Quantitative trait locus mapping using sets of near-isogenic lines: relative power comparisons and technical considerations

 The power to detect QTL using near-isogenic line (NIL) comparisons versus recombinant inbred (RI) populations was assessed. The power to detect QTL was found to be generally greater when using RI populations than when using NIL contrasts. Power to detect QTL with NIL contrasts never exceeded that of RI populations when the number of RI lines is maximized relative to replication of lines for a given number of experimental units. The relative power of NIL contrasts is highest for traits with high heritability and when a gain in precision is realized due to increased replication of entries. Although NIL populations are generally less powerful than RI populations of similar size, some practical considerations may enhance the value of these materials. Availability of NILs allows the genetic effect of a specific chromosome region to be determined by comparing two lines; all RI lines in a population need to be rescored for each new trait even if the effect of a specific chromosome region is suspected. NIL comparisons may allow genetic differences to be detected by visual inspection; genetic effects can only be expressed as means and variances with recombinant inbred populations. In summary, RI populations generally, and in some cases, substantially, provide better power for QTL detection than NIL comparisons. Practical considerations, however, indicate that many factors need to be considered when choosing a population structure to meet an experimental objective. Received: 12 December 1996 / Accepted: 21 March 1997     

Physiological routes from intra-uterine seminal contents to advancement of ovulation

Whole boar semen or seminal plasma has been demonstrated to advance the time of ovulation in gilts. As a means of clarifying this influence, the contribution of uterine lymphatics and their white cell populations has been examined. After duct visualisation with Evan's blue, lymph was sampled from a mesometrial vessel in eight pre-ovulatory gilts whose uterine lumen was infused simultaneously with whole semen in one ligated horn and saline in the contralateral ligated horn. Lymph was collected from cannulated vessels for periods of up to four hours under general anaesthesia. Thereafter, mesometrial lymph nodes, utero-tubal junction and uterine wall tissues were sampled. The proportion of nucleated cells in the sampled lymph increased towards the end of the collection period, but erythrocytes were found in all instances preventing a meaningful differentiation and identification of leukocytes. Prominent uterine lymph nodes were present in the mesometrium on both sides of the reproductive tract in 7 of 10 gilts. Differences in cellular contents were demonstrated between the side of the tract infused with semen and that infused with saline control. Two of 4 gilts had lower values for CD4 (Cluster Differentiation) and 3 of 6 gilts higher values for MHC II (Major Histocompatibility Complex) markers on the side challenged with semen. In contrast, values remained constant for CD8 but ranged widely for CD18. Immunohistochemical analysis of uterine tissue samples for MHC II+ cells revealed significant differences (P < 0.05) between the control and semen-treated ligated portions of the horns, as well as between the tissue sample of uterine wall and that from the utero-tubal junction, but there were no significant differences for CD4+ cells. It therefore remains plausible that semen-induced cytokines in the uterine lymph undergo counter-current transfer to the ipsilateral ovary and accelerate the final maturation of pre-ovulatory Graafian follicles.     

Shovelomics: high throughput phenotyping of maize (Zea mays L.) root architecture in the field

We present a method to visually score 10 root architectural traits of the root crown of an adult maize plant in the field in a few minutes. Phenotypic profiling of three recombinant inbred line (RIL) populations of maize (Zea mays L.; B73xMo17, Oh43xW64a, Ny821xH99) was conducted in 2008 in a silt loam soil in Pennsylvania and in a sandy soil in Wisconsin, and again in 2009 in Pennsylvania. Numbers, angles and branching pattern of crown and brace roots were assessed visually at flowering. Depending on the soil type in which plants were grown, sample processing took from three (sand) to 8 min (silt-loam). Visual measurement of the root crown required 2 min per sample irrespective of the environment. Visual scoring of root crowns gave a reliable estimation of values for root architectural traits as indicated by high correlations between measured and visually scored trait values for numbers (r ²?=?0.46?C0.97), angles (r ²?=?0.66?C0.76), and branching (r ²?=?0.54?C0.88) of brace and crown roots. Based on the visual evaluation of root crown traits it was possible to discriminate between populations. RILs derived from the cross NY821 x H99 generally had the greatest number of roots, the highest branching density and the most shallow root angles, while inbred lines from the cross between OH43 x W64a generally had the steepest root angles. The ranking of genotypes remained the same across environments, emphasizing the suitability of the method to evaluate genotypes across environments. Scoring of brace roots was better correlated with the actual measurements compared to crown roots. The visual evaluation of root architecture will be a valuable tool in tailoring crop root systems to specific environments.     

