Publications by authors named "Harsh Raman"

41 Publications

Modelling of gene loss propensity in the pangenomes of three Brassica species suggests different mechanisms between polyploids and diploids.

Plant Biotechnol J 2021 Dec 24;19(12):2488-2500. Epub 2021 Aug 24.

School of Biological Sciences and the Institute of Agriculture, Faculty of Science, The University of Western Australia, Crawley, WA, Australia.

Plant genomes demonstrate significant presence/absence variation (PAV) within a species; however, the factors that lead to this variation have not been studied systematically in Brassica across diploids and polyploids. Here, we developed pangenomes of polyploid Brassica napus and its two diploid progenitor genomes B. rapa and B. oleracea to infer how PAV may differ between diploids and polyploids. Modelling of gene loss suggests that loss propensity is primarily associated with transposable elements in the diploids while in B. napus, gene loss propensity is associated with homoeologous recombination. We use these results to gain insights into the different causes of gene loss, both in diploids and following polyploidization, and pave the way for the application of machine learning methods to understanding the underlying biological and physical causes of gene presence/absence.
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http://dx.doi.org/10.1111/pbi.13674DOI Listing
December 2021

The Gene, a New Player of - Interaction Maps on Chromosome C03 in Canola.

Front Plant Sci 2021 12;12:654604. Epub 2021 May 12.

Oil Crops Research Institute, Chinese Academy of Agricultural Sciences, Wuhan, China.

Canola exhibits an extensive genetic variation for resistance to blackleg disease, caused by the fungal pathogen . Despite the identification of several effectors and (race-specific) genes, specific interactions between genes are not yet fully understood in the pathosystem. In this study, we investigated the genetic basis of resistance in an F population derived from Australian canola varieties CB-Telfer ()/ATR-Cobbler () using a single-spore isolate of , PHW1223. A genetic linkage map of the CB-Telfer/ATR-Cobbler population was constructed using 7,932 genotyping-by-sequencing-based DArTseq markers and subsequently utilized for linkage and haplotype analyses. Genetic linkage between DArTseq markers and resistance to PHW1223 isolate was also validated using the 60K Illumina Infinium array. Our results revealed that a major locus for resistance, designated as , maps on chromosome C03. To date, no gene for resistance to blackleg has been reported on the C subgenome in . Twenty-four candidate genes were predicted to reside within the quantitative trait locus (QTL) region. We further resequenced both the parental lines of the mapping population (CB-Telfer and ATR-Cobbler, > 80 × coverage) and identified several structural sequence variants in the form of single-nucleotide polymorphisms (SNPs), insertions/deletions (InDels), and presence/absence variations (PAVs) near . Comparative mapping revealed that is located within the homoeologous A03/C03 region in ancestral karyotype block "R" of . Our results provide a "target" for further understanding the gene interaction as well as a valuable tool for increasing resistance to blackleg in canola germplasm.
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http://dx.doi.org/10.3389/fpls.2021.654604DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8150007PMC
May 2021

Genome-Wide Association Mapping Identifies Novel Loci for Quantitative Resistance to Blackleg Disease in Canola.

Front Plant Sci 2020 11;11:1184. Epub 2020 Aug 11.

Centre for Bioinformatics and Biometrics, National Institute for Applied Statistics Research Australia, University of Wollongong, Wollongong, NSW, Australia.

Blackleg disease, caused by the fungal pathogen , continues to be a major concern for sustainable production of canola ( L.) in many parts of the world. The deployment of effective quantitative resistance (QR) is recognized as a durable strategy in providing natural defense to pathogens. Herein, we uncover loci for resistance to blackleg in a genetically diverse panel of canola accessions by exploiting historic recombination events which occurred during domestication and selective breeding by genome-wide association analysis (GWAS). We found extensive variation in resistance to blackleg at the adult plant stage, including for upper canopy infection. Using the linkage disequilibrium and genetic relationship estimates from 12,414 high quality SNPs, GWAS identified 59 statistically significant and "suggestive" SNPs on 17 chromosomes of genome that underlie variation in resistance to blackleg, evaluated under field and shade-house conditions. Each of the SNP association accounted for up to 25.1% of additive genetic variance in resistance among diverse panel of accessions. To understand the homology of QR genomic regions with genome, we searched the synteny between QR regions with 22 ancestral blocks of Brassicaceae. Comparative analyses revealed that 25 SNP associations for QR were localized in nine ancestral blocks, as a result of genomic rearrangements. We further showed that phenological traits such as flowering time, plant height, and maturity confound the genetic variation in resistance. Altogether, these findings provided new insights on the complex genetic control of the blackleg resistance and further expanded our understanding of its genetic architecture.
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http://dx.doi.org/10.3389/fpls.2020.01184DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7432127PMC
August 2020

A copia-like retrotransposon insertion in the upstream region of the SHATTERPROOF1 gene, BnSHP1.A9, is associated with quantitative variation in pod shattering resistance in oilseed rape.

J Exp Bot 2020 09;71(18):5402-5413

Oil Crops Research Institute, Chinese Academy of Agricultural Sciences, Wuhan Hubei, P.R. China.

Seed loss resulting from pod shattering is a major constraint in production of oilseed rape (Brassica napus L.). However, the molecular mechanisms underlying pod shatter resistance are not well understood. Here, we show that the pod shatter resistance at quantitative trait locus qSRI.A9.1 is controlled by one of the B. napus SHATTERPROOF1 homologs, BnSHP1.A9, in a doubled haploid population generated from parents designated R1 and R2 as well as in a diverse panel of oilseed rape. The R1 maternal parental line of the doubled haploid population carried the allele for shattering at qSRI.A9.1, while the R2 parental line carried the allele for shattering resistance. Quantitative RT-PCR showed that BnSHP1.A9 was expressed specifically in flower buds, flowers, and developing siliques in R1, while it was not expressed in any tissue of R2. Transgenic plants constitutively expressing either of the BnSHP1.A9 alleles from the R1 and R2 parental lines showed that both alleles are responsible for pod shattering, via a mechanism that promotes lignification of the enb layer. These findings indicated that the allelic differences in the BnSHP1.A9 gene per se are not the causal factor for quantitative variation in shattering resistance at qSRI.A9.1. Instead, a highly methylated copia-like long terminal repeat retrotransposon insertion (4803 bp) in the promotor region of the R2 allele of BnSHP1.A9 repressed the expression of BnSHP1.A9, and thus contributed to pod shatter resistance. Finally, we showed a copia-like retrotransposon-based marker, BnSHP1.A9R2, can be used for marker-assisted breeding targeting the pod shatter resistance trait in oilseed rape.
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http://dx.doi.org/10.1093/jxb/eraa281DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7501816PMC
September 2020

Genetic and physical mapping of loci for resistance to blackleg disease in canola (Brassica napus L.).

