Carol D. Ryder

Aligning male and female linkage maps of apple (Malus pumila Mill.) using multi-allelic markers

Abstract Linkage maps for the apple cultivars ‘Prima’ and ‘Fiesta’ were constructed using RFLP, RAPD, isozyme, AFLP, SCAR and microsatellite markers in a ‘Prima’בFiesta’ progeny of 152 individuals. Seventeen linkage groups, putatively corresponding to the seventeen haploid apple chromosomes, were obtained for each parent. These maps were aligned using 67 multi-allelic markers that were heterozygous in both parents. A large number of duplicate RFLP loci was observed and, in several instances, linked RFLP markers in one linkage group showed corresponding linkage in another linkage group. Distorted segregation was observed mainly in two regions of the genome, especially in the male parent alleles. Map positions were provided for resistance genes to scab and rosy leaf curling aphid (Vf and Sd1, respectively) for the fruit acidity gene Ma and for the self-incompatibility locus S. The high marker density and large number of mapped codominant RFLPs and some microsatellite markers make this map an ideal reference map for use in other progenies also and a valuable tool for the mapping of quantitative trait loci.

Characterization of microsatellite markers in peach [Prunus persica (L.) Batsch]

Abstract Microsatellites have emerged as an important system of molecular markers. We evaluated the potential of microsatellites for use in genetic studies of peach [Prunus persica (L.) Batsch]. Microsatellite loci in peach were identified by screening a pUC8 genomic library, a λZAPII leaf cDNA library, as well as through database searches. Primer sequences for the microsatellite loci were tested from the related Rosaceae species apple (Malus×domestica) and sour cherry (Prunus cerasus L.). The genomic library was screened for CT, CA and AGG repeats, while the cDNA library was screened for (CT)n- and (CA)n-containing clones. Estimates of microsatellite frequencies were determined from the genomic library screening, and indicate that CT repeats occur every 100 kb, CA repeats every 420 kb, and AGG repeats every 700 kb in the peach genome. Microsatellite- containing clones were sequenced, and specific PCR primers were designed to amplify the microsatellite- containing regions from genomic DNA. The level of microsatellite polymorphism was evaluated among 28 scion peach cultivars which displayed one to four alleles per primer pair. Five microsatellites were found to segregate in intraspecific peach-mapping crosses. In addition, these microsatellite markers were tested for their utility in cross-species amplification for use in comparative mapping both within the Rosaceae, and with the un- related species Arabidopsis thaliana L.

Pairing and recombination at meiosis of Brassica rapa (AA) × Brassica napus (AACC) hybrids

Interspecific crosses contribute significantly to plant evolution enabling gene exchanges between species. The efficiency of interspecific crosses depends on the similarity between the implicated genomes as high levels of genome similarity are required to ensure appropriate chromosome pairing and genetic recombination. Brassica napus (AACC) is an allopolyploid, resulting from natural hybridization between Brassica rapa (AA) and Brassica oleracea (CC), both being diploid species derived from a common ancestor. To study the relationships between genomes of these Brassica species, we have determined simultaneously the pairing and recombination pattern of A and C chromosomes during meiosis of AAC triploid hybrids, which result from the interspecific cross between natural B. napus and B. rapa. Different AAC triploid hybrids and their progenies have been analysed using cytogenetic, BAC-FISH, and molecular techniques. In 71% of the pollen mother cells, homologous A chromosomes paired regularly, and usually one chromosome of each pair was transmitted to the progeny. C chromosomes remained mainly univalent, but were involved in homoeologous pairing in 21.5% of the cells, and 13% of the transmitted C chromosomes were either recombined or broken. The rate of transmission of C chromosomes depended on the identity of the particular chromosome and on the way the hybrid was crossed, as the male or as the female parent, to B. napus or to B. rapa. Gene transfers in triploid hybrids are favoured between A genomes of B. rapa and B. napus, but also occur between A and C genomes though at lower rates.

Homeologous Recombination Plays a Major Role in Chromosome Rearrangements That Occur During Meiosis of Brassica napus Haploids

Chromosomal rearrangements can be triggered by recombination between distinct but related regions. Brassica napus (AACC; 2n = 38) is a recent allopolyploid species whose progenitor genomes are widely replicated. In this article, we analyze the extent to which chromosomal rearrangements originate from homeologous recombination during meiosis of haploid B. napus (n = 19) by genotyping progenies of haploid × euploid B. napus with molecular markers. Our study focuses on three pairs of homeologous regions selected for their differing levels of divergence (N1/N11, N3/N13, and N9/N18). We show that a high number of chromosomal rearrangements occur during meiosis of B. napus haploid and are transmitted by first division restitution (FDR)-like unreduced gametes to their progeny; half of the progeny of Darmor-bzh haploids display duplications and/or losses in the chromosomal regions being studied. We demonstrate that half of these rearrangements are due to recombination between regions of primary homeology, which represents a 10- to 100-fold increase compared to the frequency of homeologous recombination measured in euploid lines. Some of the other rearrangements certainly result from recombination between paralogous regions because we observed an average of one to two autosyndetic A–A and/or C–C bivalents at metaphase I of the B. napus haploid. These results are discussed in the context of genome evolution of B. napus.

A SEPALLATA gene is involved in the development and ripening of strawberry (Fragaria x ananassa Duch.) fruit, a non-climacteric tissue.

