Tetraploid Crosses Research Articles

Cannabis Triploids Exhibit Reduced Fertility and Similar Growth and Flower Production Compared to Diploids

Cannabis (Cannabis sativa) grown for flowers containing cannabinoids requires all female plants, which are susceptible to seed set from exposure to pollen. Created triploids demonstrated reduced seed production compared with diploids in field and greenhouse studies in which plants were challenged with pollen from males. In the field, seed production as a percent of floral biomass ranged from 6.7% to 18.0% for triploids and from 52.6% to 57.1% for diploids. The photoperiod-insensitive triploid genotype ‘Purple Star’ × ‘Wilhelmina’ had 98.5% fewer filled (containing a developed embryo) seeds than the photoperiod-insensitive diploid genotype ‘Tsunami’ × ‘Wilhelmina’. In the greenhouse, triploid ‘Wife’ had 99.5% fewer filled seeds than diploid ‘Wife’. Plant growth and flower production were similar with eight triploid and seven diploid genotypes evaluated over three greenhouse studies. There were a few superior triploid and diploid genotypes; however, their performance was more likely attributable to the parental cultivar combination than ploidy level. The optimal cross direction for producing triploid seed in large quantities is tetraploid × diploid because the diploid × tetraploid cross exhibits triploid block caused by endosperm paternal excess. Colchicine-induced tetraploid parent plants should be tested over a prolonged period to eliminate cryptic chimeral mixoploids or tetraploid plants should be derived from seed produced by crossing two colchicine-induced putative tetraploid plants to ensure that seeds from tetraploid × diploid crosses will be triploid. The latter approach is necessary for photoperiod-insensitive cultivars because a prolonged period of ploidy testing is not possible for these plants. These findings indicate that triploid plants have significantly reduced fertility and are a suitable alternative to diploids in situations in which pollen exposure is possible.

Elimination of Chromosomes as a Mechanism for the Formation of Diploid Plants in Diploid–Tetraploid Crosses in Maize (Zea mays L.)

Elimination of Chromosomes as a Mechanism for the Formation of Diploid Plants in Diploid–Tetraploid Crosses in Maize (Zea mays L.)

Elimination of Chromosomes as a Mechanism for the Formation of Diploid Plants in Diploid–Tetraploid Crosses in Maize (<i>Zea mays</i> L.)

One of the main components of apomictic plant reproduction is the formation of unreduced embryo sacs (ESs). Heteroploid crosses, in which maternal diploid plants are pollinated by pollen of tetraploid paternal parent, can be used as an effective tool for identifying the ability to form unreduced ESs. In maize, in crosses 2n(♀) × 4n(♂), as a rule, shrunken kernels with a triploid embryo are formed, in which the development of the endosperm is impaired due to the deviation of the balance of the maternal (m) to paternal (p) genomes from the ratio of 2m : 1p. In our experiments, in several diploid maize lines, after their pollination with tetraploid pollen, the formation of large plump kernels was observed, from which diploid maternal-type plants developed, the maternal-type phenotype, as well as the hybrid endosperm phenotype, were confirmed by the expression of genetic markers. It has been suggested that the plump kernels in 2n × 4n crosses arise on the basis of unreduced embryo sacs (ESs) and pseudogamous apomixis, since the fusion of diploid sperms with diploid polar nuclei provides a 2 : 1 ratio of maternal to paternal genomes in the endosperm. In order to clarify the genetic nature of diploid plants phenotypically similar to maternal lines that developed from plump kernels in 2n × 4n crosses, we carried out their genotyping for all 10 chromosomes of the maize genome using polymorphic codominant SSR- and Indel-markers that differentiate the paternal line from maternal lines. As maternal lines, we used HPL AT, which has the ability to haploid parthenogenesis, KM, YuV-11, as well as the F2 B47/HPL AT hybrid plants; and as a paternal line – Chernaya Tetra(4n). It was found that in all the plants studied, when markers of five chromosomes (1, 2, 3, 4, and 9) were used, amplification of only maternal alleles was observed. However, in each of the studied plant, when using markers of other chromosomes, cases of amplification of alleles characteristic of the paternal line were noted. A hypothesis is put forward on the formation of diploid plants in 2n × 4n crosses in maize as a result of fertilization of the unreduced ESs and the subsequent elimination of chromosomes, predominantly of the pollen parent.

