Parental Backcross Research Articles

QTL Mapping of Melampsora Leaf Rust Resistance and Yield Component Traits in the SalixF1 Hybrid Common Parent Population

ABSTRACTThe first step in trait introgression is to identify and assess novel sources of variation. For shrub willow (Salix) breeders, there is an abundance of understudied species within a genus that readily hybridizes. Breeding targets in shrub willow center on traits contributing to biomass yield for bioenergy. These include stem biomass, insect and pathogen resistance, and leaf architecture traits. More specifically, breeding for durable resistance to willow leaf rust (Melampsora spp.) is of particular importance as the pathogen can significantly reduce biomass yields in commercial production. The Salix F1 hybrid common parent population (Salix F1 HCP) was created to characterize the variation among eight species‐hybrid families and map QTL for targeted traits. A female and male S. purpurea were used as common parents in crosses made to male S. suchowensis, S. viminalis, S. koriyanagi, and S. udensis and female S. viminalis, S. integra, S. suchowensis to produce eight families that were planted in field trials at Cornell AgriTech in Geneva, NY and phenotyped. Using 16 previously described parental backcross linkage maps and two newly generated S. purpurea consensus maps, we identified 215 QTL across all eight families and in every parent. These included 15 leaf rust severity, 61 herbivory, 65 leaf architecture, and 74 yield component QTL, resulting in 50 unique overlapping regions within the population. These genetic loci serve as an important foundation for future shrub willow breeding, and each interspecific family was identified as a novel source of useful alleles for trait introgression into high yielding cultivars.

Breeding Lettuce for Resistance against Sclerotinia minor

Lettuce drop caused by Sclerotinia minor is a damaging disease of romaine lettuce (Lactuca sativa L.) production in California. Introgression of partial resistance from wild, primitive, or heirloom accessions into modern cultivars could improve integrated management approaches to the disease. Breeding methods for lettuce drop resistance are not well developed and hinder the development of new lettuce drop–resistant cultivars. The objective of this work was to develop a pedigree-based breeding method for introgression of lettuce drop resistance into modern romaine germplasm. Progeny from crosses between the partially resistant cultivar Eruption and the susceptible romaine cultivars Darkland and Hearts Delight were selected in a modified pedigree breeding scheme. Families were evaluated for disease incidence and selected for lettuce drop resistance in artificially infested field experiments conducted in the summer and fall. Infected plants of partially resistant lines commonly do not produce seed, and therefore selection of resistant plants from infested nurseries is not possible. Noninfested field experiments were used to select individual plants with improved horticultural characteristics for seed production, but from within resistant families only. Evaluation and selection of progeny using this breeding scheme occurred from the F2:3 through the F5:6 generations. In all generations, superior resistance was identified in the ‘Eruption’ × romaine crosses. The breeding scheme generated eight green romaine-type inbred lines with better resistance than the romaine parent and better head weight than ‘Eruption’. Use of the new romaine lines as parents in backcrosses to romaine produced F2:3 families with high levels of resistance. The pedigree method used in this research can be implemented with any source of resistance, but is constrained by the use of family selection and the inability to select individual plants for resistance directly. Breeding schemes that use single seed descent or molecular markers are alternative approaches that would enable selection for resistance on individual genotypes.

Inoculum production of Phytophthora medicaginis can be used to screen for partial resistance in chickpea genotypes.

