K. K. Nkongolo

Genetic analysis of Pinus strobus and Pinus monticola populations from Canada using ISSR and RAPD markers: development of genome-specific SCAR markers

Pinus is the largest genus of conifers, containing over 100 species and is also the most widespread genus in the Northern Hemisphere. Pinus monticola and P. strobus are two closely related and economically important species in Canada. Morphological and allometric characteristics have been used to assess genetic variation within these two species but these markers are not reliable due to ecological variations. The purpose of the present study was to determine the level of genetic diversity within and among Canadian populations from the two species using molecular markers and to identify and characterize genome-specific inter-simple sequence repeats (ISSR) and random amplified polymorphic DNA (RAPD) markers. The level of genetic variation among populations was much lower for P. monticola than P. strobus. For both species, the among population variation values were smaller than within population variation. The populations from P. monticola were more closely genetically related than populations from P. strobus based on ISSR and RAPD analyses. Six ISSR and four RAPD markers specific to either P. monticola or P. strobus were cloned and sequenced. Primer pairs flanking these specific sequences were designed and genome specific SCAR markers for P. monticola and P. strobus were developed and characterized.

Comparative Soil Metal Analyses in Sudbury (Ontario, Canada) and Lubumbashi (Katanga, DR-Congo)

DR-Congo is a main world producer of copper (Cu) and cobalt (Co). Several hydrometallurgical plants and smelters also produced zinc, arsenic, and cadmium as by-products. In Sudbury (Canada), the production of nickel, copper and other metals has been maintained at high levels while industrial SO2 emissions have been reduced by approximately 90% through combination of industrial technological developments and legislated controls. Metal analysis in the present study revealed that the levels of copper and cobalt in soils from mining sites in the Lubumbashi (DR-Congo) were up to 200 fold higher compared to contaminated Sudbury sites and tailings. Zinc content in soil samples from some mining areas in Lubumbashi was at least 70 times higher compared to samples from the Sudbury area. Nickel content in soil samples from Lubumbashi were much lower compared to the Sudbury Region samples. Overall, this study confirms that the African Copper belt region is among the ten most polluted areas in the world.

Identification, characterisation, and chromosome locations of rye and wheat specific ISSR and SCAR markers useful for breeding purposes

Rye (Secale cereale L. and S. strictum) offers potential to increase the genetic variability and to introduce desirable characters for wheat improvements. Cytogenetic techniques have been used to screen wheat lines containing rye chromatin. These techniques are not adequate since they are highly technical and time consuming. They are not suitable for breeding programs that require rapid screening of large numbers of genotypes. The main objective of this study was to develop and characterize ISSR and SCAR markers that can distinguish wheat from rye genome. Total DNA from wheat, rye, and triticale accessions from different provenances were amplified with ISSR primers in PCR assays. Three wheat-diagnostic sequences were identified. In addition three rye-diagnostic ISSR markers of which, one marker specifically diagnostic for Secale strictum were characterized. Pairs of primers flanking these specific sequences were designed to produce SCAR markers. Two SCAR markers were rye genome-specific. One SCAR was present in all the seven rye chromosome, and another was specific to rye chromosomes two, three, four, and seven. These newly developed ISSR and SCAR markers should be useful to wheat breeders screening genotypes that may contain rye chromatins.

Metal Content in Soil and Black Spruce (Picea mariana) Trees in the Sudbury Region (Ontario, Canada): Low Concentration of Arsenic, Cadmium, and Nickel Detected near Smelter Sources

Several studies have reported high metal concentrations in soil within the vicinity of smelters in the Sudbury (Ontario) region. Continued investigation and monitoring of soil and vegetation are essential to the understanding of ecosystem recovery following the reduction of emissions from smelters and the establishment of a reforestation program. The concentrations of Cd, Co, Cu, Fe, Ni, and Zn, found in the present study were within the limits set by Ontario Ministry of Environment and Energy (OMEE) guidelines even in sites within the vicinity of the Falconbridge Smelters. The levels of these elements in black spruce (Picea mariana) tissues were much lower and far below the toxic levels for vegetation. This is the first documented report of metal content in black spruce populations in the Sudbury region.

