Graphical Genotyping Research Articles

HISS: Snakemake-based workflows for performing SMRT-RenSeq assembly, AgRenSeq and dRenSeq for the discovery of novel plant disease resistance genes

BackgroundIn the ten years since the initial publication of the RenSeq protocol, the method has proved to be a powerful tool for studying disease resistance in plants and providing target genes for breeding programmes. Since the initial publication of the methodology, it has continued to be developed as new technologies have become available and the increased availability of computing power has made new bioinformatic approaches possible. Most recently, this has included the development of a k-mer based association genetics approach, the use of PacBio HiFi data, and graphical genotyping with diagnostic RenSeq. However, there is not yet a unified workflow available and researchers must instead configure approaches from various sources themselves. This makes reproducibility and version control a challenge and limits the ability to perform these analyses to those with bioinformatics expertise.ResultsHere we present HISS, consisting of three workflows which take a user from raw RenSeq reads to the identification of candidates for disease resistance genes. These workflows conduct the assembly of enriched HiFi reads from an accession with the resistance phenotype of interest. A panel of accessions both possessing and lacking the resistance are then used in an association genetics approach (AgRenSeq) to identify contigs positively associated with the resistance phenotype. Candidate genes are then identified on these contigs and assessed for their presence or absence in the panel with a graphical genotyping approach that uses dRenSeq. These workflows are implemented via Snakemake, a python-based workflow manager. Software dependencies are either shipped with the release or handled with conda. All code is freely available and is distributed under the GNU GPL-3.0 license.ConclusionsHISS provides a user-friendly, portable, and easily customised approach for identifying novel disease resistance genes in plants. It is easily installed with all dependencies handled internally or shipped with the release and represents a significant improvement in the ease of use of these bioinformatics analyses.

Parent genome contribution and its relationship with grain yield in backcross‐derived lines of soybean (Glycine max)

AbstractIntrogression of yellow mosaic disease (YMD) resistance and effect of recurrent parent genome (RPG) on grain yield was studied in 84 soybean genotypes from four populations namely, F2:7, BC1F6, BC2F5 and BC3F4 derived from cross JS335 x SL525. It was observed that in F2:7, BC1F6, BC2F5 and BC3F4 derived lines, RPG contribution was 42.5%, 54.9%, 66.4% and 77.6%, respectively, which is significantly less than expected values. Linkage drag from donor parent with YMD resistance gene may be a possible reason for such deviations. Average grain yield per plant in F2:7, BC1F6, BC2F5 and BC3F4 generations was observed as 13.0, 14.3, 14.9 and 16.1 g, respectively. It was observed that genotypes with more than 80% RPG observed to have both YMD resistance and good yield potential. Graphical genotyping (GGT) analysis revealed that maximum RPG was recovered in chromosomes 8 and 10 and maximum introgression occurred in chromosomes 6 and 19. Our results demonstrated that RPG was positively associated with yield as evident from yield increase with increase in RPG.

SeSAM: software for automatic construction of order-robust linkage maps

BackgroundGenotyping and sequencing technologies produce increasingly large numbers of genetic markers with potentially high rates of missing or erroneous data. Therefore, the construction of linkage maps is more and more complex. Moreover, the size of segregating populations remains constrained by cost issues and is less and less commensurate with the numbers of SNPs available. Thus, guaranteeing a statistically robust marker order requires that maps include only a carefully selected subset of SNPs.ResultsIn this context, the SeSAM software allows automatic genetic map construction using seriation and placement approaches, to produce (1) a high-robustness framework map which includes as many markers as possible while keeping the order robustness beyond a given statistical threshold, and (2) a high-density total map including the framework plus almost all polymorphic markers. During this process, care is taken to limit the impact of genotyping errors and of missing data on mapping quality. SeSAM can be used with a wide range of biparental populations including from outcrossing species for which phases are inferred on-the-fly by maximum-likelihood during map elongation. The package also includes functions to simulate data sets, convert data formats, detect putative genotyping errors, visualize data and map quality (including graphical genotypes), and merge several maps into a consensus. SeSAM is also suitable for interactive map construction, by providing lower-level functions for 2-point and multipoint EM analyses. The software is implemented in a R package including functions in C++.ConclusionsSeSAM is a fully automatic linkage mapping software designed to (1) produce a framework map as robust as desired by optimizing the selection of a subset of markers, and (2) produce a high-density map including almost all polymorphic markers. The software can be used with a wide range of biparental mapping populations including cases from outcrossing. SeSAM is freely available under a GNU GPL v3 license and works on Linux, Windows, and macOS platforms. It can be downloaded together with its user-manual and quick-start tutorial from ForgeMIA (SeSAM project) at https://forgemia.inra.fr/gqe-acep/sesam/-/releases

