Genotypic diversity enhances recovery of hybrids and fertile backcrosses of Phaseolus vulgaris L. × P. acutifolius A. Gray

Summary Homozygous and heterozygous Phaseolus vulgaris and P. acutifolius parental genotypes were hybridized to determine if genotypic diversity would aid gene transfer between these species. Certain P. vulgaris female parents resulting from diverse intraspecific crosses increased the frequency of species hybrids obtained. From a total of 19 self-sterile hybrids, 20 backcross-1 (BC₁) lines (P. vulgaris recurrent parent) were produced from 4 partially female-fertile hybrids, each of which had a heterozygous P. vulgaris female parent. Heterozygous P. acutifolius male parents did not influence the frequency at which interspecific hybrids could be produced but apparently improved female-fertility. Fertility of the F₁BC₁ generation was highly variable. The frequency of fertile individuals increased in each subsequent backcross generation (F₁BC₂ and F₁BC₃).Purdue University Agricultural Experiment Station Publication 9946.     

Introgression of Phaseolus acutifolius A. Gray Genes into the Phaseolus vulgaris L. Genome

The tepary bean (Phaseolus acutifolius A. Gray) is a desirable genetic resource for incorporation of improved disease, pest, and stress resistance into common bean (P. vulgaris L.). Reproductive barriers separate the two species and the degree to which tepary genes may be introgressed into the common bean genome has not been well described. Greenhouse studies of gene introgression through recurrent back-crossing to common bean were performed using two first backcross (BC₁) and nine second backcross (BC₂) populations. The truncate primary leaf characteristic of tepary bean was readily observed but the short primary leaf petiole and narrow bract tepary traits were infrequently observed in both BC₁ and BC₂ populations. In one BC, population high frequencies of adaxial stomata (characteristic of P. acutifolius) and the presence of a 30 kD cotyledon polypeptide from P. acutifolius were also observed; however, a diapho-rase isozyme from P. acutifolius appeared to be eliminated from BC₁ progeny at a high rate whereas 6-phosphogluconate dehydrogenase alleles appeared to be transmitted normally. The expression of tepary primary leaf truncate morphology and bract width were correlated with decreased fertility in one of the two BC₁ populations. Given suitable parental genotypes and population sizes it should be possible to transfer genes from P. acutifolius to P. vulgaris, especially in regions of the genome which are not associated with inviability or sterility. It will be difficult to transfer factors from certain regions of the tepary genome which are preferentially eliminated during introgression.     

Genetic Control of Abnormal Seedling Development in Phaseolus vulgaris L. and P. vulgaris×P. acutifolius A. Gray Hybrids

Abnormal hybrid seedling development hinders attempts to introgress diverse germplasm in common bean (Phaseolus vulgaris L.) breeding programs. In this study, the hypothesis that DL₁ and DL₂ loci control abnormal seedling development both in Phaseolus vulgaris L. intraspecific hybrids and in P. vulgaris×P. acutifolius A. Gray interspecific hybrids was tested. Hybrids from crosses between six P. vulgaris lines (two Andean dl₁ d₁, DL₂DL₂, two Mesoamerican DL₁DL₁ dl₂ dl₂, and two facilitators dl₁dl₁, dl₂dl₂) and P. vulgaris cultivar Ex Rico 23 developed normally, indicating that Ex Rico 23 has the facilitator genotype dl₁, dl₁, dl₂dl₂. Previous studies have shown that Ex Rico 23 ×P. acutifolius results in abnormal seedlings, but ‘ICA Pijao’, also dl₁, dl₁ dl₂dl₂, ×P. acutifolius results in normal seedlings. Neither the Andean nor the Mesoamerican lines crossed successfully with P. acutifolius, but crosses between one of the two facilitators (G3807) and P. acutifolius resulted in normal seedlings. These results support the hypothesis that different loci control intra- and interspecific hybrid seedling development.     

