Utilization of marker-assisted backcrossing to generate new gynoecious cucumber lines with genetic heritage similar to the indigenous Vietnamese cucumber.

The marker-assisted backcrossing (MAB) can help to transfer an interested allele at a target locus from a donor to a recipient line. Gynoecious is a pivotal trait of cucumber since commercial F1 hybrid seeds produced with gynoecious line as one of the parents are high-yield and affordable. This study aims to transfer the F locus encoded for gynoecious trait to Vietnamese domesticated cucumbers by marker-assisted backcrossing. Two monoecious cucumber lines, A1 (Ha Giang, Vietnam) A2 (Yen Bai, Vietnam), and two gynoecious cucumber lines, B1 (Plantgene, India) and B2 (Hue, Vietnam) were utilized as the starting materials. BCAT marker (located on the F locus) and 52 SSRs (spread across seven chromosomes and tightly linked with some crucial horticultural traits) were used as the foreground and background markers, respectively. With this, phenotype selection for fruit and leaf sizes was also applied. First, using phenotypic screening and foreground marker, A1 (Ha Giang, Vietnam) and B1 (Plantgene, India) were selected as donor and recurrent parents for backcrossing. Then, after two backcrosses followed by two self-pollinations, four gynoecious C cucumber lines were created. These C lines have leaf sizes slightly bigger than the recurrent parent. Importantly, their fruit length is the same or longer than A1 (Ha Giang, Vietnam). These new gynoecious lines could be used as material lines for producing commercial F1 hybrid seeds. Supplementary Information: The online version contains supplementary material available at 10.1007/s11032-024-01481-w.

Development of an SNP marker set for marker-assisted backcrossing using genotyping-by-sequencing in tetraploid perilla.

High-quality molecular markers are essential for marker-assisted selection to accelerate breeding progress. Compared with diploid species, recently diverged polyploid crop species tend to have highly similar homeologous subgenomes, which is expected to limit the development of broadly applicable locus-specific single-nucleotide polymorphism (SNP) assays. Furthermore, it is particularly challenging to make genome-wide marker sets for species that lack a reference genome. Here, we report the development of a genome-wide set of kompetitive allele specific PCR (KASP) markers for marker-assisted recurrent selection (MARS) in the tetraploid minor crop perilla. To find locus-specific SNP markers across the perilla genome, we used genotyping-by-sequencing (GBS) to construct linkage maps of two F2 populations. The two resulting high-resolution linkage maps comprised 2326 and 2454 SNP markers that spanned a total genetic distance of 2133 cM across 16 linkage groups and 2169 cM across 21 linkage groups, respectively. We then obtained a final genetic map consisting of 22 linkage groups with 1123 common markers from the two genetic maps. We selected 96 genome-wide markers for MARS and confirmed the accuracy of markers in the two F2 populations using a high-throughput Fluidigm system. We confirmed that 91.8% of the SNP genotyping results from the Fluidigm assay were the same as the results obtained through GBS. These results provide a foundation for marker-assisted backcrossing and the development of new varieties of perilla.

Pyramiding resistance genes for bacterial leaf blight (Xanthomonas oryzae pv. Oryzae) into the popular rice variety, Pratikshya through marker assisted backcrossing.