Near-isogenic lines for resistance to blast disease, in the genetic background of the Indica Group rice (Oryza sativa L.) cultivar IR64

We developed 21 near-isogenic lines (NILs) in the genetic background of the Indica Group rice ( Oryza sativa L.) cultivar IR64, with each containing one of the 11 blast resistance genes: Pish , Piz , Piz-5 , Pi9 (t), Pii , Pi3 , Pik , Pik-h , Pik-p , Pi1 , and Pi7 (t). The NILs were confirmed to harbor these genes by their reaction patterns to standard differential blast isolates and by analyses for graphical genotypes using DNA markers located on the same chromosome regions as the target resistance genes. The agronomic traits of these NILs, such as days to heading and yield components, were also investigated and found to be very similar to those of IR64, even when grown under the dramatically different environmental conditions of the temperate and subtropical regions of Japan. These NILs and their genetic information will be used as gene sources for Indica Group rice breeding programs, to improve resistance to blast disease, and as a multiline variety that will also be useful as an alternative approach to the development of the durability of rice resistance to blast disease in tropical areas, especially in irrigated lowland ecosystems.

Comparison of Maize Genotypes Using Drought-Tolerance Indices and Graphical Analysis under Normal and Humidity Stress Conditions.

This study aimed to identify drought-tolerant genotypes and to evaluate and compare the response of genotypes under normal conditions and humidity stress. The experiment was conducted in a Randomized Complete Block Design (RCBD) on 12 commercial single cross hybrids of maize (Zea mays L.) with three replications in two separate experiments under normal and stress conditions. GT biplot was used to compare genotypes under normal conditions and humidity stress. Based on the polygon diagrams’ graphical analysis, KSC206, KSC704, KSC705 and KSC706 genotypes were identified as desirable hybrids. The ranking diagram of genotypes based on ideal genotype also showed that the KSC704 genotype had high desirability in all evaluated traits in normal and stress conditions. TOL, MP, HARM, GMP, SSI and STI indices were used to identify drought-tolerant genotypes, and the genotypes were ranked based on this index. Based on this, KSC260, SC302 and KSC400 hybrids were selected as resistant hybrids. Based on the correlation analysis between drought-tolerance indices, a positive correlation was observed between MP, GMP, HARM and STI indices. Based on the analysis of the PCA on the indices, the first and second principal components were given the titles of grain yield tolerance component under humidity stress conditions and grain yield stability component under normal humidity conditions, respectively. KSC704 was superior to other hybrids in terms of grain yield under normal conditions and stress, and the KSC260 hybrid was identified as a tolerant hybrid in terms of all studied traits under drought stress.

The genetics of vigour-related traits in chickpea (Cicer arietinum L.): insights from genomic data.