Sci Rep 2020 03 10;10(1):4416. Epub 2020 Mar 10.

NSW Department of Primary Industries, Wagga Wagga Agricultural Institute, Wagga Wagga, NSW, 2650, Australia.

Sustainable canola production is essential to meet growing human demands for vegetable oil, biodiesel, and meal for stock feed markets. Blackleg, caused by the fungal pathogen, Leptosphaeria maculans is a devastating disease that can lead to significant yield loss in many canola production regions worldwide. Breakdown of race-specific resistance to L. maculans in commercial cultivars poses a constant threat to the canola industry. To identify new alleles, especially for quantitative resistance (QR), we analyzed 177 doubled haploid (DH) lines derived from an RP04/Ag-Outback cross. DH lines were evaluated for QR under field conditions in three experiments conducted at Wagga Wagga (2013, 2014) and Lake Green (2015), and under shade house conditions using the 'ascospore shower' test. DH lines were also characterized for qualitative R gene-mediated resistance via cotyledon tests with two differential single spore isolates, IBCN17 and IBCN76, under glasshouse conditions. Based on 18,851 DArTseq markers, a linkage map representing 2,019 unique marker bins was constructed and then utilized for QTL detection. Marker regression analysis identified 22 significant marker associations for resistance, allowing identification of two race-specific resistance R genes, Rlm3 and Rlm4, and 21 marker associations for QR loci. At least three SNP associations for QR were repeatedly detected on chromosomes A03, A07 and C04 across phenotyping environments. Physical mapping of markers linked with these consistent QR loci on the B. napus genome assembly revealed their localization in close proximity of the candidate genes of B. napus BnaA03g26760D (A03), BnaA07g20240D (A07) and BnaC04g02040D (C04). Annotation of these candidate genes revealed their association with protein kinase and jumonji proteins implicated in defense resistance. Both Rlm3 and Rlm4 genes identified in this DH population did not show any association with resistance loci detected under either field and/or shade house conditions (ascospore shower) suggesting that both genes are ineffective in conferring resistance to L. maculans in Australian field conditions. Taken together, our study identified sequence-based molecular markers for dissecting R and QR loci to L. maculans in a canola DH population from the RP04/Ag-Outback cross.
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http://dx.doi.org/10.1038/s41598-020-61211-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7064481PMC
March 2020

GWAS hints at pleiotropic roles for FLOWERING LOCUS T in flowering time and yield-related traits in canola.

BMC Genomics 2019 Aug 6;20(1):636. Epub 2019 Aug 6.

School of Biological Sciences, Monash University, Clayton, VIC3800, Australia.

Background: Transition to flowering at the right time is critical for local adaptation and to maximize grain yield in crops. Canola is an important oilseed crop with extensive variation in flowering time among varieties. However, our understanding of underlying genes and their role in canola productivity is limited.

Results: We report our analyses of a diverse GWAS panel (300-368 accessions) of canola and identify SNPs that are significantly associated with variation in flowering time and response to photoperiod across multiple locations. We show that several of these associations map in the vicinity of FLOWERING LOCUS T (FT) paralogs and its known transcriptional regulators. Complementary QTL and eQTL mapping studies, conducted in an Australian doubled haploid population, also detected consistent genomic regions close to the FT paralogs associated with flowering time and yield-related traits. FT sequences vary between accessions. Expression levels of FT in plants grown in field (or under controlled environment cabinets) correlated with flowering time. We show that markers linked to the FT paralogs display association with variation in multiple traits including flowering time, plant emergence, shoot biomass and grain yield.

Conclusions: Our findings suggest that FT paralogs not only control flowering time but also modulate yield-related productivity traits in canola.
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http://dx.doi.org/10.1186/s12864-019-5964-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6685183PMC
August 2019

Stable Quantitative Resistance Loci to Blackleg Disease in Canola ( L.) Over Continents.

Front Plant Sci 2018 23;9:1622. Epub 2018 Nov 23.

IGEPP, INRA, Agrocampus Ouest, Université Rennes, Le Rheu, France.

The hemibiotrophic fungus, is the most devastating pathogen, causing blackleg disease in canola ( L). To study the genomic regions involved in quantitative resistance (QR), 259-276 DH lines from Darmor-/Yudal (DYDH) population were assessed for resistance to blackleg under shade house and field conditions across 3 years. In different experiments, the broad sense heritability varied from 43 to 95%. A total of 27 significant quantitative trait loci (QTL) for QR were detected on 12 chromosomes and explained between 2.14 and 10.13% of the genotypic variance. Of the significant QTL, at least seven were repeatedly detected across different experiments on chromosomes A02, A07, A09, A10, C01, and C09. Resistance alleles were mainly contributed by 'Darmor-' but 'Yudal' also contributed few of them. Our results suggest that plant maturity and plant height may have a pleiotropic effect on QR in our conditions. We confirmed that which is present in 'Darmor-' is not effective to confer resistance in our Australian field conditions. Comparative mapping showed that several genes coding for nucleotide-binding leucine-rich repeat (LRR) receptors map in close proximity (within 200 Kb) of the significant trait-marker associations on the reference 'Darmor-' genome assembly. More importantly, eight significant QTL regions were detected across diverse growing environments: Australia, France, and United Kingdom. These stable QTL identified herein can be utilized for enhancing QR in elite canola germplasm via marker- assisted or genomic selection strategies.
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http://dx.doi.org/10.3389/fpls.2018.01622DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6265502PMC
November 2018

A Major Locus for Manganese Tolerance Maps on Chromosome A09 in a Doubled Haploid Population of L.

Front Plant Sci 2017 12;8:1952. Epub 2017 Dec 12.

INRA, Agrocampus Ouest, Université de Rennes 1, UMR1349 Institut de Génétique, Environnement et de Protection des Plantes, Le Rheu, France.