BACKGROUND: Bacterial leaf blight (BLB) is one of the major biotic stress in rice cultivation. Management techniques, such as the development of BLB-resistant cultivars, are required to lessen the severity of the disease attack and yield losses. Pratikshya was selected in the present investigation as the recipient parent, as it is one of the popular high-yielding rice varieties of Odisha, India, which is having excellent grain as well as cooking quality. However, Pratikshya is highly susceptible to BLB which is prevalent in Eastern Indian region. METHODS AND RESULTS: Three major BLB resistance genes xa5, xa13, and Xa21 from the donor source Swarna MAS (CR Dhan 800) were attempted to introduce into Pratikshya through a marker-assisted backcross breeding program. Those markers closely linked to the target genes were employed for foreground selection in the segregating generations till BC2F3. In each backcross generation, progenies containing all three targeted resistance genes and phenotypically more similar to the recipient parent, Pratikshya were selected and backcrossed. Screening of 1,598 plants of the BC2F2 population was conducted against BLB using Xoo inoculum and 35 resistant plants similar to Pratikshya were carried forward to the next generation. In the BC2F3 generation, 31 plants were found to possess all the three resistance genes. For background selection of plants carrying resistance genes 45 polymorphic SSR markers were employed. Evaluation of the pyramided lines at BC2F4 generation exhibited that, most pyramided lines were similar to Pratikshya in terms of morphological features and yield parameters, and some lines were superior to the recurrent parent in terms of morphological features and yield parameters. CONCLUSION: The three-gene pyramided lines showed a high level of resistance to BLB infection and are anticipated to offer a significant yield advantage over the recipient parent Pratikshya. The pyramided lines can further be used for multi-location trial, so as to be released as a variety or can be used as a potential donor for BLB resistance genes.

Backcrossing as a species restoration technique.

An investigation was conducted on the phenotypic results of mouse hybridization and seven generations of backcrossing, observing reciprocal F1 hybrids and backcrosses of Mus spretus and a laboratory strain of Mus domesticus C57BL/6J. F1 hybrids, backcrosses, and pure control specimens were measured for 6 body characteristics, 4 pelage coloration characteristics, 14 behaviors, and reproduction as reflected in litter size. Backcrossing was pursued for seven generations to FBC7 (i.e., "Backcross 7" or seven generations from commencement of backcrossing from an F1 hybrid female) where species restoration is mathematically calculated to be at 99.7%. Except for a minority of FBC7 M. spretus specimens failing to conform completely to one pelage characteristic, FBC7 specimens were indistinguishable from controls both subjectively and in all areas of measurement. The M. spretus backcross line was followed generation by generation and was largely conforming to controls by FBC4 at latest. The same effect was observed in the reciprocal M. domesticus backcross line. Fertility was negatively affected in F1 hybrids but restored or improved in backcross generations. Discussion is offered on hybridization and backcrossing as it occurs in nature and how it has been used or could be used as an additional ex situ tool in wildlife conservation efforts. It is concluded that conservation-oriented backcrossing is a practical species/subspecies restoration technique and has the potential to make genetic rescue feasible with minimal gene flow at the binomial level. Backcrossing is most applicable in closely monitored ex situ settings (1) where only one sex remains of a given taxon; and (2) where inbreeding depression seriously threatens a remnant taxon's ability to recover, and the only gene flow option is from another distinct species.

Evaluation of A3 cytoplasmic male sterile forage sorghum lines for resistance to sugarcane aphid.

MAIN CONCLUSION: Three known sugarcane aphid-resistant pollinator parents were sterilized in A3 cytoplasmic male sterility and were confirmed in this study to be resistant to sugarcane aphid allowing for the development of sugarcane aphid-resistant forage hybrids. We utilized A3 cytoplasmic male sterility and converted known sugarcane aphid-resistant sorghum TX 2783, and newly released R. LBK1 (Reg. No. GP-865, PI 687244) and R. LBK2 (Reg. No. GP-866, PI 687245) into A3 sterility to determine if the sterile counterparts would also equally express tolerance and or antibiosis to sugarcane aphid. Free-choice flat screen trials and life-table demographic studies were utilized and compared to know susceptible/fertile entries KS 585, and TX 7000, and known resistant/fertile entries TX 2783 and DKS 37-07. The R. LBK1 fertile entry was more tolerant than the known susceptible entries KS 585 and TX 7000, but was not as resistant as the other resistant entries, sustaining a damage rating of 6.0 across two different screen trials. The sterile A3 R. LBK2 showed a greater tolerance and expressed higher levels of antibiosis during aphid reproductive studies when compared to the known resistant and fertile TX 2783. All other fertile (R. LBK2, TX2783) and the A3 male sterile counterparts (A3 R. LBK2, A3 TX2783) were very similar in expression of high levels of tolerance and exhibited statistically similar damage ratings of 3.3-4.3 when exposed to sugarcane aphids. No entry, either fertile or sterile, was as tolerant as DKS 37-07, a known resistant commercial hybrid. Other plant measurements including percent loss in chlorophyll content, difference in plant height, and number of true leaves for sugarcane aphid infested versus non-infested were very consistent and highly correlated with damage ratings. Antibiosis was also exhibited in both fertile and sterile versions of the resistant lines. There was a 2 × reduction in fecundity between the R. LBK1 fertile and its sterile A3 R. LBK1 when compared to the susceptible KS 585 and TX 7000; however, the remaining fertile and sterile entries had 3.8 × to 5.8 × decrease in fecundity when compared to the susceptible KS 585 and TX 7000. Other measurements in life-table statistics such as nymphs produced/female/d, and the intrinsic rates of increased were significantly lower for all fertile and sterile lines, showing that antibiosis significantly affected sugarcane aphid reproduction. In conclusion, the A3 cytoplasmic male sterility shows consistency for maintaining the single dominant trait SCA-resistant trait of TX 2783 for expressing both antibiosis and tolerance, and great utility in the development of sugarcane aphid-resistant forage sorghums.

