SSR Loci Research Articles

Assessment of genetic variability in groundnut (Arachis hypogaea L.) genotypes grown under South African conditions using agronomic and SSR markers

Groundnut (Arachis hypogeae) is a legume crop grown in arid and semi-arid regions of the world. The objective of the study was to assess the presence of genetic diversity among fifty three groundnut genotypes of diverse origin using eleven agronomic and twenty SSR markers. The analysis of variance showed that highly significant variations exist among the genotypes for all phenotypic traits measured. Five principal components showed 71% of the total phenotypic variation. The SSR loci showed high values of polymorphic information content ranging from 0.31 to 0.89, with a mean of 0.71. Heterozygosity values ranged between 0.03 and 1.00 with a mean of 0.57. The genotypes showed a wide range of allelic diversity from 3 to 16, with a mean of 8.1 alleles per locus. Analysis of molecular variance (AMOVA) indicated that larger variability (59%) was due to variation within individuals, whilst the remaining variation was accounted for variation among individuals within population. Cluster analysis grouped the genotypes into two distinct clusters, where it showed that the discrimination of the genotypes was not dependant on the origin of the genotypes. The high gene flow observed among the different geographic origin might contribute to the low differentiation among the population. The SSR and phenotypic markers were able to detect wide genetic diversity and discriminate groundnut genotypes. The two genetically distinct groups observed in this study, can be used as source of genes of novelty and parental lines for transgressive segregation and for further broadening of the genetic base of the crop

SSR analysis of modern Russian potato varieties using DNA samples of nomenclatural standards

The N.I. Vavilov All-Russian Institute of Plant Genetic Resources (VIR) is developing new approaches to documentation of national cultivars, taking into account recommendations of the International Code of Nomenclature for Cultivated Plants in parallel with methods of genetic certification. The nomenclatural standard of a particular cultivar represented by a herbarium specimen can be used as a reference for verifying authenticity and uniformity of cultivar specimens obtained from various sources. The verification requires fast and reliable methods for cultivar genotyping. This paper presents protocols for modified methods of DNA extraction, PCR-analysis and SSR-genotyping, which allow potato cultivars identification without the use of expensive reagent kits. A set of ten chromosome-specific microsatellite markers was used to study polymorphisms in 66 modern Russian potato cultivars, as well as in 11 pre-cultivars and breeding clones, represented by nomenclatural standards and voucher specimens, respectively. This subset of 77 specimens has demonstrated a high level of polymorphism in ten studied microsatellite loci. The SSR analysis identified 73 alleles; 7.3 alleles per locus were observed on average, the number of which varied from 3 (STG0025 locus) to 11 (locus StI046). The PIC values varied from 0.544 (STG0025 locus) to 0.836 (StI046 locus). The alleles, unique for this subset, were found at six studied loci. The high level of polymorphism at the SSR loci made it possible to unambiguously identify almost every cultivar, with the exception of the expected coincidence of microsatellite profiles of two cultivars, which are somaclonal variants. Using an optimized set of eight microsatellite markers, the genetic relationships of modern Russian potato cultivars were studied.

Genetic analysis of walnut cultivars from southwest China: Implications for germplasm improvement

Walnuts are highly valued for their rich nutritional profile and wide medicinal applications. This demand has led to the intensification of breeding activities in major walnut production areas such as southwest China, in order to develop more superior cultivars. With the increasing number of cultivars, accurate identification becomes fundamental to selecting the right cultivar for grafting, industrial processing or development of new cultivars. To ensure proper identification of cultivars and understand the genetic structure of wild and cultivated material, we genotyped 362 cultivated and wild individuals of walnut trees from southwest China (with two additional populations from Xinjiang, plus three cultivars from Canada, France and Belgium) using 36 polymorphic microsatellite loci. We found relatively low indices of genetic diversity (HO = 0.570, HE = 0.404, NA = 2.345) as well as a high level of clonality (>85% of cultivars), indicating reliance on genetically narrow sources of parental material for breeding. Our STRUCTURE and PCoA analyses generally delineated the two species, though considerable levels of introgression were also evident. More significantly, we detected a distinct genetic group of cultivated Juglanssigillata, which mainly comprised individuals of the popular ‘Yangbidapao’ landrace. Finally, a core set of 18 SSR loci was selected, which was capable of identifying 32 cultivars. In a nutshell, our results call for more utilization of genetically disparate material, including wild walnut trees, as parental sources to breed for more cultivars. The data reported herein will significantly contribute towards the genetic improvement and conservation of the walnut germplasm in southwest China.