QTL controlling vigour and related traits were identified in a chickpea RIL population and validated in diverse sets of germplasm. Robust KASP markers were developed for marker-assisted selection. To understand the genetic constitution of vigour in chickpea (Cicer arietinum L.), genomic data from a bi-parental population and multiple diversity panels were used to identify QTL, sequence-level haplotypes and genetic markers associated with vigour-related traits in Australian environments. Using 182 Recombinant Inbred Lines (RILs) derived from a cross between two desi varieties, Rupali and Genesis836, vigour QTL independent of flowering time were identified on chromosomes (Ca) 1, 3 and 4 with genotypic variance explained (GVE) ranging from 7.1 to 28.8%. Haplotype analysis, association analysis and graphical genotyping of whole-genome re-sequencing data of two diversity panels consisting of Australian and Indian genotypes and an ICRISAT Chickpea Reference Set revealed a deletion in the FTa1-FTa2-FTc gene cluster of Ca3 significantly associated with vigour and flowering time. Across the RIL population and diversity panels, the impact of the deletion was consistent for vigour but not flowering time. Vigour-related QTL on Ca4 co-located with a QTL for seed size in Rupali/Genesis836 (GVE = 61.3%). Using SNPs from this region, we developed and validated gene-based KASP markers across different panels. Two markers were developed for a gene on Ca1, myo -inositol monophosphatase (CaIMP), previously proposed to control seed size, seed germination and seedling growth in chickpea. While associated with vigour in the diversity panels, neither the markers nor broader haplotype linked to CaIMP was polymorphic in Rupali/Genesis836. Importantly, vigour appears to be controlled by different sets of QTL across time and with components which are independent from phenology.

Marker-Assisted Pyramiding of Downy Mildew-Resistant Gene Ppa3 and Black Rot-Resistant Gene Xca1bo in Popular Early Cauliflower Variety Pusa Meghna

Cauliflower is an important extensively grown cool season vegetable in India. Black rot and downy mildew are major devastating diseases reducing yield and quality of the crop. To tackle these through host plant resistance, a marker-assisted backcross breeding method was followed to pyramid a black rot-resistant gene (Xca1bo) and a downy mildew-resistant gene (Ppa3) from donors BR-161 and BR-2, respectively, into the background of Pusa Meghna cauliflower cultivar. Marker-assisted backcross breeding was followed up to BC2 generation using SCAR marker ScOPO-04833 and SSR marker BoGMS0624 for black rot and downy mildew resistance genes in foreground selection, respectively. In background selection, at each stage of backcrossing, 47 parental polymorphic SSR markers were used. The graphical genotyping of the five two-gene (Xca1boXca1boPpa3Ppa3) homozygous BC2F2 plants showed an average recovery of 85.44% of the Pusa Meghna genome with highest genome recovery of 91.7%. The genome contribution of donor parents (BR-161 and BR-2) was 8.26 with 6.34% of residual heterozygousity. The backcross derived pyramided lines BC2F2:3-7-16 and BC2F2:3-7-33 showed high resistance to both the diseases and exhibited higher yield and vitamin C content as compared with recipient parent Pusa Meghna. It is, therefore, evident from this study that resistant genes can be introgressed successfully into a Pusa Meghna cultivar without any yield penalty, benefitting farmers with reduced input cost and consumers with chemical residue free produce. Besides, the pyramided lines carrying dominant resistant genes can be exploited in a hybridization programme to develop hybrid(s) in cauliflower.

Construction of a high-density linkage map and graphical representation of the arrangement of transcriptome-based unigene markers on the chromosomes of onion, Allium cepa L.