Characterization of 'QTL-hotspot' introgression lines reveals physiological mechanisms and candidate genes associated with drought adaptation in chickpea.

'QTL-hotspot' is a genomic region on linkage group 04 (CaLG04) in chickpea (Cicer arietinum) that harbours major-effect quantitative trait loci (QTLs) for multiple drought-adaptive traits, and it therefore represents a promising target for improving drought adaptation. To investigate the mechanisms underpinning the positive effects of 'QTL-hotspot' on seed yield under drought, we introgressed this region from the ICC 4958 genotype into five elite chickpea cultivars. The resulting introgression lines (ILs) and their parents were evaluated in multi-location field trials and semi-controlled conditions. The results showed that the 'QTL-hotspot' region improved seed yield under rainfed conditions by increasing seed weight, reducing the time to flowering, regulating traits related to canopy growth and early vigour, and enhancing transpiration efficiency. Whole-genome sequencing data analysis of the ILs and parents revealed four genes underlying the 'QTL-hotspot' region associated with drought adaptation. We validated diagnostic KASP markers closely linked to these genes using the ILs and their parents for future deployment in chickpea breeding programs. The CaTIFY4b-H2 haplotype of a potential candidate gene CaTIFY4b was identified as the superior haplotype for 100-seed weight. The candidate genes and superior haplotypes identified in this study have the potential to serve as direct targets for genetic manipulation and selection for chickpea improvement.

Pyramiding of genes for grain protein content, grain quality, and rust resistance in eleven Indian bread wheat cultivars: a multi-institutional effort.

Improvement of grain protein content (GPC), loaf volume, and resistance to rusts was achieved in 11 Indian wheat cultivars that are widely grown in four different agro-climatic zones of India. This involved use of marker-assisted backcross breeding (MABB) for introgression and pyramiding of the following genes: (i) the high GPC gene Gpc-B1; (ii) HMW glutenin subunits 5 + 10 at Glu-D1 loci, and (iii) rust resistance genes, Yr36, Yr15, Lr24, and Sr24. GPC increased by 0.8 to 3.3%, although high GPC was generally associated with yield penalty. Further selection among high GPC lines allowed identification of progenies with higher GPC associated with improvement in 1000-grain weight and grain yield in the backgrounds of the following four cultivars: NI5439, UP2338, UP2382, and HUW468. The high GPC progenies (derived from NI5439) were also improved for grain quality using HMW glutenin subunits 5 + 10 at Glu-D1 loci. Similarly, progenies combining high GPC and rust resistance were obtained in the backgrounds of following five cultivars: Lok1, HD2967, PBW550, PBW621, and DBW1. The improved pre-bred lines developed following multi-institutional effort should prove a valuable source for the development of cultivars with improved nutritional quality and rust resistance in the ongoing wheat breeding programmes. Supplementary Information: The online version contains supplementary material available at 10.1007/s11032-022-01277-w.

Microsatellites and petal morphology reveal new patterns of admixture in Orchis hybrid zones.