Soil acidity poses a major threat to productivity of several crops; mainly due to the prevalence of toxic levels of Al and Mn. Crop productivity could be harnessed on acid soils via the development of plant varieties tolerant to phytotoxic levels of these cations. In this study, we investigated the extent of natural variation for Mn tolerance among ten parental lines of the Australian and International canola mapping populations. Response to Mn toxicity was measured on the bases of cotyledon chlorosis, shoot biomass, and leaf area in nutrient solution under control (9 μM of MnCl⋅4HO) and Mn treatment (125 μM of MnCl⋅4HO). Among parental lines, we selected Darmor- and Yudal that showed significant and contrasting variation in Mn tolerance to understand genetic control and identify the quantitative trait loci (QTL) underlying Mn tolerance. We evaluated parental lines and their doubled haploid (DH) progenies (196 lines) derived from an F cross, Darmor-/Yudal for Mn tolerance. Mn-tolerant genotypes had significantly higher shoot biomass and leaf area compared to Mn-sensitive genotypes. A genetic linkage map based on 7,805 DArTseq markers corresponding to 2,094 unique loci was constructed and further utilized for QTL identification. A major locus, . was further mapped with a SNP marker, Bn-A09-p29012402 (LOD score of 34.6) accounting for most of the variation in Mn tolerance on chromosome A09. This is the first report on the genomic localization of a Mn tolerance locus in . Additionally, an ortholog of encoding for cation efflux facilitator transporter was located within 3,991 bp from significant SNP marker associated with .. A suite of genome sequence based markers (DArTseq and Illumina Infinium SNPs) flanking the . locus would provide an invaluable tool for various molecular breeding applications to improve canola production and profitability on Mn toxic soils.
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http://dx.doi.org/10.3389/fpls.2017.01952DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5733045PMC
December 2017

Speed breeding is a powerful tool to accelerate crop research and breeding.

Nat Plants 2018 01 1;4(1):23-29. Epub 2018 Jan 1.

John Innes Centre, Norwich Research Park, Norwich, UK.

The growing human population and a changing environment have raised significant concern for global food security, with the current improvement rate of several important crops inadequate to meet future demand . This slow improvement rate is attributed partly to the long generation times of crop plants. Here, we present a method called 'speed breeding', which greatly shortens generation time and accelerates breeding and research programmes. Speed breeding can be used to achieve up to 6 generations per year for spring wheat (Triticum aestivum), durum wheat (T. durum), barley (Hordeum vulgare), chickpea (Cicer arietinum) and pea (Pisum sativum), and 4 generations for canola (Brassica napus), instead of 2-3 under normal glasshouse conditions. We demonstrate that speed breeding in fully enclosed, controlled-environment growth chambers can accelerate plant development for research purposes, including phenotyping of adult plant traits, mutant studies and transformation. The use of supplemental lighting in a glasshouse environment allows rapid generation cycling through single seed descent (SSD) and potential for adaptation to larger-scale crop improvement programs. Cost saving through light-emitting diode (LED) supplemental lighting is also outlined. We envisage great potential for integrating speed breeding with other modern crop breeding technologies, including high-throughput genotyping, genome editing and genomic selection, accelerating the rate of crop improvement.
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http://dx.doi.org/10.1038/s41477-017-0083-8DOI Listing
January 2018

Molecular Diversity Analysis and Genetic Mapping of Pod Shatter Resistance Loci in L.

Front Plant Sci 2017 30;8:1765. Epub 2017 Nov 30.

Graham Centre for Agricultural Innovation (an alliance between NSW Department of Primary Industries and Charles Sturt University), Wagga Wagga Agricultural Institute, Wagga Wagga, NSW, Australia.

Seed lost due to easy pod dehiscence at maturity (pod shatter) is a major problem in several members of Brassicaceae family. We investigated the level of pod shatter resistance in Ethiopian mustard () and identified quantitative trait loci (QTL) for targeted introgression of this trait in Ethiopian mustard and its close relatives of the genus . A set of 83 accessions of , collected from the Australian Grains Genebank, was evaluated for pod shatter resistance based on pod rupture energy (RE). In comparison to (RE = 2.16 mJ), accessions had higher RE values (2.53 to 20.82 mJ). A genetic linkage map of an F population from two contrasting selections, BC73526 (shatter resistant with high RE) and BC73524 (shatter prone with low RE) comprising 300 individuals, was constructed using a set of 6,464 high quality DArTseq markers and subsequently used for QTL analysis. Genetic analysis of the F and F derived lines revealed five statistically significant QTL (LOD ≥ 3) that are linked with pod shatter resistance on chromosomes B1, B3, B8, and C5. Herein, we report for the first time, identification of genetic loci associated with pod shatter resistance in . These characterized accessions would be useful in breeding programs for introgression of pod shatter resistance alleles in to elite breeding lines. Molecular markers would assist marker-assisted selection for tracing the introgression of resistant alleles. Our results suggest that the value of the germplasm collections can be harnessed through genetic and genomics tools.
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http://dx.doi.org/10.3389/fpls.2017.01765DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5716317PMC
November 2017

Investigation of the Genetic Diversity and Quantitative Trait Loci Accounting for Important Agronomic and Seed Quality Traits in .

Front Plant Sci 2017 24;8:615. Epub 2017 Apr 24.

National Key Laboratory of Crop Genetic Improvement, Key Laboratory of Rapeseed Genetic Improvement, Ministry of Agriculture China, Huazhong Agricultural UniversityWuhan, China.

