Sexual hybridization between Capsella bursa‐pastoris (L.) Medik (♀) and Camelina sativa (L.) Crantz (♂) (Brassicaceae)

The development of transgenic oilseed Camelina sativa (2n = 40) and the potential for hybridization with its weedy relative Capsella bursa‐pastoris (2n = 36) necessitates a careful evaluation of the reproductive compatibility between the species. Here, we conducted over 1800 crosses (emasculation and manual pollination) to examine the ability of 10 Canadian C. bursa‐pastoris (♀) accessions to hybridize with five accessions of C. sativa (♂). Seven hybrids were confirmed among 586 putative hybrids screened with species‐specific markers, indicating a hybridization rate of 1.5 hybrids per 10 000 ovules pollinated. All seven hybrids had intermediate DNA content compared to their parents, were morphologically distinct, had low (1.9%) pollen fertility and failed to produce selfed or backcrossed seed. Given the abundance of C. bursa‐pastoris along field margins, hybrids will likely be generated in the wild, but they will be unable to establish lineages unless fertility is restored. The large number of crosses and the diversity captured by the use of multiple accessions resulted in strong statistical power and a high degree of confidence in the estimated hybridization rate.     

Cytogenetics of a new trispecies hybrid in cotton: [(Gossypium hirsutum L. × G. thurberi Tod.)2 × G. longicalyx Hutch. & Lee]

A three‐species hybrid named HTL including Gossypium hirsutum L. [2n = 4 x = 52, (AD)₁ genome] was created using the pseudophyletic introgression method with G. longicalyx Hutch. & Lee (2n = 2x = 26, F₁ genome) as donor parent and G. thurberi Tod. (2n = 2x = 26, D₁ genome) as bridge species. The new hybrid was totally self‐sterile and its interspecific status was confirmed using simple sequence repeat markers and cytogenetic analysis. Cytogenetic studies showed that its chromosome configuration was 2n = 52 = 14.13 I + 15.10 II + 1.03 III + 0.9 IV + 0.03 V + 0.13 VI (where I, II, III, IV, V and VI are univalents, bivalents, trivalents, tetravalents, pentavalents and hexavalents, respectively). Prospects for successfully exploiting the HTL hybrid in breeding programmes are discussed.     

Interspecific hybridization between Vigna unguiculata (L.) Walp. and V. vexillata (L.) A. Rich. through in vitro embryo culture

Immature embryos resulting from the cross V. vexillata × V. unguiculata were cultured on MS medium supplemented with 2,4-D (2 mg/l) and resulted in embryogenic calli. Thirteen hybrid regenerants were obtained via organogenesis by subculturing the calli on MS medium supplemented with BAP (2 mg/l) + adenine sulphate (40 mg/l) + CH (500 mg/l) + cowpea tender pod extract (10%). The interspecific regenerants showed intermediate morphological traits between the parents for leaf shape, pod colour and seed coat colour. The hybrid plants inherited stem, leaf and pod hairiness of the wild species which could serve as a mechanical barrier against viral vectors. Electrophoretic studies of two isozyme systems, peroxidase and esterase, also confirms the hybrid nature of the regenerants as they expressed unique bands of both parents. Cytological study of the meiotic chromosomes revealed high frequency of univalent formation in the hybrids suggesting that the genomes of the parental species are structurally differentiated. The hybrid regenerants exhibited high enzyme activity for three enzymes viz., peroxidase, polyphenol oxidase and phenyl alanine ammonia lyase over the cultivated parent which may be useful in conferring resistance against viral pathogens. This revised version was published online in July 2006 with corrections to the Cover Date.     

Interspecific cross‐hybrids of Nicotiana tabacum L. cv. (gla.) S ‘K326’ with Nicotiana alata

In order to introduce the Tomato Spotted Wilt Virus (TSWV) resistance from Nicotiana alata into Nicotiana tabacum, a cytoplasmic male sterility (CMS) line of N. tabacum (N. tabacum L. cv. (gla.) S ‘K326’), was successfully crossed with N. alata. Despite a high DNA content variability, F₁ hybrids could be classified in two subgroups, a major one encompassing fertile hybrids morphologically similar to their tobacco maternal parent but TSWV sensitive, and a minor one displaying sterile hybrids showing an intermediate phenotype and TSWV resistant. In order to elucidate the unexpected fertility recovery of the fertile F₁ plants, some N. alata fertility restoration ppr genes were cloned and were shown to be differentially expressed between parental lineages as well as between both F₁ subgroups, suggesting that N. alata contains fertility restoring allele able to overcome the CMS of N. tabacum.     

