Data analysis for molecular characterization of plant genetic resources

Characterizations of plant genetic resources based on molecular markers have been increased in the last years. Studies using a broad range of markers applied on hundreds of plant species are the theoretical basis for inferring genetic diversity to propose both breeding and conservation strategies. Despite increased importance of molecular characterization in plant genetic resources, there is scarce information about analysis of this type of data. To fill this gap of information, this review discuss the rationale behind analyses achieved to study genetic relationship among accessions (within and between groups) and to identify accession, and also discuss the adequacy of some analyses and/or parameters for specific purposes. Genetic diversity within groups may be either quantified for the whole group (parameters to choose will depend on type of marker), or quantified and visualized for the relationships among individuals. Quantification parameters will be chosen depending on type of marker, reproduction mode and relatedness of individuals. Visualization is achieved by hierarchical and non-hierarchical methods. Genetic diversity between groups should be quantified either by analysis of molecular variance, or Nei’s parameters, or Wright’s F-statistics. Efficiency of accession identification can be evaluated by maximal probability of identical match by chance and number of resolved genotypes.     

Identification of molecular markers associated with sugar-related traits in a <Emphasis Type="Italic">Saccharum</Emphasis> interspecific cross

The cultivated sugarcane (Saccharum spp. hybrids, 2n = 100–130) is one crop for which interspecific hybridization involving wild germplasm has provided a major breakthrough in its improvement. Few clones were used in the initial hybridization event leading to a narrow genetic base for continued cultivar development. Molecular breeding would facilitate the identification and introgression of novel alleles/genes from the wild germplasm into cultivated sugarcane. We report the identification of molecular markers associated with sugar-related traits using an F₁ population derived from a cross between S. officinarum ‘Louisiana Striped’ × S. spontaneum ‘SES 147B’, the two major progenitor species of cultivated sugarcane. Genetic linkage maps of the S. officinarum and S. spontaneum parents were produced using the AFLP, SRAP and TRAP molecular marker techniques. The mapping population was evaluated for sugar-related traits namely, Brix (B) and pol (P) at the early (E) and late (L) plant growing season in the plant cane (04) and first ratoon (05) crops (04EB, 04LB, 04LP, 05EB and 05EP). For S. officinarum, combined across all the traits, a total of 30 putative QTLs was observed with LOD scores ranging from 2.51 to 7.48. The phenotypic variation (adj. R²) explained by all QTLs per trait ranged from 22.1% (04LP) to 48.4% (04EB). For S. spontaneum, a total of 11 putative QTLs was observed with LOD scores ranging from 2.62 to 4.70 and adj. R² ranging from 9.3% (04LP) to 43.0% (04LB). Nine digenic interactions (iQTL) were observed in S. officinarum whereas only three were observed in S. spontaneum. About half of the QTLs contributed by both progenitor species were associated with effects on the trait that was contrary to expectations based on the phenotype of the parent contributing the allele. Quantitative trait loci and their associated effects were consistent across crop-years and growing seasons with very few QTLs being unique to the early season. When the data were reanalyzed using the non-parametric discriminant analysis (DA) approach, significant marker-trait associations were detected for markers that were either identical to or in the vicinity of markers previously identified using the traditional QTL approach. Discriminant analysis also pointed to previously unidentified markers some of which remained unlinked on the map. These preliminary results suggest that DA could be used as a complementary approach to traditional QTL analysis in a crop like sugarcane for which saturated linkage maps are unavailable or difficult to obtain.     

The use of <Emphasis Type="Italic">Vp1</Emphasis> in real time RT-PCR to select for pre-harvest sprouting tolerance in triticale

In spite of the availability of laboratory and field tests there is still a major problem to select pre-harvest sprouting (PHS) tolerant triticale varieties in a reliable, field-independent way. One approach to minimize the influence of environmental conditions and physio-morphological traits on PHS detection is using molecular genetic tools. The ‘viviparous’ Vp1 gene has been repeatedly described to play an important role in dormancy in wheat. A quantitative RT-PCR assay based on the expression of the Vp1 gene has been developed. Specific primers were designed for detecting Vp1 in both wheat and triticale. The expression levels of Vp1 were normalized using reference genes and relatively quantified with the comparative C_t-method. However, the first results indicate that the achieved Vp1 expression levels at 50 days post anthesis are not useful to select for PHS tolerance, both in wheat and triticale. This negative outcome so far is possibly due to the existence of several splicing events or to the late assaying moment in the kernel development, when Vp1 expression is found to be low.     

