Functional relationships of <Emphasis Type="Italic">phytoene synthase 1</Emphasis> alleles on chromosome 7A controlling flour colour variation in selected Australian wheat genotypes

Thermoinhibition, or failure of seeds to germinate when imbibed at warm temperatures, can be a significant problem in lettuce (Lactuca sativa L.) production. The reliability of stand establishment would be improved by increasing the ability of lettuce seeds to germinate at high temperatures. Genes encoding germination- or dormancy-related proteins were mapped in a recombinant inbred line population derived from a cross between L. sativa cv. Salinas and L. serriola accession UC96US23. This revealed several candidate genes that are located in the genomic regions containing quantitative trait loci (QTLs) associated with temperature and light requirements for germination. In particular, LsNCED4, a temperature-regulated gene in the biosynthetic pathway for abscisic acid (ABA), a germination inhibitor, mapped to the center of a previously detected QTL for high temperature germination (Htg6.1) from UC96US23. Three sets of sister BC₃S₂ near-isogenic lines (NILs) that were homozygous for the UC96US23 allele of LsNCED4 at Htg6.1 were developed by backcrossing to cv. Salinas and marker-assisted selection followed by selfing. The maximum temperature for germination of NIL seed lots with the UC96US23 allele at LsNCED4 was increased by 2–3°C when compared with sister NIL seed lots lacking the introgression. In addition, the expression of LsNCED4 was two- to threefold lower in the former NIL lines as compared to expression in the latter. Together, these data strongly implicate LsNCED4 as the candidate gene responsible for the Htg6.1 phenotype and indicate that decreased ABA biosynthesis at high imbibition temperatures is a major factor responsible for the increased germination thermotolerance of UC96US23 seeds.     

Intracellular pH sensing is altered by plasma membrane PIP aquaporin co-expression

Seeds of most cultivated varieties of lettuce (Lactuca sativa L.) fail to germinate at warm temperatures (i.e., above 25–30°C). Seed priming (controlled hydration followed by drying) alleviates this thermoinhibition by increasing the maximum germination temperature. We conducted a quantitative trait locus (QTL) analysis of seed germination responses to priming using a recombinant inbred line (RIL) population derived from a cross between L. sativa cv. Salinas and L. serriola accession UC96US23. Priming significantly increased the maximum germination temperature of the RIL population, and a single major QTL was responsible for 47% of the phenotypic variation due to priming. This QTL collocated with Htg6.1, a major QTL from UC96US23 associated with high temperature germination capacity. Seeds of three near-isogenic lines (NILs) carrying an Htg6.1 introgression from UC96US23 in a Salinas genetic background exhibited synergistic increases in maximum germination temperature in response to priming. LsNCED4, a gene encoding a key enzyme (9-cis-epoxycarotinoid dioxygenase) in the abscisic acid biosynthetic pathway, maps precisely with Htg6.1. Expression of LsNCED4 after imbibition for 24 h at high temperature was greater in non-primed seeds of Salinas, of a second cultivar (Titan) and of NILs containing Htg6.1 compared to primed seeds of the same genotypes. In contrast, expression of genes encoding regulated enzymes in the gibberellin and ethylene biosynthetic pathways (LsGA3ox1 and LsACS1, respectively) was enhanced by priming and suppressed by imbibition at elevated temperatures. Developmental and temperature regulation of hormonal biosynthetic pathways is associated with seed priming effects on germination temperature sensitivity.     

Genetic variation for lettuce seed thermoinhibition is associated with temperature-sensitive expression of abscisic Acid, gibberellin, and ethylene biosynthesis, metabolism, and response genes

