We conducted 5 9-end sequencing of mRNAs isolated from human colon adenocarcinoma HT-29 cells treated the epigenetic agent, 5Aza, a potent inhibitor ofDNA methylation. Usingthe SOLiD platform, we generated approximately 62 million 25 bp reads (Table 1). Erroneous sequence tags were eliminated using quality values before alignment. The quality value (QV) is a well- known indicator for evaluation on error probability of a base. It is assigned by the SOLiD base (color) caller and estimates the probability each base is called correctly. The probability of a base being wrong p is given by p = 1/10 QV/10 . We removed any read which had 5 or more ofthe first 22 bases with a QV ,9. Although massively parallel sequencing technologies achieve dramatically higher throughputs than capillary sequencing, the ability ofthe short read lengths to map uniquely to a reference sequence is more sensitive to base calling errors. There is the flexibility to choose a stricter or looser QV criterion depending on how much the user wants to reduce the risk mentioned above. Filtering low quality reads yielded ,35.9 million reads, and ,23.0 million (64%) of these were aligned to the genome with at most two mismatches in order to cope with sequencing errors and SNPs. Among mapped reads, ,16.4 million (72%) were uniquely anchored on the genome. The ratios of unique reads aligned with zero, one, and two mismatches were 36%, 32%, and 32%, respectively. 73% of unique reads were located within 500 bases ofthe public representative transcription start sites (TSSs) of well- annotated protein-coding genes in the RefSeq database. Among the remaining 27% of unique reads, 11% were localized to RefSeq gene-coding regions within the genome, while 16% may represent small RNAs and annotated genes. In this study, we further analyzed the RefSeq associated reads. The 30 most highly expressed transcripts identified here in HT-29 cells are shown in Table S1. The most highly expressed genes in the untreated (control) cells encoded ribosomal proteins and ornithine decar-
DNA sequencing identifies common and rare genetic variants for association studies, but studies typically focus on variants in nuclear DNA and ignore the mitochondrial genome. In fact, analyzing variants in mitochondrial DNA (mtDNA) sequences presents special problems, which we resolve here with a general solution for theanalysisof mtDNA in next- generation sequencing studies. The new program package comprises 1) an algorithm designed to identify mtDNA variants (i.e., homoplasmies and heteroplasmies), incorporating sequencing error rates at each base in a likelihood calculation and allowing allele fractions at a variant site to differ across individuals; and 2) an estimation of mtDNA copy number in a cell directly from whole-genome sequencing data. We also apply the methods to DNA sequence from lymphocytes of ~2,000 SardiNIA Project participants. As expected, mothers and off- spring share all homoplasmies but a lesser proportion of heteroplasmies. Both homoplasmies and heteroplasmies show 5-fold higher transition/transversion ratios than variants in nuclear DNA. Also, heteroplasmy increases with age, though on average only ~1 heteroplasmy reaches the 4% level between ages 20 and 90. In addition, we find that mtDNA copy number averages ~110 copies/lymphocyte and is ~54% heritable, implying substantial genetic regula- tion ofthe level of mtDNA. Copy numbers also decrease modestly but significantly with age, and females on average have significantly more copies than males. The mtDNA copy num- bers are significantly associated with waist circumference (p-value = 0.0031) and waist-hip ratio (p-value = 2.4×10 -5 ), but not with body mass index, indicating an association with central fat distribution. To our knowledge, this is the largest population analysis to date of mtDNA dynamics, revealing the age-imposed increase in heteroplasmy, the relatively high heritability of copy number, and the association of copy number with metabolic traits.