Phytophthora root rot caused by Phytophthora medicaginis is an important disease of chickpeas (Cicer arietinum) in Australia with limited management options, increasing reliance on breeding for improved levels of genetic resistance. Resistance based on chickpea-Cicer echinospermum crosses is partial with a quantitative genetic basis provided by C. echinospermum and some disease tolerance traits originating from C. arietinum germplasm. Partial resistance is hypothesised to reduce pathogen proliferation, while tolerant germplasm may contribute some fitness traits, such as an ability to maintain yield despite pathogen proliferation. To test these hypotheses, we used P. medicaginis DNA concentrations in the soil as a parameter for pathogen proliferation and disease assessments on lines of two recombinant inbred populations of chickpea-C. echinospermum crosses to compare the reactions of selected recombinant inbred lines and parents. Our results showed reduced inoculum production in a C. echinospermum backcross parent relative to the C. arietinum variety Yorker. Recombinant inbred lines with consistently low levels of foliage symptoms had significantly lower levels of soil inoculum compared to lines with high levels of visible foliage symptoms. In a separate experiment, a set of superior recombinant inbred lines with consistently low levels of foliage symptoms was tested for soil inoculum reactions relative to control normalised yield loss. The in-crop P. medicaginis soil inoculum concentrations across genotypes were significantly and positively related to yield loss, indicating a partial resistance-tolerance spectrum. Disease incidence and the rankings for in-crop soil inoculum were correlated strongly to yield loss. These results indicate that soil inoculum reactions may be useful to identify genotypes with high levels of partial resistance.

Microsatellite Markers Reveal Unprecedented High Frequencies of Hybridization among Typha Species in the Midwestern US

In North American wetlands, two cattail species -native Typha latifolia and exotic T. angustifolia- hybridize generating T. x glauca. Typha angustifolia and the hybrid spread invasively, negatively affecting wetlands. Due to high trait variability and hybridization, Typha species are difficult to identify morphologically. Building on previous work that relied on microsatellite markers to differentiate Typha species (including hybrids, parental backcrosses, and advanced-generation hybrids) in southern Canada and in the US upper Midwest and northeast, our goals were to 1) estimate relative frequencies of parental species in additional Midwestern cattail populations, and 2) quantify their hybridization. We also assessed level of agreement between morphological identification based on leaf width and gap between inflorescences and molecular identification. Using 6 microsatellites markers (4 used previously in other populations and 2 novel ones), we identified ~25% of the samples as native T. latifolia, while ~6% were exotic T. angustifolia. Furthermore, 19% of the samples were first-generation hybrids (T. x glauca) and 50% were advanced-generation hybrids, with backcrosses to native T. latifolia being almost twice as high as those to exotic T. angustifolia, rates that are much larger than previously reported. Agreement between morphological and molecular identification was lower than expected highlighting the fact that these morphological traits can be misleading when used alone in cattail identification. We caution that the seemingly asymmetric hybridization towards the native Typha latifolia could potentially lead to its extinction in the Midwest. Cattail management may thus require efforts to preserve the native cattail through seed banking and/or other approaches.

Transpiration response of two bread wheat lines differing in drought resilience and their backcross parent under dry-down conditions.

Improving wheat productivity in drylands largely depends on how plants manage limited water resources. Using fraction of transpirable soil water threshold (FTSWTh) and drought stress response function, we characterized the water conservation traits of two wheat multiple synthetic derivative lines (MSD53 and MSD345) which both contain introgressed segments from Aegilops tauschii but differ in drought resilience. The lines and their backcross parent, ‘Norin 61’, were subjected to dry-down conditions. MSD53 had a higher FTSWTh for transpiration decrease than ‘Norin 61’ and MSD345. In terms of drought stress response function, MSD53 had the lowest threshold suction, suggesting a lower drought resilience capacity compared with MSD345. However, MSD53 exhibited an effective-water-use trait whereas MSD345 exhibited a water-saving trait under dry-down conditions. These results are consistent with the reported higher yield of MSD53 in comparison with MSD345 under drought stress in Sudan, and demonstrate that high FTSWTh supports effective water use for improved agricultural productivity in drylands. The differences in water conservation traits between the two MSD lines may be attributed to variation in introgressed segments, which can be further explored for drought resilience breeding.