Molecular and cytogenetic description of somatic hybrids between Gentiana cruciata L. and G. tibetica King

Somatic hybridization provides an opportunity to create cells with new genetic constitution. Here, the interspecific somatic hybrid plants regenerated in vitro following fusion of cell suspension–derived protoplasts of tetraploid Cross Gentian (Gentiana cruciata L., 2n = 52) with protoplasts released from mesophyll tissue of another tetraploid species, Tibetan Gentian (G. tibetica King, 2n = 52), were studied. According to the results of genome analyses with AFLP, ISSR, and CAPS markers, all somatic hybrids were genetically closer to “suspension” fusion partner G. cruciata than to “mesophyll” partner G. tibetica, but they got G. tibetica chloroplasts. Chromosome counting revealed little variation in the number of chromosomes in hybrid’s cells (2n = 88 or 2n = 90), although all plants possessed similar nuclear DNA content which remained stable even after 2 years of in vitro culture. Fluorescence in situ hybridization (FISH) showed that hybrids possessed 4 to 7 chromosomes bearing 5S rDNA sites and 6 or 7 chromosomes with 35S rDNA sites. A part of FISH signals was smaller than those observed in the parental species, which could indicate the loss of rDNA sequences. Genomic in situ hybridization (GISH) showed the predominance of the number of G. cruciata chromosomes over chromosomes of G. tibetica. However, a significant level of cross-hybridization was observed for about one-third of hybrid chromosomes, indicating a high degree of homeology between the genomes of G. cruciata and G. tibetica.

Versatility of reproductive modes and ploidy level interactions in Sorbus s.l. (Malinae, Rosaceae)

Abstract This study focuses on the frequency of reproductive modes and the role of reproductive mode variation in diversification of the genus Sorbus s.l. and evolutionary success of individual taxa. Our sampling covers all four ploidies known in Sorbus and includes stabilized taxa and recently formed biotypes. We infer reproductive modes of 3660 seeds using the flow cytometric seed screen method; we also calculated ploidy of contributing pollen. Sorbus shows high variability in reproduction pathways (sexuality, apomixis, formation of BIII hybrids, haploid parthenogenesis). Diploids are sexual, whereas polyploids show predominantly pseudogamous apomixis. In half of the polyploids studied, several reproductive modes were present and even intra-individual variation was detected. Diploids, tetraploids and pentaploids are pollinated by conspecific pollen, whereas triploids are largely, but not fully, dependent on the pollen of other species. In pseudogamous seeds, fertilization of the central cell by both sperms is a dominant pathway of endosperm origin. Flow cytometry of pollen did not detect unreduced pollen at any ploidy. The principal mechanisms of diversification in Sorbus are residual sexuality in apomicts, production of triploid taxa from diploid–tetraploid crosses and production of BIII hybrids. Evolutionary success of Sorbus taxa is largely dependent on the breeding system and is determined by self-(in)compatibility and the interplay between stabilizing apomixis and variation-generating sexuality.

Identification of a polymorphism within the Rosa multiflora muRdr1A gene linked to resistance to multiple races of Diplocarpon rosae W. in tetraploid garden roses (Rosa × hybrida).

A QTL for resistance to several races of black spot co-located with the known Rrd1 locus in Rosa. A polymorphism in muRdr1A linked to black spot resistance was identified and molecular markers were designed. Black spot, caused by Diplocarpon rosae, is one of the most serious foliar diseases of landscape roses that reduces the marketability and weakens the plants against winter survival. Genetic resistance to black spot (BS) exists and race-specific resistance is a good target to implement marker-assisted selection. High-density single nucleotide polymorphism-based genetic maps were created for the female parent of a tetraploid cross between 'CA60' and 'Singing in the Rain' using genotyping-by-sequencing following a two-way pseudo-testcross strategy. The female linkage map was generated based on 227 individuals and included 31 linkage groups, 1055 markers, with a length of 1980cM. Race-specific resistance to four D. rosae races (5, 7, 10, 14) was evaluated using a detached leaf assay. BS resistance was also evaluated under natural infection in the field. Resistance to races 5, 10 and 14 of D. rosae and field resistance co-located on chromosome 1. A unique sequence of 32bp in exon 4 of the muRdr1A gene was identified in 'CA60' that co-segregates with D. rosae resistance. Two diagnostic markers, a presence/absence marker and an INDEL marker, specific to this sequence were designed and validated in the mapping population and a backcross population derived from 'CA60.' Resistance to D. rosae race 7 mapped to a different location on chromosome 1.