(BBCC) is an allotetraploid in with unique alleles for agronomic traits and has huge potential as source for biodiesel production. To investigate the genome-wide molecular diversity, population structure and linkage disequilibrium (LD) pattern in this species, we genotyped a panel of 81 accessions of with genotyping by sequencing approach DArTseq, generating a total of 54,510 polymorphic markers. Two subpopulations were exhibited in the accessions. The average distance of LD decay ( = 0.1) in B subgenome (0.25 Mb) was shorter than that of C subgenome (0.40 Mb). Genome-wide association analysis (GWAS) identified a total of seven markers significantly associated with five seed quality traits in two experiments. To further identify the quantitative trait loci (QTL) for important agronomic and seed quality traits, we phenotyped a doubled haploid (DH) mapping population derived from the "YW" cross between two parents (Y-BcDH64 and W-BcDH76) representing from the two subpopulations. The YW DH population and its parents were grown in three contrasting environments; spring (Hezheng and Xining, China), semi-winter (Wuhan, China), and spring (Wagga Wagga, Australia) across 5 years for QTL mapping. Genetic bases of phenotypic variation in seed yield and its seven related traits, and six seed quality traits were determined. A total of 282 consensus QTL accounting for these traits were identified including nine major QTL for flowering time, oleic acid, linolenic acid, pod number of main inflorescence, and seed weight. Of these, 109 and 134 QTL were specific to spring and semi-winter environment, respectively, while 39 consensus QTL were identified in both contrasting environments. Two QTL identified for linolenic acid (B3) and erucic acid (C7) were validated in the diverse lines used for GWAS. A total of 25 QTL accounting for flowering time, erucic acid, and oleic acid were aligned to the homologous QTL or candidate gene regions in the C genome of . These results would not only provide insights for genetic improvement of this species, but will also identify useful genetic variation hidden in the C subgenome of to improve canola cultivars.
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http://dx.doi.org/10.3389/fpls.2017.00615DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5401912PMC
April 2017

Genome-wide Association Study Identifies New Loci for Resistance to in Canola.

Front Plant Sci 2016 24;7:1513. Epub 2016 Oct 24.

Marcroft Grains Pathology, Horsham VIC, Australia.

Key message "We identified both quantitative and quantitative resistance loci to , a fungal pathogen, causing blackleg disease in canola. Several genome-wide significant associations were detected at known and new loci for blackleg resistance. We further validated statistically significant associations in four genetic mapping populations, demonstrating that GWAS marker loci are indeed associated with resistance to One of the novel loci identified for the first time, , conveys adult plant resistance in canola." Blackleg, caused by , is a significant disease which affects the sustainable production of canola (). This study reports a genome-wide association study based on 18,804 polymorphic SNPs to identify loci associated with qualitative and quantitative resistance to . Genomic regions delimited with 694 significant SNP markers, that are associated with resistance evaluated using 12 single spore isolates and pathotypes from four canola stubble were identified. Several significant associations were detected at known disease resistance loci including in the vicinity of recently cloned / genes, and at new loci on chromosomes A01/C01, A02/C02, A03/C03, A05/C05, A06, A08, and A09. In addition, we validated statistically significant associations on A01, A07, and A10 in four genetic mapping populations, demonstrating that GWAS marker loci are indeed associated with resistance to . One of the novel loci identified for the first time, , conveys adult plant resistance and mapped within 13.2 kb from gene of TIR-NBS class. We showed that resistance loci are located in the vicinity of genes of and on the sequenced genome of cv. Darmor-. Significantly associated SNP markers provide a valuable tool to enrich germplasm for favorable alleles in order to improve the level of resistance to in canola.
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http://dx.doi.org/10.3389/fpls.2016.01513DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5075532PMC
October 2016

Characterization of SNP and Structural Variations in the Mitochondrial Genomes of Tilletia indica and Its Closely Related Species Formed Basis for a Simple Diagnostic Assay.

PLoS One 2016 4;11(11):e0166086. Epub 2016 Nov 4.

School of Biotechnology and Biomolecular Sciences, The University of New South Wales, Kensington, NSW, Australia.

Tilletia indica causes the disease Karnal bunt in wheat. The disease is under international quarantine regulations. Comparative mitochondrial (mt) genome analysis of T. indica (KX394364 and DQ993184) and T. walkeri (EF536375) has found 325 to 328 SNPs, 57 to 60 short InDels (from 1 to 13 nt), two InDels (30 and 61 nt) and five (>200 nt) presence/absence variations (PAVs) between the two species. The mt genomes of both species have identical gene order. The numbers of SNPs and InDels between the mt genomes of the two species are approximately nine times of the corresponding numbers between the two T. indica isolates. There are eight SNPs between T. indica and T. walkeri that resulted in amino acid substitutions in the mt genes of cob, nad2 and nad5. In contrast, there is no amino acid substitution in the mt genes of the T. indica isolates from the SNPs found. The five PAVs present in T. indica (DQ993184) are absent in T. walkeri. Four PAVs are more than 1 kb and are not present in every T. indica isolate. Analysis of their presence and absence separates a collection of T. indica isolates into 11 subgroups. Two PAVs have ORFs for the LAGLIDAG endonuclease and two have ORFs for the GIY-YIG endonuclease family, which are representatives of homing endonuclease genes (HEGs). These intron- encoded HEGs confer intron mobility and account for their fluid distribution in T. indica isolates. The small PAV of 221 bp, present in every T. indica isolate and unique to the species, was used as the genetic fingerprint for the successful development of a rapid, highly sensitive and specific loop mediated isothermal amplification (LAMP) assay. The simple procedure of the LAMP assay and the easy detection formats will enable the assay to be automated for high throughput diagnosis.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0166086PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5096740PMC
June 2017

Multi-environment QTL studies suggest a role for cysteine-rich protein kinase genes in quantitative resistance to blackleg disease in Brassica napus.

BMC Plant Biol 2016 08 24;16(1):183. Epub 2016 Aug 24.

Saskatoon Research Centre, Agriculture and Agri-Food Canada, Saskatoon, SK, S7N 0X2, Canada.

Background: Resistance to the blackleg disease of Brassica napus (canola/oilseed rape), caused by the hemibiotrophic fungal pathogen Leptosphaeria maculans, is determined by both race-specific resistance (R) genes and quantitative resistance loci (QTL), or adult-plant resistance (APR). While the introgression of R genes into breeding material is relatively simple, QTL are often detected sporadically, making them harder to capture in breeding programs. For the effective deployment of APR in crop varieties, resistance QTL need to have a reliable influence on phenotype in multiple environments and be well defined genetically to enable marker-assisted selection (MAS).

Results: Doubled-haploid populations produced from the susceptible B. napus variety Topas and APR varieties AG-Castle and AV-Sapphire were analysed for resistance to blackleg in two locations over 3 and 4 years, respectively. Three stable QTL were detected in each population, with two loci appearing to be common to both APR varieties. Physical delineation of three QTL regions was sufficient to identify candidate defense-related genes, including a cluster of cysteine-rich receptor-like kinases contained within a 49 gene QTL interval on chromosome A01. Individual L. maculans isolates were used to define the physical intervals for the race-specific R genes Rlm3 and Rlm4 and to identify QTL common to both field studies and the cotyledon resistance response.