Recently, the development ofnext-generation sequencing (NGS), also called high-throughput or deep sequencing technology has provided a powerful, highly reproducible and cost-efficient tool for transcriptomic research . The technology has been successfully applied in many advanced research areas, including resequencing , microRNA expression profiling , DNA methylation , the gene expression profiles during development [21,22] or after experimental treatments , gene discovery [24,25], SSR mining [26,27], and SNP discovery [28–30]. An advantage ofthe technology is that it could be used for gene discovery and expression profiling of organisms without reference genome by de novo assembly of short reads generated. Conse- quently, a large number of transcriptomic and genomic sequences have become available in the model or non-model organisms [31– 37]. There are several advanced and alternative NGS platforms, such as Roche’s 454 GS FLX, Illumina/Solexa Hiseq 2000 and Applied Biosystems’ SOLiD [38,39]. A full Roche/454-system run can produce about one million reads with an average read length of about 400 bp. While Solexa/Illumina or SOLiD/ABI produces nearly 20 million reads per lane, with read length ranging from 35 to100 bp, which has higher coverage and lower cost than Roche/ 454 . The greater sequence coverage obtained contributes to facilitate the assembly of transcripts and enable rare transcripts to be identified. In absence of reference genomes, a computational de novo assembly approach is required, assembly efficiency and accuracy is a commonly overlooked yet critical step. The results of de novo assembly from short reads have been improved by using increased read length, the paired-end sequencing strategy and the development of new computational tools such as Trinity, Oases and Trans-ABySS [41–43]. Especially, Trinity has been developed specifically for RNA-Seq assembly using short reads and increase the applicability of Illumina sequencing and de novo assembly.
The 454 NGS platform has become a commonly used tool for the development of genetic markers for systematic research (Mardis, 2008). In this study, we successfully iso- lated 8,856 microsatellite-containing contigs for M. gouazoubira from a total of 576,646 reads covering ~7.4% ofthe genome. From these contigs, seven polymorphic microsatellite markers were successfully characterized. We also used NGS in combination with bioinformatic tools to assemble and annotate the complete mitochondrial DNA sequence of M. gouazoubira. The low combined probabil- ity of genetic identity and thehigh power of paternity exclusion indicated that the battery of microsatellite devel- oped for M. gouazoubira will allow detailed studies of par- entage and population differentiation in this species; these topics have rarely been addressed usinghigh-resolution microsatellite markers in Neotropical deer. Furthermore, combining these markers with mitochondrial DNA se- quence analysisofthe hypervariable regions, e.g., control region and ND5, will provide a valuable resource for inves- tigating the demographic history of M. gouazoubira in response to habitat fragmentation, as well as assist in resol- ving taxonomic uncertainties in this taxon.
Identifying species from faecal DNAusing HRM analysis A conserved region ofthe 12S-rRNA gene, from base 295 to 692 ofthe Cervus elaphus mitochondrial genome, was chosen for the identification assay because of its low intraspecific variation (Table 1) and low mutational rate [37,38]. Species-specific reverse primers for each ofthe 10 species were designed so that resulting PCR products were all of different length, ranging from 93 to 399 bp, in combination with forward primer 12S-FWmod (Tables 2 and S1). Specificity ofthe primers was attained by positioning them at variable sites ofthe alignment and exploiting the 39 end SNP ofthe primer sequences. Melting behaviour ofthe fragments was predicted using uMeltbatch SM v2.0 . HRM- PCR reactions were performed in a Rotor-Gene 6000 cycler with a final volume of 10 ml, containing 5 ml of 1 6Type-it HRM PCR mix (Qiagen), 1 ml of 12S-FWmod and species-specific primer mix in the concentration shown in Table 2, and 1 ml ofDNA template. Cycling conditions consisted of an initial denaturation step of5 min at 95 uC, followed by 45 cycles of 10 s at 95 uC, 30 s at 60 uC and 10 s at 72 uC. The final melting step ramped from 70 to 90 uC, with 0.1 uC increments and 2 s at each temperature. DNA at 1 ng/ml of each ofthe 10 ungulate species was dispensed in all runs twice for technical replicates as positive controls. Large differences in concentration can affect theresolutionof HRM assays [28,40]. Preliminary testing (data not shown) indicated that a concentration of 1 ng/ml minimised variation in theresolutionof our HRM assays and hence this concentration was used for our positive controls. Raw data from the HRM-PCR were analysed using Rotor-Gene ScreenClust software (Qiagen) , which clusters the melting curves with the positive controls using a principal component analysis (PCA) with 3 dimensions. Results from the HRM-PCR were also analysed usingthe Rotor-Gene Q series software v 2.2.3 genotyping tool (HRM genotyping) that assigned species automatically based on the positive controls. Only Identifying Ungulate Species Using HRM Analysis