Mitotic instability in triploid and tetraploid one-year-old eastern oyster, Crassostrea virginica, assessed by cytogenetic and flow cytometry techniques.

For commercial oyster aquaculture, triploidy has significant advantages. To produce triploids, the principal technology uses diploid × tetraploid crosses. The development of tetraploid brood stock for this purpose has been successful, but as more is understood about tetraploids, it seems clear that chromosome instability is a principal feature in oysters. This paper is a continuation of work to investigate chromosome instability in polyploid Crassostrea virginica. We established families between tetraploids-apparently stable (non-mosaic) and unstable (mosaic)-and normal reference diploids, creating triploid groups, as well as tetraploids between mosaic and non-mosaic tetraploids. Chromosome loss was about the same for triploid juveniles produced from either mosaic or non-mosaic tetraploids or from either male or female tetraploids. However, there was a statistically significant difference in chromosome loss in tetraploid juveniles produced from mosaic versus non-mosaic parents, with mosaics producing more unstable progeny. These results confirm that chromosome instability, as manifested in mosaic tetraploids, is of little concern for producing triploids, but it is clearly problematic for tetraploid breeding. Concordance between the results from cytogenetics and flow cytometry was also tested for the first time in oysters, by assessing the ploidy of individuals using both techniques. Results between the two were non-concordant.

High proportion of diploid hybrids produced by interspecific diploid × tetraploid Sorghum hybridization

A perennial version of grain sorghum [S. bicolor (L.) Moench] would create opportunities for greatly reducing tillage and preventing soil degradation. Efforts to select for perenniality and grain production among progeny of hybrids between S. bicolor (2n = 20) and the weedy tetraploid perennial S. halepense (L.) Pers. (2n = 40) are complicated in that F1 hybrids produced by diploid × tetraploid sorghum crosses are usually tetraploid. In 2013, a set of random pollinations between 19 diploid cytoplasmic male-sterile inbred lines and 43 tetraploid perennial plants produced 165 F1 hybrid plants, more than 75% of which had highly atypical plant, panicle, and seed phenotypes. Phenotypic segregation in F2 populations derived from atypical hybrids was also anomalous. Examination of mitotic metaphase cells in F1 or F2 root tips revealed that 129 of the 165 hybrids were diploid. Parentage of the diploid progenies was confirmed using simple-sequence repeat analysis. The mechanism by which diploid hybrids arise from diploid × tetraploid crosses is unknown, but it may involve either production of monohaploid (n = 10) pollen by the tetraploid parent or chromosome elimination during early cell divisions following formation of the triploid zygote. The ability to produce diploid germplasm segregating for S. bicolor and S. halepense alleles could have great utility, both for the development of perennial sorghum and for the improvement of conventional grain sorghum.

Validating DNA Polymorphisms Using KASP Assay in Prairie Cordgrass (Spartina pectinata Link) Populations in the U.S.

Single nucleotide polymorphisms (SNPs) are one of the most abundant DNA variants found in plant genomes and are highly efficient when comparing genome and transcriptome sequences. SNP marker analysis can be used to analyze genetic diversity, create genetic maps, and utilize marker-assisted selection breeding in many crop species. In order to utilize these technologies, one must first identify and validate putative SNPs. In this study, 121 putative SNPs, developed from a nuclear transcriptome of prairie cordgrass (Spartina pectinata Link), were analyzed using KASP technology in order to validate the SNPs. Fifty-nine SNPs were validated using a core collection of 38 natural populations and a phylogenetic tree was created with one main clade. Samples from the same population tended to cluster in the same location on the tree. Polymorphisms were identified within 52.6% of the populations, split evenly between the tetraploid and octoploid cytotypes. Twelve selected SNP markers were used to assess the fidelity of tetraploid crosses of prairie cordgrass and their resulting F2population. These markers were able to distinguish true crosses and selfs. This study provides insight into the genomic structure of prairie cordgrass, but further analysis must be done on other cytotypes to fully understand the structure of this species. This study validates putative SNPs and confirms the potential usefulness of SNP marker technology in future breeding programs of this species.