Lettuce (Lactuca sativa ‘Salinas’) seeds fail to germinate when imbibed at temperatures above 25°C to 30°C (termed thermoinhibition). However, seeds of an accession of Lactuca serriola (UC96US23) do not exhibit thermoinhibition up to 37°C in the light. Comparative genetics, physiology, and gene expression were analyzed in these genotypes to determine the mechanisms governing the regulation of seed germination by temperature. Germination of the two genotypes was differentially sensitive to abscisic acid (ABA) and gibberellin (GA) at elevated temperatures. Quantitative trait loci associated with these phenotypes colocated with a major quantitative trait locus (Htg6.1) from UC96US23 conferring germination thermotolerance. ABA contents were elevated in Salinas seeds that exhibited thermoinhibition, consistent with the ability of fluridone (an ABA biosynthesis inhibitor) to improve germination at high temperatures. Expression of many genes involved in ABA, GA, and ethylene biosynthesis, metabolism, and response was differentially affected by high temperature and light in the two genotypes. In general, ABA-related genes were more highly expressed when germination was inhibited, and GA- and ethylene-related genes were more highly expressed when germination was permitted. In particular, LsNCED4, a gene encoding an enzyme in the ABA biosynthetic pathway, was up-regulated by high temperature only in Salinas seeds and also colocated with Htg6.1. The temperature sensitivity of expression of LsNCED4 may determine the upper temperature limit for lettuce seed germination and may indirectly influence other regulatory pathways via interconnected effects of increased ABA biosynthesis.     

The inheritance of resistance to Verticillium wilt caused by race 1 isolates of <Emphasis Type="Italic">Verticillium dahliae</Emphasis> in the lettuce cultivar La Brillante

Verticillium wilt of lettuce caused by Verticillium dahliae can cause severe economic damage to lettuce producers. Complete resistance to race 1 isolates is available in Lactuca sativa cultivar (cv.) La Brillante and understanding the genetic basis of this resistance will aid development of new resistant cultivars. F₁ and F₂ families from crosses between La Brillante and three iceberg cultivars as well as a recombinant inbred line population derived from L. sativa cv. Salinas 88 × La Brillante were evaluated for disease incidence and disease severity in replicated greenhouse and field experiments. One hundred and six molecular markers were used to generate a genetic map from Salinas 88 × La Brillante and for detection of quantitative trait loci. Segregation was consistent with a single dominant gene of major effect which we are naming Verticillium resistance 1 (Vr1). The gene described large portions of the phenotypic variance (R ² = 0.49–0.68) and was mapped to linkage group 9 coincident with an expressed sequence tag marker (QGD8I16.yg.ab1) that has sequence similarity with the Ve gene that confers resistance to V. dahliae race 1 in tomato. The simple inheritance of resistance indicates that breeding procedures designed for single genes will be applicable for developing resistant cultivars. QGD8I16.yg.ab1 is a good candidate for functional analysis and development of markers suitable for marker-assisted selection.     

Isolation and mapping of resistance gene analogs from the Avena strigosa genome

Degenerate primers based on conserved regions of the nucleotide binding site (NBS) domain (encoded by the largest group of cloned plant disease resistance genes) were used to isolate a set of 15 resistance gene analogs (RGA) from the diploid species Avena strigosa Schreb. These were grouped into seven classes on the basis of 60% or greater nucleic acid sequence identity. Representative clones were used for genetic mapping in diploid and hexaploid oats. Two RGAs were mapped at two loci of the linkage group AswBF belonging to the A. strigosa × A. wiestii Steud map, and ten RGAs were mapped at 15 loci in eight linkage groups belonging to the A. byzantina C. Koch cv. Kanota × A. sativa L. cv. Ogle map. A similar approach was used for targeting genes encoding receptor-like kinases. Three different sequences were obtained and mapped to two linkage groups of the hexaploid oat map. Associations were explored between already known disease resistance loci mapped in different populations and the RGAs. Molecular markers previously linked to crown rust and barley yellow dwarf resistance genes or quantitative trait loci were found in the Kanota × Ogle map linked to RGAs at a distance ranging from 0 cM to 20 cM. Homoeologous RGAs were found to be linked to loci either conferring resistance to different isolates of the same pathogen or to different pathogens. This suggests that these RGAs identify genome regions containing resistance gene clusters.     