BackgroundGenomic information for Allium cepa L. is limited as it is heterozygous and its genome is very large. To elucidate potential SNP markers obtained by NGS, we used a complete set of A. fistulosum L.-A. cepa monosomic addition lines (MALs) and doubled haploids (DHs). These were the parental lines of an A. cepa mapping population for transcriptome-based SNP genotyping.ResultsWe mapped the transcriptome sequence reads from a series of A. fistulosum-A. cepa MALs onto the unigene sequence of the doubled haploid shallot A. cepa Aggregatum group (DHA) and compared the MAL genotype call for parental bunching onion and shallot transcriptome mapping data. We identified SNP sites with at least four reads on 25,462 unigenes. They were anchored on eight A. cepa chromosomes. A single SNP site was identified on 3,278 unigenes and multiple SNPs were identified on 22,184 unigenes. The chromosome marker information was made public via the web database Allium TDB (http://alliumtdb.kazusa.or.jp/). To apply transcriptome based genotyping approach for genetic mapping, we gathered RNA sequence data from 96 lines of a DHA × doubled haploid bulb onion A. cepa common onion group (DHC) mapping population. After selecting co-dominant SNP sites, 16,872 SNPs were identified in 5,339 unigenes. Of these, at least two SNPs with identical genotypes were found in 1,435 unigenes. We developed a linkage map using genotype information from these unigenes. All unigene markers mapped onto the eight chromosomes and graphical genotyping was conducted based on the unigene order information. Another 2,963 unigenes were allocated onto the eight chromosomes. To confirm the accuracy of this transcriptome-based genetic linkage map, conventional PCR-based markers were used for linkage analysis. All SNP - and PCR-based markers were mapped onto the expected linkage groups and no inconsistency was found among these chromosomal locations.ConclusionsEffective transcriptome analysis with unique Allium resources successfully associated numerous chromosome markers with unigene information and a high-density A. cepa linkage map. The information on these unigene markers is valuable in genome sequencing and useful trait detection in Allium.

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.

Genetic mapping of Fusarium wilt resistance in a wild banana Musa acuminata ssp. malaccensis accession

Banana is an important fruit and food crop, but is threatened by Fusarium wilt, one of the most devastating soil-borne fungal diseases. Only host resistance facilitates banana cultivation in infested soils around the world, but the genetic basis of Fusarium wilt of banana (FWB) is unknown. We selfed a heterozygous wild banana accession Musa acuminata ssp. malaccensis (Mam, AA, 2n = 22) to generate a mapping population and to investigate the inheritance of resistance to Race 1 and tropical race 4 (TR4) that cause FWB. Phenotyping (N = 217) revealed segregation for resistance, and genotyping by sequencing resulted in 2802 high-quality single-nucleotide polymorphic markers (SNPs) that were used for genetic mapping. Combined analyses of these data showed that a single dominant resistance locus controls resistance to Race 1 and maps near the distal part of chromosome 10. Recombinants, together with the position of the putative resistance gene, were further analysed using graphical genotyping, which retrieved markers flanking a 360 kb genetic region that associates with Race 1 resistance. The region contains 165 putative genes on the reference genome, including 19 leucine-rich repeat receptor-like kinase-like genes. At the same position and phase, we also identified a QTL for TR4 resistance, showing that the locus for resistance against Race 1 provided partial resistance to TR4. However, this effect was far less significant and hence not included in the mapping. These data support the breeding of new banana varieties with resistance to Fusarium wilt.

GenoTypeMapper: graphical genotyping on genetic and sequence-based maps

BackgroundThe rising availability of assemblies of large genomes (e.g. bread and durum wheat, barley) and their annotations deliver the basis to graphically present genome organization of parents and progenies on a physical scale. Genetic maps are a very important tool for breeders but often represent distorted models of the actual chromosomes, e.g., in centromeric and telomeric regions. This biased picture might lead to imprecise assumptions and estimations about the size and complexity of genetic regions and the selection of suitable molecular markers for the incorporation of traits in breeding populations or near-isogenic lines (NILs). Some software packages allow the graphical illustration of genotypic data, but to the best of our knowledge, suitable software packages that allow the comparison of genotypic data on the physical and genetic scale are currently unavailable.ResultsWe developed a simple Java-based-software called GenoTypeMapper (GTM) for comparing genotypic data on genetic and physical maps and tested it for effectiveness on data of two NILs that carry QTL-regions for drought stress tolerance from wild emmer on chromosome 2BS and 7AS. Both NILs were more tolerant to drought stress than their recurrent parents but exhibited additional undesirable traits such as delayed heading time.ConclusionsIn this article, we illustrate that the software easily allows users to display and identify additional chromosomal introgressions in both NILs originating from the wild emmer parent. The ability to detect and diminish linkage drag can be of particular interest for pre-breeding purposes and the developed software is a well-suited tool in this respect. The software is based on a simple allele-matching algorithm between the offspring and parents of a crossing scheme. Despite this simple approach, GTM seems to be the only software that allows us to analyse, illustrate and compare genotypic data of offspring of different crossing schemes with up to four parents in two different maps. So far, up to 500 individuals with a maximum number of 50,000 markers can be examined with the software. The main limitation that hampers the performance of the software is the number of markers that are examined in parallel. Since each individual must be analysed separately, a maximum of ten individuals can currently be displayed in a single run. On a computer with an Intel five processor of the 8th generation, GTM can reliably either analyse a single individual with up to 12,000 markers or ten individuals with up to 3,600 markers in less than five seconds. Future work aims to improve the performance of the software so that more complex crossing schemes with more parents and more markers can be analysed.