Genetic variation in drought resilience-related traits among wheat multiple synthetic derivative lines: insights for climate resilience breeding

Twenty-four wheat lines, developed by Aegilops tauschii Coss. introgressions and previously selected for heat or salinity stress tolerance, were evaluated under a drought-rewatering-drought cycle for two years. The objective was to select breeding lines that are resilient to more than one abiotic stress. The experiment was designed in alpha lattice with three replications. Drought was imposed by withholding water during flowering. The results revealed considerable genetic variability in physio-agronomic traits, reflecting the variation in the introgressed segments. High heritability estimates (above 47%) were recorded for most traits, including days to 50% heading, plant height, and thousand-grain weight, indicating the genetic control of these traits which may be useful for cultivar development. The trait-trait correlations within and between water regimes highlighted a strong association among the genetic factors controlling these traits. Some lines exhibited superior performance in terms of stress tolerance index and mean productivity compared with their backcross parent and elite cultivars commonly grown in hot and dry areas. Graphical genotyping revealed unique introgressed segments on chromosomes 4B, 6B, 2D, and 3D in some drought-resilient lines which may be linked to drought resilience. Therefore, we recommend these lines for further breeding to develop climate-resilient wheat varieties.

Aegilops tauschii Introgressions Improve Physio-Biochemical Traits and Metabolite Plasticity in Bread Wheat under Drought Stress

With the aim of providing genetic materials for breeding drought-resistant wheat varieties, the physiological and metabolic plasticity of three drought-resistant wheat multiple synthetic derivative lines (MSDLs) and their backcross parent “Norin 61” (N61) were evaluated in response to drought stress. The results indicated that the D-genome introgressions from Aegilops tauschii into the MDSLs improved their drought-adaptive traits. Specifically, MNH5 and MSD345 showed higher photosynthesis rates and triose phosphate utilization than N61 under control conditions, resulting in greater accumulation of glucose and sucrose in the shoots. However, under drought stress, MNH5 and MSD345 had higher intrinsic water use efficiency than MSD53 and N61. The total antioxidant capacity and superoxide dismutase activity increased in all three MSDLs, whereas no significant changes were found in N61 in response to drought stress. Metabolome analysis identified six common drought-induced metabolites in all of the investigated genotypes. However, four metabolites (adenine, gamma aminobutyric acid, histidine, and putrescine) each specifically accumulated in an MSDL in response to drought stress, suggesting that these metabolites are important for MSDL drought resistance. In conclusion, MNH5 and MSD345 showed better adaptive responses to drought stress than MSD53 and N61, suggesting that these two MSDLs could be explored for breeding drought-resistant wheat lines.

Leaf mass area determines water use efficiency through its influence on carbon gain in rice mutants.

Saving water and enhancing rice productivity are consensually the most important research goals globally. While increasing canopy cover would enhance growth rates by higher photosynthetic carbon gain, an accompanied increase in transpiration would have a negative impact on saving water as well as for sustainability under water-limited conditions. Increased water use efficiency (WUE) by virtue of higher carbon assimilatory capacity can significantly circumvent this trade-off. Here, we report leaf mass area (LMA) has an important canopy architecture trait which when combined with superior carboxylation efficiency (CE) would achieve higher water productivity in rice. A set of 130 ethyl methanesulfonate induced mutants of an upland cultivar Nagina-22 (N22), was screened for leaf morphological traits leading to the identification of mutants differing in LMA. The wild-type, N22, along with a selected low-LMA (380-4-3) and two high-LMA mutants (392-9-1 and 457-1-3), all with comparable total leaf area, were raised under well-watered (100% Field Capacity (FC)) and water-limited (60% FC) conditions. Low Δ13 C and a higher RuBisCO content in high-LMA mutants indicated higher carboxylation efficiency, leading to increased carbon gain. Single parent backcross populations developed by crossing high and the low-LMA mutants with N22, separately, were screened for LMA, Δ13 C and growth traits. Comparison of dry matter accumulation per unit leaf area among the progenies differing in LMA and Δ13 C reiterated the association of LMA with CE. Results illustrated that high-LMA when combined with higher CE (low Δ13 C) lead to increased WUE and growth rates.

