BC1 Progeny Research Articles

Introgression of Herbicide-Resistant Gene from Genetically Modified Brassica napus L. to Brassica rapa through Backcrossing.

Interspecific hybridization between two different Brassicaceae species, namely Brassica rapa ssp. pekinensis (♀) (AA, 2n = 2x = 20) and genetically modified Brassica napus (♂) (AACC, 2n = 4x = 38), was performed to study the transmission of a herbicide resistance gene from a tetraploid to a diploid Brassica species. Initially, four different GM B. napus lines were used for hybridization with B. rapa via hand pollination. Among the F1 hybrids, the cross involving the B. rapa (♀) × GM B. napus (♂) TG#39 line exhibited the highest recorded crossability index of 14.7 ± 5.7. However, subsequent backcross progenies (BC1, BC2, and BC3) displayed notably lower crossability indices. The F1 plants displayed morphological characteristics more aligned with the male parent B. napus, with significant segregation observed in the BC1 generation upon backcrossing with the recurrent parent B. rapa. By the BC2 and BC3 generations, the progeny stabilized, manifesting traits from both parents to varying degrees. Cytogenetic analysis revealed a substantial reduction in chromosome numbers, particularly in backcrossing progenies. BC1 plants typically exhibited 21-25 chromosomes, while BC2 progenies showed 21-22 chromosomes, and by the BC3 generation, stability was achieved with an average of 20 chromosomes. SSR marker analysis confirmed the progressive reduction of C-genome regions, retaining minimal C-genome-specific bands throughout successive backcrossing. Despite the extensive elimination of C-genome-specific genomic regions, the glyphosate resistance gene from the male parent B. napus was introgressed into BC3 progenies, suggesting that the glyphosate resistance gene located and introgressed in A-chromosome/genome regions of the Brassica plants.

Intergeneric chromosome-specific painting reveals differential chromosomal transmission from Tripidium arundinaceum in sugarcane progeny

Sugarcane has recently attracted increasing attention for its potential as a source of sugar and bioethanol, so increasing its yield is essential to ensure the sugar security and bioenergy production. Intergeneric hybridization is a highly efficient method to produce new genetic variants of crop plants, particularly those species with high ploidy such as sugarcane (Saccharum spp.). Tripidium arundinaceum exhibits many desirable agronomic traits, and has been widely studied to produce hybrids with improved stress tolerance and other characteristics in sugarcane breeding. However, the genetic relationship between T. arundinaceum and Saccharum species, and the individual T. arundinaceum chromosomal compositions in sugarcane hybrids are still elusive. Here we used whole-genome single-nucleotide polymorphisms (SNPs) to ascertain the phylogenetic relationships between these species and found that T. arundinaceum is more closely related to Saccharum than Sorghum, in contrast to the previous narrow genetic analyses using chloroplast DNA. Additionally, oligonucleotide (oligo)-based chromosome-specific painting derived from Saccharum officinarum was able to distinctly identify the chromosomes of T. arundinaceum. We developed the oligo-genomic in situ hybridization (GISH) system for the first time, to unveil the novel chromosome translocations and the transmission of individual T. arundinaceum chromosomes in sugarcane progeny. Notably, we discovered that the chromosomal transmission of T. arundinaceum exhibited several different inheritance modes, including n, 2n, and over 2n in the BC1 progenies. Such inheritance patterns may have resulted from first division restitution (FDR) or FDR+nondisjunction of a chromosome with the sister chromatids in the second meiosis division/second division restitution (FDR+NSC/SDR) model during meiosis. These results will be of substantial benefit for the further selection of T. arundinaceum chromosomes for sugarcane genetic improvement.

Genetic enhancement of okra [Abelmoschus esculentus (L.) Moench] germplasm through wide hybridization.

