Allozyme variations in six natural populations of scots pine (<Emphasis Type="Italic">Pinus sylvestris</Emphasis>) in Turkey

Genetic variation in six natural populations of Scots pine (Pinus sylvestris L.) was determined with isoenzyme analyses. For this purpose, haploid female gametophytes of seeds and horizontal starch gel electrophoresis technique were used. A total of 17 loci and 58 alleles were observed in studying 10 enzyme systems. The average proportion of polymorphic loci for populations ranged from 58.8% to 70.6%. The average number of alleles per locus per population was 2.65. The mean estimated expected heterozygosity (He) of populations was 0.294. A rather high proportion of genetic diversity (96.4%) was due to within-population variation and the remaining (3.6%) was due to variation among populations. The level of gene flow (N_em) was found to be 6.69 per generation. Nei’s genetic distance coefficient ranged from 0.006 to 0.027 (mean 0.017) among all possible population pairs. The mean value of Nei’s genetic distance is similar to the values reported for other European Scots pine populations. The low mean value of Nei’s genetic distance among populations is enough to explain low interpopulation variation. According to genetic variation parameters, three out of six populations (Akdagmadeni-Yozgat, Refahiye-Erzincan and Vezirkopru-Samsun) appear to be preferable populations for genetic conservation and forest tree breeding programs.     

Finding loci associated to partial resistance to white pine blister rust in sugar pine (<Emphasis Type="Italic">Pinus lambertiana</Emphasis> Dougl.).

White pine blister rust (WPBR) is an exotic disease threatening five-needle pines in North America. In spite of its relatively recent introduction, some five-needle pines such as sugar pine (Pinus lambertiana) have developed both complete (major) gene resistance and partial (quantitative) resistance to WPBR. While significant effort has been dedicated to clone and locate the position of the major gene of WPBR resistance in sugar pine, the genetic basis of quantitative resistance remains largely unknown in all Strobus pines. In this work, we took a preliminary approach to identify potential genotype × phenotype associations using the results of long-term survival and symptoms of infection in both experimental and applied breeding populations. Our study found significant associations between several genes and WPBR disease symptoms such as normal active cankers and blights, important symptoms in the development of partial resistance. No significant associations were found with percentage of survival, probably due to the complex inheritance of the disease and long time to infection. With this study, we hope to lay the ground for further genome-wide association studies using large phenotypic data sets in sugar pine and other Strobus pines.     

Genetic diversity of the African poplar (<Emphasis Type="Italic">Populus ilicifolia</Emphasis>) populations in Kenya

We evaluated the genetic diversity of the African poplar (Populus ilicifolia) populations found in Kenya compared with reference samples of five poplar species from North America and one species introduced in Kenya from India (KEFRI-Kenya). Amplified fragment length polymorphism (AFLP) was used with the objective of providing important information for breeding and in situ/ex situ conservation of this species. Samples collected from three locations along the species’ natural range (Athi, Ewaso Nyiro, and Tana rivers) were compared with four samples of locally planted Populus deltoides stand introduced from India and ten reference samples from North America. Six AFLP primer combinations produced 521 clear bands for analysis. The percentage polymorphic loci were lowest in Tana (20.4 %) and highest in Athi (40.6 %). The average heterozygosity across the studied populations was between 0.07 and 0.3. AMOVA revealed more genetic variation partitioning within population (87 %; P?<?0.01) than among populations (13 %; P?<?0.01) suggesting significant genetic variation between populations. Further, UPGMA delineation showed two clusters of the Tana, Athi, and Ewaso Nyiro populations clustered together compared to the North America and India/KEFRI reference samples. Moreover, the study showed that the Athi population is more diverse than those of Tana and Ewaso Nyiro and may be important for conservation, domestication, and improvement studies. The genetic differentiation (F _ST ?=?0.134) among Kenyan P. ilicifolia populations suggests limited possibility of gene flow between these populations.     

Microsatellite versus AFLP analyses of pre-management introgression levels in loblolly pine (<Emphasis Type="Italic">Pinus taeda</Emphasis> L.) and shortleaf pine (<Emphasis Type="Italic">P. echinata</Emphasis> Mill.)

