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By the year 2050 the world population will increase to 9.7 billion people. Food production must increase by at least 70% in order to feed this population. One way to increase food production is to create crop cultivars that can produce high quality and high yielding crops without needing to increase the amount of resources required. Plant breeders are able to create new crop cultivars using high-throughput genotyping techniques, however the current bottleneck in plant breeding is in-field phenotyping. This study focuses on designing a high-throughput in-field proximal phenotyping system capable of collecting non-contact, high-resolution, multi-sensor, multi-view, phenomic data of vegetable plants.

A wild tomato species (Solanum habrochaites S. Knapp & D.M. Spooner) is tolerant to many abiotic stresses, including limited water, and can serve as a valuable genetic resource for breeding cultivated tomato (S. lycopersicum L.). Previously, we developed and used a set of 18 sub-near-isogenic lines (sub-NILs) containing introgressions from chromosome 9 of S. hab- rochaites to determine the genetic basis of tol- erance to rapid-onset water stress imposed by root chilling. In this study, we used this same set of 18 sub-NILs to investigate the genetic basis of tolerance to slow-onset water stress in the field imposed by restricted irrigation (with full water treatment as control) in replicated experiments for 2 yr in Davis, CA. The sub-NILs were evalu- ated for C isotope discrimination (D 13 C), specific leaf area (SLA), shoot dry weight (SDW), yield, and other horticultural traits. A total of 19 QTL for D 13 C, SLA, SDW, yield, and maturity traits were

Key message QTL stm9 controlling rapid-onset water stress tolerance in S. habrochaites was high-resolution mapped to a chromosome 9 region that contains genes associated with abiotic stress tolerances. Abstract Wild tomato (Solanum habrochaites) exhibits tolerance to abiotic stresses, including drought and chilling. Root chilling (6 °C) induces rapid-onset water stress by impeding water movement from roots to shoots. S. habrochaites responds to such changes by closing stomata and maintaining shoot turgor, while cultivated tomato (S. lycopersicum) fails to close stomata and wilts. This response (shoot turgor maintenance under root chilling) is controlled by a major QTL (designated stm9) on chromosome 9, which was previously fine-mapped to a 2.7-cM region. Recombinant sub-near-isogenic lines for chromosome 9 were marker-selected, phenotyped for shoot turgor maintenance under root chilling in two sets of replicated experiments (Fall and Spring), and the data were used to high-resolution map QTL stm9 to a 0.32-cM region. QTL mapping revealed a single QTL that was coincident for both the Spring and Fall datasets, suggesting that the gene or genes contributing to shoot turgor maintenance under root chilling reside within the marker interval H9–T1673. In the S. lycopersicum reference genome sequence, this chromosome 9 region is gene-rich and contains representatives of gene families that have been associated with abiotic stress tolerance. Electronic supplementary material The online version of this article (doi:10.1007/s00122-015-2540-y) contains supplementary material, which is available to authorized users.

Cultivated tomato (Solanum lycopersicum L.) is susceptible to abiotic stresses, including drought and chilling stress, while its wild relative (Solanum habrochaites) exhibits tolerance to many abiotic stresses. Chilling roots to 6°C induces rapid-onset water stress by impeding water movement from roots to shoots. Wild S. habrochaites responds to root chilling by closing stomata and maintaining shoot turgor, while cultivated tomato fails to close stomata and wilts. This phenotypic response (shoot turgor maintenance under root chilling) is controlled by a major QTL stm9 on chromosome 9 from S. habrochaites that was previously high-resolution mapped to a 0.32 cM region, but its effects on transcriptional regulation were unknown. Here we used paired near isogenic lines (NILs) differing only for the presence or absence of the S. habrochaites introgression containing stm9 in an otherwise S. lycopersicum background to investigate global transcriptional regulation in response to rapid-onset water stress induced by root chilling. NIL175 contains the S. habrochaites introgression and exhibits tolerance to root chilling stress, while NIL163 does not contain the introgression and is susceptible. RNA from roots of the two NILs was obtained at five time points during exposure to root chilling and mRNA-Seq performed. Differential expression analysis and hierarchical clustering of transcript levels were used to determine patterns of and changes in mRNA levels. Our results show that the transcriptional response of roots exposed to chilling stress is complex, with both overlapping and unique responses in tolerant and susceptible lines. In general, susceptible NIL 163 had a more complex transcriptional response to root chilling, while NIL175 exhibited a more targeted response to the imposed stress. Our evidence suggests that both the tolerant and susceptible NILs may be primed for response to root-chilling, with many of these response genes located on chromosome 9. Furthermore, serine/threonine kinase activity likely has an important role in the root chilling response of tolerant NIL175.