PREMISE: The genetic structure of hybrid zones provides insight into the potential for gene flow to occur between plant taxa. Four closely related European orchid species (Orchis anthropophora, O. militaris, O. purpurea, and O. simia) hybridize when they co-occur. We aimed to characterize patterns of hybridization in O. militaris-O. purpurea, O. purpurea-O. simia, and O. anthropophora-O. simia hybrid zones using molecular and morphological data. METHODS: We used 11 newly isolated nuclear microsatellites to genotype 695 individuals collected from seven hybrid zones and six allopatric parental populations in France. Geometric morphometric analysis was conducted using 15 labellum landmarks to capture the main aspects of petal shape. RESULTS: Backcrossing was asymmetric toward O. militaris in multiple O. militaris-O. purpurea hybrid zones. Hybrids in O. purpurea-O. simia and O. anthropophora-O. simia hybrid zones were largely limited to F1 and F2 generations, but further admixture had occurred. These patterns were reflected in labellum geometric morphometric data, which correlated strongly with nuclear microsatellite data in all three species combinations. CONCLUSIONS: The coexistence of parental and admixed individuals in these Orchis hybrid zones implies they are likely to be tension zones being maintained by a balance between gene flow into the hybrid zone and selection acting against admixed individuals. The pattern of admixture in the three species combinations suggests intrinsic selection acting on the hybrids is weaker in more closely related taxa.

Recent Advances of Genetic Resources, Genes and Genetic Approaches for Flooding Tolerance in Rice.

Flooding is one of the most hazardous natural disasters and a major stress constraint to rice production throughout the world, which results in huge economic losses. The frequency and duration of flooding is predicted to increase in near future as a result of global climate change. Breeding of flooding tolerance in rice is a challenging task because of the complexity of the component traits, screening technique, environmental factors and genetic interactions. A great progress has been made during last two decades to find out the flooding tolerance mechanism in rice. An important breakthrough in submergence research was achieved by the identification of major quantitative trait locus (QTL) SUB1 in rice chromosomes that acts as the primary contributor for tolerance. This enabled the use of marker-assisted backcrossing (MABC) to transfer SUB1 QTL into popular varieties which showed yield advantages in flood prone areas. However, SUB1 varieties are not always tolerant to stagnant flooding and flooding during germination stage. So, gene pyramiding approach can be used by combining several important traits to develop new breeding rice lines that confer tolerances to different types of flooding. This review highlights the important germplasm/genetic resources of rice to different types of flooding stress. A brief discussion on the genes and genetic mechanism in rice exhibited to different types of flooding tolerance was discussed for the development of flood tolerant rice variety. Further research on developing multiple stresses tolerant rice can be achieved by combining SUB1 with other tolerance traits/genes for wider adaptation in the rain-fed rice ecosystems.

Meiotic recombination in the offspring of Microbotryum hybrids and its impact on pathogenicity.

BACKGROUND: Hybridization is a central mechanism in evolution, producing new species or introducing important genetic variation into existing species. In plant-pathogenic fungi, adaptation and specialization to exploit a host species are key determinants of evolutionary success. Here, we performed experimental crosses between the two pathogenic Microbotryum species, M. lychnidis-dioicae and M. silenes-acaulis that are specialized to different hosts. The resulting offspring were analyzed on phenotypic and genomic levels to describe genomic characteristics of hybrid offspring and genetic factors likely involved in host-specialization. RESULTS: Genomic analyses of interspecific fungal hybrids revealed that individuals were most viable if the majority of loci were inherited from one species. Interestingly, species-specific loci were strictly controlled by the species' origin of the mating type locus. Moreover we detected signs of crossing over and chromosome duplications in the genomes of the analyzed hybrids. In Microbotryum, mitochondrial DNA was found to be uniparentally inherited from the a2 mating type. Genome comparison revealed that most gene families are shared and the majority of genes are conserved between the two species, indicating very similar biological features, including infection and pathogenicity processes. Moreover, we detected 211 candidate genes that were retained under host-driven selection of backcrossed lines. These genes and might therefore either play a crucial role in host specialization or be linked to genes that are essential for specialization. CONCLUSION: The combination of genome analyses with experimental selection and hybridization is a promising way to investigate host-pathogen interactions. This study manifests genetic factors of host specialization that are required for successful biotrophic infection of the post-zygotic stage, but also demonstrates the strong influence of intra-genomic conflicts or instabilities on the viability of hybrids in the haploid host-independent stage.