Molecular and ecological characterisation of plant populations from limed and metal-contaminated sites in Northern Ontario (Canada): ISSR analysis of white birch (Betula papyrifera) populations

The Greater Sudbury region in Northern Ontario is known as one of Canadas most ecologically disturbed regions because of the effects of heavy metal pollution. The main objectives of the present study were to assess the effects of soil liming of sites contaminated with metals on species richness and abundance, forest health in general, and to determine the level of genetic variability in white birch populations from Northern Ontario. Shannon-Wiener diversity index and tree species richness values were higher in populations from limed and control sites compared to the unlimed areas. A significant improvement in forest population health (measured using a scale of 1 to 10) in limed sites over the unlimed areas was observed. Key results revealed no significant difference for metal content in white birch (Betula papyrifera) leaves from limed compared to unlimed sites. But higher levels of Al, Ca, Mg, Mn, Ni, Sr, and Zn in leaves compared to the bioavailable amount in soil were observed. The levels of genetic variability in white birch populations were moderate to high, ranging from 30% to 79% of polymorphic loci. A high level of genetic variability such as observed in the present study is usually associated with long term sustainability in plant populations. No association was found between metal accumulation in soil or plants and the levels of genetic variation.

Total and bioavailable metals in two contrasting mining regions (Sudbury in Canada and Lubumbashi in DR-Congo): relation to genetic variation in plant populations

Knowledge of total and bioavailable metal contents in soil is important for regional risk assessment and management. The main objective of the present study was to analyse the concentrations of metals in soils in two contrasting mining regions (Sudbury in Canada and Lubumbashi in DR-Congo). Genetic variation of plant populations was investigated to assess the potential impact of metal contamination on forest sustainability. The levels of copper, cobalt, zinc, arsenic, and lead were significantly higher (up to 200-fold) in mining sites in the Lubumbashi compared to the most highly contaminated Sudbury sites. The nickel content in soil samples from Lubumbashi was much lower compared to Sudbury region samples. Only 3.5% and 4 % of total copper and nickel, respectively, were phytoavailable, with values of 6%, 5.7%, 3.6%, and 5.4% for cobalt, magnesium, manganese, and zinc, respectively. There were significant positive correlations between total metal and phytoavailable metal concentrations for copper (r=0.99), nickel (r=0.86), cobalt (r=0.72), strontium (r=0.71), and zinc (r=0.66). Although genetic variation was high in Picea glauca populations from the Sudbury region, no association was found between metal contamination levels and genetic variation within and among the P. glauca populations.

Genetic analysis of Pinus banksiana and Pinus resinosa populations from stressed sites contaminated with metals in Northern Ontario (Canada)

The Sudbury region in Canada is known for the mining and smelting of high-sulphide ores containing nickel, copper, iron and precious metals. Although reports provide information of metal levels in soil and plants, knowledge of genetic effects on plants growing in contaminated areas is limited. The main objective of this study was to characterise the level of genetic diversity in Pinus banksiana and Pinus resinosa populations from the Sudbury (Ontario) region using microsatellite markers. Soil samples were analysed for concentrations of metals. High levels of metal contents in soil were observed within short distances of the smelter compared with control sites. The level of genetic diversity was very low for P. resinosa populations and moderate for P. banksiana samples. Observed heterozygosity was fivefold higher in P. banksiana populations than P. resinosa populations studied. Overall, 17 and 24% of the total genetic diversity were attributed to differences among populations for P. banksiana and P. resinosa, respectively. In general, the inbreeding was significantly higher in P. resinosa populations than P. banksiana populations and gene flows were relatively low in both species. No significant trend of the levels of genetic diversity for metal contaminated and uncontaminated sites was found.

