Isolation and characterization of polymorphic microsatellites in European chestnut (Castanea sativa Mill.)

Thirteen polymorphic microsatellite loci were isolated from Castanea sativa (Mill.). Six contained dinucleotide repeats, six contained trinucleotide repeats, and one contained a compound microsatellite of a trinucleotide and a tetranucleotide repeat. The loci were characterized using C. sativa trees from three populations in the UK and the parents and six seedlings from a Turkish mapping population. The number of alleles revealed varied from two to 14 (mean = 5.15) per loci. Eight loci were found to be useful in the mapping family.     

Asian chestnut gall wasp in Tuscany: gall characteristics,egg distribution and chestnut cultivar susceptibility

• 1 Preliminary investigations were carried out on Dryocosmus kuriphilus Yasumatsu on Castanea sativa Miller in Tuscany to assess variations in gall characteristics in coppice and high forest at two crown heights (height < 2 or 2–6 m), influence of bud size and bud position on oviposition rates and susceptibility of three cultivars.
• 2 Gall size may depend on various factors, including wasp population density. In the present study area, small galls (with one or two cells) were the most numerous in 2008, whereas larger galls (with more than three cells) prevailed in 2009.
• 3 Dryocosmus kuriphilus oviposition occurrence was influenced by both bud size and bud position. Buds with eggs tended to be larger in size compared with bud without eggs, suggesting that D. kuriphilus females prefer to lay eggs in larger buds (approximately 6 mm³) compared with smaller buds (approximately 3 mm³). The mean number of eggs per bud tended to decrease from the apical bud toward the basal bud.
• 4 Three C. sativa cultivars, Carpinese, Fusca and Cesurone, were examined. Fusca grafts had significantly more galls compared with Carpinese and Cesurone, whereas Cesurone grafts had more larvae per bud compared with Carpinese and Fusca. Overall, the Carpinese cultivar may be less susceptible to D. kuriphilus galling compared with the Fusca and Cesurone cultivars.

     

Genetic transformation of European chestnut

Stable incorporation of the nptII gene into Castanea sativa Mill. has been achieved by Agrobacterium-mediated transformation. The transformation assays were performed by infecting wounded hypocotyls with a strain of Agrobacterium tumefaciens, LBA 4404 harbouring the plasmid p35SGUSINT. Although two schemes of selection were tested, many escapes were obtained. The best strategy to avoid this problem is the introduction of higher concentrations of kanamycin in the culture medium, immediately after coculture. PCR analysis showed of the selectable nptII gene integration in the plant genome. β-Glucuronidase histochemical assay revealed the expression of the uidA gene in shoots, regenerated from transformed explants. Received: 3 December 1996 / Revision received: 4 February 1997 / Accepted: 1 March 1997     

Plantlet formation from embryonic tissue of chestnut grown in vitro

Development of axillary shoots was induced when isolated embryonic axes of chestnut (Castanea sativa Mill.) were cultured on a defined medium containing 6-benzyl-aminopurine (BAP). The optimal concentrations of BAP were determined for development of axillary shoots from both embryonic axes and subcultured shoots. After shoot multiplication a great number of shoots have been maintained sequentially without significant change in the proliferative rate for one year. Limited rooting has been obtained with excised shoots. Indole-3-butyric acid (IBA) at 2 mg/l was used to induce root primordia. After 8 days of treatment the plantlets were transferred to an auxin-free medium for root development.     

Characterisation of Chinese elite cultivars and genetic resources of chestnut by AFLP

We selected a informative set of twelve amplified fragment length polymorphism (AFLP) primer pairs suitable for evaluation of Chinese chestnut (Castanea mollissima Blume) genotypes. Cluster analysis based on 198 polymorphic AFLP amplified by these 12 primer pairs clearly divided investigated genotypes according to their place of origin. We showed, that genetic basis of modern genotypes is narrow also in the case of this species.     

