Construction of a strawberry breeding core collection to capture and exploit genetic variation.

BACKGROUND: Genetic diversity is crucial for the success of plant breeding programs and core collections are important resources to capture this diversity. Many core collections have already been constructed by gene banks, whose main goal is to obtain a panel of a limited number of genotypes to simplify management practices and to improve shareability while retaining as much diversity as possible. However, as gene banks have a different composition and goal than plant breeding programs, constructing a core collection for a plant breeding program should consider different aspects. RESULTS: In this study, we present a novel approach for constructing a core collection by integrating both genomic and pedigree information to maximize the representation of the breeding germplasm in a minimum subset of genotypes while accounting for future genetic variation within a strawberry breeding program. Our stepwise approach starts with selecting the most important crossing parents of advanced selections and genotypes included for specific traits, to represent also future genetic variation. We then use pedigree-genomic-based relationship coefficients combined with the 'accession to nearest entry' criterion to complement the core collection and maximize its representativeness of the current breeding program. Combined pedigree-genomic-based relationship coefficients allow for accurate relationship estimation without the need to genotype every individual in the breeding program. CONCLUSIONS: This stepwise construction of a core collection in a strawberry breeding program can be applied in other plant breeding programs to construct core collections for various purposes.

Allelic variation for alpha-Glucan Water Dikinase is associated with starch phosphate content in tetraploid potato.

KEY MESSAGE: Association analysis resulted in the identification of specific StGWD alleles causing either an increase or decrease in starch phosphate content which was verified in diploid and tetraploid potato mapping populations. Potatoes are grown for various purposes like French fries, table potatoes, crisps and for their starch. One of the most important aspects of potato starch is that it contains a high amount of phosphate ester groups which are considered to be important for providing improved functionalization after derivatization processes. Little is known about the variation in phosphate content as such in different potato varieties and thus we studied the genetic diversity for this trait. From other studies it was clear that the phosphate content is controlled by a quantitative trait locus (QTL) underlying the candidate gene α-Glucan Water Dikinase (StGWD) on chromosome 5. We performed direct amplicon sequencing of this gene by Sanger sequencing. Sequences of two StGWD amplicons from a global collection of 398 commercial cultivars and progenitor lines were used to identify 16 different haplotypes. By assigning tag SNPs to these haplotypes, each of the four alleles present in a cultivar could be deduced and linked to a phosphate content. A high value for intra-individual heterozygosity was observed (Ho = 0.765). The average number of different haplotypes per individual (Ai) was 3.1. Pedigree analysis confirmed that the haplotypes are identical-by-descent (IBD) and offered insight in the breeding history of elite potato germplasm. Haplotypes originating from introgression of wild potato accessions carrying resistance genes could be traced. Furthermore, association analysis resulted in the identification of specific StGWD alleles causing either an increase or decrease in starch phosphate content varying from 12 nmol PO4/mg starch to 38 nmol PO4/mg starch. These allele effects were verified in diploid and tetraploid mapping populations and offer possibilities to breed and select for this trait.

Two different R gene loci co-evolved with Avr2 of Phytophthora infestans and confer distinct resistance specificities in potato.

Late blight, caused by the oomycete pathogen Phytophthora infestans, is the most devastating disease in potato. For sustainable management of this economically important disease, resistance breeding relies on the availability of resistance (R) genes. Such R genes against P. infestans have evolved in wild tuber-bearing Solanum species from North, Central and South America, upon co-evolution with cognate avirulence (Avr) genes. Here, we report how effectoromics screens with Avr2 of P. infestans revealed defense responses in diverse Solanum species that are native to Mexico and Peru. We found that the response to AVR2 in the Mexican Solanum species is mediated by R genes of the R2 family that resides on a major late blight locus on chromosome IV. In contrast, the response to AVR2 in Peruvian Solanum species is mediated by Rpi-mcq1, which resides on chromosome IX and does not belong to the R2 family. The data indicate that AVR2 recognition has evolved independently on two genetic loci in Mexican and Peruvian Solanum species, respectively. Detached leaf tests on potato cultivar 'Désirée' transformed with R genes from either the R2 or the Rpi-mcq1 locus revealed an overlapping, but distinct resistance profile to a panel of 18 diverse P. infestans isolates. The achieved insights in the molecular R - Avr gene interaction can lead to more educated exploitation of R genes and maximize the potential of generating more broad-spectrum, and potentially more durable control of the late blight disease in potato.