The introgression of genetic material from one species to another through wide hybridization and repeated back-crossing, plays an important role in genetic modification and enriching the cultivated gene-pool with novel genetic variations. Okra (Abelmoschus esculentus [(L.) Moench)] is a popular vegetable crop with high dietary fibre and protein, rich in essential amino acids, lysine and tryptophan. The wild Abelmoschus genepool has many desirable traits like ornamental value, short internodal length, more number of productive branches, extended bearing, perennation tendency, reduced fruit length (more consumer preferred trait), high mucilage content (medicinal value), abiotic stress tolerances such as drought, high temperature and biotic stress resistances such as okra Yellow Vein Mosaic Virus (YVMV) and Enation Leaf Curl Virus (ELCV) diseases. The repeated use of elite breeding lines led to narrowing of the genetic base of the okra crop, one of the major factors attributed to breakdown of resistance/ tolerance to biotic stresses. YVMV and ELCV are the two major diseases, causing significant yield loss in okra. Hence, wide hybridization was attempted to transfer tolerance genes from wild species to the cultivated genepool to widen the genetic base. The screening of germplasm of wild Abelmoschus species at hotspots led to the identification of tolerant species (Abelmoschus pungens var. mizoramensis, A. enbeepeegeearensis, A. caillei, A. tetraphyllus and A. angulosus var. grandiflorus), which were further used in a wide-hybridization programme to generate interspecific hybrids with the cultivated okra. Presence of pre- and post-zygotic barriers to interspecific geneflow, differences in ploidy levels and genotype specific variations in chromosome numbers led to varying degrees of sterility in F1 plants of interspecific crosses. This was overcome by doubling the chromosome number of interspecific hybrids by applying Colchicine at the seedling stage. The 113 cross derivatives generated comprising amphidiploids in the F1 generation (30), F3 (14), one each in F2 and F4 generations, back cross generation in BC1F2 (03), BC1F3 (25), and BC2F3 (02), crosses between amphidiploids (27), multi-cross combinations (07) and inter-specific cross (between A. sagittifolius × A. moschatus subsp. moschatus) selfed derivatives at F8 generation (03) were characterized in the present study. Besides they were advanced through selfing and backcrossing. The amphidiploids were found to possess many desirable genes with a considerable magnitude of linkage drag. Majority of the wide cross derivatives had an intermediate fruit morphology and dominance of wild characters viz., hispid fruits, stem, leaves, tough fruit fibre, vigorous perennial growth habit and prolonged flowering and fruiting. The fruit morphology of three BC progenies exhibited a high morphological resemblance to the cultivated okra, confirming successful transfer of useful genes to the cultivated okra genepool. The detailed morphological characteristics of the various combinations of Abelmoschus amphidiploids and the genetic enhancement of the genepool achieved in this process is reported here.

Development of Local Breeding Lines for Rust Resistance in the Common Bean (<em>Phaseolus vulgaris</em>) through the Incorporation of Rust-Resistant Genes

Bean rust is one of the major diseases of the common bean (Phaseolus vulgaris) reported in Sri Lanka and at the global level. This study aimed to develop rust-resistant snap bean breeding lines via gene pyramiding assisted by molecular markers. Resistant sources; PI 181996, BelMiNeb-RMR-8, and BelDakMi-RMR-19, enriched with the rust-resistant genes Ur-3 and Ur-11, were selected as donor parents to obtain a wide range of resistance to the rust pathogen. Resistant genotypes were crossed with popular local varieties Kappetipola nil and Galpalama Kalu (Capri) to introgress Ur-3 and Ur-11 resistant genes. Successive F1, F2, and BC (backcross) generations were obtained with the self-pollination and backcrossing processes. Standard phenotypic disease screening methods were applied to identify resistant lines. Phenotypically resistant plants obtained from these crosses were tested with sequence-characterized amplified region (SCAR) markers linked to two rust-resistant genes: SK 14 (linked to Ur-3) and SI 19 (linked to Ur-11). Molecular marker SI-19 showed higher reproducibility (50% to 80%) with the availability of relevant banding patterns for phenotypically resistant F1, F2, and BC1 progenies. However, SK 14 showed lower reproducibility (30–60%) for the same progenies. Approximately 450 genotypes introgressed with rust-resistant genes (Ur-3 and Ur-11)were produced. Among them, four advanced resistant lines obtained from the different cross combinations (Kappetipola nil x BelDakMi-RMR-19, Galpalama Kalu x BelMiNeb RMR-8, Kappetipola nil x PI 181996, and Kappetipola nil x BelMiNeb RMR-8) with preferred agronomic characters were selected for further variety development. All new genotypes will be important for future bean-resistant breeding programs in Sri Lanka.