Heteroploid mosaic tetraploids of Crassostrea virginica produce normal triploid larvae and juveniles as revealed by flow cytometry

Abstract Advantages associated with sterility in triploids have made them the standard for commercial aquaculture of Crassostrea virginica in the Chesapeake Bay. Current hatchery practices for commercial production of triploids employ tetraploids as broodstock, and thus tetraploids are a core asset for oyster culture. Tetraploids undergo reversion, losing entire sets of chromosomes, and become heteroploid mosaics comprised of triploid and tetraploid cells. The possible effects of using mosaic tetraploids for triploid production are a practical concern for commercial oyster culture. We crossed mosaic and non-mosaic tetraploids, males and females, to a reference diploid oyster and compared relative DNA content and phenotypic qualities of triploid progeny. Relative DNA content was measured in somatic and gametic tissue of tetraploid broodstock, as well as in resulting triploid larvae via flow cytometry. Size, abnormality, and survival of larvae were recorded through the rearing process. For six crosses, triploids were raised for 1 year and shell metrics, size, and somatic DNA content were compared. Flow cytometry analysis of broodstock revealed sperm from mosaic tetraploids were consistently di-haploid and virtually indistinguishable from sperm of non-mosaics. For resulting triploid progeny, there were no detectable differences between larvae or juveniles produced from mosaic and non-mosaic tetraploids. Differences existed, however, between triploid larvae produced from di-haploid sperm and those produced from tetraploid eggs: triploids from tetraploid eggs were significantly more abnormal, less viable, and grew slower. Our findings support current commercial practices of using tetraploid males as the principal parent for triploid production and demonstrate no practical consequence from using mosaic tetraploid broodstock for commercial production of triploid C. virginica . Further analysis via cytogenetic techniques may provide insight into long term consequences of using mosaic tetraploids, for example, in tetraploid × tetraploid crosses.

TECHNOLOGY APPLIED TO THE DEVELOPMENT OF NEW FLAVOUR HOP VARIETIES IN AUSTRALIA

Hop production in Australia occupies a total of approximately 500 hectares, spread across two major farms and several smaller growers. Production is based in south-eastern Australia, spread roughly evenly between Victoria and Tasmania, latitudes 37-41° south respectively. Every hop grower in Australia relies upon varieties developed locally since 1950, in a breeding program, which continues today. Since 1994, collaborative work (predominantly with the University of Tasmania - UTAS) has seen the development of technologies such as in vitro colchicine induction to produce tetraploid plants, and flow cytometry, which have led lately to the availability of tetraploid male and female plants, and the ability to conduct tetraploid by tetraploid crosses. An extensive database of gas chromatography-mass spectrometry (GC-MS) of steam distilled hop essential oil profiles has been built up, containing information on international varieties, Australian commercial varieties, and selected material. Australian hop researchers have participated actively in the international collaboration to develop Diversity Arrays Technology (DArT) markers. Future work will include quantitative genetic analysis of large progeny trials grown in both Tasmania and Victoria, the establishment of a linkage mapping population replicated in both Tasmania and Victoria, and application and further development of DArT markers in specific roles with the new cultivar development process. Active collaboration provides hop breeding efforts in Australia with the tools necessary to create, identify, and develop novel varieties with diverse and exciting flavour potential.