Marker aided introgression of opaque 2 (o2) allele improving lysine and tryptophan in maize (Zea mays L.).

Maize is the predominant food source for the world population, but lack of lysine and tryptophan in maize endosperm cannot fulfill the nutritional requirements of humans. Hence, the improvement of lysine and tryptophan content is the ultimate goal of maize biofortification programs. In the present study, the marker-assisted backcross (MABC) breeding strategy was used to enhance the lysine and tryptophan content of the elite maize inbred line UMI1230 by introgressing opaque 2 (o2) gene from the VQL1. During the transfer of the gene into UMI1230, SSR marker umc1066 tightly associated with o2 used for foreground selection. Background recovery was estimated using 168 SSR markers. Phenotype screening for morphological traits was adopted to choose plants parallel to UMI1230. As a result, four BC2F3 improved lines (DBT5-1-14/25-5/25-8/25-8/25, DBT5-1-14/25-5/25-8/25-7/25, DBT5-1-14/25-5/25-8/25-10/25 and DBT5-1-14/25-5/25-8/25-12/25) with o2 were developed. The improved line's background genome recovery varied between 90.60 and 94.80%. Also, the improved lines had better agronomic performance along with increased lysine (0.311-0.331%) and tryptophan (0.040-0.048%) contents. In summary, the MABC breeding strategy has successfully improved the levels of lysine and tryptophan in UMI1230 without affecting agronomic performance. The improved line's hold great potential as donors in biofortification programs in maize.

Application of high-throughput amplicon sequencing-based SSR genotyping in genetic background screening.

BACKGROUND: Host genetic backgrounds affect gene functions. The genetic backgrounds of genetically engineered organisms must be identified to confirm their genetic backgrounds identity with those of recipients. Marker-assisted backcrossing (MAB), transgenesis and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (CRISPR/Cas9) editing are three commonly used genetic engineering techniques. However, methods for genetic background screening between genetically engineered organisms and corresponding recipients suffer from low efficiency, low accuracy or high cost. RESULTS: Here, we improved our previously reported AmpSeq-SSR method, an amplicon sequencing-based simple sequence repeat (SSR) genotyping method, by selecting SSR loci with high polymorphism among varieties. Ultimately, a set of 396 SSRs was generated and applied to evaluate the genetic backgrounds identity between rice lines developed through MAB, transgenesis, and CRISPR/Cas9 editing and the respective recipient rice. We discovered that the percentage of different SSRs between the MAB-developed rice line and its recipient was as high as 23.5%. In contrast, only 0.8% of SSRs were different between the CRISPR/Cas9-system-mediated rice line and its recipient, while no SSRs showed different genotypes between the transgenic rice line and its recipient. Furthermore, most differential SSRs induced by MAB technology were located in non-coding regions (62.9%), followed by untranslated regions (21.0%) and coding regions (16.1%). Trinucleotide repeats were the most prevalent type of altered SSR. Most importantly, all altered SSRs located in coding regions were trinucleotide repeats. CONCLUSIONS: This method is not only useful for the background evaluation of genetic resources but also expands our understanding of the unintended effects of different genetic engineering techniques. While the work we present focused on rice, this method can be readily extended to other organisms.

Gene pyramiding for elite tomato genotypes against ToLCV (Begomovirus spp.), late blight (Phytophthora infestans) and RKN (Meloidogyne spp.) for northern India farmers.