Loblolly pine and shortleaf pine are known to naturally hybridize. In this study, we used 42 microsatellite markers and isocitrate dehydrogenase isozyme to create genetic profiles of 202 loblolly and shortleaf pine trees grown from seed collected in the 1950s for the Southwide Southern Pine Seed Source Study. Estimated Φ_PT was low in both loblolly (0.061) and shortleaf (0.080) pines, indicating that most of the diversity is accounted for within seed sources. However, both loblolly and shortleaf pines showed significant correlations between seed sources’ genetic and geographic distances, with R ² of 0.43 and 0.17, respectively. The hybridization rate was 4.0%, with more hybrids west of the Mississippi River (8.1%) than east of the river (2.1%). Additionally, about the same proportion of both species (4.5% of loblolly and 3.3% of shortleaf pine) were identified as hybrids. These results are consistent with prior studies on these two species but do contrast with the results from an amplified fragment length polymorphism (AFLP) analysis of the same samples. For example, the AFLP study concluded that 6.3% of the trees were hybrids, or 1.4 times higher than determined by this study. Of the 12 hybrids identified in the AFLP study, six were not identified as hybrids here, and of the eight hybrids identified here, only four were identified in the AFLP study. Although similar in overall results, we suggest that the microsatellite analysis is more convincing than the AFLP analysis because microsatellites provide more information per genetic locus than do AFLPs.     

Molecular mapping of stripe rust resistance gene <Emphasis Type="Italic">YrHu</Emphasis> derived from <Emphasis Type="Italic">Psathyrostachys huashanica</Emphasis>

Wheat stripe rust is a destructive disease that affects most wheat-growing areas worldwide. Resistance genes from related species and genera add to the genetic diversity available to wheat breeding programs. The stripe rust-resistant introgression line H9020-17-25-6-4 was developed from a cross of resistant Psathyrostachys huashanica with the susceptible wheat cultivar 7182. H9020-17-25-6-4 is resistant to all existing Chinese stripe rust races, including the three most widely virulent races, CYR32, CYR33, and V26. We attempted to characterize this new line by genomic in situ hybridization (GISH) and genetic analysis. GISH using P. huashanica genomic DNA as a probe indicated that the translocated segment was too small to be detected. Genetic analysis involving F₁, F₂, and F_2:3 materials derived from a cross of Mingxian 169 and H9020-17-25-6-4 indicated that a single dominant gene from H9020-17-25-6-4, temporarily designated YrHu, conferred resistance to CYR29 and CYR33. A genetic map consisting of four simple sequence repeat, two sequence-tagged site (STS), and two sequence-related amplified polymorphism markers was constructed. YrHu was located on the short arm of chromosome 3A and was about 0.7 and 1.5 cM proximal to EST-STS markers BG604577 and BE489244, respectively. Both the gene and the closely linked markers could be used in marker-assisted selection.     

Variability of nuclear microsatellite loci in the populations of Siberian dwarf pine (<Emphasis Type="Italic">Pinus pumila</Emphasis> (Pallas) Regel) from the Russian part of the range

Variability of nuclear microsatellite loci was examined in Siberian dwarf pine. Six microsatellite loci (RPS2, RPS6, RPS12, RPS124, RPS127, Pc18) demonstrated different polymorphism levels in ten populations of Siberian dwarf pine. The average number of alleles per locus was 4.88, the average observed heterozygosity was 0.465, and the average expected heterozygosity was 0.510. About 13% of total genetic variability was explained by the genetic differences between the populations (F _ST = 0.129). Genetic distances between the examined populations of Pinus pumila inferred from the data on the SSR marker frequencies statistically significantly correlated with the geographical distances between the population samples. The level of genetic variability of the populations from Kamchatka Peninsula was lower than that demonstrated by continental and island populations. The genetic differentiation of the Kamchatka–Magadan and other populations of Siberian dwarf pine observed in our study can be explained in terms of their formation from different Pleistocene refugial centers.     