The assessment of field trials in GMO research around the world and their possible integration in field trials for variety registration.

Most regulations worldwide stipulate that a new genetically modified (GM) crop event has to be compared to its closest non-GM counterpart as a corner stone of the pre-market risk assessment. To this end the GM crop and its comparator should be grown in field trials for a phenotypic comparison as well as for subsequent detailed analysis of the composition of the two crop varieties. A more in-depth globally harmonised approach for the conduct of these field trials is lacking. Only a few countries have formulated detailed protocols for the set-up of GM field trials. In some countries, commercial non-GM reference varieties need to be included in a field study to compile reliable data that indicate the range of natural variation for the compounds tested at the specific location. Detailed analysis of pre-market assessment reports have so far not shown the added value of including these reference varieties in the field trials. In all cases where specific values were found to be outside of the range of the reference varieties, it proved possible to draw conclusions on the part of the pre-market risk assessment that relates to the compositional analysis, on the basis of already available compositional data. With the increasing quality of several databases on compositional data of a growing number of crop species, it seems unlikely that reference varieties will become more important on future occasions. It was furthermore investigated whether this part of the risk assessment can be related to field trial requirements for variety registration with the explicit intention of reducing the data burden on producers of new GM plant varieties. Field trials for variety registration so far include an assessment of phenotypic characteristics that do not cover safety aspects, with the exception of establishment of the glycoalkaloid content in potatoes in the Netherlands and Sweden. It may, however, under certain conditions be relatively easy to exchange data from compositional measurements between variety registration and GM testing procedures, thus laying a foundation for testing the feasibility of combining both pre-market assessment procedures in a single pre-market evaluation path.

QTL mapping in diploid potato by using selfed progenies of the cross S. tuberosum × S. chacoense.

Usually, mapping studies in potato are performed with segregating populations from crosses between highly heterozygous diploid or tetraploid parents. These studies are hampered by a high level of genetic background noise due to the numerous segregating alleles, with a maximum of eight per locus. In the present study, we aimed to increase the mapping efficiency by using progenies from diploid inbred populations in which at most two alleles segregate. Selfed progenies were generated from a cross between S. tuberosum (D2; a highly heterozygous diploid) and S. chacoense (DS; a homozygous diploid clone) containing the self-incompatibility overcoming S locus inhibitor (Sli-gene). The Sli-gene enables self-pollination and the generation of selfed progenies. One F2 population was used to map several quality traits, such as tuber shape, flesh and skin color. Quantitative trait loci were identified for almost all traits under investigation. The identified loci partially coincided with known mapped loci and partially identified new loci. Nine F3 populations were used to validate the QTLs and monitor the overall increase in the homozygosity level.

First successful reduction of clinical allergenicity of food by genetic modification: Mal d 1-silenced apples cause fewer allergy symptoms than the wild-type cultivar.

