Theresa M. Fulton

Microprep protocol for extraction of DNA from tomato and other herbaceous plants

T he extraction of DNA from plant tissue is a critical and often very time-consuming step in many plant molecular biology procedures. This is especially true for studies of molecular genetics, QTLs, or molecular-marker-based breeding where hundreds or even thousands of plant samples need to be analyzed in a short period of time. Many protocols are laborious and are limited by the need for large amounts of plant tissue. Based on the methods originally described by Murray et al. (1980), we have developed a procedure in our laboratory that maximizes the number of plant samples one person can extract and that yields sufficient DNA for 50 to 100 PCR reactions or two to four Southern blots. The use of very small, new leaves makes it possible to extract DNA from seedlings only one to three weeks old, reducing the need for large amounts of greenhouse space. The entire procedure can be done in 1.5-mL microcentrifuge tubes, eliminating the need for large centrifuges. Using this procedure, one person can isolate DNA from several hundred plants per day. Materials and Solut ions

Advanced backcross QTL analysis in a cross between an elite processing line of tomato and its wild relative L. pimpinellifolium.

Approximately 170 BC2 plants from a cross between an elite processing inbred (recurrent parent) and the wild species Lycopersicon pimpinellifolium LA1589 (donor parent) were analyzed with segregating molecular markers covering the entire tomato genome. Marker data were used to identify QTLs controlling a battery of horticultural traits measured on BC2F1 and BC3 families derived from the BC2 individuals. Despite its overall inferior appearance, L. pimpinellifolium was shown to possess QTL alleles capable of enhancing most traits important in processing tomato production. QTL-NIL lines, containing specific QTLs modifying fruit size and shape, were subsequently constructed and shown to display the transgressive phenotypes predicted from the original BC2 QTL analysis. The potential of exploiting unadapted and wild germplasm via advanced backcross QTL analysis for the enhancement of elite crop varieties is discussed.

Identification, Analysis, and Utilization of Conserved Ortholog Set Markers for Comparative Genomics in Higher Plants

We have screened a large tomato EST database against the Arabidopsis genomic sequence and report here the identification of a set of 1025 genes (referred to as a conserved ortholog set, or COS markers) that are single or low copy in both genomes (as determined by computational screens and DNA gel blot hybridization) and that have remained relatively stable in sequence since the early radiation of dicotyledonous plants. These genes were annotated, and a large portion could be assigned to putative functional categories associated with basic metabolic processes, such as energy-generating processes and the biosynthesis and degradation of cellular building blocks. We further demonstrate, through computational screens (e.g., against a Medicago truncatula database) and direct hybridization on genomic DNA of diverse plant species, that these COS markers also are conserved in the genomes of other plant families. Finally, we show that this gene set can be used for comparative mapping studies between highly divergent genomes such as those of tomato and Arabidopsis. This set of COS markers, identified computationally and experimentally, may further studies on comparative genomes and phylogenetics and elucidate the nature of genes conserved throughout plant evolution.

QTL analysis of an advanced backcross of Lycopersicon peruvianum to the cultivated tomato and comparisons with QTLs found in other wild species

Abstract A BC3 population previously developed from a backcross of Lycopersicon peruvianum, a wild relative of tomato, into the cultivated variety L. esculentum was analyzed for QTLs. Approximately 200 BC4 families were scored for 35 traits in four locations worldwide. One hundred and sixty-six QTLs were detected for 29 of those traits. For more than half of those 29 traits at least 1 QTL was detected for which the presence of the wild allele was associated with an agronomically beneficial effect despite the inferior phenotype of the wild parent. Eight QTLs for fruit weight could be followed through the BC2, BC3, and BC4, generations, supporting the authenticity of these QTLs. Comparisons were made between the QTLs found in this study and those found in studies involving two other wild species; the results showed that while some of these QTLs can be presumed to be allelic, most of the QTLs detected in this study are ones not previously discovered.

Advanced backcross QTL analysis of a Lycopersicon esculentum ×Lycopersicon parviflorum cross

Abstract Lycopersicon parviflorum is a sexually compatible, wild tomato species which has been largely unutilized in tomato breeding. The Advanced Backcross QTL (AB-QTL) strategy was used to explore this genome for QTLs affecting traits of agronomic importance in an interspecific cross between a tomato elite processing inbred, Lycopersicon esculentum E6203, and the wild species L. parviflorum (LA2133). A total of 170 BC2 plants were genotyped by means of 133 genetic markers (131 RFLPs; one PCR-based marker, I-2, and one morphological marker, u, uniform ripening). Approximately 170 BC3 families were grown in replicated field trials, in California, Spain and Israel, and were scored for 30 horticultural traits. Significant putative QTLs were identified for all traits, for a total of 199 QTLs, ranging from 1 to 19 QTLs detected for each trait. For 19 (70%) traits (excluding traits for which effects of either direction are not necessarily favourable or unfavourable) at least one QTL was identified for which the L. parviflorum allele was associated with an agronomically favourable effect, despite the overall inferior phenotype of the wild species.

