuntia cultivars with agronomic and economic importance, classified into 12 different species using random amplified polymorphic DNA (RAPD) and inter-simple sequence repeats (ISSR) markers. Ten primers, five for each marker type, were selected to assess their ability to detect polymorphisms in this plant accesions/varieties. Both marker systems generated a total of 307 bands, of which 50.8 % were polymorphic with an average of 15.6 polymorphic bands per primer. Thus, we assume that Mexican Opuntia varieties present broad genetic variation. Based on percentage of polymorphic bands; resolving power; polymorphic information content; and Marker Index, the K-12 (RAPD) and IS-06 (ISSR) primers were the most informative ones. Clusters obtained from RAPD, ISSR and a combination of both data sets did not match the actual taxonomic classification. On the other hand, the putative varieties currently classified in the same species were not located in the same cluster. Besides, the varieties included in O. ficus-indica, O. albicarpa and O. megacantha showed broad variation but were not well defined into separate clades; these cultivars possibly have common ancestry. The results presented here support our hypothesis about the existence of a smaller number of Opuntia species in accordance with those currently described, but with high intraspecific genetic variation.     

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.     

Effective to census population size ratios in two Near Threatened Mediterranean amphibians: <Emphasis Type="Italic">Pleurodeles waltl</Emphasis> and <Emphasis Type="Italic">Pelobates cultripes</Emphasis>

Efforts to mitigate amphibian declines are hindered by a lack of information about basic aspects of their biology and demography. The effective to census population size ratio (N _e /N _c ) is one of the most important parameters for the management of wildlife populations because it combines information on population abundance and genetic diversity and helps predict population viability in the long term. Few studies have calculated this ratio in amphibians, which sometimes show low ratios, associated with a higher extinction risk. Here we integrate field-based (capture-mark-recapture studies, egg string counts) and molecular approaches (estimation of the effective number of breeders (N _b ) and the effective population size (N _e ) based on genotypes from larval cohorts and candidate parents) to produce the first estimates of the N _e /N _c and N _b /N _c ratios in two amphibians, the Iberian ribbed newt Pleurodeles waltl and the western spadefoot Pelobates cultripes. Additionally, we investigate sex-biased dispersal in both species based on direct (field observations) and indirect (genetic) evidence. Both species showed similar ratios, slightly lower in Pleurodeles (0.21–0.24) than in Pelobates (0.25–0.30). Observed displacement rates were low in both species (P. waltl?=?0.51%; P. cultripes?=?1.23%). We found no evidence for sex-biased dispersal in P. cultripes, but both direct and indirect evidences suggest a tendency for female-biased dispersal in P. waltl. We discuss differences in the genetic estimates of N _e and N _b provided by three inference methods and the implications of our findings for the management of these species, characteristic of Mediterranean wetlands in the Iberian Peninsula and listed as Near Threatened.     

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.     

Evaluation of genetic homogeneity of in vitro-raised plants of <Emphasis Type="Italic">Tecomella undulata</Emphasis> (Sm.) Seem. using molecular markers

Tecomella undulata (Sm.) Seem (family Bignoniaceae) is an economically and pharmaceutically important timber tree of arid regions of India. Overexploitation of natural stands coupled with minimal conservation and reforestation efforts has led to its incorporation in list of endangered species. This monotypic genus can be propagated only through seeds as no methods are available for its vegetative propagation. Therefore, protocol for multiplication of T. undulata via direct regeneration using nodal segments from mature trees has been standardized. Authentication of genetic homogeneity of these in vitro-raised plants is necessary for commercial-scale application of the developed micropropagation protocol. PCR-based molecular markers which have emerged as simple, fast, reliable, and labor-effective tools for testing the genetic homogeneity of in vitro-raised plants were used in the present study. Arbitrary (random amplified polymorphic DNA, RAPD), semi-arbitrary (inter-simple sequence repeat, ISSR; start codon targeted (SCoT) polymorphism), and sequence-based (simple sequence repeat, SSR) markers were used. DNA samples of shoots maintained in vitro for 2 years collected after every 4 subculture cycles (of 3 weeks each) and field-transferred plantlets were compared with the mother tree DNA using 131 primers (25 each of RAPD, ISSR, SCoT and 56 SSR). Scorable unambiguous and reproducible DNA fragments were produced by 77 (21 RAPD, 20 ISSR, 22 SCoT and 14 SSR) primers. A total of 71, 93, 94, and 42 distinct and scorable DNA fragments were produced by RAPD, ISSR, SCoT, and SSR primers respectively with an average of 3.38, 4.65, 4.27, and 3.0 DNA fragments per primer. The true-to-type nature of the in vitro-raised plants of T. undulata undergoing up to 32 subculture passages over a period of approximately 2 years was authenticated by monomorphic DNA fragments amplified with all primer combinations. Therefore, the developed micropropagation protocol can be safely used on a commercial scale for multiplying T. undulata plants.     