Previously, a Phytophthora infestans resistance QTL from Solanum habrochaites chromosome 11 was introgressed into cultivated tomato (S. lycopersicum). Fine mapping of this resistance QTL using near-isogenic lines (NILs) revealed some co-located QTL with undesirable effects on plant size, canopy density, and fruit size traits. Subsequently, higher-resolution mapping with sub-NILs detected multiple P. infestans resistance QTL within this 9.4-cM region of chromosome 11. In our present study, these same sub-NILs were also evaluated for 17 horticultural traits, including yield, maturity, fruit size and shape, fruit quality, and plant architecture traits in replicated field experiments over 2 years. The horticultural trait QTL originally detected by fine mapping each fractionated into two or more QTL at higher resolution. A total of 34 QTL were detected across all traits, with 14% exhibiting significant QTL × environment interactions (QTL × E). QTL for many traits were co-located, suggesting either pleiotropic effects or tight linkage among genes controlling these traits. Recombination in the pericentromeric region of the introgression between markers TG147 and At4g10050 was suppressed to approximately 29.7 Mbp per cM, relative to the genomewide average of 750 kbp per cM. The genetic architecture of many of the horticultural and P. infestans resistance traits that mapped within this chromosome 11 S. habrochaites region is complex. Complicating factors included fractionation of QTL, pleiotropy or tight linkage of QTL for multiple traits, pericentromeric chromosomal location(s), and/or QTL × E. High-resolution mapping of QTL in this region would be needed to determine which specific target QTL could be useful in breeding cultivated tomato.

When the allele of a wild species at a quantitative trait locus (QTL) conferring a desirable trait is introduced into cultivated species, undesirable effects on other traits may occur. These negative phenotypic effects may result from the presence of wild alleles at other closely linked loci that are transferred along with the desired QTL allele (i.e., linkage drag) and/or from pleiotropic effects of the desired allele. Previously, a QTL for resistance to Phytophthora infestans on chromosome 5 of Solanum habrochaites was mapped and introgressed into cultivated tomato (S. lycopersicum). Near-isogenic lines (NILs) were generated and used for fine-mapping of this resistance QTL, which revealed coincident or linked QTL with undesirable effects on yield, maturity, fruit size, and plant architecture traits. Subsequent higher-resolution mapping with chromosome 5 sub-NILs revealed the presence of multiple P. infestans resistance QTL within this 12.3 cM region. In our present study, these sub-NILs were also evaluated for 17 horticultural traits, including yield, maturity, fruit size and shape, fruit quality, and plant architecture traits in replicated field experiments over the course of two years. Each previously detected single horticultural trait QTL fractionated into two or more QTL. A total of 41 QTL were detected across all traits, with ∼30% exhibiting significant QTL × environment interactions. Colocation of QTL for multiple traits suggests either pleiotropy or tightly linked genes control these traits. The complex genetic architecture of horticultural and P. infestans resistance trait QTL within this S. habrochaites region of chromosome 5 presents challenges and opportunities for breeding efforts in cultivated tomato.

Quantitative disease resistance (QDR) has been observed within many crop plants but is not as well understood as qualitative (monogenic) disease resistance and has not been used as extensively in breeding. Mapping quantitative trait loci (QTLs) is a powerful tool for genetic dissection of QDR. DNA markers tightly linked to quantitative resistance loci (QRLs) controlling QDR can be used for marker-assisted selection (MAS) to incorporate these valuable traits. QDR confers a reduction, rather than lack, of disease and has diverse biological and molecular bases as revealed by cloning of QRLs and identification of the candidate gene(s) underlying QRLs. Increasing our biological knowledge of QDR and QRLs will enhance understanding of how QDR differs from qualitative resistance and provide the necessary information to better deploy these resources in breeding. Application of MAS for QRLs in breeding for QDR to diverse pathogens is illustrated by examples from wheat, barley, common bean, tomato, and pepper. Strategies for optimum deployment of QRLs require research to understand effects of QDR on pathogen populations over time.

The genetic architecture of transcript-level variation is largely unknown. The genetic determinants of transcript-level variation were characterized in a recombinant inbred line (RIL) population (n = 211) of Arabidopsis thaliana using whole-genome microarray analysis and expression quantitative trait loci (eQTL) mapping of transcript levels as expression traits (e-traits). Genetic control of transcription was highly complex: one-third of the quantitatively controlled transcripts/e-traits were regulated by cis-eQTL, and many trans-eQTL mapped to hotspots that regulated hundreds to thousands of e-traits. Several thousand eQTL of large phenotypic effect were detected, but almost all (93%) of the 36,871 eQTL were associated with small phenotypic effects (R2 < 0.3). Many transcripts/e-traits were controlled by multiple eQTL with opposite allelic effects and exhibited higher heritability in the RILs than their parents, suggesting nonadditive genetic variation. To our knowledge, this is the first large-scale global eQTL study in a relatively large plant mapping population. It reveals that the genetic control of transcript level is highly variable and multifaceted and that this complexity may be a general characteristic of eukaryotes.