Genogeography of norway spruce (Picea abies (L.) Karst.) according to the analysis of cytoplasmic DNA

Norway spruce (Picea abies (L.) Karst.) is one of the main forest-forming species in Belarus. It plays important economic, ecological and social roles. The spruce forest of the region is characterized by a complex history of the formation of its population genetic structure. The aim of this study was the genogeographic analysis of P. abies populations and the description of regional features of its gene pool in Belarus. Molecular genetic analysis of microsatellite (SSR) loci of chloroplast DNA and mt15-D02 locus of mitochondrial DNA of Norway spruce was carried out for samples from 57 naturally originated forest stands. We identified 19 allelic variants of the Pt63718, Pt26081, Pt71936 cpDNA loci and two allelic variants of mt15-D02 mtDNA. The geographical distribution of the alleles has been described and the regional features of the genogeographic differentiation of the spruce forests have been considered. The southern border of the continuous distribution area of P. abies and its island localities lie in the south of Belarus (Brest and Gomel regions). The frequency of occurrence of individual allelic variants of cpDNA SSR loci has the most pronounced deviations from the average values for the whole country in those regions. Analysis of cpDNA showed the presence of certain regional features of the genogeographic structure of the spruce forest in the “south-north” and “west-east” directions. According to mitochondrial DNA analysis higher concentration of P. abies trees of southern (Carpathian) origin is observed in the southwest of Belarus. On the contrary northern (Boreal) origin dominates in the rest of the country. The results of performed genogeographic analysis of Norway spruce populations can serve as a basis for improving the forest seed zoning of the tree species.

Assessment of genetic diversity and wilt disease resistance in hot pepper (Capsicum annuum) germplasm from Ethiopia

Abstract Hot pepper (Capsicum annuum L.) is an economically important crop in Ethiopia. Wide variability in hot pepper germplasm in Ethiopia is expected due to the presence of diverse environmental conditions and variation in farming systems. The present study was carried out to evaluate the resistance of 75 hot pepper accessions to wilt disease and assess their genetic diversity using SSR markers. Out of 75 accessions tested, the present study identified 23 accessions that showed resistance (R) with the value of 1-10% disease incidence. The genetic diversity assessment using 13 polymorphic SSR markers allowed the detection of 111 clear and scorable bands. The number of alleles per locus ranged from 5 to 13, with an average of 8.54. The PIC value ranged from 0.27 to 0.87 with an average of 0.59. The gene diversity indices were highly variable across SSR loci and ranged from 0.29 to 0.88 with mean genetic diversity of 0.62. Observed heterozygosity was also highly variable between loci (0.01-0.45) indicating that the accessions were not fixed to homozygosity. Furthermore, genetic diversity parameters were estimated among populations by grouping accessions based on their origin. Within populations, the PIC value ranged from 0.31 to 0.77. The genetic distances among the eight populations ranged from 0.15 to 0.48. The observed highest genetic diversity (0.80) in the Amhara region (Gojam) may indicate this area as the primary site for designing in situ conservation for this crop in Ethiopia. The research findings provide baseline information on disease resistance germplasm sources to be used for the breeding program, as well as the status of genetic diversity of the accessions for efficient conservation and proper utilization of the existing genetic resources in the country.

Development of genome-wide SSR markers in rapeseed by next generation sequencing.

Rapeseed (Brassica napus L.) is an important oil crop with a huge genome. This study used next generation sequencing technology to develop SSR markers in rapeseed. A total of 213,876 sequence reads were obtained in 58.8Mb. For these reads, 21,523 SSRs were recovered from 18,575 microsatellites sequences and 8,964 SSR primer pairs were identified. Di- and mono-nucleotides were the most abundant, accounting for 47.5% and 30.7% of all SSRs, respectively. A total of 8,776 SSRs were designed from contigs and 100 SSR primers were tested for validation of SSR locus amplification. Nearly all (94%) of the markers were found to produce clear amplicons and to be reproducible. For these markers, forty-three SSRs showed polymorphic bands in eight rapeseed accessions. Thirty-four SSRs were then applied to 78 rapeseed accessions from China to evaluate the genetic diversity. Result showed that the allele number varied from two to seven, with a mean value of 3.59. The effective allele number of ranged from 1.14 to 3.25, with an average of 2.09. The average values of observed heterozygosity and expected heterozygosity were 0.54 and 0.49, respectively. The Nei's gene diversity varied from 0.12 to 0.69, with a mean value of 0.48. Resulting of the markers testing showed that the identified genome-wide SSRs were useful in rapeseed genetic studies, including genetic diversity, QTL mapping and marker-assisted selection for breeding.