BACKGROUND: Genetic modification of allergenic foods such as apple has the potential to reduce their clinical allergenicity, but this has never been studied by oral challenges in allergic individuals. METHODS: We performed oral food challenges in 21 apple-allergic individuals with Elstar apples which had undergone gene silencing of the major allergen of apple, Mal d 1, by RNA interference. Downregulation of Mal d 1 gene expression in the apples was verified by qRT-PCR. Clinical responses to the genetically modified apples were compared to those seen with the wild-type Elstar using a visual analogue scale (VAS). RESULTS: Gene silencing produced two genetically modified apple lines expressing Mal d 1.02 and other Mal d 1 gene mRNA levels which were extensively downregulated, that is only 0.1-16.4% (e-DR1) and 0.2-9.9% (e-DR2) of those of the wild-type Elstar, respectively. Challenges with these downregulated apple lines produced significantly less intense maximal symptoms to the first dose (Vmax1) than with Elstar (Vmax1 Elstar 3.0 mm vs 0.0 mm for e-DR1, P = 0.017 and 0.0 mm for e-DR2, P = 0.043), as well as significantly less intense mean symptoms per dose (meanV/d) than with Elstar (meanV/d Elstar 2.2 mm vs 0.2 mm for e-DR1, P = 0.017 and 0.0 mm for e-DR2, P = 0.043). Only one subject (5%) remained symptom-free when challenged with the Elstar apple, whereas 43% did so with e-DR1 and 63% with e-DR2. CONCLUSION: These data show that mRNA silencing of Mal d 1 results in a marked reduction of Mal d 1 gene expression in the fruit and reduction of symptoms when these apples are ingested by allergic subjects. Approximately half of the subjects developed no symptoms whatsoever, and virtually all subjects wished to consume the apple again in the future.

Efficient development of highly polymorphic microsatellite markers based on polymorphic repeats in transcriptome sequences of multiple individuals.

The first hurdle in developing microsatellite markers, cloning, has been overcome by next-generation sequencing. The second hurdle is testing to differentiate polymorphic from nonpolymorphic loci. The third hurdle, somewhat hidden, is that only polymorphic markers with a large effective number of alleles are sufficiently informative to be deployed in multiple studies. Both steps are laborious and still performed manually. We have developed a strategy in which we first screen reads from multiple genotypes for repeats that show the most length variants, and only these are subsequently developed into markers. We validated our strategy in tetraploid garden rose using Illumina paired-end transcriptome sequences of 11 roses. Of 48 tested two markers failed to amplify, but all others were polymorphic. Ten loci amplified more than one locus, indicating duplicated genes or gene families. Completely avoiding duplicated loci will be difficult because the range of numbers of predicted alleles of highly polymorphic single- and multilocus markers largely overlapped. Of the remainder, half were replicate markers (i.e. multiple primer pairs for one locus), indicating the difficulty of correctly filtering short reads containing repeat sequences. We subsequently refined the approach to eliminate multiple primer sets to the same loci. The remaining 18 markers were all highly polymorphic, amplifying on average 11.7 alleles per marker (range = 6-20) in 11 tetraploid roses, exceeding the 8.2 alleles per marker of the 24 most polymorphic markers genotyped previously. This strategy therefore represents a major step forward in the development of highly polymorphic microsatellite markers.

Capturing flavors from Capsicum baccatum by introgression in sweet pepper.

KEY MESSAGE: Biochemical characterization in combination with genetic analyses in BC 2 S 1 plants and near-isogenic lines led to the detection and validation of C. baccatum loci affecting flavor, terpenoid content and Brix level. The species Capsicum baccatum includes the most common hot peppers of the Andean cuisine, known for their rich variation in flavors and aromas. So far the C. baccatum genetic variation remained merely concealed for Capsicum annuum breeding, due to post-fertilization genetic barriers encountered in interspecific hybridization. However, to exploit the potential flavor wealth of C. baccatum we combined interspecific crossing with embryo rescue, resulting in a multi-parent BC2S1 population. Volatile and non-volatile compounds plus some physical characters were measured in mature fruits, in combination with taste evaluation by a sensory panel. An enormous variation in biochemical composition and sensory attributes was found, with almost all traits showing transgression. A population-specific genetic linkage map was developed for QTL mapping. BC2S1 QTLs were validated in an experiment with near-isogenic lines, resulting in confirmed genetic effects for physical, biochemical and sensory traits. Three findings are described in more detail: (1) A small C. baccatum LG3 introgression caused an extraordinary effect on flavor, resulting in significantly higher scores for the attributes aroma, flowers, spices, celery and chives. In an attempt to identify the responsible biochemical compounds few consistently up- and down-regulated metabolites were detected. (2) Two introgressions (LG10.1 and LG1) had major effects on terpenoid content of mature fruits, affecting at least 15 different monoterpenes. (3) A second LG3 fragment resulted in a strong increase in Brix without negative effects on fruit size. The mapping strategy, the potential application of studied traits and perspectives for breeding are discussed.