Two tomato inbred backcross line (IBL) populations, derived from crosses between aphid-susceptible Lycopersicon esculentum Mill. 'Peto 95-43' X resistant wild L. pennellii Corr (D'arcy) accession LA716, and Peto 95-43 X resistant wild L. hirsutum f. glabratum Mull accession LA407, were evaluated in replicated field experiments for resistance to potato aphid, Macrosiphum euphorbiae (Thomas), and green peach aphid, Myzus persicae (Sulzer). Aphid infestation scores for each IBL and control (LA716, LA407, Peto 95-43, and susceptible 'Alta') plot were recorded weekly for 5 and 9 wk during the summers of 2000 and 2001, respectively. Aphid infestation scores from leaflets were used to calculate area under the infestation pressure curve (AUIPC), a measure of aphid infestation throughout the growing season, for each IBL and control. Score AUIPC was highly correlated with actual aphid count AUIPC, indicating that scores accurately reflected aphid infestation. Score AUIPC was also highly correlated across both years (2000 and 2001) and locations. Low score AUIPC was significantly correlated with larger plant size and sprawling, indeterminate plant growth habit. Seven IBLs, LA716, and LA407 were significantly more resistant to aphids (lower score AUIPC) than susceptible parent Peto 95-43 in both years. Two IBLs, 1034 and 1051, were not significantly different from resistant LA407 for score AUIPC in both years. The seven aphid-resistant IBLs identified here can be useful as donor parent material for resistance breeding efforts in cultivated tomato.

Cultivated tomato (L. esculentum L.) germplasm exhibits limited genetic variation compared with wild Lycopersicon species. Amplified fragment length polymorphism (AFLP) markers were used to evaluate genetic variation among 74 cultivars, primarily from California, and to fingerprint germplasm to determine if cultivar-specific patterns could be obtained. All 74 cultivars were genotyped using 26 AFLP primer combinations; of the 1092 bands scored, 102 AFLP bands (9.3%) were polymorphic. Pair-wise genetic similarity coefficients (Jaccard and Nei–Li) were calculated. Jaccard coefficients varied from 0.16 to 0.98 among cultivar pairs, and 72% of pair-wise comparisons exceeded 0.5. UPGMA (unweighted pair-group method with arithmetic averaging) clustering and principle component analysis revealed four main clusters, I–IV; most modern hybrid cultivars grouped in II, whereas most vintage cultivars grouped in I. Clusters III and IV contained three and two cultivars, respectively. Some groups of cultivars closely related by pedigree exhibited high bootstrap values, but lower values (

Quantitative trait loci (QTLs) for resistance to Phytophthora infestans (late blight) were mapped in tomato. Reciprocal backcross populations derived from cultivated Lycopersicon esculentum × wild Lycopersicon hirsutum (BC-E, backcross to L. esculentum; BC-H, backcross to L. hirsutum) were phenotyped in three types of replicated disease assays (detached-leaflet, whole-plant, and field). Linkage maps were constructed for each BC population with RFLPs. Resistance QTLs were identified on all 12 tomato chromosomes using composite interval mapping. Six QTLs in BC-E (lb1a, lb2a, lb3, lb4, lb5b, and lb11b) and two QTLs in BC-H (lb5ab and lb6ab) were most consistently detected in replicated experiments or across assay methods. Lycopersicon hirsutum alleles conferred resistance at all QTLs except lb2a. Resistance QTLs coincided with QTLs for inoculum droplet dispersal on leaves, a trait in L. hirsutum that may contribute to resistance, and dispersal was mainly associated with leaf resistance. Some P. infestans resistance QTLs detected in tomato coincided with chromosomal locations of previously mapped R genes and QTLs for resistance to P. infestans in potato, suggesting functional conservation of resistance within the Solanaceae.Key words: late blight, tomato, Lycopersicon hirsutum, QTL mapping, disease resistance, potato.