Cloning and analysis of the resistance gene fragments from silverleaf sunflower <Emphasis Type="Italic">Helianthus agrophyllus</Emphasis>

Using a combination of degenerate primers designed from the NBS domains of the resistance genes, amplification and subsequent cloning of the resistance gene fragments from sunflower (Helianthus agrophyllus) was conducted. Sequences of cloned PCR products differed from one another and displayed homology to NBS domain fragments of the already known plant resistance genes, as well as to the analogous genes from different classes. The highest homology was shown to the NBS domain regions of cultivated sunflower and the other members of the family Compositae. Two cloned fragments had open reading frames, while the other sequences carried stop codons and seemed to belong to pseudogenes. Amino acid sequences of Helianthus agrophyllus analyzed contained conservative regions typical of NBS domains of the resistance gene products.     

Disease resistance gene homologs correlate with disease resistance loci of Arabidopsis thaliana

The disease resistance genes RPS2 of Arabidopsis and N of tobacco, among other recently cloned resistance genes, share several conserved sequences. Degenerate oligonucleotide primers, based on conserved sequences in the nucleotide binding site (NBS) and a weak hydrophobic domain of RPS2 and N, were used to amplify homologous sequences from Arabidopsis thaliana. Amplification products were obtained that were similar in sequence to the disease resistance genes RPS2, RPM1, N and L6. The Arabidopsis CIC-YAC library was used to identify the position of the disease resistance homologs on the Arabidopsis genome. Their map positions could be correlated with the disease resistance loci RPS5, RAC1, RPP9, CAR1, RPP7, RPW2, RPP1, RPP10, RPP14, RPP5, RPP4, RPS2, RPW6, HRT, RPS4, RPP8, RPP21, RPP22, RPP23, RPP24 and TTR1. This method was therefore not only successful in the identification of sequences located within gene clusters that are involved in disease resistance, but could also contribute to the cloning of disease resistance genes from Arabidopsis.     

Quantitative trait loci associated with seed and seedling traits in Lactuca

Seed and seedling traits related to germination and stand establishment are important in the production of cultivated lettuce (Lactuca sativa L.). Six seed and seedling traits segregating in a L. sativa cv. Salinas x L. serriola recombinant inbred line population consisting of 103 F8 families revealed a total of 17 significant quantitative trait loci (QTL) resulting from three seed production environments. Significant QTL were identified for germination in darkness, germination at 25 and 35°C, median maximum temperature of germination, hypocotyl length at 72 h post-imbibition, and plant (seedling) quality. Some QTL for germination and early seedling growth characteristics were co-located, suggestive of pleiotropic loci regulating these traits. A single QTL (Htg6.1) described 25 and 23% of the total phenotypic variation for high temperature germination in California- and Netherlands-grown populations, respectively, and was significant between 33 and 37°C. Additionally, Htg6.1 showed significant epistatic interactions with other Htg QTL and a consistent effect across all the three seed production environments. L. serriola alleles increased germination at these QTL. The estimate of narrow-sense heritability (h²) of Htg6.1 was 0.84, indicating potential for L. serriola as a source of germination thermotolerance for lettuce introgression programs.     

QTLs for shelf life in lettuce co-locate with those for leaf biophysical properties but not with those for leaf developmental traits

Developmental and biophysical leaf characteristics that influence post-harvest shelf life in lettuce, an important leafy crop, have been examined. The traits were studied using 60 informative F9 recombinant inbed lines (RILs) derived from a cross between cultivated lettuce (Lactuca sativa cv. Salinas) and wild lettuce (L. serriola acc. UC96US23). Quantitative trait loci (QTLs) for shelf life co-located most closely with those for leaf biophysical properties such as plasticity, elasticity, and breakstrength, suggesting that these are appropriate targets for molecular breeding for improved shelf life. Significant correlations were found between shelf life and leaf size, leaf weight, leaf chlorophyll content, leaf stomatal index, and epidermal cell number per leaf, indicating that these pre-harvest leaf development traits confer post-harvest properties. By studying the population in two contrasting environments in northern and southern Europe, the genotype by environment interaction effects of the QTLs relevant to leaf development and shelf life were assessed. In total, 107 QTLs, distributed on all nine linkage groups, were detected from the 29 traits. Only five QTLs were common in both environments. Several areas where many QTLs co-located (hotspots) on the genome were identified, with relatively little overlap between developmental hotspots and those relating to shelf life. However, QTLs for leaf biophysical properties (breakstrength, plasticity, and elasticity) and cell area correlated well with shelf life, confirming that the ideal ideotype lettuce should have small cells with strong cell walls. The identification of QTLs for leaf development, strength, and longevity will lead to a better understanding of processability at a genetic and cellular level, and allow the improvement of salad leaf quality through marker-assisted breeding.     