Microsatellite analysis of genetic diversity and population genetic structure of a wild rice (<Emphasis Type="Italic">Oryza rufipogon</Emphasis> Griff.) in China

Genetic diversity and population genetic structure of natural Oryza rufipogon populations in China were studied based on ten microsatellite loci. For a total of 237 individuals of 12 populations collected from four regions, a moderate to high level of genetic diversity was observed at population levels with the number of alleles per locus ( A) ranging from 2 to 18 (average 10.6), and polymorphic loci ( P) from 40.0% to 100% (average 83.3%). The observed heterozygosity ( H(O)) varied from 0.163 to 0.550 with the mean of 0.332, and the expected heterozygosity ( H(E)) from 0.164 to 0.648 with the mean of 0.413. The level of genetic diversity for Guangxi was the highest. These results are in good agreement with previous allozyme and RAPD studies. However, it was unexpected that high genetic differentiation among populations was found ( R(ST) = 0.5199, theta = 0.491), suggesting that about one-half of the genetic variation existed between the populations. Differentiation (pairwise theta) was positively correlated with geographical distance ( r = 0.464), as expected under the isolation by distance model. The habitat destruction and degradation throughout the geographic range of O. rufipogon may be the main factor attributed to high genetic differentiation among populations of O. rufipogon in China.     

Introgression molecular analysis of a leaf rust resistance gene from <Emphasis Type="Italic">Coffea liberica</Emphasis> into <Emphasis Type="Italic">C. arabica</Emphasis> L.

Leaf rust caused by the fungus Hemileia vastatrix is the most devastating disease of arabica coffee (Coffea arabica). Therefore, developing leaf rust-resistant varieties has been a breeding objective of the highest priority in many countries. The purpose of the present work was to gain insight into the mechanism of introgression into C. arabica of a leaf rust resistance gene from C. liberica (i.e. S_H3 resistance factor) and to identify associated molecular markers. An F₂ progeny (i.e. 101 individuals) derived from a cross between Matari, an arabica accession and liberica-introgressed line S.288, was evaluated for resistance against three different races of H. vastatrix. The progeny segregated for the S_H3 gene in a 3:1 ratio, as expected for a single dominant gene. Amplified fragment length polymorphism analysis of a population subset using 80 different primer combinations revealed that at least half of the total polymorphism observed in the population is associated with introgression of C. liberica chromosome fragments. Furthermore, 15 primer combinations generating candidate marker bands associated with the S_H3 resistance gene were used to analyse the whole F₂ population. A total of 34 marker bands originating from S.288 and attributable to introgression were scored. None exhibited segregation distortion. Linkage analysis revealed only three distinct introgressed fragments corresponding to a total length of 52.8 cM. Twenty-one markers were strongly associated (LOD score >14) with the S_H3 gene and were grouped together in a single linkage group of 6.3 cM. The results are discussed in relation to the efficient use of genetic resources in arabica breeding.     

Development of microsatellite genetic markers in Siberian stone pine (<Emphasis Type="Italic">Pinus sibirica</Emphasis> Du Tour) based on the <Emphasis Type="Italic">de novo</Emphasis> whole genome sequencing

Siberian stone pine, Pinus sibirica Du Tour is one of the most economically and environmentally important forest-forming species of conifers in Russia. To study these forests a large number of highly polymorphic molecular genetic markers, such as microsatellite loci, are required. Prior to the new high-throughput next generation sequencing (NGS) methods, discovery of microsatellite loci and development of micro-satellite markers were very time consuming and laborious. The recently developed draft assembly of the Siberian stone pine genome, sequenced using the NGS methods, allowed us to identify a large number of microsatellite loci in the Siberian stone pine genome and to develop species-specific PCR primers for amplification and genotyping of 70 microsatellite loci. The primers were designed using contigs containing short simple sequence tandem repeats from the Siberian stone pine whole genome draft assembly. Based on the testing of primers for 70 microsatellite loci with tri-, tetra- or pentanucleotide repeats, 18 most promising, reliable and polymorphic loci were selected that can be used further as molecular genetic markers in population genetic studies of Siberian stone pine.     

Development of sequence characterized DNA markers linked to leaf rust (<Emphasis Type="Italic">Hemileia vastatrix</Emphasis>) resistance in coffee (<Emphasis Type="Italic">Coffea arabica</Emphasis> L.)