To understand if RNA-seq would also enhance the un- derstanding of biological principles of macrophage polarization we applied network analysis based on a priori information assessing the information content of RNA-seq data in compar- ison to array data. Genes expressed at elevated levels in M1 RNA-seq data (FC .4) were used for network generation (Fig. 5A). This primary RNA-seq based M1 network was subsequently used to visualize array-based gene expression (Fig. 5B). When genes at a lower level of differential expression (FC .2) were included 73% ofthe network was revealed in the array data and central hubs ofthe network were also categorized as being highly (FC .4) differentially expressed. However, only RNA-seq data revealed two gene clusters of immunomodulating proteins highly enriched in the M1 network, namely apolipo- proteins L (APOL) (Fig. 5A and Figure S9) and the leukocyte immunoglobulin-like receptor (LILR) family (Fig. 5A and Figure S10) [34–36]. As exemplified for LILRB1 and APOL3 both genes were clearly identified by RNA-seq, qRT-PCR, and flow cytometry respective western blotting (Fig. 5E and F) but not by microarray analysis (data not shown). Applying the RNA-seq data-based M2 network (Fig. 5C) to the array data (Fig. 5D) revealed only 54% elevated genes and major network hubs were Figure 5. Network analysisof RNA-seq data. (A) Network of genes highly expressed in M1-like macrophages (fold-change .4.0) identified by RNA-seq. (B) Data generated by microarray analysis were loaded into the M1-network established using RNA-seq. (C) Network of genes highly expressed in M2-like macrophages (fold-change .2.5) identified by RNA-seq. (D) Data generated by microarray analysis were loaded into the M2- network established using RNA-seq. All networks were generated using EGAN. (E) APOL3 and (F) LILRB1 expression in human M1- and M2-like macrophages. Far left, relative expression as determined by RNA-seq. Left, representative images of sequencing reads across genes expressed in human macrophages as described in Fig. 4. Right, relative mRNA expression by qPCR in M1- and M2-like macrophages. Far right, protein data as determined by immunoblotting, respective flow cytometry. Data are representative of three experiments (RNA-seq, qPCR, and immunoblotting resp. flow cytometry; mean and s.e.m.) each with cells derived from a different donor. Isotype controls are depicted as dotted lines. *P,0.05 (Student’s t- test).
Recent advances in next-generationDNA sequencing technologies have made possible the development ofhigh- throughput SNP genotyping platforms that allow for the simultaneous interrogation of thousands of single-nucleotide polymorphisms (SNPs). Such resources have the potential to facilitate the rapid development ofhigh-density genetic maps, and to enable genome-wide association studies as well as molecular breeding approaches in a variety of taxa. Herein, we describe the development of a SNP genotyping resource for use in sunflower (Helianthus annuus L.). This work involved the development of a reference transcriptome assembly for sunflower, the discovery of thousands ofhigh quality SNPs based on thegeneration and analysisof ca. 6 Gb oftranscriptome re-sequencing data derived from multiple genotypes, the selection of 10,640 SNPs for inclusion in the genotyping array, and the use ofthe resulting array to screen a diverse panel of sunflower accessions as well as related wild species. The results of this work revealed a high frequency of polymorphic SNPs and relatively high level of cross-species transferability. Indeed, greater than 95% of successful SNP assays revealed polymorphism, and more than 90% of these assays could be successfully transferred to related wild species. Analysisofthe polymorphism data revealed patterns of genetic differentiation that were largely congruent with the evolutionary history of sunflower, though the large number of markers allowed for finer resolution than has previously been possible.