Homoeologous Chromosome Pairing and Alien Introgression in Backcrossing Progenies Derived from Hybrids Solanum tuberosum (+) Mexican 2x (1 EBN) B-Genome Potato Species

We applied a genomic in situ hybridization (GISH) to analyze the genomic constitution of and meiotic pairing in interspecific somatic hybrids, and in a wide subset of backcrossing derivatives (BC1–BC5), from three interspecific combinations involving the cultivated potato, Solanum tuberosum (AAAA genome), and three diploid (1 EBN) wild Mexican potato species (genome BB)—S. tarnii, S. pinnatisectum, and S. bulbocastanum. The theoretically expected genomic composition was detected in the somatic hybrids (AAAABB) and in the BC1 progeny (AAAAB), whereas in the subsequent BC2–BC4 generations, the partial loss of alien chromosomes was observed and almost all the BC5 genotypes showed a complete chromosome elimination of wild species. GISH revealed a homoeologous pairing between the chromosomes of the A- and the B-genomes in each of the hybrid progenies. Using GISH, we selected introgression lines with a single chromosome of the wild species in a potato genome background, as well as introgression lines with intergenomic recombinant chromosomes. Moreover, via molecular screening, BC hybrids with diagnostic markers for the R-genes conferring resistance to late blight disease and to the quarantine pest of the potato–Columbia root-knot nematode—were selected. The potential application of the results obtained for the planning of introgressive schemes directed to the breeding of advanced lines with multiple disease and pest resistance is discussed.

Hybrid purity identification using EST-SSR markers and heterosis analysis of quantitative traits of Russian wildrye.

Russian wildrye, Psathyrostachys junceus (Fisch.) Nevski, is widely distributed in the high latitude areas of Eurasia. It plays an important role in grassland ecosystem maintenance, as well as being a valuable palatable forage species for livestock and wildlife. Russian wildrye germplasm has rich phenotypic and genetic diversity and has potential for improvement through crossbreeding. In this study, fifteen Russian wildrye hybrid combinations were produced and one F1 population with 123 putative hybrids was obtained by crossing two individual plants with significant differences in nutritional characteristics and reproductive tiller number. Twelve phenotypic traits of the F1 population were measured for three consecutive years, and ten of the twelve traits were in line with the genetic characteristics of quantitative traits. Hybrid superiority was revealed among F1 hybrids in both nutritional and reproductive traits. One non-recurrent parent plant with the highest PCA-synthesis score was selected and used to make a backcross with the 'BOZOISKY SELECT' male parent, and 143 putative BC1 hybrids were obtained. Sixteen pairs of EST-SSR primers were randomly selected from polymorphic primers derived from different expressed tiller trait related genes. Three primer pairs that amplified both the paternal and maternal characteristic band were used to assess the purity of the F1 population, and three primer pairs (with one shared primer pair) were used to identify the BC1 population. The hybrid purity was 96.75% for the F1 population and 95.80% for the BC1 population, and the results were confirmed by self-fertility test through bagging isolation. The genetic similarity coefficients between the F1 progeny and the male parent ranged from 0.500 to 0.895, and those between the BC1 progeny and the male parent ranged from 0.667 to 0.939. A subset of individuals in the BC1 population had closer genetic distance to the recurrent parent, and genetic variation within the BC1 population decreased compared to the F1 population.