Assessing trait contribution and mapping novel QTL for salinity tolerance using the Bangladeshi rice landrace Capsule

BackgroundSalinity is one of the most widespread abiotic stresses affecting rice productivity worldwide. The purpose of this study was to establish the relative importance of different traits associated with salinity tolerance in rice and to identify new quantitative trait loci (QTL) conferring tolerance to salinity at seedling stage. A total of 231 F2:3 plants derived from a cross between a sensitive variety BRRI dhan29 (BR29 hereafter) and Capsule, a salt tolerant Bangladeshi indica landrace, were evaluated under salt stress in a phytotron.ResultsOut of the 231 F2 plants, 47 highly tolerant and 47 most sensitive lines were selected, representing the two extreme tails of the phenotypic distribution. These 94 plants were genotyped for 105 simple sequence repeat (SSR) and insertion/deletion (InDel) markers. A genetic linkage map spanning approximately 1442.9 cM of the 12 linkage groups with an average marker distance of 13.7 cM was constructed. QTL were identified on the long arm of chromosome 1 for Na+ concentration, K+ concentration, Na+-K+ ratio and survival; chromosome 3 for Na+ concentration, survival and overall phenotypic evaluation using the Standard Evaluation system (SES); and chromosome 5 for SES. A total of 6 pairwise epistatic interactions were also detected between QTL-linked and QTL-unlinked regions. Graphical genotyping indicated an association between the phenotypes of the extreme families and their QTL genotypes. Path coefficient analysis revealed that Na+ concentration, survival, Na+-K+ ratio and the overall phenotypic performance (SES score) are the major traits associated with salinity tolerance of Capsule.ConclusionsCapsule provides an alternative source of salinity tolerance aside from Pokkali and Nona Bokra, the two Indian salt tolerant landraces traditionally used for breeding salt tolerant rice varieties. Pyramiding the new QTL identified in this study with previously discovered loci, such as Saltol, will facilitate breeding varieties that are highly tolerant of salt stress.

Molecular Characterization of 87 Functional Genes in Wheat Diversity Panel and Their Association With Phenotypes Under Well-Watered and Water-Limited Conditions.

Modern breeding imposed selection for improved productivity that largely influenced the frequency of superior alleles underpinning traits of breeding interest. Therefore, molecular diagnosis for the allelic variations of such genes is important to manipulate beneficial alleles in wheat molecular breeding. We analyzed a diversity panel largely consisted of advanced lines derived from synthetic hexaploid wheats for allelic variation at 87 functional genes or loci of breeding importance using 124 high-throughput KASP markers. We also developed two KASP markers for water-soluble carbohydrate genes (TaSST-D1 and TaSST-A1) associated with plant height and thousand grain weight (TGW) in the diversity panel. KASP genotyping results indicated that beneficial alleles for genes underpinning flowering time (Ppd-D1 and Vrn-D3), thousand grain weight (TaCKX-D1, TaTGW6-A1, TaSus1-7B, and TaCwi-D1), water-soluble carbohydrates (TaSST-A1), yellow-pigment content (Psy-B1 and Zds-D1), and root lesion nematodes (Rlnn1) were fixed in diversity panel with frequency ranged from 96.4 to 100%. The association analysis of functional genes with agronomic and biochemical traits under well-watered (WW) and water-limited (WL) conditions revealed that 21 marker-trait associations (MTAs) were consistently detected in both moisture conditions. The major developmental genes such as Vrn-A1, Rht-D1, and Ppd-B1 had the confounding effect on several agronomic traits including plant height, grain size and weight, and grain yield in both WW and WL conditions. The accumulation of favorable alleles for grain size and weight genes additively enhanced grain weight in the diversity panel. Graphical genotyping approach was used to identify accessions with maximum number of favorable alleles, thus likely to have high breeding value. These results improved our knowledge on the selection of favorable and unfavorable alleles through unconscious selection breeding and identified the opportunities to deploy alleles with effects in wheat breeding.