Conclusion: Through multi-environment QTL analysis we have identified and delineated four significant and stable QTL suitable for MAS of quantitative blackleg resistance in B. napus, and identified candidate genes which potentially play a role in quantitative defense responses to L. maculans.
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http://dx.doi.org/10.1186/s12870-016-0877-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4995785PMC
August 2016

Multigenic Control of Pod Shattering Resistance in Chinese Rapeseed Germplasm Revealed by Genome-Wide Association and Linkage Analyses.

Front Plant Sci 2016 21;7:1058. Epub 2016 Jul 21.

Key Laboratory of Biology and Genetic Improvement of Oil Crops, Ministry of Agriculture, Oil Crops Research Institute of the Chinese Academy of Agricultural Sciences Wuhan, China.

The majority of rapeseed cultivars shatter seeds upon maturity especially under hot-dry and windy conditions, reducing yield and gross margin return to growers. Here, we identified quantitative trait loci (QTL) for resistance to pod shatter in an unstructured diverse panel of 143 rapeseed accessions, and two structured populations derived from bi-parental doubled haploid (DH) and inter-mated (IF2) crosses derived from R1 (resistant to pod shattering) and R2 (prone to pod shattering) accessions. Genome-wide association analysis identified six significant QTL for resistance to pod shatter located on chromosomes A01, A06, A07, A09, C02, and C05. Two of the QTL, qSRI.A09 delimited with the SNP marker Bn-A09-p30171993 (A09) and qSRI.A06 delimited with the SNP marker Bn-A06-p115948 (A06) could be repeatedly detected across environments in a diversity panel, DH and IF2 populations, suggesting that at least two loci on chromosomes A06 and A09 were the main contributors to pod shatter resistance in Chinese germplasm. Significant SNP markers identified in this study especially those that appeared repeatedly across environments provide a cost-effective and an efficient method for introgression and pyramiding of favorable alleles for pod shatter resistance via marker-assisted selection in rapeseed improvement programs.
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http://dx.doi.org/10.3389/fpls.2016.01058DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4954820PMC
August 2016

Co-linearity and divergence of the A subgenome of Brassica juncea compared with other Brassica species carrying different A subgenomes.

BMC Genomics 2016 Jan 5;17:18. Epub 2016 Jan 5.

National Key Laboratory of Crop Genetic Improvement, Key Laboratory of Rapeseed Genetic Improvement, Ministry of Agriculture P. R. China, Huazhong Agricultural University, Wuhan, 430070, China.

Background: There are three basic Brassica genomes (A, B, and C) and three parallel sets of subgenomes distinguished in the diploid Brassica (i.e.: B. rapa, A(r)A(r); B. nigra, B(ni)B(ni); B. oleracea, C(o)C(o)) and the derived allotetraploid species (i.e.: B. juncea, A(j)A(j)B(j)B(j); B. napus, A(n)A(n)C(n)C(n); B. carinata, B(c)B(c)C(c)C(c)). To understand subgenome differentiation in B. juncea in comparison to other A genome-carrying Brassica species (B. rapa and B. napus), we constructed a dense genetic linkage map of B. juncea, and conducted population genetic analysis on diverse lines of the three A-genome carrying Brassica species using a genotyping-by-sequencing approach (DArT-seq).

Results: A dense genetic linkage map of B. juncea was constructed using an F2 population derived from Sichuan Yellow/Purple Mustard. The map included 3329 DArT-seq markers on 18 linkage groups and covered 1579 cM with an average density of two markers per cM. Based on this map and the alignment of the marker sequences with the physical genome of Arabidopsis thaliana, we observed strong co-linearity of the ancestral blocks among the different A subgenomes but also considerable block variation. Comparative analyses at the level of genome sequences of B. rapa and B. napus, and marker sequence anchored on the genetic map of B. juncea, revealed a total of 30 potential inversion events across large segments and 20 potential translocation events among the three A subgenomes. Population genetic analysis on 26 accessions of the three A genome-carrying Brassica species showed that the highest genetic distance were estimated when comparing A(j)-A(n) than between A(n)-A(r) and A(j)-A(r) subgenome pairs.

Conclusions: The development of the dense genetic linkage map of B. juncea with informative DArT-seq marker sequences and availability of the reference sequences of the A(r), and A(n)C(n) genomes allowed us to compare the A subgenome structure of B. juncea (A(j)) . Our results suggest that strong co-linearity exists among the three A Brassica genomes (A(r), A(n) and A(j)) but with apparent subgenomic variation. Population genetic analysis on three A-genome carrying Brassica species support the idea that B. juncea has distinct genomic diversity, and/or evolved from a different A genome progenitor of B. napus.
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http://dx.doi.org/10.1186/s12864-015-2343-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4700566PMC
January 2016

A high-throughput SNP array in the amphidiploid species Brassica napus shows diversity in resistance genes.

Funct Integr Genomics 2014 Dec 22;14(4):643-55. Epub 2014 Aug 22.

Centre for Integrative Legume Research and School of Agriculture and Food Sciences, University of Queensland, Brisbane, Australia.

Single-nucleotide polymorphisms (SNPs)are molecular markers based on nucleotide variation and can be used for genotyping assays across populations and to track genomic inheritance. SNPs offer a comprehensive genotyping alternative to whole-genome sequencing for both agricultural and research purposes including molecular breeding and diagnostics, genome evolution and genetic diversity analyses, genetic mapping, and trait association studies. Here genomic SNPs were discovered between four cultivars of the important amphidiploid oilseed species Brassica napus and used to develop a B. napus Infinium™ array containing 5,306 SNPs randomly dispersed across the genome. Assay success was high, with >94 % of these producing a reproducible, polymorphic genotype in the 1,070 samples screened. Although the assay was designed to B. napus, successful SNP amplification was achieved in the B. napus progenitor species, Brassica rapa and Brassica oleracea, and to a lesser extent in the related species Brassica nigra. Phylogenetic analysis was consistent with the expected relationships between B. napus individuals. This study presents an efficient custom SNP assay development pipeline in the complex polyploid Brassica genome and demonstrates the utility of the array for high-throughput genotyping in a number of related Brassica species. It also demonstrates the utility of this assay in genotyping resistance genes on chromosome A7, which segregate amongst the 1,070 samples.
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http://dx.doi.org/10.1007/s10142-014-0391-2DOI Listing
December 2014

Genome-wide delineation of natural variation for pod shatter resistance in Brassica napus.