Climacteric and non-climacteric fruits have traditionally been viewed as representing two distinct programmes of ripening associated with differential respiration and ethylene hormone effects. In climacteric fruits, such as tomato and banana, the ripening process is marked by increased respiration and is induced and co-ordinated by ethylene, while in non-climacteric fruits, such as strawberry and grape, it is controlled by an ethylene-independent process with little change in respiration rate. The two contrasting mechanisms, however, both lead to texture, colour, and flavour changes that probably reflect some common programmes of regulatory control. It has been shown that a SEPALLATA(SEP)4-like gene is necessary for normal ripening in tomato. It has been demonstrated here that silencing a fruit-related SEP1/2-like (FaMADS9) gene in strawberry leads to the inhibition of normal development and ripening in the petal, achene, and receptacle tissues. In addition, analysis of transcriptome profiles reveals pleiotropic effects of FaMADS9 on fruit development and ripening-related gene expression. It is concluded that SEP genes play a central role in the developmental regulation of ripening in both climacteric and non-climacteric fruits. These findings provide important information to extend the molecular control of ripening in a non-climacteric fruit beyond the limited genetic and cultural options currently available.

RFLP and RAPD markers linked to the rosy leaf curling aphid resistance gene (Sd1) in apple. T

Abstract Sd1 is a dominant gene for resistance to biotypes 1 and 2 of the rosy leaf curling aphid, Dysaphis devecta Wlk., which can cause economic damage to apple trees. This report describes the identification of three RFLP and four RAPD markers linked to Sd1 in a cross between the D. devecta susceptible variety ‘Prima’ (sd1sd1) and the resistant variety ‘Fiesta’ (Sd1sd1). Potted trees were artificially infested in the glasshouse, and the ratio of resistant:susceptible plants supported the hypothesis that the resistance was under the control of a single dominant gene. The position of the gene was mapped to a single locus on a ‘Fiesta’ chromosome, within 2 cM of three tightly linked RFLP markers (MC064a, 2B12a and MC029b); the four RAPD markers were located further away (between 13 and 46 cM). This is the first report of molecular markers for an aphid resistance gene in tree fruit crops. The potential application of these markers in a marker-assisted resistance breeding programme is discussed.

The CACTA transposon Bot1 played a major role in Brassica genome divergence and gene proliferation

We isolated and characterized a Brassica C genome-specific CACTA element, which was designated Bot1 (Brassica oleracea transposon 1). After analysing phylogenetic relationships, copy numbers and sequence similarity of Bot1 and Bot1 analogues in B. oleracea (C genome) versus Brassica rapa (A genome), we concluded that Bot1 has encountered several rounds of amplification in the oleracea genome only, and has played a major role in the recent rapa and oleracea genome divergence. We performed in silico analyses of the genomic organization and internal structure of Bot1, and established which segment of Bot1 is C-genome specific. Our work reports a fully characterized Brassica repetitive sequence that can distinguish the Brassica A and C chromosomes in the allotetraploid Brassica napus, by fluorescent in situ hybridization. We demonstrated that Bot1 carries a host S locus-associated SLL3 gene copy. We speculate that Bot1 was involved in the proliferation of SLL3 around the Brassica genome. The present study reinforces the assumption that transposons are a major driver of genome and gene evolution in higher plants.

A FRUITFULL-like gene is associated with genetic variation for fruit flesh firmness in apple ( Malus domestica Borkh.)

The FRUITFULL (FUL) and SHATTERPROOF (SHP) genes are involved in regulating fruit development and dehiscence in Arabidopsis. We tested the hypothesis that this class of genes are also involved in regulating the development of fleshy fruits, by exploring genetic and phenotypic variation within the apple (Malus domestica) gene pool. We isolated and characterised the genomic sequences of two candidate orthologous FUL-like genes, MdMADS2.1 and MdMADS2.2. These were mapped using the reference population ‘Prima x Fiesta’ to loci on Malus linkage groups LG14 and LG06, respectively. An additional MADS-box gene, MdMADS14, shares high amino acid identity with the Arabidopsis SHATTERPROOF1/2 genes and was mapped to Malus linkage group LG09. Association analysis between quantitative fruit flesh firmness estimates of ‘Prima x Fiesta’ progeny and the MdMADS2.1, MdMADS2.2 and MdMADS14 loci was carried out using a mixed model analysis of variance. This revealed a significant association (P < 0.01) between MdMADS2.1 and fruit flesh firmness. Further evidence for the association between MdMADS2.1 and fruit flesh firmness was obtained using a case–control population-based genetic association approach. For this, a polymorphic repeat, (AT)n, in the 3′ UTR of MdMADS2.1 was used as a locus-specific marker to screen 168 apple accessions for which historical assessments of fruit texture attributes were available. This analysis revealed a significant association between the MdMADS2.1 and fruit flesh firmness at both allelic (χ2 = 34, df = 9, P < 0.001) and genotypic (χ2 = 57, df = 32, P < 0.01) levels.

The genomic organization of retrotransposons in Brassica oleracea

We have investigated the copy numbers and genomic organization of five representative reverse transcriptase domains from retrotransposons in Brassica oleracea. Two non-homologous Pseudoviridae (Ty1/copia-like) elements, two Metaviridae (Ty3/gypsy-like) elements (one related to the Athila family) and one Retroposinae (LINE) element were hybridized to a gridded BAC library, “BoB”. The results indicated that the individual LTR retrotransposons (copia and gypsy-like) were represented by between 90 and 320 copies in the haploid genome, with only evidence of a single location for the LINE. Sequence analysis of the same elements against genome survey sequence gave estimates of between 60 and 570, but no LINE was found. There was minimal evidence for clustering between any of these retroelements: only half the randomly expected number of BACs hybridized to both LTR-retrotransposon families. Fluorescent in situ hybridization showed that each of the retroelements had a characteristic genomic distribution. Our results suggest there are preferential sites and perhaps control mechanisms for the insertion or excision of different retrotransposon groups.