Efficient Anchoring of Erianthus arundinaceus Chromatin Introgressed into Sugarcane by Specific Molecular Markers

Erianthus arundinaceus is a valuable gene reservoir for sugarcane improvement. However, insufficient molecular markers for high-accuracy identification and tracking of the introgression status of E. arundinaceus chromatin impede sugarcane breeding. Fortunately, suppression subtractive hybridization (SSH) technology provides an excellent opportunity for the development of high-throughput E. arundinaceus-specific molecular markers at a reasonable cost. In this study, we constructed a SSH library of E. arundinaceus. In total, 288 clones of E. arundinaceus-specific repetitive sequences were screened out and their distribution patterns on chromosomes were characterized by fluorescence in situ hybridization (FISH). A subtelomeric repetitive sequence Ea086 and a diffusive repetitive sequence Ea009, plus 45S rDNA-bearing E. arundinaceus chromosome repetitive sequence EaITS were developed as E. arundinaceus-specific molecular markers, namely, Ea086-128, Ea009-257, and EaITS-278, covering all the E. arundinaceus chromosomes for high-accuracy identification of putative progeny. Both Ea086-128 and Ea009-257 were successfully applied to identify the authenticity of F1, BC1, BC2, BC3, and BC4 progeny between sugarcane and E. arundinaceus. In addition, EaITS-278 was a 45S rDNA-bearing E. arundinaceus chromosome-specific molecular marker for rapid tracking of the inherited status of this chromosome in a sugarcane background. Three BC3 progeny had apparently lost the 45S rDNA-bearing E. arundinaceus chromosome. We reported herein a highly effective and reliable SSH-based technology for discovery of high-throughput E. arundinaceus-specific sequences bearing high potential as molecular markers. Given its reliability and savings in time and efforts, the method is also suitable for development of species-specific molecular markers for other important wild relatives to accelerate introgression of wild relatives into sugarcane.

Detection of Maternal and Cytoplasmic Effects on Resistance to Zymoseptoria tritici in Durum Wheat.

Septoria tritici blotch (STB) is a major disease problem of wheat worldwide. To optimize the introgression of resistance genes in elite genotypes throughout traditional or molecular breeding programs, a full understanding of the quantitative inheritance of resistance to Zymoseptoria tritici, plant height (PH), and thousand kernel weight (TKW) is needed. In this study, maternal and cytoplasmic effects of resistance to STB were investigated using P1 (susceptible, high-yielding line) and P2 (resistant, low-yielding line) durum wheat lines and their F1, RF1, F2, RF2, BC1, RBC1, BC2, and RBC2 progeny, assessed for resistance to STB during three growing seasons. Duncan mean's analysis revealed significant differences between generation means for STB, PH, and TKW. The two parents had an extreme pattern. The F1 and RF1 segregated close to their respective parents, suggesting the presence of cytoplasmic and maternal genetic effects for Z. tritici resistance, PH, and TKW. Separate generation mean's analysis confirmed the results of the Duncan test. A three-parameter model was found to be not adequate for all traits in all three growing years; while a digenic epistatic model with cytoplasmic or/and maternal effect was adequate for all cases. Narrow-sense heritability was in the range of 50–60%, 30–69%, and 28–31% for STB, PH, and TKW, respectively. For STB, high heritability and the presence of fixable epistatic effect is encouraging and could lead to creating varieties with the right female parent to exploit cytoplasmic and maternal effects in order to improve resistance to Z. tritici in durum wheat.

"Establishment of GISH and multi-color GISH techniques to simultaneously discriminate different genomes in Erianthus procerus x Saccharum officinarum introgressed clones "

An attempt was made to identify the chromosomes from different species genomes in BC₂ progenies of Erianthus procerus x Saccharum officinarum through multi-color GISH (MCGISH). The BC₂ population was derived from the cross between a BC1 hybrid (GU 12-14) and a commercial variety, Co 16018. The BC2 progenies were studied for their somatic chromosome number and two cytotypes were observed, i.e., 2n=92 (2) and 2n=96 (5). GISH revealed that the majority of progenies were having nine Erianthus chromosomes, except 2 clones (25-5 and 25-10) were having nine Erianthus chromosomes with one chromosome fragment. The methodology for multi-color GISH was standardized and it distinguished the chromosomes from different genomes. Interspecific recombination was also detected in somatic cells of one BC₂ clone, 25-10. The hybridity of the introgressed lines has been confirmed with amplification of Erianthus specific tandem repeat sequences.