Biochemical Evaluation of Triploid Progenies of Diploid × Tetraploid Breeding Populations of Camellia for Genotypes Rich in Catechin and Caffeine

To verify the quality of triploid varieties of Camellia tea species at the secondary metabolite level, we tested caffeine and catechin profiles of 97 F(1) segregating progenies in two breeding populations with a common tetraploid parent and diploid parents of two geographic and varietal origins. Catechin and caffeine levels of the triploid progenies were quantified and compared against their diploid parent. Some of the progenies showed better performance than their diploid parent. Most of the progenies of the diploid C. sinensis × tetraploid cross showed heterosis for caffeine and EGCG. Progenies of the C. assamica subsp. lasiocalyx × tetraploid cross showed heterosis for +C, EC, EGC, and TC. The genomic contributions of the diploid parent seem to be the main factor in the variation between the two populations. Our studies showed quantitative enhancement of some of the quality-related parameters in tea, providing a platform to refocus on this classical breeding approach for developing quality cultivars in tea.

Meiotic behavior in apomictic Brachiaria ruziziensis × B. brizantha (Poaceae) progenies

Hybrids combining desirable traits from divergent parents are the main objective of some Brachiaria (Syn. Urochloa P. Beauv.) breeding programs. There is great interest in the development of apomictic hybrid cultivars that combine desirable genes such as resistance to spittlebugs, high nutritive value, and tolerance to acid soils. Microsporogenesis of six apomictic progenies resulting from a tetraploid (2n = 4x = 36) cross between B. ruziziensis × B. brizantha was evaluated under light microscopy. Genetic recombination, ensured by multivalent chromosome association and crossing-over at prophase occurred in low frequency among progenies, and in one, recombination was almost nonexistent. The percentage of meiocytes with meiotic abnormalities among progenies ranged from 16.6 % to 85.6 %. Besides an observed irregular chromosome segregation typical of polyploid hybrids in these five progenies, putative meiotic mutations characterized as desynapsis and divergent spindle organization occurred in three progenies. These anomalies caused frequent fractionation of the genome into several microspores of different sizes. In Brachiaria, new cultivars must be apomictic to fix the genotype. However, Brachiaria is a pseudogamous apomict, and viable gametes are necessary to produce viable seeds. Considering meiotic behavior, only two progenies are promising for advancement in the breeding program.

Motility, morphology, mitochondria and ATP content of diploid spermatozoa from sex-reversed clonal diploid and neo-tetraploid loach,Misgurnus anguillicaudatus

Summary Diploid spermatozoa were obtained from sex-reversed clonal diploid males and neo-tetraploid which derived from normal diploid × natural tetraploid cross followed by the 2nd polar body release inhibition. The spermatozoa from sex-reversed clonal diploid males exhibited a significant reduction in motility when compared with those from normal diploid and neo-tetraploid males. Electron microscopy revealed that diploid spermatozoa from sex-reversed clonal diploid and neo-tetraploid males exhibited normal shape similar to haploid spermatozoa from normal diploid males, except for larger sperm-head size and longer flagellum. Considering flow-cytometric results, volume of mitochondrial mass per spermatozoon was also increased in proportion to the elevation of ploidy status from haploid to diploid sperm. Diploid spermatozoa from neo-tetraploid increased the number of mitochondria and the ATP content, but those from sex-reversed clonal diploid males did not show such results.

Factors affecting the first cleavage interval and effects of parental generation on tetraploid production in rainbow trout (Oncorhynchus mykiss)

Tetraploidy is induced in rainbow trout by applying a pressure shock at a specific time point between insemination and first cleavage, or the first cleavage interval (FCI). Previous studies suggested that variation in the FCI among individuals and populations of fish prevents the identification of a single time point that can be used for all trout. In this study we confirmed the optimal time to apply pressure is 65±5% of the FCI. In addition, we found that variation in FCI of fish from a common environment can be within limits that allow a single time point to be established for that group of fish, if ova post ovulatory aging is taken into account. Aging of ova, either in vivo or in vitro, increased FCI to a degree that is a concern for tetraploid induction. The FCI was about 12min longer at 7days post ovulation, and 30min at 10–14days, than at 1day. The FCI for a group of fish was consistent throughout the spawning season. Survival to hatching and frequency of spinal abnormalities were similar for progeny of first and second generation tetraploid males, but survival was doubled and abnormalities reduced by approximately 90% in second generation tetraploid females compared with first generation females. All progeny of tetraploid by tetraploid crosses were determined to be tetraploids based on flow cytometry of embryonic cells. In summary, attention to ova aging and use of second generation female tetraploids allows efficient production of a tetraploid rainbow trout broodstock.

