Partial purification and characterization of a zeatin Cis-trans isomerase from Phaseolus vulgaris L.

Conversion of exogenous cis-zeatin to trans-zeatin in immature seeds of Phaseolus vulgaris L. led to the isolation of a cis-trans isomerase from the endosperm. The enzyme was purified over 2000-fold by chromatography on a series of FPLC (anion exchange, gel filtration, and hydrophobic interaction) and Concanavalin A columns. Non-enzymatic isomerization occurred under standard assay conditions but the presence of the enzyme enhanced conversion significantly. The enzymatic reaction favors conversion from the cis to the trans form and requires flavin, blue light, and DTT. Optimal conditions for isomerization were identified using a Phenyl Superosepurified enzyme extract. Concentrations of 0.1-0.316 mM FAD and 0.5 mM DTT resulted in high conversion. The pH optimum for the reaction was 7.5. Although the isomerase was heat stable, a temperature of 35°C was chosen for the assay reaction in order to minimize non-enzymatic thermal isomerization. Retention on the Concanavalin A column as well as shifting in mobility of all visible protein bands on SDS-PAGE following N-glycosidase-F treatment indicated that the enzyme is a glycoprotein. The enzyme was stable for at least 8 weeks when stored at -80°C. The isomerase is capable of converting cis-zeatin riboside and trans-zeatin riboside. Changing the isoprenoid side chain location from N⁶ in zeatin to the 9-position of the adenine ring in 9-(4-hydroxy-3- methylbut-2-enyl)adenine also resulted in isomerization. The occurrence of cis-trans isomerization suggests that cis-zeatin and cis-zeatin riboside formed by tRNA degradation could be precursors of biologically active cytokinins

Genetic stability of cryopreserved shoot tips of Rubus germplasm

Abstract Questions often arise concerning the genetic stability of plant materials stored in liquid nitrogen for long time periods. This study examined the genetic stability of cryopreserved shoot tips of Rubus germplasm that were stored in liquid nitrogen for more than 12 yr, then rewarmed and regrown. We analyzed the genetic stability of Rubus grabowskii, two blackberry cultivars ("Hillemeyer" and 'Silvan'), and one raspberry cultivar ("Mandarin") as in vitro shoots and as field-grown plants. No morphological differences were observed in greenhouse-grown cryopreserved plants when compared to the control mother plants. In the field, cryopreserved plants appeared similar but were more vigorous than mother plants, with larger leaves, fruit, and seeds. Single sequence repeats (SSR) and amplified fragment length polymorphism (AFLP) analyses were performed on shoots immediately after recovery from cryopreservation and on shoots subcultured for 7 mo before analysis. Ten SSR primers developed from "Marion" and "Meeker" microsatellite-enriched libraries amplified one to 15 alleles per locus, with an average of seven alleles and a total of 70 alleles in the four genotypes tested. No SSR polymorphisms were observed between cryopreserved shoots and the corresponding mother plants regardless of subculture. Although no polymorphisms were detected in shoots analyzed immediately after recovery from cryopreservation, AFLP polymorphisms were detected in three of the four Rubus genotypes after they were subcultured for 7 mo. Field-grown plants from the polymorphic shoot tips of R. grabowskii and 'Silvan' displayed the same AFLP fingerprints as their corresponding mother plants. Only longcultured in vitro shoot tips displayed polymorphisms in vitro, and they were no longer detected when the plants were grown ex vitro. The transitory nature of these polymorphisms should be carefully considered when monitoring for genetic stability

A High-Density Linkage Map of the Ancestral Diploid Strawberry, Fragaria iinumae, Constructed with Single Nucleotide Polymorphism Markers from the IStraw90 Array and Genotyping by Sequencing

Fragaria iinumae Makino is recognized as an ancestor of the octoploid strawberry species, which includes the cultivated strawberry, Fragaria ×ananassa Duchesne ex Rozier. Here we report the construction of the first high-density linkage map for F. iinumae. The F. iinumae linkage map (Fii map) is based on two high-throughput techniques of single nucleotide polymorphism (SNP) genotyping: the IStraw90 Array (hereafter “Array”), and genotyping by sequencing (GBS). The F2 generation mapping population was derived by selfing F. iinumae hybrid F1D, the product of a cross between two divergent F. iinumae accessions collected from Hokkaido, Japan. The Fii map consists of seven linkage groups (LGs) and has an overall length of 451.7 cM as defined by 496 loci populated by 4173 markers: 3280 from the Array and 893 from GBS. Comparisons with two versions of the Fragaria vesca ssp. vesca L. ‘Hawaii 4’ pseudo-chromosome (PC) assemblies reveal substantial conservation of synteny and colinearity, yet identified differences that point to possible genomic divergences between F. iinumae and F. vesca, and/or to F. vesca genomic assembly errors. The Fii map provides a basis for anchoring a F. iinumae genome assembly as a prerequisite for constructing a second diploid reference genome for Fragaria

Crop wild relatives as germplasm resource for cultivar improvement in mint (Mentha L.)