Oh, what a tangled web we weave

Oh, what a tangled web we weave

Allele-specific analysis of single parent backcross population identifies HOX10 transcription factor as a candidate gene regulating rice root growth.

Understanding the molecular and physiological mechanisms of trait diversity is crucial for crop improvement to achieve drought adaptation. Root traits such as high biomass and/or deep rootedness are undoubtedly important drought adaptive traits. The major aim of this investigation was to functionally characterize a set of ethyl methane sulfonate-induced rice mutants for root traits. We report the identification of a high-root biomass mutant through a novel screening strategy for yield and Δ13 C measurements. The high-root mutant (392-9-1) thus identified, had a 66% higher root biomass compared to wild-type (Nagina-22). Better maintenance of leaf turgor and carbon assimilation rates resulted in lower drought susceptibility index in 392-9-1. Targeted resequencing revealed three non-synonymous single nucleotide variations in 392-9-1 for the genes HOX10, CITRATE SYNTHASE and ZEAXANTHIN EPOXIDASE. Segregation pattern of phenotype and mutant alleles in a single parent backcross F2 population revealed a typical 3:1 segregation for each of the mutant alleles. The number of F2 progeny with root biomass equal to or greater than that of 392-9-1 represented approximately one-third of the population indicating a major role played by HOX10 gene in regulating root growth in rice. Allele-specific Sanger sequencing in contrasting F2 progenies confirmed the co-segregation of HOX10 allele with the root biomass. The non-synonymous mutations in the other two genes did not reveal any specific pattern of co-segregation with root phenotype, indicating a strong role of HOX10, an upstream transcription factor, in regulating root biomass in rice.

Behavioural mechanisms of reproductive isolation between two hybridizing dung fly species

Characterization of the phenotypic differentiation and genetic basis of traits that can contribute to reproductive isolation is an important avenue to understand the mechanisms of speciation. We quantified the degree of prezygotic isolation and geographical variation in mating behaviour among four populations of Sepsis neocynipsea that occur in allopatry, parapatry or sympatry with four populations of its sister species Sepsis cynipsea. To obtain insights into the quantitative genetic basis and the role of selection against hybrid phenotypes we also investigated mating behaviour of F1 hybrid offspring and corresponding backcrosses with the parental populations. Our study documents successful hybridization under laboratory conditions, with low copulation frequencies in heterospecific pairings but higher frequencies in pairings of F1 hybrids signifying hybrid vigour. Analyses of F1 offspring and their parental backcrosses provided little evidence for sexual selection against hybrids. Longer copulation latencies in heterospecific pairings indicate species recognition, probably due to surface or volatile chemicals. The frequency of male mating attempts did not differ greatly between species or hybrid pairings, suggesting no male discrimination of mating partners. Female shaking duration, signifying female choice and/or reluctance to mate, differed strongly between the species and appears to contribute to avoiding heterospecific males; this trait is partially maternal inherited. Importantly, females of both species discriminated more strongly against males in areas of sympatry than allopatry indicating reinforcement. Shorter copulations in heterospecific parental pairings and longer copulations in F1 hybrids suggest mechanistic difficulties with sperm transfer. Overall, our study highlights an important role of character displacement affecting mating behaviour of hybridizing sepsid species in geographical areas of coexistence.

Morphological and molecular characterization of the second backcross progenies of Ogu-CMS Chinese kale and rapeseed