Genetic diversity of the house mouse <Emphasis Type="Italic">Mus musculus</Emphasis> and geographic distribution of its subspecies-specific RAPD markers on the territory of Russia

Genetic diversity and geographic distribution of taxon-specific RAPD markers was examined in ten local populations of the house mouse Mus musculus (n = 42). The house mice were generally characterized by moderate genetic variation: polymorphism P ₉₉ = 60%, P ₉₅ = 32.57%; heterozygosity H = 0.12; the observed allele number n _a = 1.6; the effective allele number n _e = 1.18; the within-population differentiation ?_s = 0.388; and Shannon index I = 0.19. The degree of genetic isolation of individual local populations was greatly variable. The genetic subdivision index G _st varied from 0.162 to 0.770 at the gene flow of Nm = 2.58?0.149, while the among-population distances D _N varied from 0.026 to 0.178. The largest part of the genetic diversity was found among the populations (H _T = 0.125), while the within-population diversity was twice lower (H _S = 0.06). The samples examined were well discriminated relative to the sets of RAPD markers. The character distribution pattern provided conditional subdivision of the mice into the “western” and the “eastern” groups with the putative boarder along the Baikal Lake. The first group was characterized by the prevalence of the markers typical of M. m. musculus and M. m. domesticus. The second group was characterized by the prevalence of the markers typical of M. m. musculus, M. m. gansuensis, M. m. castaneus, M. m. domesticus, and M. m. wagneri. The genotype of the nominative subspecies M. m. musculus was background for all populations. In the populations examined some of earlier described subspecies-specific molecular markers were found at different frequencies, pointing to the involvement of several subspecies of M. musculus in the process of hybridization.     

Assessment of genetic diversity and differentiation of <Emphasis Type="Italic">Liposcelis bostrychophila</Emphasis> (Psocoptera: Liposcelidae) in China using inter-simple sequence repeat (ISSR) fingerprinting

Liposcelis bostrychophila (Psocoptera: Liposcelidae) is a widely distributed pest that can cause considerable economic losses and pose human health risks. Rapid development of insecticide resistance has made L. bostrychophila increasingly difficult to control. To obtain information potentially useful for pest management, genetic diversity and differentiation of L. bostrychophila from five geographic locations in China was studied using inter-simple sequence repeat (ISSR). A total of 104 loci were found by ISSR markers and amplified using 9 selected primers. The percentage of polymorphic bands (PPB) was 91.4%. Shannon’s information index (I) and Nei’s gene diversity (He) indicated high genetic diversity at the species level. Population differentiation (Gst = 0.484) was average in these populations. Analysis of molecular variation (AMOVA) indicated that genetic variation was mainly distributed within populations. Gene flow (Nm = 0.534) was moderate. Cluster analysis showed that genotypes isolated from the same locations displayed higher genetic similarity and permitted the grouping of isolates of L. bostrychophila into three distinct clusters. The correlation between genetic distance and geographic distance was not significant.     