Field testing and exploitation of genetically modified cassava with low-amylose or amylose-free starch in Indonesia.

The development and testing in the field of genetically modified -so called- orphan crops like cassava in tropical countries is still in its infancy, despite the fact that cassava is not only used for food and feed but is also an important industrial crop. As traditional breeding of cassava is difficult (allodiploid, vegetatively propagated, outbreeding species) it is an ideal crop for improvement through genetic modification. We here report on the results of production and field testing of genetically modified low-amylose transformants of commercial cassava variety Adira4 in Indonesia. Twenty four transformants were produced and selected in the Netherlands based on phenotypic and molecular analyses. Nodal cuttings of these plants were sent to Indonesia where they were grown under biosafety conditions. After two screenhouse tests 15 transformants remained for a field trial. The tuberous root yield of 10 transformants was not significantly different from the control. Starch from transformants in which amylose was very low or absent showed all physical and rheological properties as expected from amylose-free cassava starch. The improved functionality of the starch was shown for an adipate acetate starch which was made into a tomato sauce. This is the first account of a field trial with transgenic cassava which shows that by using genetic modification it is possible to obtain low-amylose cassava plants with commercial potential with good root yield and starch quality.

A taste of sweet pepper: Volatile and non-volatile chemical composition of fresh sweet pepper (Capsicum annuum) in relation to sensory evaluation of taste.

In this study volatile and non-volatile compounds, as well as some breeding parameters, were measured in mature fruits of elite sweet pepper (Capsicum annuum) lines and hybrids from a commercial breeding program, several cultivated genotypes and one gene bank accession. In addition, all genotypes were evaluated for taste by a trained descriptive sensory expert panel. Metabolic contrasts between genotypes were caused by clusters of volatile and non-volatile compounds, which could be related to metabolic pathways and common biochemical precursors. Clusters of phenolic derivatives, higher alkanes, sesquiterpenes and lipid derived volatiles formed the major determinants of the genotypic differences. Flavour was described with the use of 14 taste attributes, of which the texture related attributes and the sweet-sour contrast were the most discriminatory factors. The attributes juiciness, toughness, crunchiness, stickiness, sweetness, aroma, sourness and fruity/apple taste could be significantly predicted with combined volatile and non-volatile data. Fructose and (E)-2-hexen-1-ol were highly correlated with aroma, fruity/apple taste and sweetness. New relations were found for fruity/apple taste and sweetness with the compounds p-menth-1-en-9-al, (E)-ß-ocimene, (Z)-2-penten-1-ol and (E)-geranylacetone. Based on the overall biochemical and sensory results, the perspectives for flavour improvement by breeding are discussed.

Anthocyanin production as a potential visual selection marker during plant transformation.

A mutant allele of the transcription factor gene MYB10 from apple induces anthocyanin production throughout the plant. This gene, including its upstream promoter, gene coding region and terminator sequence, was introduced into apple, strawberry and potato plants to determine whether it could be used as a visible selectable marker for plant transformation as an alternative to chemically selectable markers, such as kanamycin resistance. After transformation, red coloured calli, red shoots and red well-growing plants were scored. Red and green shoots were harvested from apple explants and examined for the presence of the MYB10 gene by PCR analysis. Red shoots of apple explants always contained the MYB10 gene but not all MYB10 containing shoots were red. Strawberry plants transformed with the MYB10 gene showed anthocyanin accumulation in leaves and roots. No visible accumulation of anthocyanin could be observed in potato plants grown in vitro, even the ones carrying the MYB10 gene. However, acid methanol extracts of potato shoots or roots carrying the MYB10 gene contained up to four times higher anthocyanin content than control plants. Therefore anthocyanin production as result of the apple MYB10 gene can be used as a selectable marker for apple, strawberry and potato transformation, replacing kanamycin resistance.