The Response of Lettuce Cultivars against Two Lettuce mosaic virus isolates,One Able to Systemically Infect the Resistant Cultivar Salinas 88

The response of seven lettuce cultivars to two geographically different Lettuce mosaic virus (LMV) isolates (LMV‐A, LMV‐T) was statistically evaluated based on infection rate, virus accumulation and symptom severity in different time trials. LMV‐A is characterized by the ability to systemically infect cv. Salinas 88 (mo1²‐carrying resistant cultivar), and inducing mild mosaic symptoms. Among lettuce cultivars, Varamin (a native cultivar) similar to cv. Salinas showed the most susceptibility to both LMV isolates, whereas another native cultivar, Varesh, was tolerant to the virus with minimal viral accumulation and symptom scores, significantly different from other cultivars at P < 0.05. LMV‐A systemically infects all susceptible lettuce cultivars more rapidly and at a higher rate than LMV‐T. This isolate accumulated in lettuce cultivars at a significantly higher level, determined by semiquantitative ELISA and induced more severe symptoms than LMV‐T isolate at 21 dpi. This is the first evidence for a LMV isolate with ability to systemically infect mo1²‐carrying resistant cultivar of lettuce from Iran. In this study, accumulation level of LMV showed statistically meaningful positive correlation with symptom severity on lettuce plants. Based on the results, three evaluated parameters differed considerably by lettuce cultivar and virus isolate.     

Characterisation and genetic mapping of resistance and defence gene analogs in cocoa (Theobroma cacao L.)

Disease resistance and defence gene analog (RGA/DGA) sequences were isolated in cocoa using a PCR approach with degenerate primers designed from conserved domains of plant resistance and defence genes: the NBS (nucleotide binding site) motif present in a number of resistance genes such as the tobacco N, sub-domains of plant serine/threonine kinases such as the Pto tomato gene, and conserved domains of two defence gene families: pathogenesis-related proteins (PR) of classes 2 and 5. Nucleotide identity between thirty six sequences isolated from cocoa and known resistance or defence genes varied from 58 to 80%. Amino acid sequences translated from corresponding coding sequences produced sequences without stop codons, except for one NBS –like sequence. Most of the RGAs could be mapped on the cocoa genome and three clusters of genes could be observed : NBS-like sequences clustered in two regions located on chromosomes 7 and 10, Pto-like sequences mapped in five genome regions of which one, located on chromosome 4, corresponded to a cluster of five different sequences. PR2-like sequences mapped in two regions located on chromosome 5 and 9 respectively. An enrichment of the genetic map with microsatellite markers allowed us to identify several co-localisations of RGAs, DGAs and QTL for resistance to Phytophthora detected in several progenies, particularly on chromosome 4 where a cluster of Pto-like sequences and 4 QTL for resistance to Phytophthora were observed. Many other serious diseases affect cocoa and the candidate genes, isolated in this study, could be of broader interest in cocoa disease management.     

Identification and comparison of natural rubber from two Lactuca species

Renewed interest in the identification of alternative sources of natural rubber to Hevea brasiliensis has focused on the Compositae family. In our search for Compositae models for rubber synthesis, we extracted latex from stems of two lettuce species: Lactuca serriola, prickly lettuce, and Lactuca sativa cv. Salinas, crisphead lettuce. Both species contained cis-1,4-polyisoprene rubber in the dichloromethane-soluble portions of their latex, and sesquiterpene lactones in their acetone-soluble portions. The rubber from both species and their progeny had molecular weights in excess of 1,000,000g/mol, and polydispersity values of 1.1. Rubber transferase activity was detected across a range of farnesyl diphosphate initiator concentrations, with decreased activity as initiator concentrations exceeded putative saturation. These results add lettuce to the short list of plant species that produce high molecular weight rubber in their latex. Due to the genomic and agronomic resources available in lettuce species, they provide the opportunity for further dissection of natural rubber biosynthesis in plants.     