Coffee leaf rust due to Hemileia vastatrix is one of the most serious diseases in Arabica coffee (Coffea arabica). A resistance gene (S_H3) has been transferred from C. liberica into C. arabica. The present work aimed at developing sequence-characterized genetic markers for leaf rust resistance. Linkage between markers and leaf rust resistance was tested by analysing two segregating populations, one F₂ population of 101 individuals and one backcross (BC₂) population of 43 individuals, derived from a cross between a susceptible and a SH3-introgressed resistant genotype. A total of ten sequence-characterized genetic markers closely associated with the S_H3 leaf rust resistance gene were generated. These included simple sequence repeats (SSR) markers, sequence-characterised amplified regions (SCAR) markers resulting from the conversion of amplified fragment length polymorphism (AFLP) markers previously identified and SCAR markers derived from end-sequences of bacterial artificial chromosome (BAC) clones. Those BAC clones were identified by screening of C. arabica genomic BAC library using a cloned AFLP-marker as probe. The markers we developed are easy and inexpensive to run, requiring one PCR step followed by gel separation. While three markers were linked in repulsion with the S_H3 gene, seven markers were clustered in coupling around the S_H3 gene. Notably, two markers appeared to co-segregate perfectly with the S_H3 gene in the two plant populations analyzed. These markers are suitable for marker-assisted selection for leaf rust resistance and to facilitate pyramiding of the S_H3 gene with other leaf rust resistance genes.     

Genetic variation of wild litchi (<Emphasis Type="Italic">Litchi chinensis</Emphasis> Sonn. subsp. <Emphasis Type="Italic">chinensis</Emphasis>) revealed by microsatellites

Understanding the amount and distribution of genetic diversity in natural populations can inform the conservation strategy for the species in question. In this study, genetic variation at eight nuclear microsatellite loci was used to investigate genetic diversity and population structure of wild litchi (Litchi chinensis Sonn. subsp. chinensis). Totally 215 individuals were sampled, representing nine populations of wild litchi. All eight loci were polymorphic, with a total of 51 alleles. The expected heterozygosity in the nine populations ranged from 0.367 to 0.638 with an average value of 0.526. Inbreeding within wild litchi populations was indicated by a strong heterozygote defect. Significant bottleneck events were detected in the populations from Yunnan and Vietnam, which could be responsible for lower levels of genetic diversity in these populations. Measures of genetic differentiation (F _ST = 0.269) indicated strong differentiation among wild litchi populations. Significant correlation was found between genetic differentiation and geographical distance (r = 0.655, P = 0.002), indicating a strong isolation by distance in these populations. Bayesian clustering suggested genetic separation among three regional groups, namely, the western group, the central group and the eastern group. Some conservation strategies for wild litchi populations were also proposed based on our results.     

Genetic diversity following demographic recovery in the insular endemic plant <Emphasis Type="Italic">Galium catalinense</Emphasis> subspecies <Emphasis Type="Italic">acrispum</Emphasis>

Galium catalinense (Rubiaceae) is a perennial shrub consisting of two subspecies endemic to California’s Channel Islands: Galium catalinense subsp. catalinense on Santa Catalina Island, and G. catalinense subsp. acrispum, a state-endangered taxon on San Clemente Island. A long history of overgrazing by introduced herbivores has contributed to population declines in G. catalinense subsp. acrispum. We surveyed 12 populations throughout the taxon’s range for genetic variation using eight polymorphic microsatellite loci to determine the genetic impact of this demographic bottleneck. At the taxon level, 65 alleles were identified with an average of 8.1 alleles per locus, although many alleles were rare; the effective number of alleles per locus averaged 2.6. Expected heterozygosity was 0.550. Individual populations had between six and eight polymorphic loci, with expected heterozygosities ranging from 0.36 to 0.60, and effective numbers of alleles ranging from 1.8 to 3.5 per locus. Populations fell into three or four genetic clusters, depending on type of analysis, which may represent refugia where the populations persisted during intense herbivory. There is little evidence of genetic bottlenecks or substantial inbreeding within populations. These findings, coupled with indications of recent migration between populations, suggest that G. catalinense subsp. acrispum is currently unlikely to be endangered by genetic factors, but small population sizes make the taxon vulnerable to future loss of genetic diversity. Management strategies based on these genetic data, population sizes, and the spatial distribution of populations are discussed.     