GpaXI ( tar ) ( l ) originating from Solanum tarijense is a major resistance locus to Globodera pallida and is localised on chromosome 11 of potato.

Resistance to Globodera pallida Rookmaker (Pa3), originating from wild species Solanum tarijense was identified by QTL analysis and can be largely ascribed to one major QTL. GpaXI ( tar ) ( l ) explained 81.3% of the phenotypic variance in the disease test. GpaXI ( tar ) ( l ) is mapped to the long arm of chromosome 11. Another minor QTL explained 5.3% of the phenotypic variance and mapped to the long arm of chromosome 9. Clones containing both QTL showed no lower cyst counts than clones with only GpaXI ( tar ) ( l ) . After Mendelising the phenotypic data, GpaXI ( tar ) ( l ) could be more precisely mapped near markers GP163 and FEN427, thus anchoring GpaXI ( tar ) ( l ) to a region with a known R-gene cluster containing virus and nematode resistance genes.

Differences in regulation of carbohydrate metabolism during early fruit development between domesticated tomato and two wild relatives.

Early development and growth of fruit in the domesticated tomato Solanum lycopersicum cultivar Money Maker and two of its wild relatives, S. peruvianum LA0385 and S. habrochaites LA1777, were studied. Although small differences exist, the processes involved and the sequence of events in fruit development are similar in all three species. The growth of developing fruits is exponential and the relative growth rate accelerates from 5 days after pollination (DAP 5) to DAP 8, followed by a decline during further development. Growth is positively correlated to the standard "Brix plus starch'' in the period DAP 8-DAP 20. Carbohydrate composition and levels of sugars and organic acids differ in fruits of the wild accessions compared to domesticated tomato. The wild accessions accumulate sucrose instead of glucose and fructose, and ripe fruits contain higher levels of malate and citrate. The enzymes responsible for the accumulation of glucose and fructose in domesticated tomatoes are soluble invertase and sucrose synthase. The regulation of initial carbohydrate metabolism in the domesticated tomato differs from that in the wild species, as could be concluded from measuring activities of enzymes involved in primary carbohydrate metabolism. Furthermore, changes in the activity of several enzymes, e.g., cell wall invertase, soluble invertase, fructokinase and phosphoglucomutase, could be attributed to changes in gene expression level. For other enzymes, additional control mechanisms play a role in the developing tomato fruits. Localization by in-situ activity staining of enzymes showed comparable results for fruits of domesticated tomato and the wild accessions. However, in the pericarp of S. peruvianum, less activity staining of phosphogluco-isomerase, phosphoglucomutase and UDP-glucosepyrophosphorylase was observed.

Isolation and characterization of a novel potato Auxin/Indole-3-Acetic Acid family member (StIAA2) that is involved in petiole hyponasty and shoot morphogenesis.

Auxin/indole-3-acetid acid (Aux/IAA) proteins are short-lived transcriptional regulators that mediate their response through interaction with auxin response factors (ARF). Although 29 Aux/IAA proteins have been identified in Arabidopsis thaliana, their individual functions are still poorly understood and are largely defined by observed growth defects in gain-of-function mutant alleles. Here we present the isolation and characterization of a novel Aux/IAA protein in potato (Solanum tuberosum) that is named StIAA2. Down regulation of StIAA2 results in distinctive phenotypes that include, increased plant height, petiole hyponasty and extreme curvature of growing leaf primordia in the shoot apex. Gene expression analysis of transgenic plants with reduced StIAA2 transcript levels resulted in the identification of a number of genes with altered expression profiles including another member of the Aux/IAA gene family (StIAA). The phenotypes that were observed in the StIAA2 suppression clones can be associated with both common as well as unique functional roles among Aux/IAA family members indicating the importance of analyzing Aux/IAA expression in different plant species.