We checked the completeness of CaTA v2 transcriptome assembly with the core eukaryotic gene-mapping approach (CEGMA) pipeline . CEGMA analysis undertakes similarity search ofthe assembly with a set of 458 highly conserved eukaryotic ubiquitous genes from the euKaryotic Orthologous Groups (KOG) database that are supposed to be present in all eukaryotes. A total of 452 (.98%) KOGs transcripts were present in CaTA v2 either completely or partially, which provides an indication ofthe completeness or comprehensiveness ofthetranscriptome assembly. Furthermore, full length transcripts present in the CaTA v2 were assessed usingthe annotated gene set (28,269) ofthe reported kabuli genome. On comparison, 11,088 TACs of CaTA v2 covers complete (100%) coding DNA sequences (CDSs) and can be considered as full length transcripts. The CaTA v2 was compared to the previously available transcriptome assemblies developed by Hiremath et al. (CaTA v1, ), Deokar et al. , Garg et al. [12,20] and Agarwal et al.  (Table 2). In this context, TACs from the CaTA v2 were clustered together with tentative unique sequences (TUSs) from CaTA v1 and contigs from other mentioned transcriptome assemblies. On clustering at 90% sequence identity, a non- redundant set of 84,754 transcripts (or unigenes) including 32,162 clusters and 52,592 singletons were defined. A total of 38,131 TACs ofthe CaTA v2 were found present in 25,580 (79.5%) clusters. Interestingly, 10,428 (47.7%) such clusters have repre- sentative sequence (longest sequence) from CaTA v2. Similarly, out of 52,592 singletons, 8,238 and 28,702 singletons are from the CaTA v2 and CaTA v1, respectively. The remaining singletons are from thetranscriptome assemblies of Agarwal et al. (13,862 singletons, ) and Garg et al. (17,90 singletons, [12,20]). These analyses in brief indicate high quality of CaTA v2 as even after combining five assemblies, 47.7% of cluster representative sequence has come from CaTA v2.
The p300, also known as E1A binding protein p300, is involved in several processes, as cellular differentiation and proliferation, cycle cell regulation, growth, apoptosis and histone acetylation (Huret 2000, Vleugel et al. 2006). Tan et al. (2009) suggested that this protein protects neuron from neurologic damages by inducing an increase of histone acetylation and preventing neuronal degeneration. Several studies have evidenced that the p300 is a transcriptional coactivator that interacts with other proteins, leading to the activation ofthe transcriptional process of several genes (Huret 2000, Tu and Luo 2007), including few genes related to cancer (Vleugel et al. 2006, Zhao et al. 2006, Chen et al. 2009) and neural pathologies development (Renoult et al. 2007, Francis et al. 2007). The relationship between p300 and GH was evidenced through an analysisofthe c-fos proto oncogene expression - the GH activates the c-fos through a stimulation ofthe p300 protein, which acts as a transcription factor, to occupy the enhancer/ promoter region ofthe gene (Cui et al. 2005).
Identification of somatic mutations in cancer is a major goal for understanding and monitoring the events related to cancer initiation and progression. Highresolution melting (HRM) curve analysis represents a fast, post-PCR high-throughput method for scanning somatic sequence alterations in target genes. The aim of this study was to assess the sensitivity and specificity of HRM analysis for tumor mutation screening in a range of tumor samples, which included 216 frozen pediatric small rounded blue-cell tumors as well as 180 paraffin-embedded tumors from breast, endometrial and ovarian cancers (60 of each). HRM analysis was performed in exons ofthe following candidate genes known to harbor established commonly observed mutations: PIK3CA, ERBB2, KRAS, TP53, EGFR, BRAF, GATA3, and FGFR3. Bi-directional sequencing analysis was used to determine the accuracy ofthe HRM analysis. For the 39 mutations observed in frozen samples, the sensitivity and specificity of HRM analysis were 97% and 87%, respectively. There were 67 mutation/variants in the paraffin-embedded samples, and the sensitivity and specificity for the HRM analysis were 88% and 80%, respectively. Paraffin-embedded samples require higher quantity of purified DNA for high performance. In summary, HRM analysis is a promising moderate- throughput screening test for mutations among known candidate genomic regions. Although the overall accuracy appears to be better in frozen specimens, somatic alterations were detected in DNA extracted from paraffin-embedded samples.