PLoS One 2014 9;9(7):e101673. Epub 2014 Jul 9.

Graham Centre for Agricultural Innovation (an alliance between NSW Department of Primary Industries and Charles Sturt University), Wagga Wagga Agricultural Institute, Wagga Wagga, NSW, Australia.

Resistance to pod shattering (shatter resistance) is a target trait for global rapeseed (canola, Brassica napus L.), improvement programs to minimise grain loss in the mature standing crop, and during windrowing and mechanical harvest. We describe the genetic basis of natural variation for shatter resistance in B. napus and show that several quantitative trait loci (QTL) control this trait. To identify loci underlying shatter resistance, we used a novel genotyping-by-sequencing approach DArT-Seq. QTL analysis detected a total of 12 significant QTL on chromosomes A03, A07, A09, C03, C04, C06, and C08; which jointly account for approximately 57% of the genotypic variation in shatter resistance. Through Genome-Wide Association Studies, we show that a large number of loci, including those that are involved in shattering in Arabidopsis, account for variation in shatter resistance in diverse B. napus germplasm. Our results indicate that genetic diversity for shatter resistance genes in B. napus is limited; many of the genes that might control this trait were not included during the natural creation of this species, or were not retained during the domestication and selection process. We speculate that valuable diversity for this trait was lost during the natural creation of B. napus. To improve shatter resistance, breeders will need to target the introduction of useful alleles especially from genotypes of other related species of Brassica, such as those that we have identified.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0101673PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4090071PMC
March 2015

Identification of QTLs associated with resistance to Phomopsis pod blight (Diaporthe toxica) in Lupinus albus.

Breed Sci 2014 May;64(1):83-9

Australian Centre for Necrotrophic Fungal Pathogens, Curtin University , Perth , Western Australia.

Phomopsis blight in Lupinus albus is caused by a fungal pathogen, Diaporthe toxica. It can invade all plant parts, leading to plant material becoming toxic to grazing animals, and potentially resulting in lupinosis. Identifying sources of resistance and breeding for resistance remains the best strategy for controlling Phomopsis and reducing lupinosis risks. However, loci associated with resistance to Phomopsis blight have not yet been identified. In this study, quantitative trait locus (QTL) analysis identified genomic regions associated with resistance to Phomopsis pod blight (PPB) using a linkage map of L. albus constructed previously from an F8 recombinant inbred line population derived from a cross between Kiev-Mutant (susceptible to PPB) and P27174 (resistant to PPB). Phenotyping was undertaken using a detached pod assay. In total, we identified eight QTLs for resistance to PPB on linkage group (LG) 3, LG6, LG10, LG12, LG17 and LG27 from different phenotyping environments. However, at least one QTL, QTL-5 on LG10 was consistently detected in both phenotyping environments and accounted for up to 28.2% of the total phenotypic variance. The results of this study showed that the QTL-2 on LG3 interacts epistatically with QTL-5 and QTL-6, which map on LG10 and LG12, respectively.
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http://dx.doi.org/10.1270/jsbbs.64.83DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4031113PMC
May 2014

Constructing a dense genetic linkage map and mapping QTL for the traits of flower development in Brassica carinata.

Theor Appl Genet 2014 Jul 14;127(7):1593-605. Epub 2014 May 14.

National Key Laboratory of Crop Genetic Improvement, Key Laboratory of Rapeseed Genetic Improvement, Ministry of Agriculture P. R. China, Huazhong Agricultural University, Wuhan, 430070, China.

Key Message: An integrated dense genetic linkage map was constructed in a B. carinata population and used for comparative genome analysis and QTL identification for flowering time. An integrated dense linkage map of Brassica carinata (BBCC) was constructed in a doubled haploid population based on DArT-Seq(TM) markers. A total of 4,031 markers corresponding to 1,366 unique loci were mapped including 639 bins, covering a genetic distance of 2,048 cM. We identified 136 blocks and islands conserved in Brassicaceae, which showed a feature of hexaploidisation representing the suggested ancestral crucifer karyotype. The B and C genome of B. carinata shared 85 % of commonly conserved blocks with the B genome of B. nigra/B. juncea and 80 % of commonly conserved blocks with the C genome of B. napus, and shown frequent structural rearrangements such as insertions and inversions. Up to 24 quantitative trait loci (QTL) for flowering and budding time were identified in the DH population. Of these QTL, one consistent QTL (qFT.B4-2) for flowering time was identified in all of the environments in the J block of the B4 linkage group, where a group of genes for flowering time were aligned in A. thaliana. Another major QTL for flowering time under a winter-cropped environment was detected in the E block of C6, where the BnFT-C6 gene was previously localised in B. napus. This high-density map would be useful not only to reveal the genetic variation in the species with QTL analysis and genome sequencing, but also for other applications such as marker-assisted selection and genomic selection, for the African mustard improvement.
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http://dx.doi.org/10.1007/s00122-014-2321-zDOI Listing
July 2014

SNP markers-based map construction and genome-wide linkage analysis in Brassica napus.

Plant Biotechnol J 2014 Sep 3;12(7):851-60. Epub 2014 Apr 3.

Graham Centre for Agricultural Innovation (an alliance between NSW Department of Primary Industries and Charles Sturt University), Wagga Wagga Agricultural Institute, Wagga Wagga, NSW, Australia.

An Illumina Infinium array comprising 5306 single nucleotide polymorphism (SNP) markers was used to genotype 175 individuals of a doubled haploid population derived from a cross between Skipton and Ag-Spectrum, two Australian cultivars of rapeseed (Brassica napus L.). A genetic linkage map based on 613 SNP and 228 non-SNP (DArT, SSR, SRAP and candidate gene markers) covering 2514.8 cM was constructed and further utilized to identify loci associated with flowering time and resistance to blackleg, a disease caused by the fungus Leptosphaeria maculans. Comparison between genetic map positions of SNP markers and the sequenced Brassica rapa (A) and Brassica oleracea (C) genome scaffolds showed several genomic rearrangements in the B. napus genome. A major locus controlling resistance to L. maculans was identified at both seedling and adult plant stages on chromosome A07. QTL analyses revealed that up to 40.2% of genetic variation for flowering time was accounted for by loci having quantitative effects. Comparative mapping showed Arabidopsis and Brassica flowering genes such as Phytochrome A/D, Flowering Locus C and agamous-Like MADS box gene AGL1 map within marker intervals associated with flowering time in a DH population from Skipton/Ag-Spectrum. Genomic regions associated with flowering time and resistance to L. maculans had several SNP markers mapped within 10 cM. Our results suggest that SNP markers will be suitable for various applications such as trait introgression, comparative mapping and high-resolution mapping of loci in B. napus.
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http://dx.doi.org/10.1111/pbi.12186DOI Listing
September 2014

Construction of integrated linkage map of a recombinant inbred line population of white lupin (Lupinus albus L.).