QTL methodology for response curves on the basis of non-linear mixed models, with an illustration to senescence in potato.

The improvement of quantitative traits in plant breeding will in general benefit from a better understanding of the genetic basis underlying their development. In this paper, a QTL mapping strategy is presented for modelling the development of phenotypic traits over time. Traditionally, crop growth models are used to study development. We propose an integration of crop growth models and QTL models within the framework of non-linear mixed models. We illustrate our approach with a QTL model for leaf senescence in a diploid potato cross. Assuming a logistic progression of senescence in time, two curve parameters are modelled, slope and inflection point, as a function of QTLs. The final QTL model for our example data contained four QTLs, of which two affected the position of the inflection point, one the senescence progression-rate, and a final one both inflection point and rate.

Post-transcriptional gene silencing of GBSSI in potato: effects of size and sequence of the inverted repeats.

In the past, silencing of granule-bound starch synthase (GBSSI) in potato was achieved by antisense technology, where it was observed that inclusion of the 3' end of the GBSSI coding region increased silencing efficiency. Since higher silencing efficiencies were desired, GBSSI inverted repeat constructs were designed and tested in potato. First, large inverted repeats comprising the 5' and the 3' half of the GBSSI cDNA were tested. The 5' IR construct gave a significantly higher silencing efficiency than the 3' IR construct. Since it was not known whether the observed difference was due to the sequence or the orientation of the inverted repeat, the GBSSI cDNA was divided into three regions, after which each region was tested in small inverted repeats in two orientations. To this end large numbers of independent transformants were produced for each construct. The results suggested that there was no effect of inverted repeat orientation on silencing efficiency. The percentage of transformants showing strong inhibition varied from 48% for a 3'-derived construct to 87% for a 5' as well as a middle region-derived construct. Similar to the large inverted repeats, the 3' sequences induced the least efficient silencing implying that the observed differences in silencing efficiency are caused by sequence differences. The small inverted repeat constructs with a repeat size of 500-600 bp and a spacer of about 150 bp were more efficient silencing inducers than the large inverted repeat constructs where the size of the repeat was 1.1 or 1.3 kb whilst the size of spacer was 1.3 or 1.1 kb. The results presented here show that size and sequence of the inverted repeat influenced silencing efficiency.

PRECISE: software for prediction of cis-acting regulatory elements.

The regulation of gene expression at the transcription initiation level is highly complex and requires the presence of multiple transcription factors. These transcription factors are often proteins or peptides that bind to the so-called cis-acting elements, which are present in the promoter regions and conserved among different species. In order to predict these cis-acting elements, a computer program called PRECISE (Prediction of REgulatory CIS-acting Elements) was developed. The power of the tool lies in its user-friendly interface and in the possibility of using empirical motif frequency tables to filter through the many discovered motifs. The tools to create the empirical motif frequency table (e.g., from a whole genome sequence) are included in the package. In the first case study, the upstream regions of all the genes in the Arabidopsis genome were used to create an empirical motif frequency table and a set of 64 upstream sequences of genes known to be involved in starch metabolism was subjected to analysis by PRECISE. The 20 motifs with the highest specificity in the selected set were analyzed in more detail. Of these 20 motifs, 15 showed a very high or complete homology to the sequences of known cis-acting elements. These cis-acting elements are regulated by light, auxin, and abscisic acid, and confer specific expression in sink organs such as leaves and seeds. All these factors have been shown to play an important role in starch biosynthesis. In the second case study, the upstream regions of 16 genes whose transcription is induced by gibberellins (GA) in Arabidopsis were analyzed with PRECISE and compared to the motifs present in the PLACE database. Among the most promising motifs found by PRECISE were 6 of the 17 known GA motifs. These results indicate the power of the PRECISE software package in the prediction of regulatory elements.