Differential response among chestnut traditional varieties to the attack of Cydia splendana

Fruit production of Castanea sativa Miller (Fagaceae) in southern Spain is carried out in traditional orchards using a large number of local varieties. The main areas of production are located in the Natural Park of Sierra de Aracena y Picos de Aroche (Huelva) and the Genal river Valley (Malaga). These agroforestry systems have shown a high genetic diversity and represent an example of in situ conservation. In this region, chestnut mainly suffers from attacks by Cydia splendana (Hübner) (Lepidoptera: Tortricidae), whose larvae feed on the fruits, thereby reducing their quality and economic value. The aim of this study was to evaluate the differential response between the chestnut local varieties to the attack of C. splendana. For that purpose, trees from the most representative varieties were evaluated on various farms. In cases where it was necessary, the trees were identified with a set of eight microsatellite markers. The infestation rate of C. splendana (percentage of infested fruits of the total fruits sampled per tree) differed among varieties, indicating a high genetic component in this effect. The results obtained in this study open the possibility of using this differential response in the control of C. splendana.     

Multilocus analysis of nucleotide variation of Oryza sativa and its wild relatives: severe bottleneck during domestication of rice

Varying degrees of reduction of genetic diversity in crops relative to their wild progenitors occurred during the process of domestication. Such information, however, has not been available for the Asian cultivated rice (Oryza sativa) despite its importance as a staple food and a model organism. To reveal levels and patterns of nucleotide diversity and to elucidate the genetic relationship and demographic history of O. sativa and its close relatives (Oryza rufipogon and Oryza nivara), we investigated nucleotide diversity data from 10 unlinked nuclear loci in species-wide samples of these species. The results indicated that O. rufipogon and O. nivara possessed comparable levels of nucleotide variation ((sil) = 0.0077 approximately 0.0095) compared with the relatives of other crops. In contrast, nucleotide diversity of O. sativa was as low as (sil) = 0.0024 and even lower ((sil) = 0.0021 for indica and 0.0011 for japonica), if we consider the 2 subspecies separately. Overall, only 20-10% of the diversity in the wild species was retained in 2 subspecies of the cultivated rice (indica and japonica), respectively. Because statistic tests did not reject the assumption of neutrality for all 10 loci, we further used coalescent to simulate bottlenecks under various lengths and population sizes to better understand the domestication process. Consistent with the dramatic reduction in nucleotide diversity, we detected a severe domestication bottleneck and demonstrated that the sequence diversity currently found in the rice genome could be explained by a founding population of 1,500 individuals if the initial domestication event occurred over a 3,000-year period. Phylogenetic analyses revealed close genetic relationships and ambiguous species boundary of O. rufipogon and O. nivara, providing additional evidence to treat them as 2 ecotypes of a single species. Lowest linkage disequilibrium (LD) was found in the perennial O. rufipogon where the r(2) value dropped to a negligible level within 400 bp, and the highest in the japonica rice where LD extended to the entirely sequenced region ( approximately 900 bp), implying that LD mapping by genome scans may not be feasible in wild rice due to the high density of markers needed.     

Changes in auxin protectors and IAA oxidases during the rooting of chestnut shoots in vitro

Auxin protectors and IAA oxidase activity were comparatively analyzed in the upper and the lower parts of shoots of chestnut ( Castanea sativa Mill.) cultivated in vitro with indolebutyric acid (IBA) pretreatment. Rhizogenesis of the shoots is accompanied by an increase in auxin protectors in the lower parts and by a decrease of these protectors in the upper parts. Besides, the IAA oxidase activity declines in the basal parts during the rooting process while it increases in the upper ones. These biochemical events would enhance the IAA level in the rooting region of the shoots. In untreated, non-rooted cuttings, the IAA oxidase activity remains low in the upper parts and high in the basal parts of the shoots. The results thus indicate that the IBA treatment may control the endogenous auxin level of the cuttings, either through a direct regulation of the IAA oxidase system or more indirectly through the transport of auxin protectors.     