Bacterial canker caused by Clavibacter michiganensis subsp. michiganensis causes significant yield losses on tomatoes grown in a humid environment. This study was conducted to identify a source of resistance that could be easily crossed to cultivated tomato and to study the inheritance of resistance. Diverse bacterial strains representative of the major DNA fingerprint classes endemic to North America were used to screen germ plasm and populations derived from wide crosses. Partial resistance to genetically characterized and distinct strains of C. michiganensis subsp. michiganensis was identified in a wild relative of cultivated tomato, Lycopersicon hirsutum Lycopersicon accession (LA)407. The level of resistance in LA407 was not significantly different from that of the resistant L. peruvianum control, LA2157. Resistance from LA407 was recovered in lines from a BC2S4 inbred backcross (IBC) population in both greenhouse and field trials. Linear correlations between field and greenhouse resistance scores were significant, though correlation coefficients tended to be low. Variance components for genetic and environmental variation in resistance were used to estimate broad-sense heritability in the IBC population. These estimates were moderate to high, ranging from 0.34 to 0.85. The number of genes contributing to resistance was estimated from four trials, with most estimates falling in the range of one to three loci. Two lines from the IBC population, IBL 2353 and IBL 2361, were identified as sources that retain resistance in a genetic background that has a theoretical L. esculentum genome content of 87.5%.

Blackmold, caused by the fungus Alternaria alternata, is a major ripe fruit disease of processing tomatoes. Previously, we found blackmold resistance in a wild tomato (Lycopersicon cheesmanii) and quantitative trait loci (QTL) for resistance were mapped in an interspecific population. Five QTLs were selected for introgression from L. cheesmanii into cultivated tomato using marker-assisted selection (MAS). Restriction fragment length polymorphism and PCR-based markers flanking, and within, the chromosomal regions containing QTLs were used for MAS during backcross and selfing generations. BC1 plants heterozygous at the QTLs, and subsequent BC1S1 and BC1S2 lines possessing different homozygous combinations of alleles at the target QTLs, were identified using DNA markers. Field experiments were conducted in 1998 (with 80 marker-selected BC1S2 lines) and 1999 (with 151 marker-selected BC1S2 and BC1S3 lines) at three California locations. Blackmold resistance was assessed during both years, and horticultural traits were evaluated in 1999. The BC1S2 and BC1S3 lines containing L. cheesmanii alleles at the QTLs were associated with a large genetic variance for resistance to blackmold and moderate heritability, suggesting that significant genetic gain may be achieved by selection in this genetic material. L. cheesmanii alleles at three of the five introgressed QTLs showed a significant, positive effect on blackmold resistance. A QTL on chromosome 2 had the largest positive effect on blackmold resistance, alone and in combination with other QTLs, and was also associated with earliness, a positive horticultural trait. The other four QTLs were associated primarily with negative horticultural traits. Fine mapping QTLs using near isogenic lines could help determine if such trait associations are due to linkage drag or pleiotropy.

Black mould lesions were caused by Alternaria alternata in 76% of 228 tomato fruit with characteristic sunken black lesions collected from fields of processing tomatoes in California. Analysis of 29 RAPD primers revealed a high level of genetic diversity among the 69 isolates tested. Two major phenetic groups (Group 1 with 55 isolates and Group 2 with 14) were identified independently by PCA and by UPGMA of Jaccard similarity coefficients. Only 34 of 137 RAPD markers were monomorphic for all isolates and the genetic similarity between the two groups was 50%. Co-infection of black mould lesions by genetically distinct strains of A. alternata occurred in two of 10 isolates tested. There was no evidence for geographic clustering of isolates with high levels of genetic similarity, suggesting that isolates are widely dispersed across California. Only one isolate was identified which also caused stem canker disease on a susceptible tomato cv., suggesting that these strains play a minor role in causing black mould on processing tomatoes in California. This isolate and two other known stem canker isolates were clustered together with 11 other isolates in Group 2. Group 2-specific bands were also identified in a survey of seven additional isolates known to produce host-specific toxins.

To improve the efficiency of introgressing genes from Lycopersicon hirsutum (H) into L. esculentum (E), environmental and genetic variation for the number of progeny per fruit from E × H crosses was quantified. Over three dates in a year, 36 H accessions were crossed to seven E accessions in a greenhouse. The proportion of total variation for the number of E × H progeny per fruit due to environment (dates, location, and error), H accession, E accession, interactions between E and H, and interactions between accessions and environments was 0.42, 0.26, 0.12, 0.11, and 0.09, respectively. Sampling greater numbers of fruit on a single date improved the efficiency of recovering progeny more than increased sampling over time. The specific combination of E and H parents can profoundly affect the number of E × H progeny recovered and therefore the efficiency of gene introgression. Accessions of H from the southern edge of the species' natural geographic range generally yielded few to zero progeny per fruit in crosses with E. In contrast to the southern H accessions, most northern accessions produced greater than 40 E × H progeny per fruit. Most genes within H should be readily accessible for tomato breeding but genes that are found only in southern H accessions may be challenging to introgress.