Mega variety with multiple disease and pest tolerance is one of the major research objectives to protect the crop from biotic stresses in current scenario of climate change. Three tomato leaf curl virus resistance genes (Ty-1, Ty-2 and Ty-3), two late blight resistance genes (Ph-2 and Ph-3) and one root knot nematodes resistance gene (Mi-1.2) were pyramided from different donor parents with the help of marker-assisted backcrossing followed by phenotypic selection. During 2016-2017 (August-March), 100 lines of BC1F4 mapping population were evaluated against these diseases and pest screening. Punjab Chhuhara (a popular north Indian tomato cultivar) was used as a recipient parent that is susceptible for all these diseases and pest. Whereas the resistant parents included CLN3022F2-154-11-11-0 for tomato leaf curl virus, LBR-15 for late blight and NR-5 for root knot nematodes. The study results evaluated, 13 advanced backcrossed lines which possessed multiple disease and pest resistance with better horticultural traits over the parents as well as checks. This investigation showed that the introgression of Ty-1, Ty-2, Ty-3, Ph-2, Ph-3 and Mi-1.2 genes significantly improved the resistance against leaf curl, late blight and root knot nematodes diseases in selected advanced backcrossed lines of tomato. These improved multiple diseases resistant lines may play an important role in future pre-breeding tomato improvement projects.

Increased susceptibility to mammary carcinogenesis and an opposite trend in endometrium in Trp53 heterozygous knockout female mice by backcrossing the BALB/c strain onto the background C3H strain.

Patients with dominantly inherited Li-Fraumeni syndrome have a loss-of-function mutation in TP53 and develop diverse mesenchymal and epithelial neoplasms at multiple sites. Trp53 +/- female mice with the BALB/c background provide unique characteristics for the study of breast cancer in Li-Fraumeni syndrome; however, we previously found that female C3H-Trp53+/ - mice did not spontaneously develop mammary tumors. Therefore, we obtained F1 and N2-N4 female mice by backcrossing the BALB/c strain and examined the incidence of mammary and other tumors in lifetime studies. Malignant lymphomas, osteosarcomas, and uterine adenocarcinomas spontaneously developed in approximately 20% or more of Trp53+/ - mice with the C3H background. In contrast, the incidence of uterine adenocarcinomas showed a tendency to decrease, while that of mammary adenocarcinomas gradually increased in mice with the BALB/c strain backcross. Wild-type BALB/c female mice are predisposed to a wide spectrum of neoplasms, including mammary tumors, partly due to genetic factors, whereas uterine tumors are uncommon not only in BALB/c mice but also C3H mice. Thus, genetic factors appear to contribute to a strain-specific predisposition to malignant neoplasms in Trp53+/- mice, and further studies are needed to clarify the detailed mechanisms.

Marker-assisted introgression of resistance to fusarium wilt race 2 in Pusa 256, an elite cultivar of desi chickpea.

Fusarium wilt caused by F. oxysporum f. sp. ciceris causes extensive damage to chickpea (Cicer arietinum L.) in many parts of the world. In the central part of India, pathogen race 2 (Foc 2) causes severe yield losses. We initiated molecular marker-assisted backcrossing (MABC) using desi cultivar, Vijay, as a donor to introgress resistance to this race (Foc2) in Pusa 256, another elite desi cultivar of chickpea. To confirm introgression of resistance for this race, foreground selection was undertaken using two SSR markers (TA 37 and TA110), with background selection to observe the recovery of recurrent parent genome using 45 SSRs accommodated in 8 multiplexes. F1 plants were confirmed with molecular markers and backcrossed with Pusa 256, followed by cycles of foreground and background selection at each stage to generate 161 plants in BC3F2 during the period 2009-2013. Similarly, 46 BC3F1 plants were also generated in another set during the same period. On the basis of foreground selection, 46 plants were found homozygotes in BC3F2. Among them, 17 plants recorded >91% background recovery with the highest recovery percentage of 96%. In BC3F1 also, 14 hybrid plants recorded a background recovery of >85% with the highest background recovery percentage of >94%. The identified plants were selfed to obtain 1341 BC3F3 and 2198 BC3F2 seeds which were screened phenotypically for resistance to fusarium wilt (race 2) besides doing marker analysis. Finally, 17 BC3F4 and 11 BC3F3 lines were obtained which led to identification of 5 highly resistant lines of Pusa 256 with Foc 2 gene introgressed in them. Development of these lines will help in horizontal as well as vertical expansion of chickpea in central part of India.