Mentha is a strongly scented herb of the Lamiaceae (formerly Labiatae) and includes about 30 species and hybrid species that are distributed or introduced throughout the globe. These fragrant plants have been selected throughout millennia for use by humans as herbs, spices, and pharmaceutical needs. The distilling of essential oils from mint began in Japan and England but has become a significant industrial product for the US, China, India, and other countries. The US Department of Agriculture (USDA), Agricultural Research Service, National Clonal Germplasm Repository (NCGR) maintains a mint genebank in Corvallis, Oregon. This facility preserves and distributes about 450 clones representing 34 taxa, hybrid species, advanced breeder selections, and F1 hybrids. Mint crop wild relatives are included in this unique resource. The majority of mint accessions and hybrids in this collection were initially donated in the 1970s by the A.M. Todd Company, located in Kalamazoo, Michigan. Other representatives of diverse mint taxa and crop wild relatives have since been obtained from collaborators in Australia, New Zealand, Europe, and Vietnam. These mints have been evaluated for cytology, oil components, verticillium wilt resistance, and key morphological characters. Pressed voucher specimens have been prepared for morphological identity verification. An initial set of microsatellite markers has been developed to determine clonal identity and assess genetic diversity. Plant breeders at private and public institutions are using molecular analysis to determine identity and diversity of the USDA mint collection. Evaluation and characterization includes essential oil content, disease resistance, male sterility, and other traits for potential breeding use. These accessions can be a source for parental genes for enhancement efforts to produce hybrids, or for breeding new cultivars for agricultural production. Propagules of Mentha are available for distribution to international researchers as stem cuttings, rhizome cuttings, or seed, which can be requested through the GRIN-Global database of the US National Plant Germplasm System, subject to international treaty and quarantine regulations

Mapping a Novel Black Spot Resistance Locus in the Climbing Rose Brite Eyes™ (‘RADbrite’)

Rose black spot, caused by Diplocarpon rosae, is one of the most devastating foliar diseases of cultivated roses (Rosa spp.). The globally distributed pathogen has the potential to cause large economic losses in the outdoor cultivation of roses. Fungicides are the primary method to manage the disease, but are often viewed unfavorably by home gardeners due to potential environmental and health impacts. As such, rose cultivars with genetic resistance to black spot are highly desired. The tetraploid climbing rose Brite EyesTM (‘RADbrite’) is known for its resistance to black spot. To better characterize the resistance present in Brite EyesTM, phenotyping was conducted on a 94 individual F1 population developed by crossing Brite EyesTM to the susceptible tetraploid rose ‘Morden Blush’. Brite EyesTM was resistant to all D. rosae races evaluated except for race 12. The progeny were either resistant or susceptible to all races (2, 3, 8, 9, 10, 11, and 13) evaluated. The segregation ratio was 1:1 (χ2 = 0.3830, P = 0.5360) suggesting resistance is conferred by a single locus. The roses were genotyped with the WagRhSNP 68K Axiom array and the ‘polymapR’ package was used to construct a map. A single resistance locus (Rdr4) was identified on the long arm of chromosome 5 homoeolog 4. Three resistance loci have been previously identified (Rdr1, Rdr2, and Rdr3). Both Rdr1 and Rdr2 are located on a chromosome 1 homoeolog. The chromosomal location of Rdr3 is unknown, however, races 3 and 9 are virulent on Rdr3. Rdr4 is either a novel gene or an allele of Rdr3 as it provides resistance to races 3 and 9. Due to its broad resistance, Rdr4 is an excellent gene to introgress into new rose cultivars

A roadmap for research in octoploid strawberry

The cultivated strawberry (Fragaria × ananassa) is an allo-octoploid species, originating nearly 300 years ago from wild progenitors from the Americas. Since that time the strawberry has become the most widely cultivated fruit crop in the world, universally appealing due to its sensory qualities and health benefits. The recent publication of the first high-quality chromosome-scale octoploid strawberry genome (cv. Camarosa) is enabling rapid advances in genetics, stimulating scientific debate and provoking new research questions. In this forward-looking review we propose avenues of research toward new biological insights and applications to agriculture. Among these are the origins of the genome, characterization of genetic variants, and big data approaches to breeding. Key areas of research in molecular biology will include the control of flowering, fruit development, fruit quality, and plant–pathogen interactions. In order to realize this potential as a global community, investments in genome resources must be continually augmented.Peer reviewe

