Holger Budahn

Interspecific hybrids between onion (Allium cepa L.) with S-cytoplasm and leek (Allium ampeloprasum L.)

Abstract Interspecific hybrids between Allium cepa and A. ampeloprasum have been generated as a first step for the introduction of S-cytoplasm from onion into leek. Pre-zygotic barriers of crossability were observed after the arrival of pollen tubes at the end of the style when entering the cavity. Nevertheless, micropyle penetration of pollen tubes and the formation of hybrid embryos were also observed. After accomplishing in vitro culture of ovaries and ovules successively, triploid hybrid plants with 24 chromosomes were obtained. Their hybrid nature was confirmed by RAPD analysis, genomic in situ hybridization, and morphological analysis. Southern hybridization with a cytoplasmic probe indicated the transfer of unaltered S-cytoplasm into the hybrid plants.

Characterization of centromeric histone H3 (CENH3) variants in cultivated and wild carrots (Daucus sp.)

In eukaryotes, centromeres are the assembly sites for the kinetochore, a multi-protein complex to which spindle microtubules are attached at mitosis and meiosis, thereby ensuring segregation of chromosomes during cell division. They are specified by incorporation of CENH3, a centromere specific histone H3 variant which replaces canonical histone H3 in the nucleosomes of functional centromeres. To lay a first foundation of a putative alternative haploidization strategy based on centromere-mediated genome elimination in cultivated carrots, in the presented research we aimed at the identification and cloning of functional CENH3 genes in Daucus carota and three distantly related wild species of genus Daucus varying in basic chromosome numbers. Based on mining the carrot transcriptome followed by a subsequent PCR-based cloning, homologous coding sequences for CENH3s of the four Daucus species were identified. The ORFs of the CENH3 variants were very similar, and an amino acid sequence length of 146 aa was found in three out of the four species. Comparison of Daucus CENH3 amino acid sequences with those of other plant CENH3s as well as their phylogenetic arrangement among other dicot CENH3s suggest that the identified genes are authentic CENH3 homologs. To verify the location of the CENH3 protein in the kinetochore regions of the Daucus chromosomes, a polyclonal antibody based on a peptide corresponding to the N-terminus of DcCENH3 was developed and used for anti-CENH3 immunostaining of mitotic root cells. The chromosomal location of CENH3 proteins in the centromere regions of the chromosomes could be confirmed. For genetic localization of the CENH3 gene in the carrot genome, a previously constructed linkage map for carrot was used for mapping a CENH3-specific simple sequence repeat (SSR) marker, and the CENH3 locus was mapped on the carrot chromosome 9.

Mapping genes governing flower architecture and pollen development in a double mutant population of carrot

A linkage map of carrot (Daucus carota L.) was developed in order to study reproductive traits. The F2 mapping population derived from an initial cross between a yellow leaf (yel) chlorophyll mutant and a compressed lamina (cola) mutant with unique flower defects of the sporophytic parts of male and female organs. The genetic map has a total length of 781 cM and included 285 loci. The length of the nine linkage groups (LGs) ranged between 65 and 145 cM. All LGs have been anchored to the reference map. The objective of this study was the generation of a well-saturated linkage map of D. carota. Mapping of the cola-locus associated with flower development and fertility was successfully demonstrated. Two MADS-box genes (DcMADS3, DcMADS5) with prominent roles in flowering and reproduction as well as three additional genes (DcAOX2a, DcAOX2b, DcCHS2) with further importance for male reproduction were assigned to different loci that did not co-segregate with the cola-locus.

Construction of a chromosome-assigned, sequence-tagged linkage map for the radish, Raphanus sativus L. and QTL analysis of morphological traits

The radish displays great morphological variation but the genetic factors underlying this variability are mostly unknown. To identify quantitative trait loci (QTLs) controlling radish morphological traits, we cultivated 94 F4 and F5 recombinant inbred lines derived from a cross between the rat-tail radish and the Japanese radish cultivar ‘Harufuku’ inbred lines. Eight morphological traits (ovule and seed numbers per silique, plant shape, pubescence and root formation) were measured for investigation. We constructed a map composed of 322 markers with a total length of 673.6 cM. The linkage groups were assigned to the radish chromosomes using disomic rape-radish chromosome-addition lines. On the map, eight and 10 QTLs were identified in 2008 and 2009, respectively. The chromosome-linkage group correspondence, the sequence-specific markers and the QTLs detected here will provide useful information for further genetic studies and for selection during radish breeding programs.