Assessment of genetic diversity on tomato (Lycopersicon esculentum Mill.) landraces using SSR molecular markers in Turkey

Genetic diversity is getting have been increasingly narrowed by the dense bred cultivar usage for production, on the other hand, genetic variation created by the landraces is known as the raw material of plant breeding. A collection of 21-landrace and three-commercial tomato (Lycopersicon esculentum Mill.) genotypes were screened for allele profile, genetic definition and genetic similarity with the aim of identifying genetic variability in genetic level with 40 SSR loci. Number of alleles per locus ranged from 3 (SSR50, SSR80, SSR9, LEtat002) to 13 (LEat018). Expected and observed heterozygosity values were scored between He: 0.753 and Ho: 0.714 respectively and the mean value of polymorphism information content (PIC) value of these loci was found as 0.694. Considering distribution of alleles at loci, the highest allele frequency was observed from SSR75 with % 62.5 LEat014 with % 58.3 LEga004 with % 50.0 locus, while the lowest was in AI491065 with 16.6% LEta024 locus. UPGMA (Unweighted Pair-Group Method using Arithmetic Means) method was conducted for cluster analysis. The dendrogram was consisted 2 main groups; Group 2 was the largest and contained many sub-groups. The highest genetic similarity level of genotypes from Bafra-Merkez and Sivas-Yukarıkale Village was found as 0.776. The lowest similarity ratio was observed between Sivas-Yıldızeli-Emirler Village and Samsun-Carsamba genotypes with 0.136. The obtained results are indicated that genetic diversity information of landraces with effective SSR loci will help to manage tomato genetic resources for tomato breeding.

Development of genic-SSR markers and their application in revealing genetic diversity and population structure in an Eastern and North-Eastern Indian collection of Jack (Artocarpus heterophyllus Lam.)

Jack (Artocarpus heterophyllus) is a multi-purpose out-breeding tree species of the family Moraceae. We generated 42,928,887 high-quality expressed sequence reads, assembled them into 89,356 unigenes, and discovered 16,853 unigene-based perfect SSRs in A. heterophyllus. Thirty-eight polymorphic SSRs were used to analyze the genetic diversity and population structure of 224 germplasm accessions of A. heterophyllus constituting three populations from three agro-climatic zones, namely Eastern Plateau and Hills, Middle Gangetic Plain Region, and Eastern Himalayan Region, encompassing five Eastern and North-Eastern states of India. At the 38 SSR loci, we detected 142 alleles with a mean of 3.74 alleles per locus. The PIC values for the loci ranged from 0.25 to 0.69. The maximum genetic diversity was recorded in Eastern Plateau and Hills (I = 0.98, He = 0.52). The ANOVA analysis indicated significantly higher within-population variation (90%) than between populations (10%). The indirect estimation of gene flow (Nm) from PhiPT indicated significant gene flow among all three populations. The population structure analysis showed at least four distinct groups among the three populations with different introgression degrees. The NJ-based clustering grouped the 224 germplasm accessions into three main clusters, each with three sub-clusters. However, we did not observe distinct geographical structure among populations except some clustering among the germplasm accessions of the populations of geographically close locations. The transcriptome dataset and the SSR markers developed in the study would boost the species' molecular characterization, conservation, and specific need-based improvement.

A comparison of microsatellites and genome-wide SNPs for the detection of admixture brings the first molecular evidence for hybridization between Mustela eversmanii and M.putorius (Mustelidae, Carnivora).