A genetic linkage map of black raspberry (Rubus occidentalis) and the mapping of Ag (4) conferring resistance to the aphid Amphorophora agathonica

Black raspberry (Rubus occidentalis L.) is a high-value crop in the Pacific Northwest of North America with an international marketplace. Few genetic resources are readily available and little improvement has been achieved through breeding efforts to address production challenges involved in growing this crop. Contributing to its lack of improvement is low genetic diversity in elite cultivars and an untapped reservoir of genetic diversity from wild germplasm. In the Pacific Northwest, where most production is centered, the current standard commercial cultivar is highly susceptible to the aphid Amphorophora agathonica Hottes, which is a vector for the Raspberry mosaic virus complex. Infection with the virus complex leads to a rapid decline in plant health resulting in field replacement after only 3–4 growing seasons. Sources of aphid resistance have been identified in wild germplasm and are used to develop mapping populations to study the inheritance of these valuable traits. We have constructed a genetic linkage map using single-nucleotide polymorphism and transferable (primarily simple sequence repeat) markers for F1 population ORUS 4305 consisting of 115 progeny that segregate for aphid resistance. Our linkage map of seven linkage groups representing the seven haploid chromosomes of black raspberry consists of 274 markers on the maternal map and 292 markers on the paternal map including a morphological locus for aphid resistance. This is the first linkage map of black raspberry and will aid in developing markers for marker-assisted breeding, comparative mapping with other Rubus species, and enhancing the black raspberry genome assembly

Autopolyploid inheritance and a heterozygous reciprocal translocation shape chromosome genetic behavior in tetraploid blueberry (Vaccinium corymbosum)

Understanding chromosome recombination behavior in polyploidy species is key to advancing genetic discoveries. In blueberry, a tetraploid species, the line of evidences about its genetic behavior still remain poorly understood, owing to the inter-specific, and inter-ploidy admixture of its genome and lack of in depth genome-wide inheritance and comparative structural studies. Here we describe a new high-quality, phased, chromosome-scale genome of a diploid blueberry, clone W85. The genome was integrated with cytogenetics and high-density, genetic maps representing six tetraploid blueberry cultivars, harboring different levels of wild genome admixture, to uncover recombination behavior and structural genome divergence across tetraploid and wild diploid species. Analysis of chromosome inheritance and pairing demonstrated that tetraploid blueberry behaves as an autotetraploid with tetrasomic inheritance. Comparative analysis demonstrated the presence of a reciprocal, heterozygous, translocation spanning one homolog of chr-6 and one of chr-10 in the cultivar Draper. The translocation affects pairing and recombination of chromosomes 6 and 10. Besides the translocation detected in Draper, no other structural genomic divergences were detected across tetraploid cultivars and highly inter-crossable wild diploid species. These findings and resources will facilitate new genetic and comparative genomic studies in Vaccinium and the development of genomic assisted selection strategy for this cro

Construction of a SNP and SSR linkage map in autotetraploid blueberry using genotyping by sequencing

The construction of the first genetic map in autotetraploid blueberry has been made possible by the development of new SNP markers developed using genotyping by sequencing in a mapping population created from a cross between two key highbush blueberry cultivars, Draper × Jewel (Vaccinium corymbosum). The novel SNP markers were supplemented with existing SSR markers to enable the alignment of parental maps. In total, 1794 single nucleotide polymorphic (SNP) markers and 233 simple sequence repeat (SSR) markers exhibited segregation patterns consistent with a random chromosomal segregation model for meiosis in an autotetraploid. Of these, 700 SNPs and 85 SSRs were utilized for construction of the ‘Draper’ genetic map, and 450 SNPs and 86 SSRs for the ‘Jewel’ map. The ‘Draper’ map comprises 12 linkage groups (LG), associated with the haploid chromosome number for blueberry, and totals 1621 cM while the ‘Jewel’ map comprises 20 linkage groups totalling 1610 cM. Tentative alignments of the two parental maps have been made on the basis of shared SSR alleles and linkages to double-simplex markers segregating in both parents. Tentative alignments of the two parental maps have been made on the basis of shared SSR alleles and linkages to double-simplex markers segregating in both parents.This is the publisher’s final pdf. The published article is copyrighted by Springer and can be found at: http://link.springer.com/journal/11032Keywords: Vaccinium corymbosum, Tetraploid Map, Genotyping by sequencing, Autotetraploi