Phenotypic and seed structural comparison in hybrids of different Brassica rapa and B. oleracea

Brassica napus is an important oil species with short history and narrow genetic background. Interspecific hybrids from crosses between B. oleracea and different B. rapa were obtained. We found the hybrids with white petal resembling B. oleracea, the flavonoid and phenolic content decreased in hybrids, agreeing with the expressional changes of flavonoid biosynthesis genes. Seed coat of hybrids resembled diploid parents, or partly resembled to each parent with a clear outline. The palisade layer in hybrids was thicker than parents, with similar pigment accumulation as B. oleracea but more than B. rapa. Differentially sized protein bodies (PBs) were found in hybrids. The radical and inner cotyledon of all hybrids were identified with larger but less PBs than parents. The average size of PBs in outer cotyledon of resynthesized B. napus was also larger than parents, but the number of PBs was not significantly reduced. The phenotypic and seed structural variations after polyploidization of B. napus would be interesting for genetic broadening and breeding of rapeseed.     

Interspecific hybridization of a multiploid mutant of durum wheat with rye and Triticum monococcum L. results in pentaploid hybrids

The multiploid mutant of durum wheat is a genotype that produces unreduced gametes. Our objective was to test the recovery of pentaploid hybrids in crosses of the mutant with rye and Triticum monococcum L. Compared with check crosses, the mutant had a two‐third reduction in percent seed set for rye crosses, but had only a slight decrease in crossability with T. monococcum. Pentaploid hybrids were associated with plump seeds of the mutant/rye cross, and with shrivelled seeds of the mutant/T. monococcum cross. We suggest that the endosperm balance number hypothesis explains the association of pentaploid hybrids with endosperm type. This association made for easy recovery of pentaploid hybrids from crosses to both species. Mature, plump seeds from the mutant/rye cross were germinated and pentaploid hybrids were recovered. One pentaploid hybrid was recovered for every 50.5 and 15.1 florets pollinated with rye and T. monococcum, respectively. Unreduced gametes in the multiploid mutant will facilitate interspecific hybridization by reducing the time to produce pentaploid plants.     

Chromosome pairing and fertility of interspecific hybrids between Trifolium repens L. and T. occidentale Coombe

Trifolium occidentale is a diploid wild relative with the potential to improve the drought and salt tolerances of white clover (Trifolium repens). Previous work has shown that it is possible to efficiently produce large T. repens × T. occidentale breeding populations using colchicine‐doubled (4×) T. occidentale. For effective introgression (backcross) breeding, it is also essential that interspecific chromosome pairing and recombination occur. In this study, it was apparent that chromosome pairing was occurring not only between T. occidentale and T. repens subgenomes, but also between the ancestral subgenomes of T. repens. Thus, interspecific hybridization has the potential for major genome recombination and opens the way for introgression of traits from T. occidentale into white clover.     

Inheritance of resistance to the peach root‐knot nematode (Meloidogyne floridensis) in interspecific crosses between peach (Prunus persica) and its wild relative (Prunus kansuensis)

The peach root‐knot nematode, Meloidogyne floridensis (MF), infects majority of available nematode‐resistant peach rootstocks which are mostly derived from peach (Prunus persica) and Chinese wild peach (P. davidiana). Interspecific hybridization of peach with its wild relative, Kansu peach (P. kansuensis), offers potential for broadening the resistance spectrum in standard peach rootstocks. We investigated the inheritance of resistance to MF in segregating populations of peach (‘Okinawa’ or ‘Flordaguard’) × P. kansuensis. A total of 379 individuals from 13 F₂ and BC₁F₁ families were challenged with a pathogenic MF isolate “MFGnv14” and were classified as resistant (R) or susceptible (S) based on root galling intensity. Segregation analyses in F₂ progeny revealed the involvement of a major locus with a dominant or recessive allele determining resistance in progeny segregating 3R:1S and 1R:3S, respectively. Testcrosses with a homozygous‐susceptible peach genotype (‘Flordaguard’ or ‘UFSharp’) confirmed P. kansuensis as a source of new resistance and the heterozygous allelic status of P. kansuensis at the locus conferring resistance to MF. We propose a single‐locus dominant/recessive model for the inheritance of resistance.     