Introgressive hybridization can pose a serious threat to endangered species which have an overlapping distribution such as in the case of two polecat species, Mustela eversmanii and M. putorius, in Europe. The population size of steppe polecat is known to continuously shrink, whereas its sister species, the European polecat, is still somehow widespread. In this study, we perform an analysis using microsatellite (SSR) and genomic (SNP) data sets to identify natural hybrids between polecats. Four populations were genotyped for eight polymorphic SSR loci, and thousands of unlinked SNPs were generated using a reduced‐representation sequencing approach, RADseq, to characterize the genetic make‐up of allopatric populations and to identify hybrids in the sympatric area. We applied standard population genetic analyses to characterize the populations based on their SSR allelic frequency. Only a single sample out of 48 sympatric samples showed exact intermediacy that we identified as an F1 hybrid. Additionally, one specimen was indicated in the genomic data sets as backcrossed. Other backcrosses, indicated by SSRs, were not validated by SNPs, which highlights the higher efficacy of the genomic method to identify backcrossed individuals. The low frequency of hybridization suggests that the difference in habitat preference of the two species may act as a barrier to admixture. Therefore, it is apparently unlikely that polecat populations are threatened by significant introgression. The two species showed a clear genetic differentiation using both techniques. We found higher genetic diversity values in the sympatric steppe polecat population than in the other studies on polecat populations. Although M. putorius is a hunted species in most countries, genetic diversity values indicate worse conditions in Europe than in the protected sibling species M. eversmanii. Suspending hunting and providing protected status of the former seems to be reasonable and timely.

Genetic diversity and genetic structures of original plants of Viticis Fructus:an SSR markers-based analysis

The present study aimed to provide the protection strategies for wild germplasm resources of original plants of Viticis Fructus and a theoretical basis for the sustainable use of Viticis Fructus. The genetic diversity and genetic structures of the 232 indivi-duals in 19 populations of Vitex rotundifolia and V. trifolia were analyzed by eight SSR markers with tools such as Popgene32, GenAlex 6.502, and STRUCTURE. Bottleneck effect was detected for the population with more than 10 individuals. The results indicated that 42 and 26 alleles were detected from the populations of V. rotundifolia and V. trifolia, respectively, with average expected heterozygo-sities of 0.448 6 and 0.583 9, which are indicative of low genetic diversity. AMOVA revealed the obvious genetic variation of V. rotundifolia and V. trifolia within population(84.43%, P<0.01; 60.37%, P<0.01). Furthermore, in eight SSR loci, six from V. rotundifolia populations and two from V. trifolia populations failed to meet Hardy-Weinberg equilibrium expectations(P<0.05), which confirmed that the populations experienced bottleneck effect. As assessed by Mantel test, geographical distance posed slight impacts on the genetic variation between the populations of V. rotundifolia and V. trifolia. Principal component analysis(PCA) and STRUCTURE analysis demonstrated evident introgression of genes among various populations. The original plants of Viticis Fructus were confirmed low in genetic diversity and genetic differentiation level. Therefore, the protection of wild resources of original plants of Viticis Fructus should be strengthened to ensure its sustainable use.

Structural and Functional Genomics of the Resistance of Cacao to Phytophthora palmivora.

Black pod disease, caused by Phytophthora spp., is one of the main diseases that attack cocoa plantations. This study validated, by association mapping, 29 SSR molecular markers flanking to QTL (Quantitative Trait Loci) associated with Phytophthora palmivora Butler (Butler) (PP) resistance, in three local ancient varieties of the Bahia (Comum, Pará, and Maranhão), varieties that have a high potential in the production of gourmet chocolate. Four SSR loci associated with resistance to PP were detected, two on chromosome 8, explaining 7.43% and 3.72% of the Phenotypic Variation (%PV), one on chromosome 2 explaining 2.71%PV and one on chromosome 3 explaining 1.93%PV. A functional domains-based annotation was carried out, in two Theobroma cacao (CRIOLLO and MATINA) reference genomes, of 20 QTL regions associated with cocoa resistance to the pathogen. It was identified 164 (genome CRIOLLO) and 160 (genome MATINA) candidate genes, hypothetically involved in the recognition and activation of responses in the interaction with the pathogen. Genomic regions rich in genes with Coiled-coils (CC), nucleotide binding sites (NBS) and Leucine-rich repeat (LRR) domains were identified on chromosomes 1, 3, 6, 8, and 10, likewise, regions rich in Receptor-like Kinase domain (RLK) and Ginkbilobin2 (GNK2) domains were identified in chromosomes 4 and 6.

Assessment of genetic diversity and population structure of oil palm (Elaeis guineensis Jacq.) field genebank: A step towards molecular-assisted germplasm conservation.