Double Haploid Development and Assessment of Androgenic Competence of Balkan Pepper Core Collection in Bulgaria

This study was designed to assess the androgenic potential of 180 pepper accessions and 11 progenies (four F1 and seven BC) possessing PMMoV resistance in order to complement an ongoing pepper breeding program. The experiment was carried out in 10 replications with 20 anthers for each accession in two different induction mediums from 2017 to 2019. The highest androgenic response was observed in culture medium 17-2 but differences between two mediums were nonsignificant. From a total of 191 genotypes, 102 genotypes expressed a potential for direct embryogenesis. Embryo induction was seen to be genotype-dependent and decreased in the following order: Pumpkin > Conical > Bell or blocky > Round > Elongate as the most responsive genotypes with over 10% reacted anthers being observed in CAPS-23, CAPS-29, CAPS-127, CAPS-157, CAPS-169, F1 and BC 887 derived from CAPS-23. The number of regenerated plants was higher in the conical group and least in the round varietal group. Regenerated plants were examined visually and by flow cytometry for identification of spontaneous doubled haploids (DH) and haploids. Those originating from F1 and BC progenies were additionally evaluated by a CAPS marker targeting L4 allele for resistance against PMMoV. Obtained results revealed two groups consisting of homozygous susceptible and resistant plants. Therefore, use of anther culture in ongoing breeding will greatly facilitate the pepper genetic improvement.

Characterization of Ogura CMS fertility-restored interspecific hybrids and backcross progenies from crosses between broccoli and rapeseed

Interspecific F1 hybrids were obtained from crosses between two Ogura cytoplasmic male sterility (Ogura CMS) broccoli lines (B14 and 137) and a rapeseed restorer line to introgress the Ogura CMS’s fertility-restoration gene Rfo from rapeseed into broccoli. Ten Rfo-positive interspecific, triploid plant progenies, phenotypically between rapeseed and broccoli, were obtained but still contained multiple rapeseed genes, with reduced fertility and seed setting potential under natural pollination. For fertility and seed setting improvement, successive backcrosses with broccoli were carried out to yield BC2 progenies. Screening revealed six plants with Rfo among 25 BC2 progenies, which were investigated for agronomic traits, ploidy determination, pollen viability and SSR (simple sequence repeat) markers to evaluate for ideal individuals closer to broccoli. Pollen viability tests were carried out at different stages for various hybrid parents. The results were as follows: 137-F1 (68.54%) > B14-F1 (65.32%) > 137-BC1 (48.15%) > B14-BC1 (32.41%). Although there were significant differences among the BC1 and BC2 individuals, the genetic background of BC2 was closer to the parent broccoli compared with that of BC1 plants. The existence of the Rfo gene in broccoli not only facilitates broccoli germplasm innovation, combination of interspecific hybridization and backcrossing, but may also shed light on trait segregation in different generations.

Intergeneric hybidization of sunflower (Helianthus annuus L.) with spiny plumeless thistle (Carduus acanthoides L.)

Abstract Carduus acanthoides L (spiny plumeless thistle) a biannual wild species with 2n = 22 chromosomes was crossed with Helianthus annuus L. When crossing, pollen from C. acanthoides germinated and pollinated the stigmas of sunflower lines HA 89A after 48 h. The crossability rate was low, but seeds and hybrid plants were obtained. The F1 plants strongly resembled the cultivated sunflower with the most important bio-morphological characters, even though they had an intermediate type of heritability. The hybrid nature was confirmed by RAPD markers. The polymorphism between H. annuus, C. acanthoides, and their F1 hybrids was studied using RAPD. The result showed introgression of C. acanthoides in the hybrid progeny. It was established that the wild species carried Rf genes for the CMS PET-1. After self-pollination and sib-pollination of the F1 plants and back-crossing with cultivated sunflower, F2, BC1 and next generation hybrid progenies were obtained. The investigation encompassed the period 2000–2007 and 2014–2018. Some of the new lines have been included in a heterosis breeding program for developing hybrids for the sunflower market.