Coping Mechanisms of Plants to Metal Contaminated Soil

Metals such as cobalt (Co), copper (Cu), iron (Fe) and nickel (Ni) are essential for nor‐ mal plant growth and development since they contribute to the function of many en‐ zymes and proteins. However, metals can potentially become toxic to plants when they are present at high levels in their bioavailable forms (Hall, 2002). Phytotoxic levels of one or more inorganic ions in soil can be found in various parts of the world. These toxic sites occurred through natural processes or by anthropogenic effects. Naturally toxic soils include saline, acidic and serpentine soils, while anthropogenic polluted soils occur through mining activities, aerial fallout, and the run-off from galvanized sources of electricity pylons or motorway verges polluted by vehicle exhaust fumes (Bradshaw, 1984). The biochemical effect of metals on plants varies and the excess metal usually results in oxidative damage which affects their phenotype (Kachout et al., 2009)

Effects of rs6234/rs6235 and rs6232/rs6234/rs6235 PCSK1 single-nucleotide polymorphism clusters on proprotein convertase 1/3 biosynthesis and activity.

BACKGROUND Proprotein convertase 1/3 (PC1/3) is one of the endoproteases initiating the proteolytic activation of prohormones and proneuropeptides in the secretory pathway. It is produced as a zymogen that is subsequently modified by activity-determining cleavages at the amino and the carboxyl termini. In human, it is encoded by the PCSK1 locus on chromosome 5. Spontaneous inactivating mutations in its gene have been linked to obesity. Minor alleles of the common non-synonymous single-nucleotide polymorphisms (SNPs) rs6232 (T>C, N221D), rs6234 (G>C, Q665E) and rs6235 (C>G, S690T) have been associated with increased risk of obesity. We have shown that the variations associated with these SNPs are linked on minor PCSK1 alleles. GOAL In this study, we examined the impact of amino acid substitutions specified by the minor PCSK1 alleles on PC1/3 biosynthesis and prohormone processing activity in cultured cells. METHODS The common and variant isoforms of PC1/3 were expressed in transfected rat pituitary GH4C1 cells with or without proopiomelanocortin (POMC) as a substrate. Secreted PC1/3- or POMC-related proteins and peptides were analyzed by immunoblotting and immunoprecipitation. RESULTS When expressed in GH4C1 cells, the triple-variant PC1/3 underwent significantly more proteolytic processing at the amino and carboxyl termini than the common and double-variant isoforms. However, there was no detectable difference among these isoforms in their ability to process POMC in the transfected cells. CONCLUSIONS Since truncation of PC1/3 in its C-terminal region reportedly renders the enzyme unstable, we speculate that the accentuated processing of the triple variant in this region may, in vivo, create a subtle deficit of PC1/3 enzymatic activity in endocrine and neuroendocrine cells, causing impaired processing of prohormones and proneuropeptides to their bioactive forms.

Genetic and metal analyses of fragmented populations of Betula papyrifera (Marsh) in a mining reclaimed region: identification of population–diagnostic molecular marker

White birch (Betula papyrifera) is an open pollinate species that is, dominant in the Northern Ontario after land reclamation. In fact, this species represents 65% of all trees in the region. We hypothesized that the exchange of genetic information between fragmented populations by range-wide paternal introgression is possible in wind-pollinated species such as B. papyrifera. On the other hand, the effects of heavy metal contamination from the mining activities on plant growth and population dynamics are well documented. The main objectives of this study were (1) to assess the level of genetic variation, gene flow, and population sustainability of B. papyrifera after land reclamation; and (2) to determine the level of phytoavailable metals in soil and their accumulation in trees. We found that B. papyrifera is a Ni and Zn accumulator with a translocation factor of 6.4 and 81, respectively, and an indicator of Cu and Pb. The level of polymorphic loci, Shannon index, Neis genetic diversity, observed number of alleles, and gene flow were determined for the fragmented populations within the targeted region. The percent of polymorphic loci ranged from 28% to 56%; the gene flow was also low with a value of 0.89, and the population differentiation was very high with a value of 0.36. Two population–diagnostic ISSR markers were identified. They were cloned, sequenced, and converted to SCAR markers. Overall, the fragmented populations of B. papyrifera in Northern Ontario are genetically sustainable based on the moderate level of intrapopulation variability.