Identification of RFLP and NBS/PK profiling markers for disease resistance loci in genetic maps of oats

Two of the domains most widely shared among R genes are the nucleotide binding site (NBS) and protein kinase (PK) domains. The present study describes and maps a number of new oat resistance gene analogues (RGAs) with two purposes in mind: (1) to identify genetic regions that contain R genes and (2) to determine whether RGAs can be used as molecular markers for qualitative loci and for QTLs affording resistance to Puccinia coronata. Such genes have been mapped in the diploid A. strigosa × A. wiestii (Asw map) and the hexaploid MN841801-1 × Noble-2 (MN map). Genomic and cDNA NBS-RGA probes from oat, barley and wheat were used to produce RFLPs and to obtain markers by motif-directed profiling based on the NBS (NBS profiling) and PK (PK profiling) domains. The efficiency of primers used in NBS/PK profiling to amplify RGA fragments was assessed by sequencing individual marker bands derived from genomic and cDNA fragments. The positions of 184 markers were identified in the Asw map, while those for 99 were identified in the MN map. Large numbers of NBS and PK profiling markers were found in clusters across different linkage groups, with the PK profiling markers more evenly distributed. The location of markers throughout the genetic maps and the composition of marker clusters indicate that NBS- and PK-based markers cover partly complementary regions of oat genomes. Markers of the different classes obtained were found associated with the two resistance loci, PcA and R-284B-2, mapped on Asw, and with five out of eight QTLs for partial resistance in the MN map. 53 RGA-RFLPs and 187 NBS/PK profiling markers were also mapped on the hexaploid map A. byzantina cv. Kanota × A. sativa cv. Ogle. Significant co-localization was seen between the RGA markers in the KO map and other markers closely linked to resistance loci, such as those for P. coronata and barley yellow dwarf virus (Bydv) that were previously mapped in other segregating populations.     

PCR-based approach for mining of resistant gene analogues in taro (Colocasia esculenta)

Primers based on the conserved motifs were used to isolate nucleotide-binding sites (NBS) type sequences in taro (Colocasia esculenta). Cloning and sequencing identified three taro NBS-type sequences called resistance gene analogues (RGAs) that depicted similarity to other cloned RGA sequences. The deduced amino acid sequences of the RGAs detected the presence of conserved domains, viz. P-loop, categorising them with the NBS–leucine-rich repeat class gene family. Phylogenetic characterisation of the taro RGAs along with RGAs of other plant species grouped them with the non-toll interleukin receptor subclasses of the NBS sequences. The isolation and characterisation of taro RGAs have been reported for the first time in this study. This will provide a starting point towards characterisation of candidate resistance genes in taro and can act as a reference guide for future studies.     

Identification and characterization of NBS-encoding disease resistance genes in Lotus japonicus

Nucleotide-binding site (NBS) disease resistance genes play an important role in defending plants from a range of pathogens and insect pests. Consequently, NBS-encoding genes have been the focus of a number of recent studies in molecular disease resistance breeding programs. However, little is known about NBS-encoding genes in Lotus japonicus. In this study, a full set of disease resistance (R) candidate genes encoding NBS from the complete genome of L. japonicus was identified and characterized using structural diversity, chromosomal locations, conserved protein motifs, gene duplications, and phylogenetic relationships. Distinguished by N-terminal motifs and leucine-rich repeat motifs (LRRs), 92 regular NBS genes of 158 NBS-coding sequences were classified into seven types: CC-NBS-LRR, TIR-NBS-LRR, NBS-LRR, CC-NBS, TIR-NBS, NBS, and NBS-TIR. Phylogenetic reconstruction of NBS-coding sequences revealed many NBS gene lineages, dissimilar from results for Arabidopsis but similar to results from research on rice. Conserved motif structures were also analyzed to clarify their distribution in NBS-encoding gene sequences. Moreover, analysis of the physical locations and duplications of NBS genes showed that gene duplication events of disease resistance genes were lower in L. japonicus than in rice and Arabidopsis, which may contribute to the relatively fewer NBS genes in L. japonicus. Sixty-three NBS-encoding genes with clear conserved domain character were selected to check their gene expression levels by semi-quantitative RT-PCR. The results indicated that 53 of the genes were most likely to be acting as the active genes, and exogenous application of salicylic acid improved expression of most of the R genes.     