Identification of pre-zygotic reproductive and morphological barriers limiting controlled crossed seed production of triploid Acacia mearnsii

A study of the reproductive biology of black wattle (Acacia mearnsii de Wild) was conducted, specifically with regard to the compatibility of the diploid×tetraploid cross to produce a triploid variety, together with identifying ways of increasing triploid seed production. Fluorescent microscopy was used to determine pollination and fertilisation rates and to identify any pre-zygotic barriers at the stigma, style and ovary. Both diploid and tetraploid families were tested as maternal parents to establish if this was imperative to producing a triploid. Morphological measurements were documented in order to determine any incompatibilities in the cross to produce a triploid. The in vivo results showed that successful fertilisation of the ovary was possible whether one used a diploid or tetraploid maternal parent. When the maternal parent was a tetraploid, however, the pollination rate (polyads adhering to the stigma) and ovary fertilisation rates were significantly (p<0.05) greater. Morphological measurements and observations also revealed that tetraploid floral parts were significantly (p<0.05) larger than the diploids. The morphological size differences between the diploid and tetraploid polyads and pistils did not appear to influence the fertilisation of the ovaries and thus did not pose any identifiable barrier in the cross to produce a triploid. When considering the results from the cross to produce a triploid (2n×4n or 4n×2n), the diploid polyads were significantly (p<0.05) more vigorous and suitable in fertilising the tetraploid ovaries as opposed to the reverse. Possible pre-zygotic barriers at the stigma, style or ovary were investigated and the only area that could be identified limiting seed production was within the ovary.

Mapping of FHB resistance QTLs in tetraploid wheat

Triticum turgidum L var. durum is known to be particularly susceptible to infection by Fusarium graminearum, the causal agent for Fusarium head blight (FHB), which results in severe yield losses and grain contaminated with mycotoxins. This research was aimed at identifying FHB resistance in tetraploid wheat and mapping the location of FHB resistance genes. A tetraploid cross of durum wheat ('Strongfield') x Triticum carthlicum ('Blackbird') was used to generate a doubled-haploid (DH) population. This population was evaluated for type II resistance to F. graminearum in replicated greenhouse trials, in which heads were innoculated and the percent of infected spikelets was determined 21 days later. The population was also genotyped with microsatellite markers to construct a map of 424 loci, covering 2 052 cM. The FHB reaction and genotypic data were used to identify FHB resistance quantitative trait loci (QTLs). It was determined that 2 intervals on chromosomes 2BL and 6BS controlled FHB resistance in this tetraploid cross. The FHB resistance allele on chromosome 2BL (r2=0.26, logarithm of odds (LOD)=8.5) was derived from 'Strongfield', and the FHB resistance allele on chromosome 6BS (r2=0.23, LOD=6.6) was derived from 'Blackbird'. Two other loci, on chromosomes 5AS and 2AL, were shown to regulate FHB infection and to have an epistatic effect on the FHB resistance QTL on chromosome 6BS. Further, the FHB resistance QTL peak on chromosome 6BS was clearly coincident with the known FHB resistance gene Fhb2, derived from Sumai 3. The results show that FHB resistance can be expressed in durum wheat, and that T. carthlicum and Triticum aestivum likely share a common FHB resistance gene on chromosome 6BS.

INHERITANCE OF VARIEGATED AND PURPLE FOLIAGE PHENOTYPES IN Hypericum androsaemum L.

Inheritance of two mutant foliage types (purple and mottled variegated) was investigated for diploid, triploid, and tetraploid tutsan (Hypericum androsaemum). Segregation ratios were determined for diploid crosses in reciprocal dihybrid F1 and F2, BC1P1, and BC1P2 families. F2 tetraploids were derived from autotetraploid F1s. Triploid segregation ratios were determined from crosses between autotetraploid F1s and diploid F1s. Diploid di-hybrid crosses fit the expected 9:3:3:1 ratio for a single, simple recessive gene for both traits, with no evidence of linkage between each trait. Data from backcross and triploid crosses generally supported this model. In tetraploid crosses we observed twice as many variegated phenotypes as predicted which was not explained by random chromosome or chromatid assortment. Inheritance of purple foliage did not deviate from random chromosome assortment at the tetraploid level.