Reconstruction of molecular phylogeny of closely related <Emphasis Type="Italic">Amorphophallus</Emphasis> species of India using plastid DNA marker and fingerprinting approaches

Plastid DNA markers sequencing and DNA fingerprinting approaches were used and compared for resolving molecular phylogeny of closely related, previously unexplored Amorphophallus species of India. The utility of individual plastid markers namely rbcL, matK, trnH–psbA, trnLC–trnLD, their combined dataset and two fingerprinting techniques viz. RAPD and ISSR were tested for their efficacy to resolves Amorphophallus species into three sections specific clades namely Rhaphiophallus, Conophallus and Amorphophallus. In the present study, sequences of these four plastid DNA regions as well as RAPD and ISSR profiles of 16 Amorphophallus species together with six varieties of two species were generated and analyzed. Maximum likelihood and Bayesian Inference based construction of phylogenetic trees indicated that among the four plastid DNA regions tested individually and their combined dataset, rbcL was found best suited for resolving closely related Amorphophallus species into section specific clades. When analyzed individually, rbcL exhibited better discrimination ability than matK, trnH–psbA, trnLC–trnLD and combination of all four tested plastid markers. Among two fingerprinting techniques used, the resolution of Amorphophallus species using RAPD was better than ISSR and combination of RAPD +ISSR and in congruence with resolution based on rbcL.     

Genetic Diversity and Phylogenetic Relationships of Two Closely Related Northeast China <Emphasis Type="Italic">Vicia</Emphasis> Species Revealed with RAPD and ISSR Markers

RAPD and ISSR analyses revealed genetic diversity and relationships among 11 populations of two closely related northeast China Vicia species, Vicia ramuliflora and V. unijuga. Both methods yielded similar and complementary results, showing high genetic diversity. Vicia ramuliflora had 100% polymorphic loci in both RAPD and ISSR, and V. unijuga had 100% polymorphic loci for RAPD and 98.96% for ISSR. Genetic differentiation was moderate among populations of each species. Genetic variation was distributed mainly within populations for the two species. The high level of gene flow was important for the allocation of genetic variation. The UPGMA dendrogram and principal coordinates analysis at the level of individuals and populations showed that V. ramuliflora and V. unijuga were more closely related than either of them was to the outgroup species, V. cracca. The small molecular variance of V. ramuliflora and V. unijuga supports the conclusion that these two species had a common ancestor.     

Isolation of Polymorphic RAPD-SSR Markers from Xinjiang Arctic Grayling (<Emphasis Type="Italic">Thymallus arcticus grubei</Emphasis>) and a Test of Cross-Species Amplification

A total of 18 polymorphic microsatellite loci were isolated and characterized from RAPD products in the Xinjiang Arctic Grayling (Thymallus arcticus grubei). The number of alleles (N_a) per locus varied from 2 to 10. Observed (H_o) and expected (H_e) heterozygosities ranged from 0.64 to 0.92, and from 0.63 to 0.88, respectively. Considerable differences were found among HBH, FH and FY populations in the number of alleles, effective number of alleles, number of genotypes at all of these loci. These new RAPD-SSR markers have provided a helpful tool for genetic analyses and resources conservation of T. arcticus grubei. Five additional fish species, Amur grayling (Thymallus grubii), Taimen (Hucho taimen), Sea perch (Lateolabrax japonicus), Lenok (Brachymystax lenok) and Red seam bream (Pagrosomus major) were assessed for cross-species amplification. Three of the five species showed at least one polymorphic locus. In addition, seven loci were found to be polymorphic in at least one species.     

Genetic Diversity in Various Accessions of Pineapple [<Emphasis Type="Italic">Ananas comosus</Emphasis> (L.) Merr.] Using ISSR and SSR Markers