Identification and characterization of sources for photo‐ and thermo‐insensitivity in Vigna species

Cultivation of the same varieties of mungbean and blackgram across different seasons and locations is constrained by their photo‐ and thermo‐sensitive behaviour. Developing insensitive genotypes, which can fit well across all seasons, requires robust donors which would provide genes imparting this trait. This study was undertaken to identify such donors in the Vigna species. Forty‐eight accessions belonging to 13 Vigna species and eight released cultivars were evaluated under natural field conditions. Among these, two accessions, viz. V. umbellata (IC251442) and V. glabrescens (IC251372) were found photo‐ and thermo‐insensitive as these were able to flower and set pods at temperatures as high as 43.9°C and as low as 2.7°C. Pollen viability studies indicated viable pollen (>75% at 2.7°C and >85% at 41.9°C) and normal pollen tube growth at both the extremes of temperature. The identified V. glabrescens accession has long, constricted pods and dark green, mottled seeds while V. umbellata has smooth, curved pods and shining, oval, large seeds. Both these accessions can be utilized in developing photo–thermo insensitive genotypes in cultivated Vigna species.     

Development and utilization of genomic and genic microsatellite markers in Assam tea (Camellia assamica ssp. assamica) and related Camellia species

Microsatellite or simple sequence repeat (SSR) markers are valuable tools for many purposes, such as phylogenetic, fingerprinting and molecular breeding studies. However, such marker resources are unavailable in Assam tea (Camellia assamica ssp. assamica; Masters). With an objective to enrich the repertoire of microsatellite markers in traditional tea, 185 novel microsatellite (150 genomic and 35 genic) markers were identified from (GA)n‐enriched genomic libraries and public expressed sequence data in Assam tea. High‐quality 0.412‐Mb non‐redundant (NR) genomic data set derived from nucleotide sequencing of 1297 (GA)n‐enriched genomic positive clones and 2723 unigenes (1.33 Mb) predicted from 10 803 random public expressed sequence tags (ESTs) in C. assamica ssp. assamica were utilized for identification of genomic and genic microsatellite markers, respectively. The average number of alleles and polymorphic information content (PIC) recorded for the newly developed SSR markers were 6.17 and 0.398, respectively. The average observed (H_o) and expected (H_e) heterozygosity varied from 0.626 to 0.697, respectively. These markers were found to be highly transferable (74.5–100%) to cultivated (C. sinensis, C. assamica ssp. lasiocalyx) and five wild Camellia species. Genetic diversity coefficient detected a high level of divergence in 24 cultivated tea accessions (69.3%). Phylogenetic analysis revealed that major groupings were broadly in accordance with taxonomic classification of tea, and all the wild Camellia species remained as an out‐group. The high polymorphic content coupled with high rate of cross‐transferability demonstrates wider applicability of novel microsatellite markers in genotyping, genetic diversity, genome mapping and evolutionary studies in various Camellia species.     

Comparative Study of CaMsrB2 Gene Containing Drought‐Tolerant Transgenic Rice (Oryza sativa L.) and Non‐Transgenic Counterpart

Concerns regarding the safety of transgenic foods have been raised because of possibility of undesirable effects development during genetic engineering. Analysis of phenotypic traits can increase the likelihoods of identifying those unintended effects in dietary composition of the GM crops. Objective of this study was to compare the transgenic lines with their non‐transgenic counterpart. Different vegetative and reproductive traits as well as antioxidant properties were considered to evaluate the transgenic (HV8 and HV23) lines containing CaMsrB2 gene and their non‐transgenic (Ilmi) parent line. Grain size and weight, seed germination, root length, root and shoot dry weight, length and width of flag leaf, plant height, and ligule, stamen and carpel length were not significantly different. Onset and completion of heading in each line occurred almost during the same period. The antioxidant properties in terms of DPPH (1,1‐diphenyl‐2‐picrylhydrazyl) radical scavenging activity and polyphenol content were not statistically different under same treatment condition. The results suggested that the transgenic rice lines containing CaMsrB2 gene were equivalent to their non‐transgenic counterpart without any visible unintended effects.     

Interspecific hybridization in Cucumis (L.). I. Need for genetic variation,biosystematic relations and possibilities to overcome crossability barriers

Summary The genetic variation in pickling and slicing cucumbers (C. sativus L.) seems insufficient to enable the breeder to solve serious cultivation problems. Wild allies of C. sativus possess a wide range of interesting characters which could be incorporated by means of species crosses. In the past, attempts to achieve such crosses have failed. Important characters of Cucumis species are mentioned and biosystematic problems discussed. Results of previous research on species crosses prompted an examination of the prospects of further research.     

Drought‐induced osmotic adjustment and changes in carbohydrate distribution in leaves and flowers of cotton (Gossypium hirsutum L.)