 Premise of the study: Cultivated tomato, Solanum lycopersicum , suffers chilling induced wilting because water movement through its roots decreases with declining soil temperatures. Certain wild tomato species exhibit resistance to chilling-induced wilting, but the extent of this chilling tolerance in wild tomatoes is not known.  Methods: We measured shoot wilting during root chilling in wild Solanum accessions from habitats differing in elevation, temperature, and precipitation. We also measured shoot wilting during root chilling in introgression lines (ILs) with chromosome 9 segments collinear to the shoot turgor maintenance QTL stm9 region from chilling-tolerant S. habrochaites , chilling and drought-tolerant S. lycopersicoides, or drought-tolerant S. pennellii .  Key results: Wild tomato species, which experience chilling temperatures (

Cultivated tomato (Solanum lycopersicum) is susceptible to late blight, a major disease caused by Phytophthora infestans, but quantitative resistance exists in the wild tomato species S. habrochaites. Previously, we mapped several quantitative trait loci (QTL) from S. habrochaites and then introgressed each individually into S. lycopersicum. Near-isogenic lines (NILs) were developed, each containing a single introgressed QTL on chromosome 5 or 11. NILs were used to create two recombinant sub-NIL populations, one for each target chromosome region, for higher-resolution mapping. The sub-NIL populations were evaluated for foliar and stem resistance to P. infestans in replicated field experiments over two years, and in replicated growth chamber experiments for resistance to three California isolates. Each of the original single QTL on chromosomes 5 and 11 fractionated into between two and six QTL for both foliar and stem resistance, indicating a complex genetic architecture. The majority of QTL from the field experiments were detected in multiple locations or years, and two of the seven QTL detected in growth chambers were co-located with QTL detected in field experiments, indicating stability of some QTL across environments. QTL that confer foliar and stem resistance frequently co-localized, suggesting that pleiotropy and/or tightly linked genes control the trait phenotypes. Other QTL exhibited isolate-specificity and QTL × environment interactions. Map-based comparisons between QTL mapped in this study and Solanaceae resistance genes/QTL detected in other published studies revealed multiple cases of co-location, suggesting conservation of gene function.

The oomycete Pythium ultimum is homothallic, thus a single isolate completes the sexual stage in pure culture. It has been generally assumed that homothallic oomycetes are predominantly inbreeding. In P. ultimum, antheridia occasionally develop from hyphae not directly connected to the oogonium and appear to participate in fertilization, suggesting a possible mechanism for outcrossing. We have used molecular markers to confirm that outcrossing can occur between isolates of P. ultimum. Genetic markers based on randomly amplified polymorphic DNA (RAPD) and restriction fragment length polymorphisms (RFLP) were used to distinguish isolates in a collection of P. ultimum. Two isolates displaying a high level of polymorphism were mixed and placed on media which allows the development of the sexual stage. RAPD markers were used to screen single oospore progeny to identify potential hybrids between the two parental isolates. Subsequent self-fertilization of one putative F1 yielded a F2 population which demonstrated segregation and independent assortment of RAPD and RFLP markers. A similar strategy was used to show that an isolate which is incapable of producing oospores in pure culture can outcross when mixed with a homothallic isolate. These results suggest that other homothallic oomycetes may be capable of outcrossing, and sexual reproduction may, therefore, play an important role in the generation of variation in homothallic oomycetes.

Little is known about how gene expression variation within a given species controls phenotypic variation under different treatments or environments. Here, we surveyed the transcriptome response of seven diverse Arabidopsis thaliana accessions in response to two treatments: the presence and absence of exogenously applied salicylic acid (SA), an important signaling molecule in plant defense. A factorial experiment was conducted with three biological replicates per accession with and without applications of SA and sampled at three time points posttreatment. Transcript level data from Affymetrix ATH1 microarrays were analyzed on both per-gene and gene-network levels to detect expression level polymorphisms associated with SA response. Significant variation in transcript levels for response to SA was detected among the accessions, with relatively few genes responding similarly across all accessions and time points. Twenty-five of 54 defined gene networks identified from other microarray studies (pathogen-challenged Columbia [Col-0]) showed a significant response to SA in one or more accessions. A comparison of gene-network relationships in our data to the pathogen-challenged Col-0 data demonstrated a higher-order conservation of linkages between defense response gene networks. Cvi-1 and Mt-0 appeared to have globally different SA responsiveness in comparison to the other five accessions. Expression level polymorphisms for SA response were abundant at both individual gene and gene-network levels in the seven accessions, suggesting that natural variation for SA response is prevalent in Arabidopsis.