Rapid and targeted introgression of fgr gene through marker-assisted backcrossing in rice (Oryza sativa L.).

While it is crucial for developing countries like Malaysia to achieve self-sufficiency in rice (Oryza sativa L.), it is equally critical to be able to produce high-quality rice, specifically fragrant rice, which demands are often met through importation. The present study was aimed at developing high-yielding fragrant rice, in a timely and cost-effective manner. A marker-assisted backcross (MABC) approach was optimised to introgress the fragrance gene (fgr) into two high-yielding Malaysian varieties, MR84 and MR219, within two years utilising less than 50 molecular markers. Coupled with phenotypic screening, one single foreground marker (fgr-SNP) and 48 background markers were selected and utilised, revealing recovery of at least 90% of recurrent parent genome (RPG) in merely two backcross generations. Collectively, the yield potential of the developed BC2F2 lines (BLs) was higher (P > 0.05) than the donor parent, MRQ74, and similar (P < 0.05) to both the recurrent parents, MR84 and MR219. In addition, some of the developed BLs showed good grain quality, such as having long grain. We believe that this is the first report comprising the validation and utilisation of the single functional marker system (fgr-SNP) in introgressing the fgr gene into different rice varieties.

The TaDREB3 transgene transferred by conventional crossings to different genetic backgrounds of bread wheat improves drought tolerance.

Drought tolerance of the wheat cultivar Bobwhite was previously enhanced by transformation with a construct containing the wheat DREB3 gene driven by the stress-inducible maize Rab17 promoter. Progeny of a single T2 transgenic line were used as pollinators in crosses with four elite bread wheat cultivars from Western Australia: Bonnie Rock, IGW-2971, Magenta and Wyalkatchem, with the aim of evaluating transgene performance in different genetic backgrounds. The selected pollinator line, BW8-9-10-3, contained multiple transgene copies, had significantly improved drought tolerance compared with wild-type plants and showed no growth and development penalties or abnormalities. A single hybrid plant was selected from each cross-combination for three rounds of backcrossing with the corresponding maternal wheat cultivar. The transgene was detected in all four F1 BC3 combinations, but stress-inducible transgene expression was found in only three of the four combinations. Under well-watered conditions, the phenotypes and grain yield components of the F2 BC3 transgene-expressing lines were similar to those of corresponding recurrent parents and null-segregants. Under severe drought conditions, the backcross lines demonstrated 12-18% higher survival rates than the corresponding control plants. Two from four F3 BC3 transgenic lines showed significantly higher yield (18.9% and 21.5%) than control plants under limited water conditions. There was no induction of transgene expression under cold stress, and therefore, no improvement of frost tolerance observed in the progenies of drought-tolerant F3 BC3 lines.

Evaluation of virus resistance and agronomic performance of rice cultivar ASD 16 after transfer of transgene against Rice tungro bacilliform virus by backcross breeding.

Severe losses of rice yield in south and southeast Asia are caused by Rice tungro disease (RTD) induced by mixed infection of Rice tungro bacilliform virus (RTBV) and Rice tungro spherical virus (RTSV). In order to develop transgene-based resistance against RTBV, one of its genes, ORF IV, was used to generate transgenic resistance based on RNA-interference in the easily transformed rice variety Pusa Basmati-1, and the transgene was subsequently introgressed to rice variety ASD 16, a variety popular in southern India, using transgene marker-assisted selection. Here, we report the evaluation of BC3F4 and BC3F5 generation rice plants for resistance to RTBV as well as for agronomic traits under glasshouse conditions. The BC3F4 and BC3F5 generation rice plants tested showed variable levels of resistance, which was manifested by an average of twofold amelioration in height reduction, 1.5-fold decrease in the reduction in chlorophyll content, and 100- to 10,000-fold reduction in the titers of RTBV, but no reduction of RTSV titers, in three backcrossed lines when compared with the ASD 16 parent. Agronomic traits of some of the backcrossed lines recorded substantial improvements when compared with the ASD 16 parental line after inoculation by RTBV and RTSV. This work represents an important step in transferring RTD resistance to a susceptible popular rice variety, hence enhancing its yield in areas threatened by the disease.