A consensus map for <Emphasis Type="Italic">Cucurbita pepo</Emphasis>

Using random amplified polymorphic DNA (RAPD), amplified fragment length polymorphism (AFLP), simple sequence repeats (SSR), and morphological traits, the first genetic maps for Cucurbita pepo (2n=2x=40) were constructed and compared. The two mapping populations consisted of 92 F₂ individuals each. One map was developed from a cross between an oil-seed pumpkin breeding line and a zucchini accession, into which genes for resistance to Zucchini Yellow Mosaic Virus (ZYMV) from a related species, C. moschata, had been introgressed. The other map was developed from a cross between an oil-seed pumpkin and a crookneck variety. A total of 332 and 323 markers were mapped in the two populations. Markers were distributed in each map over 21 linkage groups and covered an average of 2,200 cM of the C. pepo genome. The two maps had 62 loci in common, which enabled identification of 14 homologous linkage groups. Polyacrylamide gel analyses allowed detection of a high number of markers suitable for mapping, 10% of which were co-dominant RAPD loci. In the Pumpkin-Zucchini population, bulked segregant analysis (BSA) identified seven markers less than 7 cM distant from the locus n, affecting lignification of the seed coat. One of these markers, linked to the recessive hull-less allele (AW11-420), was also found in the Pumpkin-Crookneck population, 4 cM from n. In the Pumpkin-Zucchini population, 24 RAPD markers, previously introduced into C. pepo from C. moschata, were mapped in two linkage groups (13 and 11 markers in LGpz1 and LGpz2, respectively), together with two sequence characterized amplified region (SCAR) markers linked to genes for resistance to ZYMV.     

Genetic characterization of grey wolves (<Emphasis Type="Italic">Canis lupus</Emphasis> L. <Emphasis Type="Italic">1758</Emphasis>) from Bosnia and Herzegovina: implications for conservation

The grey wolves of Bosnia and Herzegovina form a subpopulation of the Dinaric-Balkan wolf population and represent one of Europe’s least studied wolf populations. Since the Dinaric-Balkan population is a valuable source of genetic diversity for neighboring populations, comprehensive assessments are warranted. We aimed to determine the genetic variability and structure of the grey wolf population from Bosnia and Herzegovina, as well as estimate levels of gene flow and inbreeding and evaluate genetic signals of a bottleneck. To do this, we analyzed the variability of eighteen microsatellite loci. We found moderately high genetic heterozygosity for wolves from Bosnia and Herzegovina, as described for other Dinaric-Balkan wolf populations. We reveal weak genetic structuring with two genetic clusters identified. Wolves from the eastern part of the region formed a relatively distinct cluster, whereas individuals in the second cluster overlapped quite considerably with admixed individuals. Despite the signal of genetic structure being weak, clustering of individuals from the eastern part of the country extended through all analyses. Thus, this cluster could be considered a separate management unit, perhaps requiring specific conservation attention.     

Molecular mapping of a rust resistance gene <Emphasis Type="Italic">R</Emphasis><Subscript><Emphasis Type="Italic">14</Emphasis></Subscript> in cultivated sunflower line PH 3

Sunflower, the fifth largest oilseed crop in the world, plays an important role in human diets. Recently, sunflower production in North America has suffered serious yield losses from newly evolved races of sunflower rust (Puccinia helianthi Schwein.). The rust resistance gene, designated R ₁₄ , in a germplasm line PH 3 originated from a wild Helianthus annuus L. population resistant to 11 rust races. PH 3 has seedling with an extraordinary purple hypocotyl color. The objectives of this study were to map both the R ₁₄ rust resistance gene and the purple hypocotyl gene-designated PHC in PH 3, and to identify molecular markers for marker-assisted breeding for sunflower rust resistance. A set of 517 mapped SSR/InDel and four SNP markers was used to detect polymorphisms between the parents. Fourteen markers covering a genetic distance of 17.0 cM on linkage group (LG) 11 were linked to R ₁₄ . R ₁₄ was mapped to the middle of the LG, with a dominant SNP marker NSA_000064 as the closest marker at a distance of 0.7 cM, and another codominant marker ORS542 linked at 3.5 cM proximally. One dominant marker ZVG53 was linked on the distal side at 6.9 cM. The PHC gene was also linked to R ₁₄ with a distance of 6.2 cM. Chi-squared analysis of the segregation ratios of R ₁₄ , PHC, and ten linked markers indicated a deviation from an expected 1:2:1 or 3:1 ratio. The closely linked molecular or morphological markers could facilitate sunflower rust-resistant breeding and accelerate the development of rust-resistant hybrids.     