Oil palm (Elaeis guineensis) germplasm is exclusively maintained as ex situ living collections in the field for genetic conservation and evaluation. However, this is not for long term and the maintenance of field genebanks is expensive and challenging. Large area of land is required and the germplasms are exposed to extreme weather conditions and casualty from pests and diseases. By using 107 SSR markers, this study aimed to examine the genetic diversity and relatedness of 186 palms from a Nigerian-based oil palm germplasm and to identify core collection for conservation. On average, 8.67 alleles per SSR locus were scored with average effective number of alleles per population ranging from 1.96 to 3.34 and private alleles were detected in all populations. Mean expected heterozygosity was 0.576 ranging from 0.437 to 0.661 and the Wright’s fixation index calculated was -0.110. Overall moderate genetic differentiation among populations was detected (mean pairwise population FST = 0.120, gene flow Nm = 1.117 and Nei’s genetic distance = 0.466) and this was further confirmed by AMOVA analysis. UPGMA dendogram and Bayesian structure analysis concomitantly clustered the 12 populations into eight genetic groups. The best core collection assembled by Core Hunter ver. 3.2.1 consisted of 58 palms accounting for 31.2% of the original population, which was a smaller core set than using PowerCore 1.0. This core set attained perfect allelic coverage with good representation, high genetic distance between entries, and maintained genetic diversity and structure of the germplasm. This study reported the first molecular characterization and validation of core collections for oil palm field genebank. The established core collection via molecular approach, which captures maximum genetic diversity with minimum redundancy, would allow effective use of genetic resources for introgression and for sustainable oil palm germplasm conservation. The way forward to efficiently conserve the field genebanks into next generation without losing their diversity was further discussed.

Authentication, genetic fingerprinting and assessing relatedness of rice (Oryza Sativa) genotypes by SSR molecular markers

Rice (Oryza sativa L.), is a staple food and cash crop in many countries and studies on geneticstructure and differentiation patterns of rice land races along with the cultivated rice, provide important data for future rice breeding. Therefore, the aims of present investigation were 1-To study the genetic diversity present withinIranian rice genotypes, 2-To study genetic relatedness of these rice genotypes, and 3-To providebarcoding of the rice genotypes based on SSR molecular markers and produce data for rice varieties authentication. In total, 201 rice samples originated from 10 geographical regions of Iran were studied in this project. All rice samples underwent fragment analysis in every 64 SSR loci and different clustering and ordination methods performed. In general four major clusters were formed. Both landraces as well as rice cultivars were distributed in different clusters due to their genetic difference. STRUCTURE analysis of the studied genotypes followed by Evanno test produced the optimal number of genetic groups K = 2. The mean Nm = 13.6, for the studied genotypes indicates that a high degree of gene flow/ancestral common alleles are present in the rice genotypes studied. Mantel test indicated a significant positive association between genetic distance and geographic distance of the rice genotype studied and presence of an overall isolation by distance (IBD) model of differentiation across the geographical regions of Iran. Overall, the significant genetic difference observed between rice landraces and rice cultivars ofthe country may be used in future hybridization and breeding of rice in the country. The landracerice genotypes may contain useful genes to be transferred to the popular rice cultivars. Moreover, SSR loci that can differentiate rice genotypes are identified and can be used in rice cultivars authentication.

Molecular Profiling of Chilli Germplasm By Using SSR Marker

The molecular characterization of chilli germplasm was done based on estimation of genetic diversity among the germplasm by using SSR markers. Forty chilli germplasms were analyzed using eight SSR primers. The SSR primers produced 30 SSR loci with an average value of 3.75 alleles per SSR locus. The similarity index matrix ranged from zero to 2.74. Polymorphic information content (PIC) of the SSR primers ranged from 0.543 to 0.735 with an average value of 0.658. The highest number (five) of allele was observed in primer CAMS-647, whereas the primers CAMS-864, CAMS-880 and CAMS-885 showed lowest number (three) of allele. The smallest allele was found in case of primer CAMS- 236 (176 bp), while the longest allele was detected for the primer CAMS- 864 (288 bp). Based on similarity matrix using the un-weighed Pair Group Method of Arithmetic Means (UPGMA) dendrogram, chilli germplasms were grouped into four main clusters. SSR markers showed genetic variability in the studied chilli germplasm. SAARC J. Agric., 19(1): 1-13 (2021)

Identification of unique alleles and assessment of genetic diversity of soybean genotypes using SSR markers and seed traits