Identification of candidate genes responsible for the susceptibility of apple (Malus \xd7 domestica Borkh.) to Alternaria blotch

BackgroundThe mechanism underlying the interaction between host plant and host-selective toxin (HST)-producing Alternaria alternata during infection is of particular interest for sustainable crop production. Alternaria blotch of apple (Malus × domestica Borkh.) caused by A. alternata apple pathotype is a major disease particularly in East Asia, which is the largest producer of apples globally. A single dominant gene, Alt, controls the susceptibility of the apple cultivar ‘Delicious’ to Alternaria blotch. In this study, we fine mapped the Alt locus and characterized three potential candidate genes.ResultsWe used 797 F1 individuals derived from 15 crosses between apple accessions susceptible (Alt/alt) and resistant (alt/alt) to Alternaria blotch to construct physical and genetic maps of the Alt locus located on the top of chromosome 11. Susceptible accessions were derived from ‘Delicious.’ To fine map the Alt locus, we constructed a BAC library of ‘Starking Delicious,’ a sport of ‘Delicious,’ and used graphical genotyping to delimit the Alt locus to a region of 43 kb. Three genes predicted within the candidate Alt region were potentially involved in plant defense response, among which the gene encoding a coiled coil-nucleotide binding-leucine rich repeat (CC-NB-LRR) type disease resistance protein was the most promising. Moreover, a 12-bp insertion was uniquely identified in the 5′ untranslated region of the Alt-associated allele of this gene, the presence or absence of which co-segregated with the susceptibility or resistance to A. alternata apple pathotype, respectively, among 43 tested cultivars including old ones and founders of modern apple breeding.ConclusionA disease resistance protein has been suggested as a determinant of susceptibility/resistance to HST-producing A. alternata for the first time. Our finding provides new insight into the mechanism of HST-mediated disease control used by A. alternata against host plants.

Genetic diversity and population structure among 133 elite genotypes of sugarcane (Saccharum spp.) for use as parents in sugarcane varietal improvement

A measure of genetic diversity of genotypes to be used as parents is imperative to use them prudently in crop improvement. In this study, genetic diversity and population structure of 133 sugarcane hybrid derivatives were quantified using 20 sequence-tagged microsatellite sites (STMS) primers. The number of alleles ranged from 9 to 27 with the average of 17.95 alleles per primer, while the polymorphism information content values of the primers ranged from 0.29 to 0.78. Cophenetic correlation coefficient value observed as 0.84 by STMS markers revealed that the cluster result was acceptable for the calculation of genetic similarity matrix. Principal component analysis showed that 133 genotypes fell in two groups, first and second components associated 8.34 and 3.22% with eigen values of 5.61 and 2.17, respectively. Similar trend was observed with principal coordinate analysis, wherein, the first and second component accounted to 8.34 and 3.22% with eigen values of 741.29 and 286.11. The similarity index values ranged from 0.50 to 0.87 for the possible 8778 combinations from 133 genotypes, of which 8069 combinations exhibited less/moderate genetic similarity indicating the availability of sufficient genetic diversity in the experimental material and hence their value in the genetic improvement of sugarcane. Dissimilarity analysis using DARwin of 133 genotypes could distinguish two major clusters and into five subclusters and the results matched with those of the population structure which also showed five subpopulations. The bigger group SP1 was predominantly comprised of clones developed at the main sugarcane-breeding place in India, located at Coimbatore. The subpopulation SP4 was formed largely with clones from research stations other than at Coimbatore and interspecific hybrids, while SP5 comprised of clones of early origin. These observations were similar to the radial tree based on the DARwin software in that 81.95% of the genotypes of each cluster were similar in the two analyses. The results thus showed that location and time of origin were two major factors that contributed to diversity. Based on analysis of molecular variance, subpopulations SP2 and SP4 were more variable from the rest. SP2 (comprising of Co 99008, Co 99006, Co 94012, Co 93023, CoC 671, Co 89034, Co 91003, Co 06022, Co 98017, Co 87044, Co 06018, Co 89003, Co 98014, and Co 86032) exhibited maximum genetic variation, the least gene flow, and the lowest heterozygosity value and would serve as the best group for utilization in genetic improvement. Graphical genotyping (GGT) image of each genotype was distinctly different, indicating the genetic uniqueness of sugarcane genotypes under study as revealed through STMS technology. A core set of 40 genotypes was identified using GGT 2.0 software program for the easiness of harnessing the available genetic diversity of 133 genotypes, through hybridization in sugarcane improvement programs.