Transfer of a male-sterility-inducing cytoplasm from onion to leek (Allium ampeloprasum)

Abstract.Two interspecific triploid (AAC) hybrids (84/1-94 and 99/1-94) from crosses between onion [Allium cepa (2n=2x=16, CC)] and leek [A. ampeloprasum (2n=4x=32, AAAA)] were backcrossed to leek in order to transfer a male-sterility-inducing cytoplasm from onion that would enable the production of hybrid leek. GISH evaluations of meiosis in the interspecific hybrids revealed irregularities due to univalent onion chromosomes producing micronuclei from onion chromatin, whereas the pairing of the two sets of leek chromosomes was nearly normal. Attempts to use colchicine to double the chromosome number of the hybrids failed. Backcrosses of 84/1-94 to leek as the pollen parent were not successful. The first backcross of 99/1-94 to tetraploid leek produced 11 BC1 plants with chromosome numbers between 38 and 41. Identification of parental chromosomes by GISH showed that all eight onion chromosomes and 30–33 leek chromosomes were transmitted to the backcross progenies due to unreduced egg cells. Onion chromosomes were eliminated during the second backcross. Southern hybridization confirmed the transfer of the T-cytoplasm like source of CMS from onion to the BC2 progenies. After the third backcross to leek, 158 plants were obtained with varying numbers of onion chromosomes and some intergenomic recombinant chromosomes. Alloplasmic leek plants without onion chromatin were selected for further characterization of male sterility and quality traits.

The Terpene Synthase Gene Family of Carrot (Daucus carota L.): Identification of QTLs and Candidate Genes Associated with Terpenoid Volatile Compounds

Terpenes are an important group of secondary metabolites in carrots influencing taste and flavor, and some of them might also play a role as bioactive substances with an impact on human physiology and health. Understanding the genetic and molecular basis of terpene synthases (TPS) involved in the biosynthesis of volatile terpenoids will provide insights for improving breeding strategies aimed at quality traits and for developing specific carrot chemotypes possibly useful for pharmaceutical applications. Hence, a combination of terpene metabolite profiling, genotyping-by-sequencing (GBS), and genome-wide association study (GWAS) was used in this work to get insights into the genetic control of terpene biosynthesis in carrots and to identify several TPS candidate genes that might be involved in the production of specific monoterpenes. In a panel of 85 carrot cultivars and accessions, metabolite profiling was used to identify 31 terpenoid volatile organic compounds (VOCs) in carrot leaves and roots, and a GBS approach was used to provide dense genome-wide marker coverage (>168,000 SNPs). Based on this data, a total of 30 quantitative trait loci (QTLs) was identified for 15 terpenoid volatiles. Most QTLs were detected for the monoterpene compounds ocimene, sabinene, β-pinene, borneol and bornyl acetate. We identified four genomic regions on three different carrot chromosomes by GWAS which are both associated with high significance (LOD ≥ 5.91) to distinct monoterpenes and to TPS candidate genes, which have been identified by homology-based gene prediction utilizing RNA-seq data. In total, 65 TPS candidate gene models in carrot were identified and assigned to known plant TPS subfamilies with the exception of TPS-d and TPS-h. TPS-b was identified as largest subfamily with 32 TPS candidate genes.

Use of CACTA Mobile Genetic Elements for Revision of Phylogenetic Relationships in Brassica rapa L. Species

To supplement our previous studies of genetic diversity in Brassica rapa L. by SSR analysis, for the first time, we used Class II transposable elements to clarify the phylogenetic relationships. The core collection of 96 accessions stored at the Vavilov Institute of Plant Industry is used in the study. Experiments with 12 S-SAP primer pairs gave rise to 123 polymorphic markers. Using TE markers, we divide B. rapa crops into two major clusters: East Asian vegetables and Indo-European-Asian oilseed and turnip. The first cluster is divided into the subclusters of pak-choi and bok choi according to botanical classification and the results of SSR analysis. The earlier classification is improved by subclassification of pak-choi into groups, including identification of the separate group of headed cabbages, revision of the positions of the Hiroshimana and Mizuna forms, and subcategorization of oilseed by geographical origin. However, more accurate positions of accessions in the system of the species are obtained by construction of a SAHN dendrogram with 149 SSR and 123 S-SAP markers. The diagram shows that only a few accessions of European turnip are dispersed among the accessions of the other cluster but some accessions form a separate group in the Nepal-Indian subcluster. They are assumed to be the oldest forms closely related to the first domesticated Central-Asian B. rapa form.