Root peroxidases and Rhizobium, Bradyrhizobium nodulation affinities of Phaseolus vulgaris L. and P. acutifolius A. Gray congruity backcross populations

Four parents [P. acutifolius var.acutifolius (A₁₉), P. a.var. latifolius `Serowi' (A<sub>9</sub>),P. a. var. latifolius(A<sub>10</sub>), and P. vulgaris `Red Cloud' kidney (V₁)] were used to create six interspecific recurrent and congruity backcross pedigrees (V₁ × A₉, A₉ × V₁, V₁ × A₁₀, A₁₀ × V₁, V₁ × A₁₉, A₁₉ × V₁) for evaluation of Rhizobium, Bradyrhizobium nodulation affinities and root peroxidase banding patterns. Most primary hybrids nodulated with all Rhizobium, Bradyrhizobium strains, while recurrent generations nodulated with strains of the recurrent parent, suggesting a number of independent loci and random assortment for strain-specific nodulation. A proximity matrix of nodulation phenotypes produced a cluster with V₁, two recurrent and two congruity backcross hybrids with V₁ as the cytoplasmic parent; a separate cluster with all primary interspecific and balanced, congruity backcross hybrids. A₉ and A₁₀were clustered the farthest away from V₁. Root peroxidase zymograms of primary hybrids were the summation of the parents and controlled by independent loci. Advanced hybrids exhibited new root peroxidases, matching those found for otherPhaseolus spp., including ancestralP. coccineus. A proximity matrix of root peroxidase phenotypes produced a cluster with only A₉ ³V₁F₁, a cluster containing only A₁₉ ³V₁ F₂, and a large cluster with all P. acutifoliusparents and hybrids with P. acutifolius as the cytoplasmic parent in balanced hybrids or the majority parent in unbalanced hybrids. Estimation of genetic distances showed primary, interspecific hybrids to be more closely related with the cytoplasmic parent. Recurrent and congruity backcross generations varied in genetic distances between each respective parent. This is the first report of congruity backcrossing creating hybrids with biochemical traits of nonparental species. This revised version was published online in August 2006 with corrections to the Cover Date.     

Interspecific hybridization between cultivated american species of the genusphaseolus

Summary Attempted interspecific hybridization between the speciesPhaseolus vulgaris, P. coccineus, P. acutifolius andP. lunatus has indicated that a close genetic relationship exists betweenP. vulgaris andP. coccineus. Viable hybrids of varying fertility can be made reciprocally between these species. Affinity is indicated withP. acutifolius by the formation of viable but sterile hybrids. Less affinity is apparent between these three species andP. lunatus with which they do not produce viable interspecific hybrids. Nevertheless morphological resemblances between the four species justify their retention within the same section of the genus.Effects of cytoplasmic constitution on both fertility and viability were shown in the F₂ and backcross progenies ofP. vulgaris () ×P. coccineus () hybrids and on viability in theP. vulgaris () ×P. acutifolius () and reciprocal hybrids. In slightly fertileP. vulgaris () ×P. coccineus () hybrids it was concluded that distorted F₂ segregations were due to differential loss ofP. coccineus genes in sporogenesis and embryogenesis occurring in aP. vulgaris plasmon.Formerly at The School of Agriculture, University of Cambridge.     

Pre- and post-fertilization barriers to backcrossing the interspecific hybrid between Allium fistulosum L. and A. cepa L. with A. cepa

Summary To facilitate the introgression of desirable traits of Allium fistulosum into the genome of A. cepa, several accessions of the hybrid between these species were pollinated with A. cepa as the recurrent parent, and in vitro ovary and ovule culture were performed to obtain an increase in the recovery of backcross progeny. Compared to the results obtained from seed development in planta, the increase in the number of backcross progeny was generally very limited, and in some cases even a decrease was found. Raising the sucrose concentration in the ovary culture medium resulted in a higher frequency of ovules developing back seed coats but this was not followed by an increase in the number of backcross progeny obtained. Pollen tube growth of A. cepa was disturbed in the styles of the interspecific hybrids. Per ovule, frequencies of micropylar penetration exceeded frequencies of backcross progeny only to a limited extent. Hence, it was concluded that in the tested interspecific hybrid accessions the attainable gain in viable backcross progeny by the application of in vitro culture techniques is limited by strong pre-fertilization barriers acting at the level of stylar incongruity.     