The domestication of Amazonia before European conquest

During the twentieth century, Amazonia was widely regarded as relatively pristine nature, little impacted by human history. This view remains popular despite mounting evidence of substantial human influence over millennial scales across the region. Here, we review the evidence of an anthropogenic Amazonia in response to claims of sparse populations across broad portions of the region. Amazonia was a major centre of crop domestication, with at least 83 native species containing populations domesticated to some degree. Plant domestication occurs in domesticated landscapes, including highly modified Amazonian dark earths (ADEs) associated with large settled populations and that may cover greater than 0.1% of the region. Populations and food production expanded rapidly within land management systems in the mid-Holocene, and complex societies expanded in resource-rich areas creating domesticated landscapes with profound impacts on local and regional ecology. ADE food production projections support estimates of at least eight million people in 1492. By this time, highly diverse regional systems had developed across Amazonia where subsistence resources were created with plant and landscape domestication, including earthworks. This review argues that the Amazonian anthrome was no less socio-culturally diverse or populous than other tropical forested areas of the world prior to European conquest.     

Polypheolic auxin protectors in buds of juvenile and adult chestnut

Both qualitative and quantitative estimates of the relative amounts of several poly-phenols in buds of juvenile and adult plants of chestnut ( Castanea sativa x C. crenata ) were carried out. In buds of both types of plants, chesnatin, crenatin, cretanin and (+)-catechin were identified. Crenatin and cretanin showed inhibitory activity of peroxidase-catalysed indoleacetic acid oxidation. Auxin protection capacity was greater in extracts of the juvenile tissues which also were richer in active phenols. The phenolic content and its possible relationship to the easier establishment in vitro of juvenile tissue is discussed.     

Etiolation as a pretreatment for in vitro establishment and multiplication of mature chestnut

Localized etiolation of branches in the crown of a 30-year-old chestnut tree produced plant material that responded much better to establishment and multiplication in vitro than unetiolated material, whose cultures were very difficult to maintain (response being measured in terms of the percentage of cultures established, the mean number of shoots formed per explant, the number of 8-mm segments per new shoot, the length of longest shoot in each culture, and the coefficient of multiplication). Only 22% of the initial explants from unetiolated material were successfully established, as against 79% for etiolated material, with similar differences between the coefficients of multiplication of the two lines in successive cultures. Accordingly, partial etiolation of branches is proposed as a suitable pretreatment for in vitro propagation of selected mature trees, when physiologically juvenile materials such as stump sprouts, epicormic shoots or root suckers are not available.     

Grinding up wheat: a massive loss of nucleotide diversity since domestication

Several demographic and selective events occurred during the domestication of wheat from the allotetraploid wild emmer (Triticum turgidum ssp. dicoccoides). Cultivated wheat has since been affected by other historical events. We analyzed nucleotide diversity at 21 loci in a sample of 101 individuals representing 4 taxa corresponding to representative steps in the recent evolution of wheat (wild, domesticated, cultivated durum, and bread wheats) to unravel the evolutionary history of cultivated wheats and to quantify its impact on genetic diversity. Sequence relationships are consistent with a single domestication event and identify 2 genetically different groups of bread wheat. The wild group is not highly polymorphic, with only 212 polymorphic sites among the 21,720 bp sequenced, and, during domestication, diversity was further reduced in cultivated forms--by 69% in bread wheat and 84% in durum wheat--with considerable differences between loci, some retaining no polymorphism at all. Coalescent simulations were performed and compared with our data to estimate the intensity of the bottlenecks associated with domestication and subsequent selection. Based on our 21-locus analysis, the average intensity of domestication bottleneck was estimated at about 3--giving a population size for the domesticated form about one third that of wild dicoccoides. The most severe bottleneck, with an intensity of about 6, occurred in the evolution of durum wheat. We investigated whether some of the genes departed from the empirical distribution of most loci, suggesting that they might have been selected during domestication or breeding. We detected a departure from the null model of demographic bottleneck for the hypothetical gene HgA. However, the atypical pattern of polymorphism at this locus might reveal selection on the linked locus Gsp1A, which may affect grain softness--an important trait for end-use quality in wheat.     