Removal of undesirable genes using yeast backcrossing.

4-Vinylguaiacol (4-VG) is one of the causative compounds for the phenolic odor characteristics of brewed liquor. In the case of Japanese sake brewing, 4-VG is formed through the decarboxylation of ferulic acid produced by rice koji enzymes from steamed rice. PAD1 (phenylacrylic acid decarborxylase gene) and FDC1 (ferulic acid decarboxylase gene) genes are essential for decarboxylation of ferulic acid in Saccharomyces cerevisiae and the single polymorphisms of both genes show a relationship with ferulic acid decarboxylation ability. While most of the Kyokai yeasts distributed by the Brewing Society of Japan have homozygous non-function fdc1 alleles, many newly isolated natural yeasts for local specialities carry the wild-type FDC1 gene. In our previous research, we found that a crossbreed strain lost a significant amount of chromosomal DNA as it underwent meiosis-like adaptation. Here, we established a breeding approach to exclude undesirable gene alleles (such as the wild-type FDC1 genes) from yeast strains of interest by backcrossing using Kyokai yeasts. Homozygous fdc1 crossbreeds were generated through the three rounds of crossing procedures, and we confirmed the reduction of 4-VG in the culture supernatant of the homozygous fdc1 hybrid strain compared to the parental strain. Importantly, this approach does not include growth selection for the mutation of interest (the fdc1 mutant in the current case). Using various yeast strains generated throughout human history, it may become possible to design and build any yeast strains according to the purpose.

Pyramiding aphid resistance genes into the elite cowpea variety, Zaayura, using marker-assisted backcrossing.

The cowpea aphid (Aphis cracivora) is a cosmopolitan insect pest that causes economic damage on cowpea. Although the pest persists at all the growth stages of the crop, in West Africa, aphids are the only major insect pests that farmers regularly control at the vegetative stage. Thus, deploying aphid-resistant crop varieties can reduce farmers' expenditure on insecticide. The availability of different biotypes of the pest and reports of resistance breakdown necessitates pyramiding of sources of aphid resistance to develop a more robust genotype for durable resistance. Two aphid-resistance genes, sourced from SARC-1-57-2 and IT97K-556-6, were introgressed through gene pyramiding technique into a farmers' preferred cowpea variety, Zaayura, using marker-assisted backcrossing. A simple sequence repeat (SSR) marker, CP 171F/172R, and an allele-specific single nucleotide polymorphism (SNP) marker, 1_0912, were used for foreground selection of the SARC-1-57-2 and IT97K-556-6 aphid resistance genes, respectively. A stepwise backcross approach was used to introgress the major aphid resistance QTL (QAc-vu7.1) from IT97K-556-6 into Zaayura using the marker 1_0912 coupled with intermittent screening under artificial aphid infestation. After the fourth backcross generation, three heterozygous BC4F1 of Zaayura/TT97K-556-6 were intercrossed to Zaayura Pali to develop intercross F1 (ICF1). Three true ICF1 hybrids allowed to self to produce ICF2. Five (5) out of 48 ICF2 plants which were genotyped with the two foreground markers had the two aphid resistance genes fixed in the double homozygous dominant state. For background selection, out of 192 allele-specific markers screened, only 47 polymorphic markers were identified and used for the background analysis of the pyramided lines. The recurrent parent genome recovery ranged from 72 to 93.8 %. ICF2_Zaa/556/SARC-P6 had the highest recurrent parent genome and the least heterozygosity among the five improved lines. The five pyramided lines showed superior resistance under artificial aphid infestation as compared to the two donor parents with damage scores ranging from 2.0 to 2.3. On the field, however, there were no significant differences between the pyramided lines and their recurrent parent for all the agronomic traits measured except for grain yield. The pyramided lines do not only stand the chance of being released as new varieties but are also valuable genetic resources for other breeding programs that seek to improve cowpea for aphid resistance.