Simultaneous Improvement and Genetic Dissection of Salt Tolerance of Rice (Oryza sativa L.) by Designed QTL Pyramiding.

Breeding of multi-stress tolerant rice varieties with higher grain yields is the best option to enhance the rice productivity of abiotic stresses prone areas. It also poses the greatest challenge to plant breeders to breed rice varieties for such stress prone conditions. Here, we carried out a designed QTL pyramiding experiment to develop high yielding “Green Super Rice” varieties with significantly improved tolerance to salt stress and grain yield. Using the F4 population derived from a cross between two selected introgression lines, we were able to develop six mostly homozygous promising high yielding lines with significantly improved salt tolerance and grain yield under optimal and/or saline conditions in 3 years. Simultaneous mapping using the same breeding population and tunable genotyping-by-sequencing technology, we identified three QTL affecting salt injury score and leaf chlorophyll content. By analyzing 32M SNP data of the grandparents and graphical genotypes of the parents, we discovered 87 positional candidate genes for salt tolerant QTL. According to their functional annotation, we inferred the most likely candidate genes. We demonstrated that designed QTL pyramiding is a powerful strategy for simultaneous improvement and genetic dissection of complex traits in rice.

Graphical genotyping as a method to map Ny(o,n)sto and Gpa5 using a reference panel of tetraploid potato cultivars

Key messageThe method of graphical genotyping is applied to a panel of tetraploid potato cultivars to visualize haplotype sharing. The method allowed to map genes involved in virus and nematode resistance. The physical coordinates of the amount of linkage drag surrounding these genes are easily interpretable.Graphical genotyping is a visually attractive and easily interpretable method to represent genetic marker data. In this paper, the method is extended from diploids to a panel of tetraploid potato cultivars. Application of filters to select a subset of SNPs allows one to visualize haplotype sharing between individuals that also share a specific locus. The method is illustrated with cultivars resistant to Potato virus Y (PVY), while simultaneously selecting for the absence of the SNPs in susceptible clones. SNP data will then merge into an image which displays the coordinates of a distal genomic region on the northern arm of chromosome 11 where a specific haplotype is introgressed from the wild potato species S. stoloniferum (CPC 2093) carrying a gene (Ny(o,n)sto) conferring resistance to two PVY strains, PVYO and PVYNTN. Graphical genotyping was also successful in showing the haplotypes on chromosome 12 carrying Ry-fsto, another resistance gene derived from S. stoloniferum conferring broad-spectrum resistance to PVY, as well as chromosome 5 haplotypes from S. vernei, with the Gpa5 locus involved in resistance against Globodera pallida cyst nematodes. The image also shows shortening of linkage drag by meiotic recombination of the introgression segment in more recent breeding material. Identity-by-descent was found to be a requirement for using graphical genotyping, which is proposed as a non-statistical alternative method for gene discovery, as compared with genome-wide association studies. The potential and limitations of the method are discussed.