There is increasing interest in understanding the molecular basis of complex traits. Initially, the genetic dissection of quantitative traits involved measurements of gross phenotypes. Subsequently, specific physiological and developmental components of individual traits have been dissected. Most recently, the underlying mechanisms of inheritance have been studied through various approaches that are supported by modern technological and methodological advances, namely quantitative trait locus/loci (QTL) analysis (Mackay, 2001; Mauricio, 2001; Doerge, 2002) and mutant analysis (Rossant and Spence, 1998; Hughes et al., 2000) in genetics; genome sequencing (Jang et al., 1999; The Arabidopsis Genome Initiative, 2000; Mouse Genome Sequencing Consortium, 2002) and gene expression analysis (Duggan et al., 1999; Lipshutz et al., 1999) in genomics; and protein structure analysis (Service, 1999) and protein assay (Kodadek, 2001; MacBeath, 2002)

Background Gene expression microarrays allow the quantification of transcript accumulation for many or all genes in a genome. This technology has been utilized for a range of investigations, from assessments of gene regulation in response to genetic or environmental fluctuation to global expression QTL (eQTL) analyses of natural variation. Current analysis techniques facilitate the statistical querying of individual genes to evaluate the significance of a change in response, also known as differential expression. Since genes are also known to respond as groups due to their membership in networks, effective approaches are needed to investigate transcriptome variation as related to gene network responses. Results We describe a statistical approach that is capable of assessing higher-order a priori defined gene network response, as measured by microarrays. This analysis detected significant network variation between two Arabidopsis thaliana accessions, Bay-0 and Shahdara. By extending this approach, we were able to identify eQTLs controlling network responses for 18 out of 20 a priori-defined gene networks in a recombinant inbred line population derived from accessions Bay-0 and Shahdara. Conclusion This approach has the potential to be expanded to facilitate direct tests of the relationship between phenotypic trait and transcript genetic architecture. The use of a priori definitions for network eQTL identification has enormous potential for providing direction toward future eQTL analyses.

Expression microarrays hybridized with RNA can simultaneously provide both phenotypic (gene expression) and genotypic (marker) data. We developed two types of genetic markers from Affymetrix GeneChip expression data to generate detailed haplotypes for 148 recombinant inbred lines (RILs) derived from Arabidopsis thaliana accessions Bayreuth and Shahdara. Gene expression markers (GEMs) are based on differences in transcript levels that exhibit bimodal distributions in segregating progeny, while single feature polymorphism (SFP) markers rely on differences in hybridization to individual oligonucleotide probes. Unlike SFPs, GEMs can be derived from any type of DNA-based expression microarray. Our method identifies SFPs independent of a gene’s expression level. Alleles for each GEM and SFP marker were ascertained with GeneChip data from parental accessions as well as RILs; a novel algorithm for allele determination using RIL distributions capitalized on the high level of genetic replication per locus. GEMs and SFP markers provided robust markers in 187 and 968 genes, respectively, which allowed estimation of gene order consistent with that predicted from the Col-0 genomic sequence. Using microarrays on a population to simultaneously measure gene expression variation and obtain genotypic data for a linkage map will facilitate expression QTL analyses without the need for separate genotyping. We have demonstrated that gene expression measurements from microarrays can be leveraged to identify polymorphisms across the genome and can be efficiently developed into genetic markers that are verifiable in a large segregating RIL population. Both marker types also offer opportunities for massively parallel mapping in unsequenced and less studied species.

Differential gene expression controls variation in numerous plant traits, such as flowering time and plant/pest interactions, but little is known about the genomic distribution of the determinants of transcript levels and their associated variation. Affymetrix ATH1 GeneChip microarrays representing 22,810 genes were used to survey the transcriptome of seven Arabidopsis thaliana accessions in the presence and absence of exogenously applied salicylic acid (SA). These accessions encompassed ∼80% of the moderate- to high-frequency nucleotide polymorphisms in Arabidopsis. A factorial design, consisting of three biological replicates per accession for the two treatments at three time points (4, 28, and 52 hr post-treatment), and a total of 126 microarrays were used. Between any pair of Arabidopsis accessions, we detected on average 2234 genes (ranging from 1428 to 3334) that were significantly differentially expressed under the conditions of this experiment, using a split-plot analysis of variance. Upward of 6433 genes were differentially expressed between at least one pair of accessions. These results suggest that analysis of additional genetic, developmental, and environmental conditions may show that a significant fraction of the Arabidopsis genome is differentially expressed. Examination of sequence diversity demonstrated a significant positive association with diversity in gene expression.