With the development of noninvasive neuroimaging techniques, Yacoub and colleagues showed the ability to map the ocular dominance columns ofthe human visual cortex using fMRI (Yacoub et al., 2007). In the same study, a difference between the performance of two techniques (spin-echo (SE) and gradient-echo (GE)) was evaluated. Their conclusion points that SE signal can uniformly resolve columnar patterns better than GE, mainly due to their different susceptibility regarding the vascular signal: while SE BOLD corresponds to the intravascular signal, GE BOLD accounts all the vascular components (intra and extra vascular) (Ogawa et al., 1993). This susceptibility difference allied with the demonstration that large vessels influence the columnar specificity of cerebral blood flow(Duong et al., 2001); indicate the way to be successful in mapping columnar structures usingthe positive BOLD effect, which is the avoidance of large vessel contributions in the data. This approach was used in earlier studies that attempted to map columns in humans using GE. (Dechent and Frahm, 2000; Goodyear and Menon, 2001; Menon et al., 1997)
Mutation scanning of all samples was per- formed using a high-resolution melting (HRM) technique in a real-time PCR thermal cycler (LightCycler 480, Roche Diagnostics). The primers were constructed according to the com- mon HRM specifications [http://www.gene- quantification.de/LC480-Technical-Note-01- HRM.pdf], usingthe following online software and databases: UCSC Genome Browser (http://genome.ucsc.edu/cgi-bin/hgGateway), Primer 3 (http://frodo.wi.mit.edu/), DINAMelt (http://dinamelt.bioinfo.rpi.edu/), Poland service (http://www.biophys.uni-duesseldorf.de/local /POLAND/poland.html) and Primer-BLAST (http://www.ncbi.nlm. nih.gov/tools/primer-blast). The HPLC grade primers were purchased from Metabion (Germany). The amplification, melting and fluorescence detection conditions were as indicated by the supplier (Roche Diagnostics). Briefly, after amplification (35 cycles of 95 ºC for 10 seconds, 60 ºC for 15 seconds and 72 ºC for 10 seconds), the PCR products were heat- ed to 95 ºC and cooled to 40 ºC (for heterodu- plex formation), and melting was monitored (by fluorescence emission) from 65 ºC to 95 ºC. Table I lists the exon-intron boundaries covered in theanalysis (fragments inclu-
address a specific research question, namely whether RNA polymerase II (RNAPII) kinetics decrease near splice sites. In light ofthe finding that exons are ‘marked’ by nucleosomes [29– 35], we speculated that as RNAPII approaches an exon/intron junction, the presence ofthe nucleosome may reduce transcrip- tional rates , allowing time for the precise assembly of spliceosomal components over the exon/intron junction [36–39]. To detect whether exon-intron junctions were enriched in transcriptionally-engaged polymerase, we aligned a dataset of .23 million GRO-seq reads from lung fibroblasts to the human genome; we retained .11 million reads uniquely mapping the genome, similar to the number obtained by Core et al. . As in , the5 9 most coordinate ofthe read was considered to reflect the position of transcriptionally engaged RNAPII. Each genomic position was allocated a score equal to the number of reads beginning at that position. We then used the above-constructed dataset of exon-intron quintets, and plotted the mean read densities in the regions surrounding the two ends ofthe exons, i.e., the 3 9 and 59 splice sites (39ss and 59ss respectively). Since the number of reads obtained from a given transcript is highly correlated with the expression level ofthe gene coding that transcript, we divided the exons into five equally sized groups based on the expression levels ofthe genes in which they are located; gene expression levels in lung fibroblasts were obtained from . In our initial analysis we adopted a naı¨ve approach, and did not take into account the nucleotide per cycle or the mappability biases.