Ogura cytoplasmic male sterility (Ogu-CMS) is widely used in the production of commercial hybrids of Brassica oleracea. However, the widespread application of the Ogu-CMS system in B. oleracea has hindered the germplasm innovation of Ogu-CMS resources due to the lack of a natural restorer line. Previously, the Ogu-CMS fertility-restored interspecific hybrids between rapeseed 15Y403 (2n = 38, AACC) and Chinese kale JL1 (2n = 18, CC) have been successfully produced. However, these progenies, which still contained a large proportion of rapeseed genomic components, showed poor fertility and a low seed setting rate under natural pollination. To improve fertility and seed setting, a successive backcross with JL1 was performed to produce BC2 progenies. Screening with the Rfo-specific marker, five individuals harboring the Rfo gene were identified among 98 BC2 progenies. These five individuals underwent background marker screening and an evaluation of agronomic traits and fertility. One individual (code: 15Q23) was identified with higher pollen viability, better seed setting under natural pollination, and a closer genetic background to the parent Chinese kale JL1. Many morphological traits showed no significant differences (P < 0.05) between 15Q23 and the backcross parent JL1. However, the average seed setting of 15Q23 under natural pollination was 0.72 seeds per pod, which was 50 times higher than that of BC1 progenies, and the average pollen viability was 87.07%, which was significantly better than that of the F1 and BC1 plants (P < 0.01). The genetic background of 15Q23 was more closer to the parent JL1 than that of BC1 plants and another BC2 fertility-restored individual, with 82% of the polymorphic alleles being the same as those of the parent Chinese kale JL1. Thus, the individual 15Q23 could be used as a donor plant for further backcrosses. This study lays the foundation for the development of Ogu-CMS restorer material in B. oleracea.

Inheritance of Perpetual Blooming in Rosa chinensis ‘Old Blush’

Perpetual blooming is one of the most important biological and economical traits in modern rose, while the genetic basis underlining the control of this trait is poorly investigated. With an aim in dissecting the genetic determinism of perpetual blooming, we developed six rose populations (OB, W, F1, F2, BC1OB and BC1W) derived from a WOB population [interspecific diploid hybridization between Rosa chinensis ‘Old Blush’ (OB) and R. wichuriana ‘Basye’s Thornless’ (W)]. Perpetual blooming is absent both in a F1 population with 296 individuals and a BC1W population (W as the backcross parent) with 150 individuals. However, the perpetual blooming trait showed a typical 3:1 segregation in a backcross population BC1OB with OB as the backcross parent. In this population with 300 individuals, 83 plants had the perpetual blooming phenotype while the other 217 featured non-perpetual blooming, indicating that the perpetual blooming trait is very likely controlled by two recessive genes in R. chinensis (rpb1 and rpb2). These genetic data suggest that the inheritance of rose perpetual blooming may be controlled by a complex mechanism.

Sour passion fruit breeding: Strategy applied to individual selection in segregating population of Passiflora resistant to Cowpea aphid-born mosaic virus (CABMV)

The Restricted Maximum Likelihood/Best Linear Unbiased Prediction (REML/BLUP) method has been widely used in plant breeding programs, since besides estimating and predicting the genetic values, it takes into account several experimental designs. Therefore, this study aimed to estimate genetic parameters and predict gain via selection in a segregating population derived from interspecific crosses of Passiflora. Five quantitative characteristics of fruit were evaluated in 77 individuals during two growing seasons, through the REML/BLUP methodology. The experiment was arranged in an incomplete block design with two replications. Both individual and within plot heritabilities presented the highest values for number of fruits (0.32 and 0.30, respectively) and pulp mass (0.30 and 0.46, respectively), which favors gains in both yield and pulp yield. The 30 best genotypes for each evaluated trait were selected. The genetic correlation between harvests was less than the unity, for all the characteristics observed, which indicates different genetic control in different harvests. Progenies 5 and 9 were considered the most promising due to their higher percentage of selected genotypes over the harvest seasons for the traits number for fruits, pulp mass and production, with 50, 47 and 28%, respectively. The genotypes H5-1, H9-11, H9-57 and H2-20 stood out over the harvest seasons as the best individuals for number for fruits, yield and pulp mass and,therefore, are the best suited for being used as parents in backcrosses with sour passion fruit.

Disrupting ER-associated protein degradation suppresses the abscission defect of a weak hae hsl2 mutant in Arabidopsis.