Development and molecular-genetic characterization of a stable Brassica allohexaploid.

We report first-time synthesis of a stable Brassica allohexaploid. It may evolve into a new species and also advance our understanding of pairing regulation and genome evolution in complex allopolyploids. Crop Brassicas include both monogenomic and digenomic species. A trigenomic Brassica (AABBCC) is not known to exist in nature. Past attempts to synthesize a stable allohexaploid were not successful due to aberrant meiosis and very high proportion of aneuploid plants in the selfed progenies. We report the development of a stable allohexaploid Brassica (2n=54; AABBCC). Genomic in situ hybridization confirmed the complete assemblage of three genomes. Only allohexaploids involving B. rapa cv. R01 (2n=20; AA) as pollinator with a set of B. carinata (2n=34; BBCC) were stable. These exhibited a high proportion (0.78-0.94) of pollen mother cells with normal meiosis and an excellent hexaploid ratio (0.80-0.94) in the selfed progenies. Stability of two allohexaploid combinations was demonstrated from H1 to H4 generations at two very diverse locations in India. Graphical genotyping of allohexaploids allowed detection of chromosome fragment exchanges among three genomes. These were much smaller for meiotically stable allohexaploids as compared to unstable ones. The putative hexaploids were morphologically closer to the female donor, B. carinata, for leaf morphology, inflorescence structure and flower shape. The newly formed allohexaploid may also provide unique opportunities to investigate the immediate genetic and genomic consequences of a Brassica allohexaploid with three resident genomes.

Estimation of gas exchange parameters in backcross introgressed lines of rice (Oryza sativaL.) with different combinations of drought qtls

The present investigation was carried out to study the gas exchange parameters in backcross inbred lines of ‘Improved White Ponni’ (IWP) introgressed with different drought yield QTLs and their parents raised under control and stress conditions. Various gas exchange parameters and single plant yield were observed using portable photosynthetic system (infra red gas analyser -IRGA). Among the genotypes, three combination of QTL line IWP-4-2 recorded significantly higher photosynthetic rate (22.97 μ mol CO2 m−2 s−1), stomatal conductance (0.36 mol H2O m−2 s−1), transpiration rate (6.25 mmol H2O m−2 s−1), internal CO2 concentration (240.82) ratio between internal and atmospheric CO2 concentration (0.25) and single plant yield (15.11) which was on par with the tolerant parent Apo (an high yielding indica cultivar moderately tolerant to drought stress) under severe moisture stress regime. Overall study clearly revealed that, the performance of introgressed line with three drought QTLs (IWP-4-2) was better than two drought QTL introgressed lines of IWP-1-52 and IWP-1-57. Results from graphical genotyping using three foreground and 36 polymorphic background markers revealed that, the three QTL introgressed line IWP-4-2 had 72.20% genome recovery from Improved White Ponni, 13.89% from APO and 13.89% heterozygous region.

Identification and genetic characterization of a quantitative trait locus for adventitious rooting from apple hardwood cuttings

Vegetative propagation enables the multiplication of plants that are genetically identical to the original. Hardwood cuttings have a low cost advantage and are used widely for the propagation of JM series apple rootstocks, which root well from hardwood cuttings. To breed new apple rootstocks with similarly high rooting capabilities, the development of a marker-assisted selection method is desirable. Therefore, it is important to understand the genetic mechanisms behind the rooting capabilities of hardwood cuttings. We identified quantitative trait loci (QTLs) for the rooting capability of ‘JM7’ using a QTL analysis with phenotypic data obtained under two environmental conditions. The QTLs for rooting rate and root quantity co-located. The QTL for rooting rate explained 66 % of the genetic variance and 57 % of the phenotypic variance. Graphical genotyping and database searching revealed candidate genes, including three related to auxin response, one related to ethylene response, and a gene encoding a WRKY transcription factor.