Breed Sci 2013 Sep 1;63(3):292-300. Epub 2013 Sep 1.

Graham Centre for Agricultural Innovation (an alliance between NSW Department of Primary Industries and Charles Sturt University), Wagga Wagga Agricultural Institute , Wagga Wagga, NSW 2650 , Australia.

We report the development of a Diversity Arrays Technology (DArT) marker panel and its utilisation in the development of an integrated genetic linkage map of white lupin (Lupinus albus L.) using an F8 recombinant inbred line population derived from Kiev Mutant/P27174. One hundred and thirty-six DArT markers were merged into the first genetic linkage map composed of 220 amplified fragment length polymorphisms (AFLPs) and 105 genic markers. The integrated map consists of 38 linkage groups of 441 markers and spans a total length of 2,169 cM, with an average interval size of 4.6 cM. The DArT markers exhibited good genome coverage and were associated with previously identified genic and AFLP markers linked with quantitative trait loci for anthracnose resistance, flowering time and alkaloid content. The improved genetic linkage map of white lupin will aid in the identification of markers for traits of interest and future syntenic studies.
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http://dx.doi.org/10.1270/jsbbs.63.292DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3770556PMC
September 2013

Identifying genetic diversity of avirulence genes in Leptosphaeria maculans using whole genome sequencing.

Funct Integr Genomics 2013 Aug 21;13(3):295-308. Epub 2013 Jun 21.

School of Agriculture and Food Sciences and Centre for Integrative Legume Research, University of Queensland, Brisbane, Queensland 4072, Australia.

Next generation sequencing technology allows rapid re-sequencing of individuals, as well as the discovery of single nucleotide polymorphisms (SNPs), for genomic diversity and evolutionary analyses. By sequencing two isolates of the fungal plant pathogen Leptosphaeria maculans, the causal agent of blackleg disease in Brassica crops, we have generated a resource of over 76 million sequence reads aligned to the reference genome. We identified over 21,000 SNPs with an overall SNP frequency of one SNP every 2,065 bp. Sequence validation of a selection of these SNPs in additional isolates collected throughout Australia indicates a high degree of polymorphism in the Australian population. In preliminary phylogenetic analysis, isolates from Western Australia clustered together and those collected from Brassica juncea stubble were identical. These SNPs provide a novel marker resource to study the genetic diversity of this pathogen. We demonstrate that re-sequencing provides a method of validating previously characterised SNPs and analysing differences in important genes, such as the disease related avirulence genes of L. maculans. Understanding the genetic characteristics of this devastating pathogen is vital in developing long-term solutions to managing blackleg disease in Brassica crops.
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http://dx.doi.org/10.1007/s10142-013-0324-5DOI Listing
August 2013

A consensus map of rapeseed (Brassica napus L.) based on diversity array technology markers: applications in genetic dissection of qualitative and quantitative traits.

BMC Genomics 2013 Apr 23;14:277. Epub 2013 Apr 23.

EH Graham Centre for Agricultural Innovation (an alliance between NSWDPI and Charles Sturt University), Wagga Wagga Agricultural Institute, Wagga Wagga, NSW 2650, Australia.

Background: Dense consensus genetic maps based on high-throughput genotyping platforms are valuable for making genetic gains in Brassica napus through quantitative trait locus identification, efficient predictive molecular breeding, and map-based gene cloning. This report describes the construction of the first B. napus consensus map consisting of a 1,359 anchored array based genotyping platform; Diversity Arrays Technology (DArT), and non-DArT markers from six populations originating from Australia, Canada, China and Europe. We aligned the B. napus DArT sequences with genomic scaffolds from Brassica rapa and Brassica oleracea, and identified DArT loci that showed linkage with qualitative and quantitative loci associated with agronomic traits.

Results: The integrated consensus map covered a total of 1,987.2 cM and represented all 19 chromosomes of the A and C genomes, with an average map density of one marker per 1.46 cM, corresponding to approximately 0.88 Mbp of the haploid genome. Through in silico physical mapping 2,457 out of 3,072 (80%) DArT clones were assigned to the genomic scaffolds of B. rapa (A genome) and B. oleracea (C genome). These were used to orientate the genetic consensus map with the chromosomal sequences. The DArT markers showed linkage with previously identified non-DArT markers associated with qualitative and quantitative trait loci for plant architecture, phenological components, seed and oil quality attributes, boron efficiency, sucrose transport, male sterility, and race-specific resistance to blackleg disease.

Conclusions: The DArT markers provide increased marker density across the B. napus genome. Most of the DArT markers represented on the current array were sequenced and aligned with the B. rapa and B. oleracea genomes, providing insight into the Brassica A and C genomes. This information can be utilised for comparative genomics and genomic evolution studies. In summary, this consensus map can be used to (i) integrate new generation markers such as SNP arrays and next generation sequencing data; (ii) anchor physical maps to facilitate assembly of B. napus genome sequences; and (iii) identify candidate genes underlying natural genetic variation for traits of interest.
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http://dx.doi.org/10.1186/1471-2164-14-277DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3641989PMC
April 2013

Predicting polymorphic EST-SSRs in silico.

Mol Ecol Resour 2013 May 11;13(3):538-45. Epub 2013 Feb 11.

Melbourne eResearch Group, University of Melbourne, Parkville, Vic, 3010, Australia.