In Arabidopsis thaliana, the process of abscission, or the shedding of unwanted organs, is mediated by two genes, HAESA (HAE) and HAESA-LIKE 2 (HSL2), encoding receptor-like protein kinases (RLKs). The double loss-of-function mutant hae-3 hsl2-3 is completely deficient in floral abscission, but, interestingly, the hae-3 hsl2-9 mutant displays a less severe defect. This mutant was chosen for an ethyl methanesulfonate (EMS) screen to isolate enhancer and suppressor mutants, and two such suppressors are the focus of this study. Pooled DNA from the F2 generation of a parental backcross was analyzed by genome sequencing to reveal candidate genes, two of which complement the suppressor phenotype. These genes, EMS-MUTAGENIZED BRI1 SUPPRESSOR 3 (EBS3) and EBS4, both encode mannosyltransferases involved in endoplasmic reticulum (ER)-associated degradation (ERAD) of proteins. Further analysis of these suppressor lines revealed that suppressor mutations are acting solely on the partially functional hsl2-9 mutant receptor to modify the abscission phenotype. Expressing a hsl2-9-yellow fluorescent protein (YFP) transgene in ebs3 mutants yields a higher fluorescent signal than in EBS3/ebs3, suggesting that these mutants restore abscission by disrupting ERAD to allow accumulation of the hsl2-9 receptor, which probably escapes degradation to be trafficked to the plasma membrane to regain signaling.

Interspecific hybrids between Paspalum plicatulum and P. oteroi: a key tool for forage breeding

Grama-tio-pedro (Paspalum oteroi Swallen) is a rare stoloniferous grass of the Plicatula group of Paspalum, well adapted to continuous grazing in areas subject to seasonal flooding in the Pantanal region, in central western Brazil. The species is a facultative apomictic (asexual reproduction by seed) tetraploid, sporadically cultivated on Pantanal farms, propagated either by cuttings or seed. Due to its potential for extensive cultivation and forage quality, Grama-tio-pedro appears as a candidate for genetic improvement within the Plicatula group through plant breeding. We used a colchicine-induced sexual autotetraploid genotype of P. plicatulum Michx. to obtain interspecific hybrids using the apomictic species, P. oteroi, as pollen donor. The very similar meiotic chromosome behavior observed in both parents, with main quadrivalent and bivalent associations, suggested that P. oteroi is a natural autotetraploid. The hybrids showed less irregular meiotic behavior with fewer quadrivalents and more bivalents than either parent. Fertility among interspecific hybrids varied from complete sterility in some of them to seed productions in others that were approximately twice as much as for either parent. The great variability of seed set performance may well be a drastic genetic consequence of joining two homologous chromosome sets of P. plicatulum together with two homologous sets of P. oteroi that, in turn, have some homeology between them. Most hybrids reproduce by sexual means, thus, they could be used as female parents in backcrosses and in crosses with other species of the Plicatula group for interspecific gene transferring in breeding programs.

Phenotype and Fertility of Intersectional Hybrids between Tetraploid Highbush Blueberry and Colchicine-treated Vaccinium stamineum

Several hundred hybrid seedlings were produced by pollinating flowers of tetraploid highbush blueberry cultivars with pollen from 13 plants of Vaccinium stamineum that were selected as tetraploids following colchicine treatment. The hybrids were intermediate between the parents in many characteristics. They were less vigorous than the parents, but 46 plants flowered when 1.3 years old from seed. The F1 hybrids produced pollen abundantly, but only 30% of the microspores appeared potentially viable when viewed at 250X. F1 flowers that were pollinated with pollen from either parent taxon or with pollen from a different F1 hybrid produced thousands of well-developed seeds. The F1 hybrids were less successful when used as male parents in backcrosses to highbush, but 4790 well-developed seeds were obtained by pollinating 3250 highbush flowers. Flowers on F1 plants had long peduncles and pedicels, giving an open raceme. The flowers were open in the bud and had anther awns, two characteristics from V. stamineum. Berries on the F1 plants had black skins, and the ripe berries of 11 F1 plants had red to purple pulp like their V. stamineum parent. Berries on the hybrids were juicy. They had little or no bitterness typical of V. stamineum, and most had a pleasing balance of sugar and acid.