Hybrid plant of Phaseolus vulgaris L. and P. lunatus L. obtained by means of embryo rescue and confirmed by restriction endonuclease analysis of rDNA

Summary Hybridizations between three lines of the common bean (Phaseolus vulgaris L.) and two lines of the lima bean (P. lunatus L.) were attempted in order to transfer characters from the lima bean to the common bean. A total of 115 interspecific hybrid embryos were rescued and cultured, and 7 plantlets were eventually transferred to soil. The most compatible cross was Edogawa XPI164891, which had a high proportion of expanded ovules, and from which we obtained one mature interspecific hybrid. In general, morphological characters of the hybrid were intermediate between the parents, and the chromosome number of the hybrid was 2n=22 the same as that of both parents. The hybrid nature of the progeny plant was confirmed by restriction endonuclease analysis of rDNA. Species specific fragments were detected when total DNA was digested by BamHI, and BamHI-digested total DNA of the hybrid contained all fragments from both parents. Selves and backcrosses were attempted, but no progeny were obtained. The only hybrid obtained was completely sterile and meiosis highly irregular.     

Congruity backcrossing as a means of creating genetic variability in self pollinated crops: seed morphology of Phaseolus vulgaris L. and P. acutifolius A. Gray hybrids

Summary Seeds of early generations of three reciprocal congruity-backcross (CBC) pedigrees, developed by backcrossing Phaseolus vulgaris-P. acutifolius hybrids to each of the parent species in alternate generations, exhibited a preponderance of traits (size, shape, color, and pattern) of the cytoplasmic parent. The large size of Red Cloud (V₁), the P. vulgaris parent common to all of the pedigrees, dominated pedigrees with V₁ as the cytoplasmic parent, while the small size and rounded or square shapes of the tepary parents, wild P. acutifolius var. acutifolius PI 263590 or G400445 (A₁₉), wild P. acutifolius var. latifolius PI 406622 (A₁₀), or cultivated P. acutifolius var. latifolius Serowi PI 319443 (A₉), were the majority phenotypes when P. acutifolius was the cytoplasmic parent. Continuing through the second cycle of CBC, that is the second backcross with each of the parent species or the fourth backcross, began an amelioration of the apparent cytoplasmic effect on gene expression, as reciprocal pedigrees became more alike, usually with intermediate expression of parental traits or the appearance of new traits. The large seed size of V₁ was recovered in hybrids with P. acutifolius cytoplasm and the kidney shape of V₁ became rare in hybrids with P. vulgaris cytoplasm. Although the tepary-bean parents represented two subspecies and both cultivated and wild P. acutifolius, the three sets of reciprocal-hybrid pedigrees with P. vulgaris Red Cloud are surprisingly similar. It may be that the exotic parent used to develop a CBC pedigree should be selected more for combining ability in the interspecific cross than for specific economic traits. While the number of generations (six or more) required to produce fertile, intermediate CBC hybrids (that did not require embryo rescue) may preclude routine use of this method by practical plant breeders, the crossability of advanced hybrids with both parental species and the amount of variability apparent in advanced-hybrids progenies suggests that CBC would be valuable for maintaining exotic germplasm in immediately useful forms.     

Transfer of powdery mildew resistance from Brassica carinata to Brassica oleracea through embryo rescue

Interspecific hybrid plants and backcross 1 (BC₁) progeny were produced through sexual crosses and embryo rescue between Brassica carinata accession PI 360883 and B. oleracea cvs Titleist’and‘Cecile’to transfer resistance to powdery mildew to B. oleracea. Four interspecific hybrids were obtained through application of embryo rescue from crosses with B. carinata as the maternal parent, and their interspecific nature confirmed through plant morphology and random amplified polymorphic DNA (RAPD) analysis. Twenty‐one BC₁ plants were obtained through sexual crosses and embryo rescue although embryo rescue was not necessary to produce first backcross generation plants between interspecific hybrids and B. oleracea. All interspecific hybrids and eight of the BC₁ plants were resistant to powdery mildew.     