Research has indicated osmotic adjustment as a mechanism by which leaves and roots of cotton plants overcome a drought period. However, the relevance of this mechanism in reproductive tissues of modern cultivars under drought has not been fully investigated. The objectives of this study were to measure osmoregulation and carbohydrate balance in reproductive tissues and their subtending leaves grown under water‐deficit conditions. Two cotton cultivars were grown under controlled environment and field conditions. Plants were exposed to water‐deficit stress at peak flowering, approximately 70 days after planting. Measurements included stomatal conductance, proline concentration, soluble carbohydrates and starch concentration, and water potential components. Stomatal conductance of drought‐stressed plants was significantly lower compared to control, while osmotic adjustment occurred in reproductive tissues and their subtending leaves by different primary mechanisms. Pistils accumulated higher sucrose levels, maintaining cell turgor in plants exposed to drought at similar levels to those in well‐watered plants. However, subtending leaves lowered osmotic potential and maintained cell turgor by accumulating more proline. Soluble carbohydrates and starch concentration in leaves were more affected by drought than those of floral tissues, with corresponding reduction in dry matter, suggesting that flowers are more buffered from water‐deficit conditions than the adjacent leaves.     

Hydrogen peroxide reduces heat‐induced yield losses in cotton (Gossypium hirsutum L.) by protecting cellular membrane damage

We investigated the effect of various growth substances such as hydrogen peroxide, salicylic acid (SA), moringa leaf‐extract (MLE) and ascorbic acid (ASA) on leaf physiology and seed cotton yield (SCY) of heat‐stressed cotton. Cotton plants were exposed to elevated temperatures at three reproductive stages, either by staggering planting time in the field or by increasing growth cabinet temperatures (38/24°C and 45/30°C) in glasshouse. Elevated temperature at any reproductive phase significantly damaged cellular membrane and reduced SCY. Plants exposed to 38/24°C and 45/30°C in glasshouse produced 63% and 22% lower SCY, respectively, compared with plants under optimal temperature ((32/20°C). In response to high temperature, cotton plants up‐regulated activities of anti‐oxidative enzymes e.g. peroxidase and ascorbic acid. However, this defensive system could not protect cellular membrane of stressed plants from extreme temperature (38 and 45°C). In contrast, growth substances such as H2O2, ASA and MLE significantly increased anti‐oxidative enzymes activity to an extent, which reduced heat‐induced damage to cellular membrane. No significant effect of any regulator was observed on SCY under optimum temperatures; although H2O2, MLE and ASA significantly increased SCY of heat‐stressed cotton. Hydrogen peroxide increased SCY of April and May thermal regimes crops by 16% (averaged across both sowing dates) under field, while it caused 14% and 20% increase in SCY of plants exposed to sub (38/24°C) and supra optimal (45/30°C) thermal regimes under glasshouse. We concluded that growth regulators, specifically, H2O2 can protect cotton crops from heat‐induced cellular membrane damage by up‐regulating antioxidant defense system.     

Integration of AFLPs,SSRs and SNPs markers into a new genetic map of industrial chicory (Cichorium intybus L. var. sativum)

A chicory genetic map of 1208 cM has been created using 247 F₂ plants and 237 markers (170 AFLP, 28 SSR, 27 EST‐SNP and 12 EST‐SSR). This map covers 84% of the chicory genome. The chicory‐genic‐markers‐associated sequences were used to find potential orthologs in mapped lettuce ESTs from the Compositae Genome Project Database. Twenty‐seven putative orthologous pairs were retained, pinpointing seven putative blocks of synteny that covered 11% of the chicory genome and 13% of the lettuce genome, opening new perspectives for the analysis of these two species.     

Phenotypic and marker‐assisted pyramiding of genes for resistance to zucchini yellow mosaic virus in oilseed pumpkin (Cucurbita pepo)

We report on the identification of phenotypic and molecular markers for genes introgressed into oilseed pumpkin Cucurbita pepo from C. moschata germplasm originating in Nigeria, Portugal and Puerto Rico, which provide resistance against zucchini yellow mosaic virus (ZYMV) and on pyramiding these genes for improved and long‐lasting field protection of oilseed pumpkins. One SCAR and two SSR markers have been found for three dominant resistance genes, Zym‐0, Zym‐1 and Zym‐2. Characteristic reactions to ZYMV inoculation of plants carrying the recessive genes for resistance zym‐4* and zym‐6 have been defined. Described are procedures and results of pyramiding various combinations of these genes in oilseed pumpkin using the three markers and the specific phenotypic reactions to infection of some of these genes. The putative combination of all six resistance genes in one genotype resulted in a resistance that appeared to be at least as strong as or even stronger than that of the resistance source germplasm in C. moschata.     