Genetic and environmental control of dormancy in white-grained wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis> L.)

Grain dormancy in wheat is an important component of resistance to preharvest sprouting and hence an important trait for wheat breeders. The significant influence of environment on the dormancy phenotype makes this trait an obvious target for marker-assisted-selection. Closely related breeding lines, SUN325B and QT7475, containing a major dormancy QTL derived from AUS1408 located on chromosome 4A, but substantially different in dormancy phenotype, were compared with a non-dormant cultivar, Hartog, in a range of controlled environments. As temperature increased, dormancy at harvest-ripeness decreased particularly for QT7475. The dormancy phenotypes of reciprocal F₁ grains involving all possible combinations of Hartog, QT7475 and SUN325B were also compared in two environments with different temperatures. The results were consistent with the presence of QTL in addition to 4A in SUN325B, compared with QT7475, at least one of which was associated with the seed coat. Genetic analysis of a doubled haploid population derived from SUN325B × QT7475 identified a highly significant QTL located on chromosome 3BL, close to the expected position of the mutant allele of the red seed coat colour gene in white-grained wheat, R-B1a. When the lines in the population were grouped according to the parental alleles at marker loci flanking the 3B QTL, the dormancy phenotype frequency distribution for the SUN325B group was shifted towards greater dormancy compared with the QT7475 group. However, significant variation for dormancy phenotype remained within each group. Lines representing the extremes of the range of phenotypes within each group maintained their relative ranking across seven environments consistent with the presence of another unidentified QTL contributing to dormancy in SUN325B.     

Grain dormancy in fixed lines of white-grained wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis> L.) grown under controlled environmental conditions

Pre-harvest sprouting (PHS) in wheat (Triticum aestivum L.) can be a significant problem, causing deleterious effects on grain quality. However, the adverse impacts of PHS can be reduced by introgressing genes controlling grain dormancy into white-grained bread wheat. Screening for grain dormancy typically involves germination testing of harvest-ripe grain grown in a glasshouse or field. However, the more uniform environmental conditions provided by temperature controlled glasshouses (i.e. controlled environmental conditions—CEC) may provide significant benefits for the assessment of grain dormancy. In this study, the dormancy phenotype of grain grown under CEC incorporating an extended photoperiod, was compared with 2 years of data from field grown material. Four dormant double haploid lines (derived from SW95-50213 and AUS1408) and two locally adapted non-dormant cultivars EGA Gregory and EGA Wills were compared in three replicated experiments grown under CEC (22 ± 3°C and 24 h photoperiod). The germination response of harvest-ripe grain was examined to assess the expression of grain dormancy. Two measures of germination, the predicted time to 50% germination (G ₅₀) and a weighted germination index, both clearly differentiated dormant and non-dormant lines grown under CEC. In addition, levels of grain dormancy were similar to field-grown plants. These results demonstrated that CEC with an extended photoperiod can be used for rapid and reliable characterisation of grain dormancy in fixed lines of bread wheat.     

Promoter anchored amplified polymorphism based on random amplified polymorphic DNA (PAAP-RAPD) in cotton