Thirty polymorphic SSR markers and seven seed traits were used to determine the genetic diversity among twenty five soybean genotypes that differ with respect to seed coat color. A total of 133 alleles were detected and the number of alleles for each SSR locus varied from two to seven with an average of 4.0. Polymorphic information content varied from 0.81 to 0.07. Jaccard’s similarity coefficient grouped the genotypes into two major clusters. To test the goodness of fit of clusters to SSR markers, cophenetic correlation was estimated. Cophenetic value of 0.98 indicated a very good fit. A combination of SSR and seed traits data grouped the genotypes into two major clusters with grouping of all the brown seeded genotypes into one subcluster. A total of 21 unique alleles were identified in twenty five soybean genotypes which is a valuable resource for DNA fingerprinting studies. A set of nine SSR markers (Satt 600, Satt 463, Satt 371, Satt 193, Satt 538, Satt 126, Satt 286, Satt 281 and Satt 656) could differentiate all the soybean genotypes. Key WordsGenetic diversity, SSR markers, Soybean, unique alleles, seed traits

Deciphering the global phylogeography of a coastal shrub (Scaevola taccada) reveals the influence of multiple forces on contemporary population structure

AbstractThe phylogeography of coastal plant species is heavily influenced by past sealevel fluctuations, dispersal barriers, and life‐history traits, such as long‐distance dispersal ability of the propagules. Unlike the widely studied mangroves, phylogeographic patterns have remained mostly obscure for other coastal plant species. In this study, we sampled 42 populations of Scaevola taccada (Gaertn.) Roxb., a coastal shrub of the family Goodeniaceae, from 17 countries across its distribution range. We used five chloroplast DNA (cpDNA) and 14 nuclear microsatellite (simple sequence repeat [SSR]) markers to assess the influence of abiotic factors and population genetic processes on the phylogeographic pattern of the species. Geographical distribution of cpDNA haplotypes suggests that the species originated in Australia, followed by historical dispersal and expansion of its geographic range. Multiple abiotic factors, including the sealevel changes during the Pleistocene, the presence of landmasses like the Malay Peninsula, and contemporary oceanic circulation patterns, restricted gene flow between geographically distinct populations, thereby creating low haplotype diversity and a strong population structure. Population genetic processes acted on these isolated populations, leading to high nuclear genetic diversity and population differentiation, as revealed from analyzing the polymorphic SSR loci. Although genetic divergence was mostly concordant between cpDNA and SSR data, asymmetrical gene flow and ancestral polymorphism could explain the discordance in the detailed genetic structure. Overall, our findings indicate that abiotic factors and population genetic processes interactively influenced the evolutionary history and current phylogeographic pattern of S. taccada across its distribution range.

Body Remains Left by Bird Predators as a Reliable Source for Population Genetic Studies in the Great Capricorn Beetle Cerambyx cerdo, a Veteran Oak Specialist.

Simple SummaryMolecular genetic techniques can support species conservation by providing information about processes critical to population survival. Unfortunately, obtaining biological material for DNA extraction is often associated with an adverse impact on the animals under study. In legally protected or threatened species, non-invasive sampling (i.e., sampling without injuring or disturbing an animal) is preferred as it carries no risk to the population’s survival. Here, we tested the possibility of using body remains left by bird predators for microsatellite genotyping in Cerambyx cerdo, a veteran oak specialist. We compared results obtained from such remains samples with samples of fresh beetle tarsi (i.e., invasive and destructive but non-lethal samples). We found that the sample type had no significant effect on PCR amplification efficiency; instead, it was strongly affected by allele length and individual heterozygosity. Allele frequencies were perfectly correlated for both sample types (R2 = 0.94). Although point estimates of individual inbreeding were higher in remains than fresh samples (medians 0.08 vs. 0.02, respectively), both groups were not significantly different from each other and zero. Our study demonstrated that non-invasive remains samples could provide satisfactory data for population–genetic studies. However, we highlight the problem of potentially biased inbreeding estimates, which may result from allelic dropout.Obtaining biological material for DNA extraction is often challenging in organisms of conservation interest. Non-invasive sampling (i.e., sampling without injuring or disturbing an animal) is preferred as it carries no risk to the population’s survival. Here, we tested the possibility of using the body remains left by bird predators for microsatellite genotyping in Cerambyx cerdo, a veteran oak specialist. We compared results obtained from such potentially degraded samples with samples of fresh beetle tarsi (i.e., invasive and destructive but non-lethal samples). Using 10 SSR loci, we genotyped 28 fresh, and 28 remains samples. The analysis indicated that PCR amplification efficiency was not influenced by sample type but allele length and individual heterozygosity. Allele frequencies were perfectly correlated for both sample types (R2 = 0.94). Additionally, null allele frequencies and genotyping failure rates were not significantly different from zero. Although the point estimates of individual inbreeding rates (fi) were higher in remains than fresh samples (medians 0.08 vs. 0.02, respectively), both groups were not significantly different from each other and zero. Our study demonstrated that non-invasive remains samples could provide satisfactory data for population–genetic studies. However, we highlight the problem of biased inbreeding estimates, which may result from samples affected by allelic dropout.