Inter simple sequence repeat (ISSR) and simple sequence repeat (SSR) markers were used to assess the genetic diversity of 36 pineapple accessions that were introduced from 10 countries/regions. Thirteen ISSR primers amplified 96 bands, of which 91 (93.65%) were polymorphic, whereas 20 SSR primers amplified 73 bands, of which 70 (96.50%) were polymorphic. Nei’s gene diversity (h = 0.28), Shannon’s information index (I = 0.43), and polymorphism information content (PIC = 0.29) generated using the SSR primers were higher than that with ISSR primers (h =  0.23, I = 0.37, PIC = 0.24), thereby suggesting that the SSR system is more efficient than the ISSR system in assessing genetic diversity in various pineapple accessions. Mean genetic similarities were 0.74, 0.61, and 0.69, as determined using ISSR, SSR, and combined ISSR/SSR, respectively. These results suggest that the genetic diversity among pineapple accessions is very high. We clustered the 36 pineapple accessions into three or five groups on the basis of the phylogenetic trees constructed based on the results of ISSR, SSR, and combined ISSR/SSR analyses using the unweighted pair-group with arithmetic averaging (UPGMA) method. The results of principal components analysis (PCA) also supported the UPGMA clustering. These results will be useful not only for the scientific conservation and management of pineapple germplasm but also for the improvement of the current pineapple breeding strategies.     

Genetic diversity and structure of <Emphasis Type="Italic">Capparis spinosa</Emphasis> L. in Iran as revealed by ISSR markers

Capparis spinosa L. (caper bush) is an economically and ecologically important perennial shrub that grows across different regions of Iran. In this study, the genetic diversity and population structure of Iranian genepool of C. spinosa is evaluated using Inter Simple Sequence Repeat (ISSR) markers. Using 10 ISSR primers, 387 DNA fragments (bands) were amplified from the genomic DNA of 92 individuals belonging to twenty-one populations of C. spinosa, of which 378 (97.7%) were polymorphic. High level of genetic diversity (percentage of polymorphic loci = 98.2%, h = 0.1382, I = 0.243), high genetic differentiation (G_st = 0.5234) and low gene flow (Nm = 0.4553) among populations were observed. Caper bush populations were divided into 4 groups in the dendrogram, PCoA plot and Bayesian clustering results, mostly corresponded to their geographic regions. The results showed that there are value in sampling Iranian caper bush populations to look for valuable alleles for use in plant breeding programs.     

Variance in Female Reproductive Success Differentially Impacts Effective Population Size in the Short-Nosed Fruit Bat, <Emphasis Type="Italic">Cynopterus sphinx</Emphasis>

Effective population size (N _e) quantifies the effects of micro-evolutionary processes and the rate of loss of genetic diversity in a population. Several demographic and mating parameters reduce N _e. Theoretical studies elucidate the impacts of various demographic and mating system parameters on N _e, while empirical studies illustrate realized N _e for species with differing life histories and mating systems. However, effect of intra-specific variation in mating system on effective size remains largely unexplored. In this paper we investigated the effect of promiscuous and polygynous mating on N _e in two wild populations of the short-nosed fruit bat, Cynopterus sphinx. N _e/N (ratio of effective population size to census size) was lower than unity in both populations, and much lower for the polygynous population compared to promiscuous population. Elasticity analyses reveal that N _e/N was sensitive to deviations in the sex ratio. Variance in female reproductive success had a higher impact on N _e compared to variance in male reproductive success in the promiscuous population. However, for the polygynous population, impact of variance in male reproductive success on N _e was higher than that of variance in female reproductive success. Our results suggest that depending on mating system, different populations of the same species could have alternate evolutionary trajectories. The rate of loss of genetic diversity would be lower for the promiscuous population compared to the polygynous population. Our study is the first to highlight which parameters would most significantly impact population specific N _e under different mating systems.     

AFLP,RAPD, and ISSR analysis of intraspecific polymorphism and interspecific differences of allotetraploid species <Emphasis Type="Italic">Aegilops kotschyi</Emphasis> Boiss. and <Emphasis Type="Italic">Aegilops variabilis</Emphasis> Eig