Phosphate response of Trifolium uniflorum compared with T. repens and some T. repens×T. uniflorum hybrids

Introgression of genes from Trifolium uniflorum L. into T. repens L. (white clover) is being investigated as a method to improve phosphorus (P) use efficiency in white clover; however, little is known about the edaphic adaptations or P physiology of T. uniflorum. Growth responses to added P of T. uniflorum, T. repens and some T. repens × T. uniflorum hybrids were determined in a glasshouse experiment in pots of soil. Trifolium uniflorum showed traits consistent with adaptation to low-P soils: slow growth rate, small leaves, relatively high leaf-tissue P concentrations, and sequestration of P in its roots when soil P levels were increased. The response of Kopu II, one of the hybrid backcross parents, was quite different; it showed high growth rate, large leaves, much lower leaf P concentrations, and a large decrease in root : shoot P allocation as soil P increased. Tahora, the other backcross parent, exhibited several characteristics that were intermediate between Kopu II and T. uniflorum, probably reflecting its breeding origins from New Zealand hill-country ecotypes. This study confirms the potential for interspecific hybridisation with T. uniflorum to increase the tolerance of white clover to low soil P levels, through incorporation of traits related to edaphic adaptations. Variation among the hybrid families in their response to changing soil P confirmed previously published conclusions about the need to screen widely in hybrid material.

Fertility and Other Characteristics of F1 and Backcross1 Progeny from an Intersectional Blueberry Cross [(highbush cultivar × Vaccinium arboreum) × highbush cultivar

Fertility and morphological traits were studied in the F1 and BC1 generations of intersectional crosses between tetraploid highbush blueberry cultivars (Vaccinium section Cyanococcus) and colchicine-induced tetraploid V. arboreum (Vaccinium section Batodendron). The goal of the introgression project was to combine desirable plant characteristics from V. arboreum with the large fruit and high fruit quality of highbush cultivars. Highbush × V. arboreum crosses were hard to make, but large numbers of BC1 seedlings were easily obtained using the most fertile F1 plants as parents in backcrosses to highbush. Anther awns, a character from V. arboreum, were present in all F1 seedlings, but fruit sclerids, another V. arboreum trait, were absent in most seedlings. Berry size in the BC1 generation was twice as large as in the F1 generation and was twice as large in the F1 as in V. arboreum. The BC1 generation was extremely variable in vigor and berry quality. Although berries of most BC1 plants were smaller, darker, and less desirable in texture and flavor than highbush berries, the high fertility of BC1 plants and the high variability among plants indicate that useful clones could be selected or developed by further breeding.

Facile mutant identification via a single parental backcross method and application of whole genome sequencing based mapping pipelines

Forward genetic screens have identified numerous genes involved in development and metabolism, and remain a cornerstone of biological research. However, to locate a causal mutation, the practice of crossing to a polymorphic background to generate a mapping population can be problematic if the mutant phenotype is difficult to recognize in the hybrid F2 progeny, or dependent on parental specific traits. Here in a screen for leaf hyponasty mutants, we have performed a single backcross of an Ethane Methyl Sulphonate (EMS) generated hyponastic mutant to its parent. Whole genome deep sequencing of a bulked homozygous F2 population and analysis via the Next Generation EMS mutation mapping pipeline (NGM) unambiguously determined the causal mutation to be a single nucleotide polymorphisim (SNP) residing in HASTY, a previously characterized gene involved in microRNA biogenesis. We have evaluated the feasibility of this backcross approach using three additional SNP mapping pipelines; SHOREmap, the GATK pipeline, and the samtools pipeline. Although there was variance in the identification of EMS SNPs, all returned the same outcome in clearly identifying the causal mutation in HASTY. The simplicity of performing a single parental backcross and genome sequencing a small pool of segregating mutants has great promise for identifying mutations that may be difficult to map using conventional approaches.