Genetic Characterization of Brassica oleracea L. var. acephala DC cultivars from Bosnia and Herzegovina


 
 Fifteen Brassica oleracea L. var. acephala DC accessions were collected and evaluated for their genetic diversity and molecular characteristics using 10 previously developed and used codominant SSR markers. The SSR loci used revealed a total of 51 alleles. The domestic kale varieties were collected locally and the hybrids kales were found in markets. The averages of total heterozygosity (H) and polymorphic information content (PIC) was 0.6774 and 0.6201, respectively. The average number of alleles was 4.2 per locus, where 8 of 10 markers showed high polymorphic information content (PIC), being more than 0.5. The genetic relationships among the populations revealed by Unweighted Pair Group Method with Arithmetic mean (UPGMA), showed a clear clustering into three distinct groups. As expected, all local varieties were separated within two separated clusters, whereas only two domestic varieties shared one cluster with the group of hybrids, indicating similar genetic background shared by domestic and hybrid varieties. These results indicate and confirm the regional and physiological differences between the kale varieties and confirm the SSR marker efficiency for genetic characterization assays
 
 
 
 

Molecular evidence of introgression of Asian germplasm into a natural Castanea sativa forest in Spain

Abstract Evidence has been documented in Europe of introgression, the transfer of genetic material, between Asian chestnut species (Castanea crenata and C. mollissima) and the native C. sativa through spontaneous hybridization and subsequent backcrossing. However, the extent of this introgression has not been monitored in a particular forest stand or in adult and juvenile trees simultaneously. We assessed introgression in a natural C. sativa forest in northwest Spain and developed a reliable method to detect the presence of Asian germplasm of Castanea spp. A total of 34 adult trees and 42 saplings were genotyped at 13 SSRs in a forest where ink-disease-resistant C. crenata and C. mollissima seedlings had been planted in the 1940s. The 13 SSR loci selected were highly polymorphic and 115 different alleles were detected for the individuals sampled. Bayesian clustering identified two groups for C. sativa and one group each for C. mollissima and C. crenata. Within the forest, 70.6 per cent of adults and 28.6 per cent of juveniles were classified as pure C. sativa. Most juveniles were C. sativa × C. crenata (>40 per cent) and ca. 10 per cent were C. crenata × C. mollissima hybrids. Six new alleles private to C. crenata are reported here. The study quantifies, for the first time in Europe, introgression of Asian germplasm into a natural C. sativa forest. It also examines the extent of introgression in offspring and provides a method to detect exotic germplasm in C. sativa forests. We discuss why the forest studied may benefit from transfer of alleles involved in ink-disease resistance and why introgression will be detrimental to drought tolerance in offspring.

The complete chloroplast genome of a purple Ethiopian rape (Brassica carinata: Brassicaceae) from Guizhou Province, China and its phylogenetic analysis

Brassica carinata A. Braun (Ethiopian rape), which was derived from the interspecific hybridization between B. nigra and B. oleracea, is used as both an oilseed and a leafy vegetable. The complete chloroplast (cp) genome of a purple B. carinata was obtained. This cp genome has a typical quadripartite structure and is 153,641 bp in length. The GC content of the cp genome is 36.39%. A total of 113 genes were predicted on this cp genome, including 79 protein coding, 4 rRNA, and 30 tRNA genes. Among these genes, 18 genes were duplicated (7 tRNAs, 4 rRNAs, and 7 protein coding genes). Sixty-eight SSR loci, including 11 compound SSRs, were identified in this cp genome by MISA. The phylogenetic tree analysis fully resolved B. carinata in a clade with B. nigra. This study provides important information for future evolution, genetic and molecular biology studies of B. carinata.