To evaluate genetic variation, 27 accessions of allotetraploid species Aegilops kotschyi and Ae. variabilis with the US genome were analyzed using the AFLP, RAPD, and ISSR methods. A total of 316 polymorphic RAPD fragments, 750 polymorphic AFLP fragments, and 234 polymorphic ISSR fragments were obtained. It was demonstrated that the analyzed species were characterized by a considerable level of nuclear genome variation. According to the data of ISSR and RAPD analysis, the average value of the Jaccard similarity coefficient for the accessions of Ae. variabilis from different geographical regions was slightly lower than that for the accessions of Ae. kotschyi. At the same time, AFLP analysis showed no considerable differences in the levels of intraspecific variation of the studied species. Analysis of the summarized RAPD, ISSR, and AFLP marking data in the Structure software program showed that most of the analyzed accessions with high degree of probability could be assigned to one of two groups, the first of which corresponded to Ae. kotschyi and the second corresponded to Ae. variabilis, thereby confirming the species independence of Ae. kotschyi and Ae. variabilis. Accessions k900, k907, k908, and v90 could not with a sufficiently high degree of probability be assigned to one of the species, which possibly was the result of interspecific hybridization. Analysis of the species diversity using different molecular markers made it possible to identify the accessions that were notably different from other accessions of its species.     

Conservation genetics of the threatened catfish <Emphasis Type="Italic">Conorhynchos conirostris</Emphasis> (Siluriformes: <Emphasis Type="Italic">incertae sedis</Emphasis>), an evolutionary relict endemic to the São Francisco River Basin,Brazil

Analysis of genetic datasets can be particularly useful in providing guidelines for conservation management of understudied species targeted by commercial activities. Here we used population genetic approaches to inform on the conservation status of the Neotropical long-nose pirá catfish, Conorhynchos conirostris. Pirá is a large migratory fish endemic to the São Francisco River Basin (SFRB). It is an evolutionarily divergent and relict species, being the sole representative of an incertae sedis family. The species is considered locally extinct in the upper and lower SFRB, listed as vulnerable on the IUCN Red List and as endangered on the Brazilian Red List (ICMBIO). Fishing prohibition has received severe criticism from middle SFRB fisheries that claim that this understudied species is relatively abundant in that region. We used information from 13 microsatellite markers and COI mitochondrial sequences to clarify the genetic diversity of this enigmatic species in the middle SFRB, to estimate contemporary effective population size (N_e), and to assess its conservation status. Results from bottleneck analyses indicated that the species has experienced recent reductions in population size, which is consistent with small estimates of contemporary N_e. The predicted amount of heterozygosity loss (H_t) in t generations ranged from 0.1152 (for an estimated N_e of 26.4; t?=?100) to 0.7573 (for an estimated N_e of 169.9; t?=?10). Our study supports the conservation status proposed by the ICMBIO to the remaining pirá population. Moreover, we highlight the need for demographic data and the re-assessment of the current IUCN classification for this evolutionary relict lineage.     

A novel set of EST-InDel markers in <Emphasis Type="Italic">Eucalyptus</Emphasis> L’Hérit.: polymorphisms,cross-species amplification,physical positions and genetic mapping

Insertion/deletion (InDel) markers are valuable for genetic applications in plant species, and the public databases of expressed sequence tags (ESTs) have facilitated the development of genic InDel markers. In this study, we developed a novel set of 144 InDel markers in an important tree genus Eucalyptus L’Hérit. using the ESTs of GenBank. Amplicon sequencing against two parents of a mapping population (Eucalyptus urophylla S. T. Blake × E. tereticornis Smith) revealed that the InDel size ranged from 2 to 44 bases, and the dinucleotide type was the most abundant (37.3 %). The cross-species/subgenus amplification rate ranged from 62.5 % in E. tessellaris F. Muell. (subgenus Blakella) to 99.3 % in E. grandis Hill ex Maiden (subgenus Symphyomyrtus) with an average of 85.4 %. There were 121 EST-InDels (84.0 %) polymorphic among 12 individuals of E. grandis, and the mean number of alleles per polymorphic locus (N _a), observed heterozygosity (H _o), expected heterozygosity (H _e) and polymorphic information content (PIC) were 4.0, 0.278, 0.538 and 0.465, respectively. Physical positions of 143 EST-InDels were predicted on the E. grandis genome sequence. A total of 81 EST-InDels were incorporated into prior dense genetic maps of E. urophylla and E. tereticonis, and extensive synteny and colinearity were observed between E. grandis genome sequence and the mapped EST-InDel markers. These EST-InDels will provide a valuable resource of functional markers for genetic diversity evaluation, genome comparison, QTL mapping and marker-assisted breeding in Eucalyptus.