Quantitative trait loci (QTL) affecting sugars, organic acids and other biochemical properties possibly contributing to flavor, identified in four advanced backcross populations of tomato

Although the Advanced Backcross strategy has proven very useful for QTL detection in tomato, it has been used mainly in identifying QTL for agronomic traits such as yield, color, etc. Tomato flavor is an important quality characteristic, yet it has been difficult to assess flavor or traits that affect it. In this study the AB-QTL strategy was applied to four advanced backcross populations to identify QTL for biochemical properties that may contribute to the flavor of processed tomatoes, such as sugars and organic acids. A total of 222 QTL were identified for 15 traits, including flavor as assessed by a taste panel. Correlations of certain biochemicals with flavor and possible methods of assessing and improving flavor are discussed. In particular, QTL with very significant effects associated with the ratio of sugars/glutamic acid, a trait highly correlated with improved flavor, have been identified as good targets for future work in improving the flavor of tomatoes.

Advanced backcross QTL analysis of a Lycopersicon esculentum × L. pennellii cross and identification of possible orthologs in the Solanaceae

In this study, the advanced backcross QTL (AB-QTL) mapping strategy was used to identify loci for yield, processing and fruit quality traits in a population derived from the interspecific cross Lycopersicon esculentum E6203 × Lycopersicon pennellii accession LA1657. A total of 175 BC2 plants were genotyped with 150 molecular markers and BC2F1 plots were grown and phenotyped for 25 traits in three locations in Israel and California, U.S.A. A total of 84 different QTLs were identified, 45% of which have been possibly identified in other wild-species-derived populations of tomato. Moreover, three fruit-weight/size and shape QTLs (fsz2b.1, fw3.1/fsz3.1 and fs8.1) appear to have putative orthologs in the related solanaceous species, pepper and eggplant. For the 23 traits for which allelic effects could be deemed as favorable or unfavorable, 26% of the identified loci had L. pennellii alleles that enhanced the performance of the elite parent. Alleles that could be targeted for further introgression into cultivated tomato were also identified.

Introgression and DNA marker analysis of Lycopersicon peruvianum, a wild relative of the cultivated tomato, into Lycopersicon esculentum, followed through three successive backcross generations

Abstract Segregation of the Lycopersicon peruvianum genome was followed through three generations of backcrossing to the cultivated tomato L. esculentum cv ‘E6203’ using molecular markers. Thirteen BC1 plants were genotyped with 113 markers, 67 BC2 plants with 84 markers, and finally 241 BC3 plants were genotyped with 177 markers covering the entire genome and a BC3 map constructed. Several segments of the genome, including parts of chromosomes 3, 4, 6, and 10, quickly became fixed for esculentum alleles, possibly due to sterility problems encountered in the BC1. Observed overall heterozygosity and chromosome segment lengths at each generation were very near the expected theoretical values. Markers located near the top telomeric region of chromosome 9 showed segregation highly skewed towards the wild allele through all generations, suggesting the presence of a gamete promoter gene. One markers, TG9, mapped to a new position on chromosome 9, implying an intrachromosomal translocation event. Despite the great genetic distance between the two parents, overall recombination was only 25% less than that observed in a previous tomato cross, indicating that L. peruvianum genes may be more readily introgressed into cultivated germplasm than originally believed.

Dissecting quantitative trait variation : examples from the tomato

Quantitative variation is prevalent in nature and in plant breeding. However, it wasn’t until the past two decades that it became technically feasible to begin probing the individual loci and genetic causes of natural, quantitative trait variation. Understanding the basis of quantitative variation is key not only to understanding evolution, but also in developing new insights and methodologies to improve traits such as yield, resistance to abiotic stress, and nutritional quality. In plants, tomato was one of the first species in which significant inroads were made into understanding the genetic and molecular bases of quantitative trait variation. Summarized herein are some of the lessons learned from tomato with regards to quantitative trait variation.

Advanced backcross QTL analysis: a method for the systematic use of exotic germplasm in the improvement of crop quality

Most traits of agronomic interest, including those related to quality, are polygenically inherited. Exotic germplasm represents a valuable and unique source of genetic variation, but it has rarely been used for the genetic improvement of quantitative traits. We have efficiently screened for new and beneficial quantitative trait loci (QTL) able to improve the agronomic performance of elite varieties. We refer to this method as Advanced Backcross QTL (AB-QTL) analysis. The strategy has been tested in tomato over the past 5 years (using 5 different wild species donors) and in rice for 2 years (one wild species donor). In each case, QTL from the wild species have been identified which are able of substantially increasing quality or productivity of the cultivated species. We have created lines, in the genetic background of an elite processing tomato cultivar, which contain wild QTL alleles for several quality attributes important for the tomato processing industry including soluble solids content, fruit colour, viscosity and firmness. These lines have been subjected to replicated field trials in five locations world-wide. A number of these lines significantly outperformed the elite variety by 22%, 33% and 48% for soluble solids, fruit colour and yield, respectively.