Whole-genome assembly and resequencing reveal genomic imprint and key genes of rapid domestication in narrow-leafed lupin

Shifting from a livestock-based protein diet to a plant-based protein diet has been proposed as an essential requirement to maintain global food sustainability, which requires the increased production of protein-rich crops for direct human consumption. Meanwhile, the lack of sufficient genetic diversity in crop varieties is an increasing concern for sustainable food supplies. Countering this concern requires a clear understanding of the domestication process and dynamics. Narrow-leafed lupin (Lupinus angustifolius L.) has experienced rapid domestication and has become a new legume crop over the past century, with the potential to provide protein-rich seeds. Here, using long-read whole-genome sequencing, we assembled the third-generation reference genome for the narrow-leafed lupin cultivar Tanjil, comprising 20 chromosomes with a total genome size of 615.8 Mb and contig N50 = 5.65 Mb. We characterized the original mutation and putative biological pathway resulting in low seed alkaloid level that initiated the recent domestication of narrow-leafed lupin. We identified a 1133-bp insertion in the cis-regulatory region of a putative gene that may be associated with reduced pod shattering (lentus). A comparative analysis of genomic diversity in cultivars and wild types identified an apparent domestication bottleneck, as precisely predicted by the original model of the bottleneck effect on genetic variability in populations. Our results identify the key domestication genetic loci and provide direct genomic evidence for a domestication bottleneck, and open up the possibility of knowledge-driven de novo domestication of wild plants as an avenue to broaden crop plant diversity to enhance food security and sustainable low-carbon emission agriculture.     

Utilization of Response Surface Methodology in Optimization of Proline Extraction from Castanea sativa Honey

Proline constitutes approximately 85 % of the amino acid composition of honey. Therefore, the quantitative determination of this amino acid in honey samples is used by many national/international authorities to evaluate the quality of honey types. In this study, it was aimed to achieve maximum proline amino acid extraction from honey samples whose botanical origins were confirmed by melissopalynological analysis. For this reason, based on three different spectrophotometric methods used in the literature for proline analysis, proline extraction was optimized with the Response Surface Method (RSM) and Box-Behnken experimental design. Three independent variables were determined as treatment time (2, 6, and 10 min), treatment temperature (22, 46, and 70 °C), and cooling time (5, 25, and 45 min). As a result of the optimization, it was seen that only significantly effective independent variable on the proline content of honey was the processing temperature. The optimum conditions obtained as a result of the RSM were found to be 2 min for the treatment time, 70 °C for the treatment temperature and 45 min for the cooling time. The composite desirability of the optimum conditions (R²) was found to be 1.00. It was determined that the method proposed by International Honey Commission (IHC) is efficient for proline analysis, but it provides more proline extraction by reducing of time from 10 min to 2 min in hold time in boiling water bath only during the extraction step. As a result, the conditions to be used in order to achieve maximum proline extraction with different spectrophotometric methods were determined and optimum values were determined. In addition, since the botanical origin of honey samples significantly affects the proline content of honey, it can be suggested that this study be optimized for different monofloral honey samples as well.     

A genetic linkage map of European chestnut (Castanea sativa Mill.) based on RAPD, ISSR and isozyme markers

A genetic linkage map of European chestnut (Castanea sativa Mill.) based on RAPD, ISSR and isozyme markers was constructed using the two-way pseudo-testcross strategy. A total of 96 individuals from a F₁ full-sib family was genotyped with 381 molecular markers (311 RAPDs, 65 ISSRs, 5 isozymes). Markers in testcross configuration, segregating 1:1, were used to establish two separate maternal and paternal maps including 187 and 148 markers, respectively. The markers identified 12 linkage groups based on the haploid number of chestnut. The female and male framework maps reached a total length of 720 and 721 cM (Kosambi), respectively, representing a 76% and 68% coverage of the overall genome. A total of 46 markers, found in intercross configuration, segregating 3:1 and 1:2:1, were used to identify homologous linkage groups between parental maps; out of 12 linkage groups 11 could be joined. RAPD and ISSR markers showed a good and comparable reliability, allowing for the first time the establishment of a saturated linkage map for European chestnut. These maps will be a starting point for studies on the structure, evolution and function of the chestnut genome. Identification of QTLs for adaptive traits in chestnut will be the primary target. Received: 3 July 2000 / Accepted: 16 October 2000     