Unreduced 3x gamete formation of allotriploid hybrid derived from the cross of Primula denticulata (4x)?×?P. rosea (2x) as a causal factor for producing pentaploid hybrids in the backcross with pollen of tetraploid P. denticulata

A triploid hybrid, which was obtained from interspecific crosses between tetraploid Primula denticulata (2n = 4x = 44) and P. rosea (2n = 2x = 22), successfully produced 11 plants by backcrossing with pollen of tetraploid P. denticulata. Analysis of ploidy level using flow cytometry and chromosome counting in the 11 BC₁ plants revealed that all progeny had much larger DNA contents and chromosome number than both parents. In this triploid-tetraploid (3x–4x) crossing, progeny was predominantly true or near pentaploid presumably produced by the fertilization between true or near triploid female gamete produced from triploid hybrid and diploid pollen of tetraploid P. denticulata. These results suggest that unreduced (3x) or near triploid female gametes were partially produced by single step meiosis, either first-division restitution or second-division restitution process. The zygotes formed by the fertilization between true or near triploid egg produced by single step meiosis in triploid hybrid and diploid pollen produced by normal meiosis of tetraploid P. denticulata might be the only survivors in embryogenesis.     

Effect of maternal genotype on development ofPhaseolus vulgaris L. ×P. lunatus L. interspecific hybrid embryos

Summary CertainPhaseolus vulgaris L. ×P. lunatus L. crosses were performed to study the effect of maternal heterozygosity on development and growth of the interspecific hybrid embryos. Interspecific embryos had a much slower growth rate in vitro compared with embryos derived from self-pollination ofP. vulgaris parents. Thus, interspecific embryos could be identified by growth rate in vitro. TheP. vulgaris maternal genotype affected both the number and size of 15-day-old interspecific embryos. Specifically, 76 Spartan Arrow produced significantly more interspecific embryos than did Great Northern as the seed parent, while maternal intraspecific hybrids produced smaller embryos than did maternal pure lines. There were no reciprocal differences between hybrid maternal parents for embryo number or size. Embryo size at excision and final size after culturing were closely correlated (r²=+0.93). The crossP. vulgaris 76 Spartan Arrow ×P. lunatus P.I. 214170 produced both the largest mean size at excision and the fastest growth in culture, indicating that specific combining ability affected both characteristics.Journal Paper No. J-12208 of the Iowa Agriculture and Home Economics Experiment Station, Ames, Iowa. Project No. 2495     

Introduction of a self-compatible gene of Lolium temulentum L. to perennial ryegrass (Lolium perenne L.) for the purpose of the production of inbred lines of perennial ryegrass

Interspecific hybrids between self-compatible species, Lolium temulentum L. and self-incompatible species, L. perenne L. were obtained using embryo rescue. Two cycles of backcrossing of interspecific hybrids with L. perenne were carried out. A 1: 1 segregation ratio of self comatibility and incompatibility was observed in backcross generations. These segregation data confirmed that self-compatibility of L. temulentum was controlled by a single gene. It suggested that the self-compatible gene of L. temulentum could be introduced to self-incompatible plants of L. perenne through interspecific hybridization. It appears that utilization of a self-compatible gene of L. temulentum would be useful for production of inbred lines of L. perenne. The possibility of breeding procedures of perennial ryegrass using a self-compatible gene was discussed. This revised version was published online in August 2006 with corrections to the Cover Date.     

Inheritance of Resistance to Downy Mildew (Peronospora destructor [Berk.] Casp.) from Allium roylei Stearn in the Backcross Allium cepa L. × (A. roylei ×A. cepa)

Allium roylei Stearn and both reciprocals of the interspecific hybrid between Allium cepa L. and A. roylei displayed no symptoms of Peronospora destructor (Berk.) Casp. after artificial or natural inoculations, whereas A. cepa was susceptible. In the offspring from the backcross A. cepa× (A. roylei ×A. cepa) resistant and susceptible plants segregated after artificial inoculations, fitting a 1: 1-ratio. This suggests that resistance is controlled by a single, dominantly-inherited locus (designated Pd₁) from the nuclear genome of A. roylei. During a severe epidemic in the field, plants from the same backcross segregated resistant and susceptible individuals in a 3: 1-ratio. Escapes may explain the deviation from the 1: 1-segregation, but the presence of a second resistance locus segregating independently from Pd₁ cannot be excluded, implicating possible differences in virulence between populations of P. destructor. The occurrence in the backcross offspring of plants having a morphology similar to A. cepa without showing symptoms of downy mildew opens perspectives for breeding P. destructor-resistant onion varieties.     