Creation of fertility-restored materials for Ogura CMS in Brassica oleracea by introducing Rfo gene from Brassica napus via an allotriploid strategy

Key messageOgura CMS fertility-restored materials, with 18 chromosomes, normal seed setting, stable fertility and closer genetic background to the parent Chinese kale, were successfully developed in B. oleracea via a triploid strategy for the first time.Ogura cytoplasmic male sterility (CMS) is the most widely used sterile type in seed production for commercial hybrids of Brassica oleracea vegetables. However, the natural Ogura CMS restorer line has not been found in B. oleracea crops. In this study, the triploid strategy was used with the aim to create euploid B. oleracea progenies with the Rfo gene. The allotriploid AAC hybrid YL2 was used as a male parent to backcross with Ogura CMS Chinese kale. After successive backcrosses, the BC2Rfo-positive individual 16CMSF2-11 and its BC3 progenies, with 18 chromosomes, were developed, which were morphologically identical to the parent Chinese kale. Compared with F1 and BC1 plants, it showed stable fertility performance, and regular meiosis behavior and could produce seeds normally under natural pollination. The genomic composition analysis of Rfo-positive progenies by using molecular markers showed that more than 87% of the C-genome components of BC3Rfo-progenies recovered to the parent Chinese kale, while most or all of the An-genome segments were lost in 16CMSF2-11 and its progenies. The results suggested that the genetic background of Rfo-positive individuals was closer to that of the parent Chinese kale along with backcrossing. Hereof, the Ogura CMS fertility-restored materials of Chinese kale were successfully created via triploid strategy for the first time, providing a bridge for utilizing the Ogura CMS B. oleracea germplasm in the future. Moreover, our study indicates that the triploid strategy is effective for transferring genes from B. napus into B. oleracea.

Introgression of Two Broad-Spectrum Late Blight Resistance Genes, Rpi-Blb1 and Rpi-Blb3, From Solanum bulbocastanum Dun Plus Race-Specific R Genes Into Potato Pre-breeding Lines

There is a wealth of resistance genes in the Mexican wild relative of cultivated Solanum, but very few of these species are sexually compatible with cultivated Solanum tuberosum. The most devastating disease of potato is late blight caused by the oomycete Phytophthora infestans (Pi). The wild hexaploid species S. demissum, which it is able to cross with potato, was used to transfer eleven race-specific genes by introgressive hybridization that were subsequently widely used in potato breeding. However, there are now more virulent races of Pi that can overcome all of these genes. The most sustainable strategy for protecting potatoes from late blight is to pyramid or stack broad-spectrum resistance genes into the cultivars. Recently four broad-spectrum genes (Rpi) conferring resistance to Pi were identified and cloned from the sexually incompatible species S. bulbocastanum: Rpi-blb1 (RB), Rpi-blb2, Rpi-blb3, and Rpi-bt1. For this research, a resistant S. bulbocastanum accession was selected carrying the genes Rpi-blb1 and Rpi-blb3 together with race-specific R3a and R3b genes. This accession was previously used to produce a large number of somatic hybrids (SHs) with five commercial potato cultivars using protoplast electrofusion. In this study, three SHs with cv. ‘Delikat’ were selected and backcross generations (i.e., BC1 and BC2) were obtained using cvs. ‘Baltica’, ‘Quarta’, ‘Romanze’, and ‘Sarpo Mira’. Their assessment using gene-specific markers demonstrates that these genes are present in the SHs and their BC progenies. We identified plants carrying all four genes that were resistant to foliage blight in greenhouse and field trials. Functionality of the genes was shown by using agro-infiltration with the effectors of corresponding Avr genes. For a number of hybrids and BC clones yield and tuber number were not significantly different from that of the parent cultivar ‘Delikat’ in field trials. The evaluation of agronomic traits of selected BC2 clones and of their processing qualities revealed valuable material for breeding late blight durable resistant potato. We show that the combination of somatic hybridization with the additional use of gene specific markers and corresponding Avr effectors is an efficient approach for the successful identification and introgression of late blight resistance genes into the potato gene pool.