Non-coding sequences account for a majority of the higher plant genome, some of which have important effects in gene regulation and plant development. In an effort to develop molecular marker systems to search for polymorphisms associated with high fiber yield and quality in cotton, we have developed a methodology that could specifically target the regulatory regions of the cotton genome. In this study we designed 10-nucleotide degenerate promoter primers based on conserved core promoter sequences and tested their applicability in PCR amplifications in combination with 10-mer random amplified polymorphic DNA (RAPD) primers. The amplified markers are called promoter anchored amplified polymorphism based on RAPD (PAAP-RAPD). Forty cotton genotypes with diverse genetic and geographical backgrounds were used to test the PAAP-RAPD system using polyacrylamide gel electrophoresis. Based on PAAP-RAPD markers amplified from 12 primer combinations, the 40 genotypes were classified into five distinctive groups: two Upland cotton (Gossypium hirsutum) groups from China, another two Upland cotton groups from the USA, and one group from American Pima cotton (G. barbadense). The groupings are in general consistent with their genetic and geographical origins. Thirty-six PAAP-RAPD and RAPD fragments were cloned and four of them were further subjected to sequence analysis. Signal scanning using software PLACE confirmed that they contained an array of cis-regulatory sequences in addition to the core promoter sequences. The results demonstrate the potential application of PAAP-RAPD as a new marker system specifically targeting regulatory regions of the plant genome.     

In vitro polyploidisation of <Emphasis Type="Italic">Helleborus</Emphasis> species

Helleborus species are members of the family of the Ranunculaceae. These popular perennials are all diploids (2n = 2x = 32). This study investigates polyploidy induction by different antimitotic agents. Colchicine, oryzalin and trifluralin were tested in vitro on shoots of Helleborus niger, H. orientalis and H. × nigercors. Furthermore the effect of the antimitotic agents on the viability and the multiplication rate of cultured plantlets were analyzed. Flow cytometry demonstrated that polyploidisation was genotype dependent: using H. niger, tetraploids were obtained using either oryzalin (3 μM) or trifluralin (3 or 10 μM), whereas for H. × nigercors only trifluralin (3 or 10 μM) induced polyploidisation. For H. orientalis neither treatment was effective to produce tetraploids or mixoploids. For these three species, colchicine (100 μM) was ineffective. The polyploidisation events in H. niger and H. × nigercors were confirmed by chromosome counts of mounted nuclei derived from root tips (2n = 4x = 64).     

Downy mildew resistance in opium poppy: resistance sources,inheritance pattern,genetic variability and strategies for crop improvement

Downy mildew (DM) caused by Peronospora arborescens is the most destructive disease of opium poppy which assumes considerable importance in India and other poppy growing countries. The present study was aimed at identification and evaluation of stable resistance sources of DM in opium poppy. Furthermore, genetic variability and inheritance pattern of DM resistance has also been studied which can help in making strategy for crop improvement. Evaluation of 35 selected germplasm accessions of opium poppy under glasshouse and field conditions during the three consecutive years (2004–2005 to 2006–2007) resulted in identification of two genotypes (I-14 and Pps-1) as highly resistant and stable sources for DM resistance. Genetic studies of DM resistance revealed polygenic control with the dominance of susceptibility over resistance. Significant reciprocal differences were found largely due to maternal transmission of DM resistance indicating the involvement of cytoplasmic genes in addition to nuclear control. Analysis of genetic variability and selection parameters indicated predominance of additive effects for DM resistance and other yield contributing traits. Multivariate analysis resulted in classification of 35 selected accessions into 11 different clusters revealing very high level of diversity among the genotypes. Cluster analysis suggested that hybridization program involving genotypes from cluster V (which included highly resistant genotypes Pps-1 and I-14) and cluster IX (which included highly susceptible Jawahar-16 having good economically important traits like seed yield) could be expected to give best recombinants for improvement in terms of DM resistance and high seed and straw yield in opium poppy. Analysis of selection parameters like heritability and genetic advance also suggested that simple selection methods will be effective in stabilizing resistance traits following hybridization with high yielding genotypes.     

Flow over rectangular sharp-crested weirs

Sharp-crested weirs are the simplest form of over-flow spillway that commonly used to determine the flow rate in hydraulic laboratories, industry and irrigation systems, where highly accurate discharge measurements are needed. In this study, the experimental upper and lower nappe profiles in rectangular sharp-crested weirs are fitted by quadratic and cubic equations, respectively. In addition, free-vortex theory is used to simulate flow over this kind of weirs and determine discharge coefficient. Physical models of sharp-crested weirs with various widths and heights were considered. The proposed method agrees well with the experimental observations. Also, the experimental data indicate that the suggested equation presents reasonable results for the range of 0 < h/P < 9.