Heterosis for symbiotic nitrogen fixation in Phaseolus interspecific hybrids

Summary Interspecific hybrids of Phaseolus vulgaris and Phaseolus acutifolius or Phaseolus filiformis have been evaluated for evidence of heterosis for symbiotic nitrogen fixation activity. Hybrids showing composite leghemoglobin profiles were selected for comparison. Rooted cuttings from hybrids and their respective parents were used to estimate nitrogen fixation (acetylene reduction) rates and to determine nitrogen accumulation during the growth period. Some hybrids of Phasolus vulgaris × Phaseolus acutifolius and Phaseolus vulgaris × Phaseolus filiformis had significantly (P<0.01) higher nitrogen fixation rates and in the latter case, accumulated more total nitrogen, than either parent. The interspecific hybrids also showed more complex leghemoglobin profiles than either parent.     

Dissection of genetic variance of fibre quality in advanced generations from an interspecific cross of Gossypium hirsutum and G. barbadense

The interspecific genetic introgression approach has been shown to facilitate the detection and dissection of quantitative trait loci (QTL). A population consisting of 121 F₆ recombinant inbred lines was developed by crossing Gossypium hirsutum cv. ‘Handan 208’ and G. barbadense cv. ‘Pima 90’ via single‐seed descent. Genotyping indicated that the mean ‘Pima 90’ allele frequency at each locus was 21%. Phenotyping and phenotypic distribution indicated a trend of return of individual lines’ characters to ‘Handan 208’ coupled with a wide variance for each trait. Significant loci influencing fibre quality were detected by one‐way analysis of variance (anova; P < 0.005) and association analysis [?log₁₀(P) ≥ 3]: five and three markers for fibre length, four and one marker(s) for uniformity, two and one marker(s) for micronaire, 13 and 15 markers for strength, six and 10 markers for elongation, respectively. Two‐way anova based on genotypes of all marker loci combination showed that 807 two‐locus combinations were significant, and two‐way anova based on marker genotypes of QTL markers combination showed five significant digenic interactions (P < 0.01).     

Allozyme Variability in the Tepary Bean, Phaseolus acutifolius A. Gray

The tepary bean (Phaseolus acutifolius) is the object of renewed interest because it possesses some interesting agronomic attributes such as tolerance to drought and salinity. In order to gain a better understanding of the organisation of its genetic diversity, we have examined patterns of diversity for nine polymorphic enzyme systems representing 12 loci, in a sample of 55 wild and 8 cultivated accessions. Several geographic patterns were identified, including an East-West differentiation, (across the Sierra Madre Occidental mountains), a North-South differentiation, and a localized differentiation. Wild teparies displayed a higher number of polymorphic loci, as well as a higher number of alleles per polymorphic locus compared to cultivars, consistent with previous observations indicating a reduction of phaseolin diversity on domestication. No clearcut separation with respect to isozyme differentiation was observed between P. acutifolius var. tenuifolius and var. acutifolius, further questioning the validity of this taxonomic separation based on leaflet shape.     

Alteration of the genomic composition of Solanum nigrum (+) potato backcross derivatives by somatic hybridization: selection of fusion hybrids by DNA measurements and GISH

Fusion experiments were performed with a first (BC₁‐6738) and a second (BC₂‐9017) generation backcross hybrid of 6x Solarium nigrum (+) 2x potato somatic hybrids with potato cultivars. Because no progeny was obtained from the BC₂ genotypes, alternative approaches were sought to overcome the sexual crossing barrier. Five potato genotypes, one of which contains the hygromycin resistance gene, were used in the fusion experiments. All vigorous regenerants were used for the estimation of nuclear DNA content using flow cytometry. Plants with a DNA content higher than that of the BC₁‐6738 or BC₂ genotypes were considered potential somatic hybrids. Forty‐nine potential somatic hybrids resulted from fusion experiments with BC₁‐6738, from which 20 grew vigorously in the greenhouse and flowered. After pollination with several 4x potato cultivars, eight genotypes produced seeded berries and five genotypes gave seedless berries. In addition, 11 of these 13 somatic hybrids were selected for genomic in situ hybridization (GISH) analysis to determine their genomic composition. Nine had exactly or approximately the expected number of 36 S. nigrum and 60 potato chromosomes. In one genotype, only 22 instead of 36 S. nigrum chromosomes were found and one potato chromosome was possibly missing. Only five potential somatic hybrids were detected among the 79 regenerants from BC₂‐9017 (+) 2x potato fusion experiments that were analysed by flow cytometry. Two of these hybrids were rather vigorous and did flower, but pollinations with potato have not yet set any berries.     