Biosynthesis of avenanthramides in suspension cultures of oat (<Emphasis Type="Italic">Avena sativa</Emphasis>)

Oats produce a group of secondary metabolites termed avenanthramides (avn). These compounds are biosynthesized through the action of the enzyme hydroxycinnamoyl CoA: hydroxyanthranilate N-hydroxycinnamoyl transferase (HHT) which catalyzes the condensation of one of several cinnamate CoA thioesters with the amine functionality of anthranilic acid, 4-hydroxy- or 5-hydroxy-anthranilic acid. In oat leaf tissue the biosynthesis of avenanthramides appears to result from elicitation by fungal infection. Here we demonstrate the biosynthesis of several avenanthramides in suspension cultures of oat apical meristem callus tissue. This phenomenon appears as a generalized pathogen response, evidenced by the production of PR-1 mRNA, in response to elicitation with chitin (poly-N-acetyl glucosamine). The suspension cultures also produce relatively large quantities of avnA and G in response to chitin elicitation. Under certain culture conditions avnB and C are also produced as well as three additional metabolites tentatively identified as avnH, O and R. These findings portend the utility of oat suspension culture as a tool for more detailed investigation of the mechanisms triggering their biosynthesis as well as the factors dictating the particular types of avenanthramides biosynthesized.     

Genetic Perturbation of the Maize Methylome

DNA methylation can play important roles in the regulation of transposable elements and genes. A collection of mutant alleles for 11 maize (Zea mays) genes predicted to play roles in controlling DNA methylation were isolated through forward- or reverse-genetic approaches. Low-coverage whole-genome bisulfite sequencing and high-coverage sequence-capture bisulfite sequencing were applied to mutant lines to determine context- and locus-specific effects of these mutations on DNA methylation profiles. Plants containing mutant alleles for components of the RNA-directed DNA methylation pathway exhibit loss of CHH methylation at many loci as well as CG and CHG methylation at a small number of loci. Plants containing loss-of-function alleles for chromomethylase (CMT) genes exhibit strong genome-wide reductions in CHG methylation and some locus-specific loss of CHH methylation. In an attempt to identify stocks with stronger reductions in DNA methylation levels than provided by single gene mutations, we performed crosses to create double mutants for the maize CMT3 orthologs, Zmet2 and Zmet5, and for the maize DDM1 orthologs, Chr101 and Chr106. While loss-of-function alleles are viable as single gene mutants, the double mutants were not recovered, suggesting that severe perturbations of the maize methylome may have stronger deleterious phenotypic effects than in Arabidopsis thaliana.     

Routine utilization of green fluorescent protein as a visual selectable marker for cereal transformation

Summary Development of new selectable markers is needed to increase the efficiency and flexibility of plant transformation, and to overcome drawbacks sometimes associated with use of existing markers. A useful alternative to chemical-based selection systems would be a system using visual screening to obtain transgenic lines. Investigations were carried out to determine if the green fluorescent protein (gfp) gene could be utilized alone as a visual screenable marker to produce stably transformed, fertile oat plants. Twelve experiments were conducted in which gfp-based selection was utilized to obtain routinely stable transgenic lines in oat. A synthetic gfp gene under the control of the maize ubiquitin promoter was delivered into embryogenic oat callus via microprojectile bombardment. Cell clusters (1–3 mm) expressing gfp were visually identified using epifluorescence microscopy and physically isolated approximately 3 wk post-bombardment. Fertile, gfp-expressing T0 plants were regenerated from 78% of the glowing cell sectors placed on regeneration medium. T0 plants from 55% of the events produced gfp-expressing progeny in a 3∶1 Mendelian ratio. Southern blot and PCR analysis confirmed transgene integration and transmission to progeny. Expression of gfp did not reduce plant growth or fertility. Transgene copy number and integration patterns were similar to those in transgenic plants derived from chemical-based selection systems. The mean transformation frequency, based on fertile events obtained per bombarded plate, was 1.8%. Over 180 independent transgenic oat lines were produced, to date, using only visual screening for expression of gfp, demonstrating efficiency and repeatability of the selection system. Mention of a trademark or proprietary product does not constitute a guarantee or warranty by the University of Wisconsin or the US Department of Agriculture and does not imply its approval to the exclusion of other products that may be suitable.     

QTL mapping and phenotypic variation for root architectural traits in maize (Zea mays L.)

Key message

QTL were identified for root architectural traits in maize.

Abstract

Root architectural traits, including the number, length, orientation, and branching of the principal root classes, influence plant function by determining the spatial and temporal domains of soil exploration. To characterize phenotypic patterns and their genetic control, three recombinant inbred populations of maize were grown for 28 days in solid media in a greenhouse and evaluated for 21 root architectural traits, including length, number, diameter, and branching of seminal, primary and nodal roots, dry weight of embryonic and nodal systems, and diameter of the nodal root system. Significant phenotypic variation was observed for all traits. Strong correlations were observed among traits in the same root class, particularly for the length of the main root axis and the length of lateral roots. In a principal component analysis, relationships among traits differed slightly for the three families, though vectors grouped together for traits within a given root class, indicating opportunities for more efficient phenotyping. Allometric analysis showed that trajectories of growth for specific traits differ in the three populations. In total, 15 quantitative trait loci (QTL) were identified. QTL are reported for length in multiple root classes, diameter and number of seminal roots, and dry weight of the embryonic and nodal root systems. Phenotypic variation explained by individual QTL ranged from 0.44 % (number of seminal roots, NyH population) to 13.5 % (shoot dry weight, OhW population). Identification of QTL for root architectural traits may be useful for developing genotypes that are better suited to specific soil environments.