Somatic hybrids of Sinapis alba + Brassica juncea: study of backcross progenies for morphological variations, chromosome constitution and reaction to Alternaria brassicae

The wild relatives of crops represent a rich reservoir of genes for introducing resilience to climate change into cultivated crops. To introgress genes from Sinapis alba, a wild relative of Brassicaceae, into Brassica juncea, a cultivated member of this family, we initially produced somatic hybrids between the two species and then produced a large number of backcross populations involving the two somatic hybrids (H1 and H2) with Brassica juncea. BC1 progeny were morphologically very similar. However, when they were challenge inoculated with a highly virulent Alternaria brassicae (ITCC No. 2542) culture under in vivo and in vitro conditions in two growing seasons, they showed wide variations in their disease reaction. Of the 40 BC1 lines tested in one season, 36 showed a resistant reaction. BC1F2 progenies derived from these resistant BC1 plants also showed resistance to Alternaria brassicae, indicating stable inheritance of the resistant phenotype. However, BC1F2 progenies showed a wide variation in morphological traits, including plant height, basal branching, leaf thickness, trichome density on leaves and stem. BC1 plants were examined by genomic in-situ hybridization (GISH) to determine their chromosome constitution. All five plants were found to possess 12 strong hybridization signals upon hybridization with a FITC-labeled S. alba-specific probe. GISH studies on BC1F2 plants indicated localized signals in addition to 12 full chromosome hybridization signals, suggesting alien introgressions into B. juncea that requires further validation. The BC2 generation was found to possess half of the haploid set of alien chromosomes. The BC1F2 and BC2 generations were further screened against A. Brassiceae and found to be resistant/tolerant.

Fast-track breeding system to introduce CTV resistance of trifoliate orange into citrus germplasm, by integrating early flowering transgenic plants with marker-assisted selection

BackgroundGlobal warming will expand the range of new and invasive pathogens in orchards, and subsequently increase the risk of disease epidemics and economic losses. The development of new resistant plant varieties can help to reduce the impact of pathogens, however, the breeding speed can be extremely slow, due to the growth rates of the plants, and the availability of resistance genes. Citrus trees are suffering immense damage from serious diseases such as citrus canker (XCC), huanglongbing (HLB), and citrus tristeza virus (CTV). A fast-track breeding system, that aimed at shortening the duration for disease resistance breeding by incorporating the resistance genes from related species to commercial varieties, has been developed using the integration of precocious transgenic trifoliate orange with the overexpression of CiFT and MAS. It was applied here to incorporate CTV resistance of trifoliate orange into citrus germplasm.ResultsOne generation of backcrossed breeding, that would normally take at least 5 years, was achieved in a single year by fast-track breeding system. Linkage analysis using the corresponding DNA markers revealed that CTV resistance and T-DNA integrated regions were found in different linkage groups, and they were independently segregated in the BC progenies. The CTV resistant null segregants, in which the T-DNA integrated region was removed from their genome, were feasibly obtained by MAS in each generation of the BC progenies, and their CTV resistance was confirmed by immunological analysis. Several BC3 null segregants, whose genetic backgrounds had been substituted into citrus germplasm, except for the haplotype block of CTV resistance, were successfully obtained. CGH and NGS analyses revealed that the T-DNA integrated region was safely segregated out in null segregants.ConclusionFast-track breeding systems are expected to shorten the required breeding time by more than one-fifth in comparison with conventional cross breeding techniques. Using this system, we obtained BC3–8, whose genetic background was successfully substituted except for the CTV resistance locus, and could be a novel mandarin breeding material. The fast-track breeding system will be useful to introduce important traits from related species to citrus germplasm while also drastically reducing the time required for breeding.

Genetic analysis of resistance to Fusarium oxysporum f. sp. niveum race 2 in cultivated watermelon [Citrullus lanatus (Thunb.) Matsum & Nakai

The genetics and nature of gene action involved in resistance to Fusarium oxysporum f. sp. niveum race 2 causing wilt in watermelon was studied. Five resistant accessions were crossed with a common susceptible parent to develop six generations for each combination. The populations were screened in two consecutive assays during June to September 2019. Based on the segregation of F2, the resistance in IC0523048, EC794455 and EC759804 was found to be governed by two major complementary dominant genes. However, the deviation from the expected ratio in BC1 progeny suggested the involvement of modifying genetic factors. The F2 and backcross progenies of BIL-53 × IIHR-9 and BIL-53 × EC794421 did not follow any of the ratios tested. Allelism test revealed the absence of any allelic relationship between IIHR9 and IC0523048 for resistance. Generation mean analysis revealed presence of epistasis in BIL-53 × EC794455 and BIL-53 × EC759804 while in the other three crosses, simple additive-dominance interaction was observed. These resistant accessions and the inheritance information generated in this study would help in incorporation of resistance to Fusarium wilt race 2 into elite commercial backgrounds.