Cloning and analysis of the resistance gene fragments from silverleaf sunflower Helianthus agrophyllus

Using a combination of degenerate primers designed from the NBS domains of the resistance genes, amplification and subsequent cloning of the resistance gene fragments from sunflower (Helianthus agrophyllus) was conducted. Sequences of cloned PCR products differed from one another and displayed homology to NBS domain fragments of the already known plant resistance genes, as well as to the analogous genes from different classes. The highest homology was shown to the NBS domain regions of cultivated sunflower and the other members of the family Compositae. Two cloned fragments had open reading frames, while the other sequences carried stop codons and seemed to belong to pseudogenes. Amino acid sequences of Helianthus agrophyllus analyzed contained conservative regions typical of NBS domains of the resistance gene products.     

Cloning and characterisation of a family of disease resistance gene analogs from wheat and barley

 The most common class of plant disease resistance (R) genes cloned so far belong to the NBS-LRR group which contain nucleotide-binding sites (NBS) and a leucine-rich repeat (LRR). Specific primer sequences derived from a previously isolated NBS-LRR sequence at the Cre3 locus, which confers resistance to cereal cyst nematode (CCN) in wheat (Triticum aestivum L.) were used in isolating a family of resistance gene analogs (RGA) through a polymerase chain reaction (PCR) cloning approach. The cloning, analysis and genetic mapping of a family of RGAs from wheat (cv ‘Chinese Spring’) and barley (Hordeum vulgare L. cvs ‘Chebec’ and ‘Harrington’) are presented. The wheat and barley RGAs contain other conserved motifs present in known R genes from other plants and share between 55–99% amino acid sequence identity to the NBS-LRR sequence at the Cre3 locus. Phylogenetic analysis of the RGAs with other cloned R genes and RGAs from various plant species indicate that they belong to a superfamily of NBS-containing genes. Two of the barley derived RGAs were mapped onto loci on chromosomes 2H (2), 5H (7) and 7H (1) using barley doubled haploid (DH) mapping populations. Some of these loci identified are associated with regions carrying resistance to CCN and corn leaf aphid. Received: 6 January 1998 / Accepted: 1 April 1998     

Cloning and characterization of NBS-LRR class resistance-gene candidate sequences in citrus

Numerous disease resistance gene-like DNA sequences were cloned from an intergeneric hybrid of Poncirus and Citrus, using a PCR approach with degenerate primers designed from conserved NBS (nucleotide-binding site) motifs found in a number of plant resistance genes. Most of the cloned genomic sequences could be translated into polypeptides without stop codons, and the sequences contained the characteristic motifs found in the NBS-LRR class of plant disease resistance genes. Pairwise comparisons of these polypeptide sequences indicated that they shared various degrees of amino-acid identity and could be grouped into ten classes (RGC1–RGC10). When the sequences of each class were compared with known resistance-gene sequences, the percentage of amino-acid identity ranged from 18.6% to 48%. To facilitate genetic mapping of these sequences and to assess their potential linkage relationship with disease resistance genes in Poncirus, we developed CAPS markers by designing specific primers based on the cloned DNA sequences and subsequently identifying restriction enzymes that revealed genetic polymorphisms. Three of the amplified DNA fragment markers (designated as 18P33a, Pt9a, and Pt8a) were associated with the citrus tristeza virus resistance gene (Ctv), and one fragment (Pt8a) was associated with the major gene responsible for the citrus nematode resistance (Tyr1); both genes are from Poncirus and of importance to citrus survival and production. These polymorphic fragments were located on two local genetic linkage maps of the chromosome region from Ctv to Tyr1. These results indicate that resistance-gene candidate sequences amplified with the NBS-derived degenerate primers are valuable sources for developing markers in disease resistance-gene tagging, mapping, and cloning. Received: 25 October 1999 / Accepted: 27 March 2000