An SSR-based genetic linkage map for perennial ryegrass (<Emphasis Type="Italic">Lolium perenne</Emphasis> L.)

A simple sequence repeat (SSR)-based linkage map has been constructed for perennial ryegrass (Lolium perenne L.) using a one-way pseudo-testcross reference population. A total of 309 unique perennial ryegrass SSR (LPSSR) primer pairs showing efficient amplification were evaluated for genetic polymorphism, with 31% detecting segregating alleles. Ninety-three loci have been assigned to positions on seven linkage groups. The majority of the mapped loci are derived from cloned sequences containing (CA)_n-type dinucleotide SSR arrays. A small number (7%) of primer pairs amplified fragments that mapped to more than one locus. The SSR locus data has been integrated with selected data for RFLP, AFLP and other loci mapped in the same population to produce a composite map containing 258 loci. The SSR loci cover 54% of the genetic map and show significant clustering around putative centromeric regions. BLASTN and BLASTX analysis of the sequences flanking mapped SSRs indicated that a majority (84%) are derived from non-genic sequences, with a small proportion corresponding to either known repetitive DNA sequence families or predicted genes. The mapped LPSSR loci provide the basis for linkage group assignment across multiple mapping populations.     

Protecting evolutionary significant units for the remnant populations of <Emphasis Type="Italic">Berchemiella wilsonii</Emphasis> var. <Emphasis Type="Italic">pubipetiolata</Emphasis> (Rhamnaceae)

Berchemiella wilsonii var. pubipetiolata (Rhamnaceae) is an endangered tree in eastern China. Habitat destruction has resulted in fragmentation of remnant populations and extinction of local populations. AFLP and cpDNA markers were used to determine the population structure of remnant populations of B. wilsonii var. pubipetiolata. Moderate nuclear genomic diversity was found within each of the four remnant populations (H _S = 0.141–0.172), while the cpDNA haplotype diversity in each population ranged from 0.356 to 0.681. Six haplotypes were identified by a combined cpRFLP and cpSSR analysis in a total of 89 individuals. AMOVA revealed significantly AFLP genetic differentiation within and between regions (Φ_SC = 0.196, Φ_CT = 0.396, respectively), and a high cpDNA haplotype differentiation between regions (Φ_CT = 0.849). The results suggest low gene flow between populations of B. wilsonii var. pubipetiolata. Strong genetic divergence between two regional populations as revealed by both AFLP and cpDNA markers provided convincing evidence that two distinct evolutionary lineages existed, and should be recognized as ‘evolutionary significant units’ (ESUs) for conservation concerns.     

Allozyme diversity in pheasants (<Emphasis Type="Italic">Phasianus</Emphasis> spp.) from breeding stations in Serbia

The pheasant breeds are widely used for restocking of natural populations depleted by hunting. The pheasant population number decline was detected during the 1970s in many hunting areas of Europe. One of its possible reasons might be the loss of adaptability in populations originating from breeding stations, which was caused by inbreeding depression. The aim of this paper was the analysis of genetic variability in pheasant populations from three breeding stations in Vojvodina province (Serbia) by means of allozyme diversity detection. The allozyme variability analysis of pheasants from all three breeding stations revealed polymorphisms at nine loci: Ldh-1, Mor-1, Mor-2, Es-1, Mod-2, Pgd, Gpi-2, Odh, and Sod. The analysis of individuals from three different breeding stations showed mean values of observed heterozygosity of H _o=0.137, polymorphism P _95%=30%, and H/P ratio H/P=0.430, which indicate a normal level of genetic variability for bird populations. Comparative analysis of three pheasant populations showed a high level of interpopulation differentiation.