A methodology to evaluate disease severity: a case study of chestnut blight in El Bierzo region (northwestern Spain)

Correct knowledge of the incidence and severity of disease is essential for implementation of timely and effective management control strategies. In this article, multiple correspondence analysis (MCA) is applied to evaluate the severity of chestnut blight incited by the ascomyceteous fungus Cryphonectria parasitica. This economically important bark disease leads to the loss of an important part of the chestnut production and the progressive death of the tree. A total of 7240 living European chestnut (Castanea sativa) trees across 452 plots were surveyed in El Bierzo, NW Spain. For each tree, the main stem and canopy were visually assessed for signs of the pathogen and/or symptoms of the disease and the extent of the disease was classified on a qualitative ordinal scale consisting of six levels. The statistical procedure is useful because it quickly analyses measurable, discrete observations from assessed individuals to provide a disease severity measure related to tree features and disease extension inside the tree. The results indicated that the penetration of the pathogen is located in the lower part of the crown and spreads to the tips of the branches in the upper part of the crown. Thus, our results suggest that man‐made wounds, when the tree was grafted or pruned, are the main channel of pathogen penetration in El Bierzo region. Disease severity estimates and incidence data for C. parasitica across the region were compared. From the perspective of the management and control of the disease, a disease severity value provides a more accurate indication of the scenario of the disease in a region than presence or absence data alone.     

Assessing losses of genetic diversity due to translocation: long-term case histories in Merriam's turkey (<Emphasis Type="Italic">Meleagris gallopavo merriami</Emphasis>)

Translocation is a widely used tool in wildlife management, but populations established as a result of translocations may be subject to a range of genetic problems, including loss of genetic diversity and founder effects. The genetic impact of single translocation events can be difficult to assess because of complex management histories in translocated or source populations. Here we use molecular markers to assess the genetic impact of three well-documented translocation events, each occurring between 42 and 53 years ago and each originating from a native, extant source population that we also included in our study. Comparing translocated populations to their sources, we found genetic evidence of a recent bottleneck in all three translocated populations, including one which is now a very large, productive population. Based on our results, we recommend caution in (1) using short term census data to assess the long term success of a translocation and (2) conducting serial translocations (i.e., using translocated populations as the source for other translocations), which could exacerbate a genetic bottleneck. We also used the data on translocated populations to investigate the relative utility of three bottleneck detection methods. With this dataset, only assessment of the modal allele frequency distribution, described by Luikart etal. [Journal of Heredity, 89, 238–247 (1998)], provided evidence of a bottleneck in the absence of source population data.     

Superoxide dismutases of chestnut leaves, Castanea sativa: Characterization and study of their involvement in natural leaf senescence

Superoxide dismutases (SOD; EC 1.15.1.1) in chestnut ( Castanea sativa Mill., cv. 431) leaves were characterized by native polyacrylamide gel electrophoresis. The three molecular forms of SOD were distinguished from each other by their different sensitivity to cyanide and H₂O₂ Three CuZn-containing SODs were detected (CuZn-SOD I, II. and III), and all the isozymes had a molecular mass of 33 kDa. CuZn-SOD III was the most abundant isozyme. whereas CuZn-SOD II was present in a minor amount. In leaves showing typical symptoms of senescence increases of 2.5-. 7- and 4-fold in the specific activities of CuZn-SODs I, II, and III. respectively, were found. In addition, the pattern of the three isozymes was modified by the age of leaves, a rise in the CuZn-SOD II and a decrease in the CuZn-SOD 1 percentages being found in senescent leaves compared to green leaves. As to other activated oxygen-related enzymes, an increase in the superoxide-generating xanthine oxidase activity and a decline in both catalase and peroxidase activities during natural senescence of chestnut leaves were observed. Results obtained suggest that in natural senescence of chestnut leaves activated oxygen species are involved, and an overproduction of hydrogen peroxide and superoxide radicals probably takes place.