Chromosomal assignment of oil radish resistance to Meloidogyne incognita and M. javanica using a set of disomic rapeseed-radish chromosome addition lines

Root-knot nematodes cause severe damage to a great number of crops worldwide. The use of nematicides is restricted due to environmental and toxicological risks and control of the pest by crop rotation is difficult because root-knot nematodes have a very wide range of host plants. To verify the strategy of converting rapeseed from a tolerant host for Meloidogyne incognita and M. javanica to a resistant catch crop, a complete set of nine disomic rapeseed-radish chromosome addition lines (lines A to I) was tested for resistance against these Meloidogyne species. Thirty plants of each addition line and the rapeseed and radish parents as control were infected with 2500 second-stage juveniles per plant. The presence of the alien radish chromosome was confirmed by chromosome-specific microsatellite markers. After cultivation of the inoculated plants for 10 weeks in a climatic chamber the root systems were washed. The egg masses were stained with Cochenille Red and counted. The radish parent A24 was found to be resistant to M. incognita (2.4 egg masses (g root)−1) and M. javanica (0.4 egg masses (g root)−1) compared to 53.3 and 33.1 egg masses (g root)−1 for the susceptible rapeseed parent cv. Madora. The radish chromosome e was shown to be the carrier of radish root-knot nematode resistance with an average number of <1 egg mass (g root)−1 for M. incognita and M. javanica. The disomic addition lines B, C, D, G, H and I and the parental radish line A107 were classified as highly susceptible, whereas the addition lines A and F showed significantly reduced susceptibility for M. incognita but not for M. javanica. To our knowledge this is the first study on resistance effects of individual radish chromosomes in a rapeseed background against these root-knot nematodes.

Nematode resistance of rape-radish chromosome addition lines

Oilseed radish is resistant to the beet cyst nematode ( Heterodera schachtii Schmidt), interrupting the life cycle of this sedentary pathogen by blocking feeding cell development in the root. A complete set of nine disomic rape-radish chromosome additions, a to i , derived from a susceptible rapeseed parent as recipient and a resistant radish as chromosome donor, was assayed for nematode resistance. The addition line d exhibited the resistance level of the radish parent, confirming previous results that radish chromosome d carries a dominant gene, Hs1 Rph , for nematode resistance. It was investigated if Hs1 Rph is effective against a further important sedentary parasite, the northern root-knot nematode Meloidogyne hapla . The set of chromosome addition lines and the parents, rape and radish, were inoculated with second-stage juveniles (J2) of M. hapla and the plant reaction was evaluated by counting the number of egg masses per root system. By contrast to the situation in H. schachtii , the radish parent as well as addition line d showed no resistance against M. hapla and was even more susceptible than rape. It was concluded that the resistance gene Hs1 Rph , which inhibits syncytium development of H. schachtii , is ineffective against M. hapla , a nematode inducing giant cell formation. Most added radish chromosomes significantly changed the number of egg masses in the recipient rape towards higher susceptibility. Two chromosomes enhanced the egg mass number beyond that of the chromosome donor radish. However, one radish chromosome decreased the egg mass production in the corresponding addition line below that in rape. This wide range of effects of the individual radish chromosomes in the rape background indicates a quantitative inheritance of host suitability to M. hapla and a complex interaction between the pathogen and radish.

Evaluation of genetic resources in the genus Asparagus for resistance to Asparagus virus 1 (AV-1)

Forty-four Asparagus officinalis cultivars, gene bank accessions and breeding lines as well as thirty-four accessions of wild relatives of Asparagus were evaluated for resistance to Asparagus virus 1. Three different test strategies were developed for the assessment of individual plants: (1) natural infection under field conditions, or two vector-mediated infection assays using the green peach aphid Myzus persicae (2) in an insect-proof gauze cage or (3) in a climate chamber. The AV-1 infections were verified by DAS-ELISA and RT-PCR approaches. All tested 660 individual plants of A. officinalis germplasm were susceptible to AV-1 infection. In contrast, in 276 plants of 29 Asparagus wild accessions no virus infection could be detected. These resistant accessions comprised of nineteen diploid, tetraploid and hexaploid species of both the Eurasian clade and the African clade of the asparagus germplasm. Data of the AV-1 resistance evaluation are discussed in relation to the genetic distance of the resistance carrier and potential application in breeding.