Many plants of tropical or subtropical origin, such as tomato, suffer damage under chilling temperatures (under 10 degrees C but above 0 degrees C). An earlier study identified several quantitative trait loci (QTLs) for shoot turgor maintenance (stm) under root chilling in an interspecific backcross population derived from crossing chilling-susceptible cultivated tomato (Lycopersicon esculentum) and chilling-tolerant wild L. hirsutum. The QTL with the greatest phenotypic effect on stm was located in a 28 cM region on chromosome 9 (designated stm 9), and enhanced chilling-tolerance was conferred by the presence of the Lycopersicon hirsutum allele at this QTL. Here, near-isogenic lines (NILs) were used to verify the effect of stm 9, and recombinant sub-NILs were used to fine map its position. Replicated experiments were performed with NILs and sub-NILs in a refrigerated hydroponic tank in the greenhouse. Sub-NIL data was analyzed using least square means separations, marker-genotype mean t-tests, and composite interval mapping. A dominant QTL controlling shoot turgor maintenance under root chilling was confirmed on chromosome 9 using both NILs and sub-NILs. Furthermore, sub-NILs permitted localization of stm 9 to a 2.7 cM interval within the original 28 cM QTL region. If the presence of the L. hirsutum allele at stm 9 also confers chilling-tolerance in L. esculentum plants grown under field conditions, it has the potential to expand the geographic areas in which cultivated tomato can be grown for commercial production.

Random amplified polymorphic DNA (RAPD) and restriction fragment length polymorphism (RFLP) markers were used to assess the variability in tomato germplasm (Lycopersicon esculentum Mill.), which included 46 accessions from the following groups: vintage cultivars, modern cultivars, South American regional cultivars, and wild L. esculentum van cerasiforme. Two L. cheesmanii accessions served as an outgroup. The 48 accessions were monomorphic at 135 of the 215 RAPDs loci and 31 of the 48 RFLP loci that were assayed. Alleles were identified that distinguished the five groups and many of the cultivars. The frequency of polymorphic loci was low in vintage cultivars, with less than 2.8% of the loci being variable within the group. In contrast, introgression of wild germplasm into modern cultivars has increased the polymorphic loci to 11.6%, whereas within the group of regional cultivars linkage drag and outcrossing may be responsible for the further increase to 20.3%. These levels of genetic variability were lower in comparison to the 24.5% polymorphic loci of cerasiforme, a group that may contain unutilized desirable traits. The small genetic distance from the vintage cultivars to several of the widely distributed regionals and cerasiformes indicated that proximity of vintage cultivars in Latin America may have resulted in intercrossing of these materials with the wilder germplasm. RAPDs appear promising for both germplasm fingerprinting and as a predictor of genetic diversity for plant breeding applications.Key words: tomato, restriction fragment length polymorphism (RFLP), random amplified polymorphic DNA (RAPD), genetic distance.

The influence of cryogenic pollen storage on fruit set and seed production in tomato (Lycopersicon esculentum Mill.) was investigated. Flowers pollinated with pollen samples stored for 5 weeks at –80C, with or without 20 h precooling at 4C, had similar fruit set and number of viable seed per fruit as those pollinated with fresh pollen. Pollen samples, which were repeatedly cooled (–80C) and warmed (to 22 to 24C) for up to six cycles, continuously maintained the same viability as the fresh pollen. When cryogenically stored pollen of L. esculentum 2-837, LA359, LA3198, and LA3199 were used to pollinate LA359, the number of viable seed formed per fruit differed significantly. Results of this study suggest that pollen cryopreservation can be used successfully for tomato breeding and germplasm storage.

The inbred backcross (IBC) breeding method is being used to introgress genes controlling high fruit soluble solids from a wild tomato species (Lycopersicon cheesmanii f. minor) into a California processing tomato cultivar (Lycopersicon esculentum cv. UC204B). One IBC tomato population (i.e. P1: 106 lines) is being used to map quantitative trail loci (QTL) for soluble solids and other traits. A genetically related but independently generated IBC population (i.e. P2: 96 lines) is being used to lest the efficiency of QTL-linked RPLPs for indirect marker-assisted selection (MAS) to improve soluble solids. P1 was analyzed for fruit quality traits in a replicated field design over 2 years. Twelve P1 lines were significantly greater than UC204B for soluble solids and also had acceptable fruit weights and horticultural traits. All twelve lines have been publicly released for further breeding efforts. In P1. we have identified RPLP markers that have significant correlations to QTL. Some of these markers map to regions previously reported by other researchers to contain QTL for the same traits. We will use 70-80 markers spaced approximately 10-20 cM apart across the genome to screen PI and map QTL. The RPLP analyses are currently in progress. P2 was replicated for one year using the same field design as P1. and analyzed for the same traits. P2 will be screened with QTL-linked RFLPs identified in P1 to test the consistency of QTL locations between independently derived populations. P2 lines selected using RFLP data will be compared to P2 lines identified by classical selection indices. This will indicate if MAS for QTL is effective in a population (P2) genetically independent from the mapping population (P1).