Characterization and high-resolution mapping of a late blight resistance locus similar to R2 in potato.

Identification of resistance (R) genes to Phytophthora infestans is an essential step in molecular breeding of potato. We identified three specific R genes segregating in a diploid mapping population. One of the R genes is located on chromosome 4 and proved phenotypically indistinguishable from the Solanum demissum-derived R2, although S. demissum is not directly involved in the pedigree of the population. By bulked segregant analysis combined with a resistance assay, a genetic linkage map of the R2-like locus was constructed with 30 coupling and 23 repulsion phase AFLP markers. Two markers flanking the R2-like locus were applied to screen an extended population of 1,586 offspring. About 103 recombinants were selected, and an accurate high-resolution map was constructed. The R2-like resistance was localized in a 0.4 cM interval and was found co-segregating with four AFLP markers, which can be used to isolate the R2-like gene by map-based gene cloning. By analyzing race-specificity and R gene-specific molecular markers, we also found that an R1-like gene and an additional unknown R gene are segregating in the population.

Intergenomic recombination in F1 lily hybrids (Lilium) and its significance for genetic variation in the BC1 progenies as revealed by GISH and FISH.

Intergenomic recombination was assessed in a BC1 population of Oriental (O)xAsiatic (A) lilies (Lilium) backcrossed to Asiatic parents. This population consisted of 38 plants generated from the 2n gametes from 2 genotypes (951502-1 and 952400-1) of the diploid F1, Orientalx Asiatic lilies (2n=2x=24) as parents. In the majority of BC1 plants, there was evidence that first division restitution, with and without crossovers, resulted in functional gametes. However, there were 5 BC1 plants in which 2n gametes originated from indeterminate meiotic restitution (IMR). Based on the number of recombinant chromosomes for a particular homoeologous pair, 3 types of plants were identified: (i) those with both the reciprocal product of a crossover (O/A, A/O, where O represents the centromere of the O genome and A the recombinant segment of Asiatic chromosome, and vice versa); (ii) those with 1 normal chromosome of the O genome and a recombinant chromosome (O, A/O); and (iii) those with 1 normal chromosome of the A genome and a recombinant chromosome (A, O/A). An important feature of A x OA backcross progeny is the occurrence of substitutions for the segment distal in the crossover wherever the recombinant chromosome O/A was present. In the case of IMR, the substitution occurred for both proximal and distal recombinant segments. The significance of these substitutions is that they offer the potential for the phenotypic expression of recessive genes in polyploids (i.e., nulliplex genotype).

Molecular characterization and physical localization of highly repetitive DNA sequences from Brazilian Alstroemeria species.

Highly repetitive DNA sequences were isolated from genomic DNA libraries of Alstroemeria psittacina and A. inodora. Among the repetitive sequences that were isolated, tandem repeats as well as dispersed repeats could be discerned. The tandem repeats belonged to a family of interlinked Sau3A subfragments with sizes varying from 68-127 bp, and constituted a larger HinfI repeat of approximately 400 bp. Southern hybridization showed a similar molecular organization of the tandem repeats in each of the Brazilian Alstroemeria species tested. None of the repeats hybridized with DNA from Chilean Alstroemeria species, which indicates that they are specific for the Brazilian species. In-situ localization studies revealed the tandem repeats to be localized in clusters on the chromosomes of A. inodora and A. psittacina: distal hybridization sites were found on chromosome arms 2PS, 6PL, 7PS, 7PL and 8PL, interstitial sites on chromosome arms 2PL, 3PL, 4PL and 5PL. The applicability of the tandem repeats for cytogenetic analysis of interspecific hybrids and their role in heterochromatin organization are discussed.