Quantitative Trait Loci (QTL) Analysis of Fruit and Agronomic Traits of Tropical Pumpkin (Cucurbita moschata) in an Organic Production System

Interest in the development of organically grown vegetable crops has risen over the past decades due to consumer preferences. However, most crops that have desirable consumer traits have been bred in conventional growing conditions, and their transfer to an organic setting is challenging. Here, the organically grown Hawaiian pumpkin (Cucurbita moschata) accession ‘Shima’ was crossed with the conventionally grown Puerto Rican variety ‘Taina Dorada’ to develop a backcross (BC1) population, where ‘Shima’ was the recurrent parent. A total of 202 BC1 (‘Shima’ X F1) progenies were planted in a certified organic field, and twelve traits were evaluated. We used genotype-by-sequencing (GBS) to identify the Quantitative Trait Loci (QTL) associated with insect tolerance along with commercially desirable traits. A total of 1582 single nucleotide polymorphisms (SNPs) were identified, from which 711 SNPs were used to develop a genetic map and perform QTL mapping. Reads associated with significant QTLs were aligned to the publicly available Cucurbita moschata genome and identified several markers linked to genes that have been previously reported to be associated with that trait in other crop systems, such as melon (Cucumis melo L.). This research provides a resource for marker-assisted selection (MAS) efforts in Cucurbita moschata, as well as serving as a model study to improve cultivars that are transitioning from a conventional to an organic setting.

Markör destekli piramit potivirüs direnç genleri, Ruanda fasulye (Phaseolus vulgaris L.) genotipleri

Bean common mosaic virus and Bean common mosaic necrosis virus belong to Potyvirus and the disease poses significant threat to bean yield especially in East and Central Africa. Combining the dominant and recessive resistance genes is the most effective disease control against all known strains of the virus, a strategy to enhance the durability and stabilize the yield. The aim of the study was to transfer both the dominant I and recessive bc-3 genes into susceptible Rwandan common beans via marker-assisted backcross breeding. The markers specific for I gene (BCMV-48289723-CAPS) and for bc-3 gene (ENM-CAPS) were used for selection in BC progenies. Successful gene combination was established in 33% of BC1F1 and BC2F1, 43% of BC3F1 and 16% of BC3F2 progenies. The seed color was fully recovered on some of the BC3 progenies with combined resistance genes. The advanced lines are expected to shield the crop against both BCMV and BCMNV under East and Central African conditions.

Elevation of Condensed Tannins in the Leaves of Ta-MYB14-1 White Clover (Trifolium repens L.) Outcrossed with High Anthocyanin Lines.

Condensed tannins (CT) are highly desirable in forage as they sequester dietary protein and reduce bloat and methane emissions in ruminants. However, the widely used forage legume white clover (Trifolium repens) only produces CTs in flowers and trichomes and at levels too low to achieve therapeutic effects. Genetic transformation with transcription factor Ta-MYB14-1 from Trifolium arvense was effective in inducing CTs to 0.6% of leaf dry matter. CT synthesis has been elevated further by crossing the primary white clover transgenic line with wild type genotypes producing the related phenylpropanoids, anthocyanins. CT levels in leaves were highest under the anthocyanin leaf marks associated with the "red midrib" trait; however, there was no evidence for CT accumulation in leaf sections with the "red V" anthocyanin marking. Ta-MYB14-1 was stably inherited in two generations of crosses, and T2 progeny produced up to 3.6-fold higher CTs than the T0 parent. The profile of small CT oligomers such as dimers and trimers was consistent in T0, T1, T2, and BC2 progeny and consisted predominantly of prodelphinidins (PD), with lesser amounts of procyanidins (PC) and mixed PC:PD oligomers.