Characterization of interspecific hybrids and first backcross generations from crosses between two cultivated eggplants (Solanum melongena and S. aethiopicum Kumba group) and implications for eggplant breeding

Common (Solanum melongena L.) and scarlet (S. aethiopicum L.) eggplants are cultivated for their fruits and form part of the same genepool. We have studied plant and fruit characteristics, pollen viability and seed set, phenolics content, and fruit flesh browning in accessions of S. melongena and S. aethiopicum Kumba group, as well as interspecific hybrids between these species and first backcross generations to each parental species. Respective genotypes were also characterized with seven polymorphic SSR markers. The results demonstrate that many differences exist for plant and fruit morphology among S. melongena, S. aethiopicum and the interspecific hybrids. The latter are very vigorous and generally intermediate between the two parents, except for fruit size which is smaller (and parthenocarpic due to a high pollen sterility) than those of any of the parents. Backcross progenies also exhibited morphological variation with moderate heritability values for the attributes evaluated. Variation for fruit size was present in the backcross generations but fruits were small resulting in little variation for fruit shape. Backcross plants with moderate fertility produced seeded fruits. Primary hybrids had fruit phenolics content similar to that of S. aethiopicum, the parent with lowest phenolics concentration, and were heterotic for fruit flesh browning. Backcross progenies were quite variable for both traits. SSR markers did not reveal segregation distortion in the backcross generations for these interspecific hybrids. The results demonstrate that generations derived from sexual interspecific hybridization can be a powerful tool for S. melongena and S. aethiopicum Kumba group breeding.     

Development of self-incompatible Brassica napus: (I) introgression of S-alleles from Brassica oleracea through interspecific hybridization

Cultivars in Brassica napus var. oleifera, a self‐pollinating, self‐compatible species, have traditionally been developed as open‐pollinated lines or populations. Significant yield gains in this species have been realized through the exploitation of heterosis. Commercial hybrid production has been possible as a result of the development of a number of pollination control systems. Self‐incompatibility was transferred from B. oleracea var. italica to B. napus var. oleifera through interspecific hybridization. The response to interspecific pollination, as measured by pod elongation and initial stages of ovule development, was genotype dependent, and two highly responsive B. napus genotypes were identified. Embryo rescue was used to produce the interspecific hybrids. Isoelectric focusing of stigma proteins was used to identify S‐alleles in the interspecific hybrids to facilitate backcrossing. Segregation of the S‐locus through a series of back‐crosses to B. napus was complicated by aneuploidy; however, the S‐locus was found to segregate as a single gene. Usefulness of B. oleracea as a source of S‐alleles for pollination control in B. napus is discussed.     

Development of interspecific somatic hybrid cell lines in cultivated jute and their early characterization using jute chloroplast RFLP marker

A strong sexual incompatibility barrier that exists between the two cultivated jute species, Corchorus capsularis and Corchorus olitorius, limits the scope for improvement through genetic introgression. Protoplast fusion was carried out to generate interspecific hybrid cell lines. Cotyledonary cell protoplasts of C. capsularis and anthocyaninpigmented hypocotyl protoplasts of C. olitorius were used in the fusion experiments, which appeared to be visually useful in the early selection of the fused products. A chloroplast DNA (cpDNA) marker was developed in jute, which showed species‐specific hybridization patterns with EcoRI‐digested total genomic DNA of C. capsularis and C. olitorius. This cpDNA marker was used in the characterization of the somatic hybrid cell lines at their early stages of growth. Evidence for the presence of both types of cpDNA in the hybrid cell lines was obtained when the total genomic DNA of 4‐ to 7‐month‐old hybrid cell lines was challenged with the chloroplast DNA marker through Southern analysis. It was shown that the early segregation of the parental chloroplasts did not occur in jute, although this is common in other plant species. The hybrid nature of the fused cell lines could also be identified through peroxidase isozyme analysis. Isozyme banding patterns were complex and varied among the hybrid cell lines.