sleeping chironomid Polypedilum vanderplanki, the largest anhydrobiotic animal known up to now . Previous studies have yielded unequivocal findings about the transcriptional response of Hsps to desiccation in tardigrades. For instance, Jo¨nsson and Schill  have detected a down-regulation of Hsp70 in desiccated specimens of eutardigrade Richtersius coronifer, whereas Altiero et al.  reported that the expression levels of Hsp70 are similar between dehydrated and active specimens of eutardigrade Bertolanius volubilis. In eutardigrade M. tardigradum, one isoform ofthe Hsp70 family gene shows an up-regulation during the induction ofthe desiccated state, while the other two isoforms have significantly lower levels of mRNA expression in the dehydated state than in the hydrated state . In the Bertolanius volubilis investigated by Altiero et al. , the dehydration stress does not induce an increase in Hsp90 expression either. In contrast to this finding, Hsp90 in eutardigrade M. tardigradum has been found to be significantly up-regulated in the anhydrobiotic state . Hydrophilic LEA proteins were first reported in resurrection plants and later in non-plant organisms . For instance, in nematodes [27–29], rotifers  as well as in plants , the induction of LEA proteins has been associated with dehydration tolerance. The evaluation of LEA proteins in tardigrades is very recent and leads to some contradictory data too. The presence of LEA-like transcripts and protein was detected by a few studies of tardigrades using expressed sequence tag (EST) or proteomics [31–33]. LEA proteins are heat-soluble molecules considering their biochemical property. Recently, a heat-soluble proteomics study has showed that abundant heat-soluble proteins bear no sequence similarity with LEA proteins and stress-induced novel protein families have distinct sub-cellular localizations in an anhydrobiotic tardigrade Ramazzottius varieornatus .
This is an extremely important report ofthe psychological evaluation of children with cloacal exstrophy, with particular emphasis on 6 patients with 46 XY chromosomes who were raised as females. The researcher was blinded as to the chromosomal diagnosis in the cases and there was a control group of patients with cloacal anomalies, but not cloacal exstrophy or gender conversion. The authors found no statistical differences between the 2 groups, with scores comparable to those reported for other chronic illnesses.
genotypes not representing unique and diverse alleles, or trying to explain, in theory, methodologies with applied im- pact. Molecular markers were originally proposed to solve breeding challenges with quantitative traits with challenging phenotyping. Besides, active cooperation with industry should provide access to latest technology without having to invest in obsolete academic labs (Guimaraes et al. 2006). There is a need to target newer technologies on traits that are genetically complex, dificult to measure, and carry unique alleles based on maximum sampling. Breeding programs should develop not only products but also applied breeding methodologies accurately screening thousands of genotypes fast. These considerations will ill the gap between geneticists, physiolo- gists, and breeders to develop thenextgenerationof products with maximum eficiency. Breeding programs should adapt, improve, and develop elite and unique germplasm through most eficient (old and new) breeding strategies depending on cost and impact. Stratiied mass selection (Gardner 1961), for instance, haves been extremely successful (making maize products 2-4 days earlier per year independent from geno- type) and very cost/effective for adapting genetic materials to northern latitudes in the U.S. by utilizing large samples and selecting for earliness visually (Hallauer and Carena 2009).