High temperatures during flowering have been implicated in reducing seed set and fruit set in tomatoes (Lycopersicon esculentum). Pollen viability and vigor were studied by measuring in vitro germination and pollen tube development in pollinated pistils of four processing tomato cultivars under normal (25° C day/15° C night) and high (32° C day/23° C night) temperatures. Preliminary studies were carried out to determine the length of pollen tubes in styles collected in times ranging from 3 to 48 hours after pollination. Under normal temperatures the pollen tubes reach the end of the style between 12 and 18 hours. At high temperatures there are fewer pollen tubes moving through the style and the time to reach the end of the style is longer. In pollen vigor studies, crosses were made between pollen and pistils of plants grown under different temperature treatments, then pollinated pistils were collected at 4, 8 and 12 hours after pollination. There were differences in in vitro pollen germination percentage and pollen tube length in the pollinated pistils, suggesting that high temperatures act to slow down pollen activity.

Background Several high-throughput technologies can measure in parallel the abundance of many mRNA transcripts within a sample. These include the widely-used microarray as well as the more recently developed methods based on sequence tag abundances such as the Massively Parallel Signature Sequencing (MPSS) technology. A comparison of microarray and MPSS technologies can help to establish the metrics for data comparisons across these technology platforms and determine some of the factors affecting the measurement of mRNA abundances using different platforms. Results We compared transcript abundance (gene expression) measurement data obtained using Affymetrix and Agilent microarrays with MPSS data. All three technologies were used to analyze the same set of mRNA samples; these samples were extracted from various wild type Arabidopsis thaliana tissues and floral mutants. We calculated correlations and used clustering methodology to compare the normalized expression data and expression ratios across samples and technologies. Abundance expression measurements were more similar between different samples measured by the same technology than between the same sample measured by different technologies. However, when expression ratios were employed, samples measured by different technologies were found to cluster together more frequently than with abundance expression levels. Furthermore, the two microarray technologies were more consistent with each other than with MPSS. We also investigated probe-position effects on Affymetrix data and tag-position effects in MPSS. We found a similar impact on Affymetrix and MPSS measurements, which suggests that these effects were more likely a characteristic of the RNA sample rather than technology-specific biases. Conclusion Comparisons of transcript expression ratios showed greater consistency across platforms than measurements of transcript abundance. In addition, for measurements based on abundances, technology differences can mask the impact of biological differences between samples and tissues.

Background Nucleotide binding site-leucine rich repeat (NBS-LRR)-encoding genes comprise the largest class of plant disease resistance genes. The 149 NBS-LRR-encoding genes and the 58 related genes that do not encode LRRs represent approximately 0.8% of all ORFs so far annotated in Arabidopsis ecotype Col-0. Despite their prevalence in the genome and functional importance, there was little information regarding expression of these genes. Results We analyzed the expression patterns of ~170 NBS-LRR-encoding and related genes in Arabidopsis Col-0 using multiple analytical approaches: expressed sequenced tag (EST) representation, massively parallel signature sequencing (MPSS), microarray analysis, rapid amplification of cDNA ends (RACE) PCR, and gene trap lines. Most of these genes were expressed at low levels with a variety of tissue specificities. Expression was detected by at least one approach for all but 10 of these genes. The expression of some but not the majority of NBS-LRR-encoding and related genes was affected by salicylic acid (SA) treatment; the response to SA varied among different accessions. An analysis of previously published microarray data indicated that ten NBS-LRR-encoding and related genes exhibited increased expression in wild-type Landsberg erecta (Ler) after flagellin treatment. Several of these ten genes also showed altered expression after SA treatment, consistent with the regulation of R gene expression during defense responses and overlap between the basal defense response and salicylic acid signaling pathways. Enhancer trap analysis indicated that neither jasmonic acid nor benzothiadiazole (BTH), a salicylic acid analog, induced detectable expression of the five NBS-LRR-encoding genes and one TIR-NBS-encoding gene tested; however, BTH did induce detectable expression of the other TIR-NBS-encoding gene analyzed. Evidence for alternative mRNA polyadenylation sites was observed for many of the tested genes. Evidence for alternative splicing was found for at least 12 genes, 11 of which encode TIR-NBS-LRR proteins. There was no obvious correlation between expression pattern, phylogenetic relationship or genomic location of the NBS-LRR-encoding and related genes studied. Conclusion Transcripts of many NBS-LRR-encoding and related genes were defined. Most were present at low levels and exhibited tissue-specific expression patterns. Expression data are consistent with most Arabidopsis NBS-LRR-encoding and related genes functioning in plant defense responses but do not preclude other biological roles.