Interspecific hybridization in sexual diploid Allium senescens var. minor× apomictic tetraploid A. nutans and sexual diploid A. senescens var. minor× apomictic hexaploid A. senescens

Fourteen interspecific hybrids in sexual diploid Allium senescens var. minor× apomictic tetraploid Allium nutans L. crosses, and eight interspecific hybrids in sexual diploid A. senescens var. minor× apomictic hexaploid A. senescens L. crosses were produced. The number of chromosomes was 2n= 24 in interspecific hybrids of diploid × tetraploid, and 2n= 32 in diploid × hexaploid crosses. Triploid and tetraploid interspecific hybrids showed intermediate parental morphological characteristics. Tetraploid interspecific hybrids of A. senescens var. minor×A. senescens crosses formed two groups based on leaf colour and leaf width. Seeds were formed in 11 out of 14 triploid interspecific hybrids under natural conditions. In cytological observations of parthenogenesis, three out of 12 triploid interspecific hybrids and five out of eight interspecific tetraploid hybrids were observed. Parthenogenesis ranged from 26.0% to 86.0% in five tetraploid interspecific hybrids. Non-parthenogenesis to parthenogenesis segregated in a 3:5 ratio in A. senescens var. minor×A. senescens crosses.     

Oligogenic control of resistance to soil‐borne viruses SBCMV and WSSMV in rye (Secale cereale L.)

Rye production in European growing areas is constrained by the soilborne cereal mosaic virus (SBCMV) and the wheat spindle streak mosaic virus (WSSMV). To date, no European rye cultivars are known to exhibit resistance against these viruses. In this study, we pursued a quantitative trait locus (QTL) mapping strategy to identify genomic regions for resistance to SBCMV and WSSMV in rye. Three populations, each comprising 100 lines segregating for resistance to SBCMV and/or WSSMV, were evaluated for disease response at two years in three locations in Germany where soils are naturally infested with SBCMV and WSSMV. In the combined analysis across environments, one QTL for SBCMV resistance on chromosome 5R explained 31.9% of the phenotypic variation in one of the populations. For WSSMV resistance, one QTL explaining up to 64.0% of the phenotypic variation was detected on chromosome 7R in each of the three populations. On the Triticeae homoeologous group 5, we found evidence for synteny of the major QTL for SBCMV resistance between the wheat and rye genomes.     

Development of chloroplast‐specific microsatellite markers for molecular characterization of alloplasmic lines and phylogenetic analysis in wheat

Wheat (Triticum aestivum L.) is strictly a self‐pollinated crop, where hybrid breeding requires well‐characterized cytoplasmic male sterile (CMS) lines. The CMS has mostly been developed by substituting nuclear genome of wheat into the cytoplasm from wild relatives. Molecular characterization of 90 genotypes including 82 CMS lines originating from five different species, namely Aegilops speltoides, Ae. kotschyi, Ae. variabilis, Triticum araraticum and T. timopheevii, and eight popular varieties was carried out. Consequently, a set of 25 microsatellite markers specific to chloroplast (cpSSRs) were designed and successfully validated for specificity of amplification. A total of 15 cpSSRs (60%) were found polymorphic, of which three cpSSRs (TaCM7, TaCM8 and TaCM11) in genic region and twelve cpSSRs were located in intergenic region. Phylogenetic analysis of genotypes using cpSSRs revealed two major groups well in accordance with respective origin. A set of cpSSRs and phylogeny of CMS belonging to different origins developed, which will be helpful for the improvement in CMS system in wheat. The genic cpSSRs can be used for the allele mining and evolutionary studies.     

Development of AS‐PCR marker based on a key mutation confirmed by resequencing of Wx‐mp in Milky Princess and its application in japonica soft rice (Oryza sativa L.) breeding

Soft rice with low amylose content (AC) ranging by 5–15% is a unique type with special eating and appearance quality and has become popular in the rice market. We resequenced the Wx‐mp, a key allele from Milky Princess, a Japanese low AC variety, and found that the +473 mutation in exon 4 is the key mutation in both Wx‐mp and its ancestor allele, Wx‐mq from Milky Queen. Based on this functional mutation, an allele‐specific PCR (AS‐PCR) marker was developed and proven in a breeding population derived from a cross between a Chinese late variety Nan Keng 46 (Wx‐mp/Wx‐mp) and an early line Ning 63121(Wx‐b/Wx‐b). Based on the marker‐aided selection by our newly developed AS‐PCR marker for Wx‐mp and the known ST10 marker for Stvb‐i, a total of 12 Wx‐mp homozygotes were selected from 198 F₂ progenies, and four of them were immune to rice stripe virus (RSV) with averagely 11.3 days earlier heading than Nan Keng 46 without significant change in grain yield.