Absorção da radiação solar UV pelas células da pele altera a estrutura química do DNA e causa estresse oxidativo (BIESALSKI et al., 2003; ICHIHASHI et al., 2003; YAAR; GILCHREST, 2007). Essas alterações ativam vias de sinalização celular que regulam múltiplas funções celulares na pele (FISHER et al., 2002; QUAN et al., 2005; RITTIÉ; FISHER, 2002). Células de mamíferos respondem à radiação UV com várias alterações bioquímicas incluindo a expressão de genes distintos chamados de genes de resposta UV, e já foi mostrado que esses genes estão relacionados à transdução de sinal, defesa antioxidante e controle do ciclo celular (SCHARFFETTER –KOCHANEK et al., 2000; SHAULIAN et al., 2000). Ainda, genes que respondem à radiação UVB compreendem muitas proteases, dentre elas as metaloproteases de matriz (MMPs) (QUAN et al., 2009), as quais degradam os componentes da matriz extracelular (HO et al., 2005), bem como reduzem a produção de pro colágeno tipo I (FISHER et al., 2002).
As we know that to find the area of sector the angle made by the chord (that is chord which divides the circle) is required. But in the below method we find the ratio ofthe segments ofthe circle. Thus by relating the area of segment to the area of sector the area of sector could be found. The ratio of area of segments is related to tangents that are drawn through diameter on either side.
reported that the reduction of zein protein synthesis is associ- ated with an increased percentage of lysine in the endosperm. The potential use of opaque-2 maize has been hampered by its poor agronomic performance. Plant breeders have made a big effort to improve the nutritional quality of maize with the opaque-2 gene but pleiotropic effects associated with this mutation, however, have resulted in seeds with inferior agronomic properties (Vasal 1994, Lopes and Larkins 1995). Modifier genes have been described that convert the soft, starchy endosperm of mutants to a hard, vitreous (hard and translucent) phenotype, while maintaining the enhanced lysine content ofthe grain. This conversion occurs naturally in certain maize genotypes (Lopes et al. 1995) and the genes that condition this change are generally called opaque-2 modi- fiers. Modified genotypes, which resemble normal maize, both in kernel phenotype and agronomic performance, are known as Quality Protein Maize (QPM) genotypes. CIM- MYT is one ofthe institutions that yielded a broad array of QPM germplasm adapted to tropical and subtropical areas. Their work initially emphasized the conversion to O 2 geno- types of various genetically broad based germplasm pools, which were subsequently improved by breeding programs that included backcrossing and recurrent selection (Pixley and Bjarnason 1993). As a consequence, vitreous kernels and agronomic performance similar to normal-endosperm maize were achieved by accumulating modifier genes (Vasal et al. 1980). Gupta et al. (1975) suggested that line selection with this characteristic may be important for the production of improved QPM hybrids. South Africa developed QPM hybrids with higher yields than the best hybrids used in that region. Some ofthe initial problems have been solved. The grain phenotype has been improved and yield was expressed because the more accumulation of dry matter in grain. QPM hybrids are more resistant to insects and mold during stor- age and they keep the protein quality. However, adaptation of QPM genotypes to temperate environments and higher latitudes has been challenging.
phenotypes. It is based on a linear classifier and can be trained to enable thehigh-content soft- ware to recognise and classify cells based on phenotype. Building block 9 ([Select Population (2)]) was used to train two classes of cells—class A: Singlets and class B: Multiplets using approximately 150 cells from each class. The linear classifier generated from this analysis used morphological properties to separate the two cell populations namely, nucleus axial length ratio, nucleus symmetry, nucleus axial small length and nucleus roundness with a “goodness” of 2.24. The “goodness” value indicates the quality ofthe separation but does not provide infor- mation about the distribution of classification results. This approach worked well for cells that have a tendency to grow as clumps such as HT29 and U87MG cells. For cells that grow as a more uniform monolayer (such as SKOV-3 cells) finding sufficient class B cells to train the lin- ear classifier required theanalysisof multiple wells and fields. In general, the linear classifier only required training when applied to a new cell line. However, if a compound induces signifi- cant changes to nuclear morphology, additional re-training using examples of treated cells may be necessary to ensure accurate cell separation. Using PhenoLOGIC to separate the cell populations had the added advantage of also filtering out any fluorescent debris from subse- quent analyses.