Acid Citrus Fruit Improvement via Interploid Hybridization Using Allotetraploid Somatic Hybrid and Autotetraploid Breeding Parents

Interploid hybridization was conducted using `Key' lime [Citrus aurantifolia (Cristm.) Swing.], `Lakeland' limequat hybrid [C. aurantifolia × Fortunella japonica (Thumb.) Swing.], Palestine sweet lime (C. limettioides Tan.), `Etrog' citron (C. medica L.), and seven lemon [C. limon (L.) Burm. F.] varieties as female progenitors and five allotetraploid somatic hybrids {`Hamlin' sweet orange [C. sinensis (L.) Osbeck] × `Femminello' lemon (C. limon)]; `Key' lime × `Valencia' sweet orange (C. sinensis); `Valencia' sweet orange × rough lemon (C. jambhiri Lush); Milam lemon (purported C. jambhiri hybrid) × `Femminello' lemon (C. limon); and `Valencia' sweet orange × `Femminello' lemon} and two autotetraploids [`Giant Key' lime (C. aurantifolia) and `Femminello' lemon] as pollen progenitors. A few tetraploid × diploid crosses were also performed. Thirty-five parental cross combinations were accomplished in 2000, 2001, and 2002. The breeding targets were seedlessness, cold-tolerance, and disease resistance. Triploid hybrids were recovered through embryo culture. Generation of triploid citrus hybrids was affected by several factors including sexual compatibility, cross direction, embryo developmental stage, pollen viability, as well as horticultural practices and climatic conditions. Efficiency of triploid hybrid production was higher in diploid × tetraploid crosses than the reciprocal. Many more triploid hybrids were generated from lemon seed progenitors compared to the other acid citrus fruit progenitors. `Todo el Año', `Lisbon', and `Limonero Fino 49' showed the highest sexual compatibility. Embryo germination rate and normal plant recovery were also higher in lemons as compared to the other seed progenitors. Low winter temperatures might have affected the hybrid production efficiency from tropical acid fruit progenitors. A total of 650 hybrids (mostly triploid) were transferred to soil. The novel genetic combinations of these progenies should be valuable for the genetic improvement of acid citrus fruit (lemons and limes).

A fluorogenic 5' nuclease (TaqMan) assay to assess dosage of a marker tightly linked to red skin color in autotetraploid potato.

We have recently identified an allele of dihydroflavonol 4-reductase ( dfr) that cosegregates with the ability of potato ( Solanum tuberosum L) to produce red pelargonidin-based anthocyanin pigments. A rapid assay to assess dosage of this allele in cultivated potato, an autotetraploid, would be useful for breeding programs that develop red-skinned cultivars. To identify regions of dfr that are conserved between alleles, as well as regions that are variable, a portion of the gene was sequenced from several cultivated and wild potato clones. In one region the sequence of the 'red' dfr allele differed at two nucleotide positions from the three other sequence classes observed. A fluorogenic oligonucleotide probe labeled with 6-FAM was designed to anneal specifically to the red allele in this region, while a second probe labeled with VIC was designed to anneal to the 'not-red' dfr alleles. PCR primers that annealed to conserved sequences flanking the variable region were also developed. When subjected to a fluorogenic 5' nuclease (TaqMan) allelic discrimination assay all diploid clones tested clustered into three distinct groups based on the relative amounts of FAM and VIC released. These three groups represented clones homozygous for the red allele, heterozygous for the red allele, and homozygous for the not-red allele(s). When tetraploid clones were tested they separated into five distinct clusters, three of which were shared with diploid clones. The five clusters were interpreted to represent clones quadruplex, triplex, duplex, simplex and nulliplex for the red dfr allele. This interpretation was supported by monitoring the segregation of red allele dosage in several tetraploid crosses. To the best of our knowledge this is the first report of a fluorogenic 5' nuclease assay being used for allelic discrimination in an autopolyploid.