The public availability of large quantities of gene sequence data provides a valuable resource of the mining of Simple Sequence Repeat (SSR) molecular genetic markers for genetic analysis. These markers are inexpensive, require minimal labour to produce and can frequently be associated with functionally annotated genes. This study presents the characterization of barley EST-SSRs and the identification of putative polymorphic SSRs from EST data. Polymorphic SSRs are distinguished from monomorphic SSRs by the representation of varying motif lengths within an alignment of sequence reads. Two measures of confidence are calculated, redundancy of a polymorphism and co-segregation with accessions. The utility of this method is demonstrated through the discovery of 597 candidate polymorphic SSRs, from a total of 452 642 consensus expressed sequences. PCR amplification primers were designed for the identified SSRs. Ten primer pairs were validated for polymorphism in barley and for transferability across species. Analysis of the polymorphisms in relation to SSR motif, length, position and annotation is discussed.
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http://dx.doi.org/10.1111/1755-0998.12078DOI Listing
May 2013

A Tourist-like MITE insertion in the upstream region of the BnFLC.A10 gene is associated with vernalization requirement in rapeseed (Brassica napus L.).

BMC Plant Biol 2012 Dec 15;12:238. Epub 2012 Dec 15.

National Key Laboratory of Crop Genetic Improvement, Huazhong Agricultural University, Wuhan, 430070, China.

Background: Rapeseed (Brassica napus L.) has spring and winter genotypes adapted to different growing seasons. Winter genotypes do not flower before the onset of winter, thus leading to a longer vegetative growth period that promotes the accumulation and allocation of more resources to seed production. The development of winter genotypes enabled the rapeseed to spread rapidly from southern to northern Europe and other temperate regions of the world. The molecular basis underlying the evolutionary transition from spring- to winter- type rapeseed is not known, however, and needs to be elucidated.

Results: We fine-mapped the spring environment specific quantitative trait locus (QTL) for flowering time, qFT10-4,in a doubled haploid (DH) mapping population of rapeseed derived from a cross between Tapidor (winter-type) and Ningyou7 (semi-winter) and delimited the qFT10-4 to an 80-kb region on chromosome A10 of B. napus. The BnFLC.A10 gene, an ortholog of FLOWERING LOCUS C (FLC) in Arabidopsis, was cloned from the QTL. We identified 12 polymorphic sites between BnFLC.A10 parental alleles of the TN-DH population in the upstream region and in intron 1. Expression of both BnFLC.A10 alleles decreased during vernalization, but decreased more slowly in the winter parent Tapidor. Haplotyping and association analysis showed that one of the polymorphic sites upstream of BnFLC.A10 is strongly associated with the vernalization requirement of rapeseed (r2 = 0.93, χ2 = 0.50). This polymorphic site is derived from a Tourist-like miniature inverted-repeat transposable element (MITE) insertion/deletion in the upstream region of BnFLC.A10. The MITE sequence was not present in the BnFLC.A10 gene in spring-type rapeseed, nor in ancestral 'A' genome species B. rapa genotypes. Our results suggest that the insertion may have occurred in winter rapeseed after B. napus speciation.

Conclusions: Our findings strongly suggest that (i) BnFLC.A10 is the gene underlying qFT10-4, the QTL for phenotypic diversity of flowering time in the TN-DH population, (ii) the allelic diversity caused by MITE insertion/deletion upstream of BnFLC.A10 is one of the major causes of differentiation of winter and spring genotypes in rapeseed and (iii) winter rapeseed has evolved from spring genotypes through selection pressure at the BnFLC.A10 locus, enabling expanded cultivation of rapeseed along the route of Brassica domestication.
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http://dx.doi.org/10.1186/1471-2229-12-238DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3562271PMC
December 2012

Comparative analysis of FLC homologues in Brassicaceae provides insight into their role in the evolution of oilseed rape.

PLoS One 2012 27;7(9):e45751. Epub 2012 Sep 27.

National Key Laboratory of Crop Genetic Improvement, Huazhong Agricultural University, Wuhan, China.

We identified nine FLOWERING LOCUS C homologues (BnFLC) in Brassica napus and found that the coding sequences of all BnFLCs were relatively conserved but the intronic and promoter regions were more divergent. The BnFLC homologues were mapped to six of 19 chromosomes. All of the BnFLC homologues were located in the collinear region of FLC in the Arabidopsis genome except BnFLC.A3b and BnFLC.C3b, which were mapped to noncollinear regions of chromosome A3 and C3, respectively. Four of the homologues were associated significantly with quantitative trait loci for flowering time in two mapping populations. The BnFLC homologues showed distinct expression patterns in vegetative and reproductive organs, and at different developmental stages. BnFLC.A3b was differentially expressed between the winter-type and semi-winter-type cultivars. Microsynteny analysis indicated that BnFLC.A3b might have been translocated to the present segment in a cluster with other flowering-time regulators, such as a homologue of FRIGIDA in Arabidopsis. This cluster of flowering-time genes might have conferred a selective advantage to Brassica species in terms of increased adaptability to diverse environments during their evolution and domestication process.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0045751PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3459951PMC
February 2013

Genetic and physical mapping of flowering time loci in canola (Brassica napus L.).

Theor Appl Genet 2013 Jan 7;126(1):119-32. Epub 2012 Sep 7.

EH Graham Centre for Agricultural Innovation (an alliance between NSWDPI and Charles Sturt University), Wagga Wagga, Australia.

We identified quantitative trait loci (QTL) underlying variation for flowering time in a doubled haploid (DH) population of vernalisation-responsive canola (Brassica napus L.) cultivars Skipton and Ag-Spectrum and aligned them with physical map positions of predicted flowering genes from the Brassica rapa genome. Significant genetic variation in flowering time and response to vernalisation were observed among the DH lines from Skipton/Ag-Spectrum. A molecular linkage map was generated comprising 674 simple sequence repeat, sequence-related amplified polymorphism, sequence characterised amplified region, Diversity Array Technology, and candidate gene based markers loci. QTL analysis indicated that flowering time is a complex trait and is controlled by at least 20 loci, localised on ten different chromosomes. These loci each accounted for between 2.4 and 28.6% of the total genotypic variation for first flowering and response to vernalisation. However, identification of consistent QTL was found to be dependant upon growing environments. We compared the locations of QTL with the physical positions of predicted flowering time genes located on the sequenced genome of B. rapa. Some QTL associated with flowering time on A02, A03, A07, and C06 may represent homologues of known flowering time genes in Arabidopsis; VERNALISATION INSENSITIVE 3, APETALA1, CAULIFLOWER, FLOWERING LOCUS C, FLOWERING LOCUS T, CURLY LEAF, SHORT VEGETATIVE PHASE, GA3 OXIDASE, and LEAFY. Identification of the chromosomal location and effect of the genes influencing flowering time may hasten the development of canola varieties having an optimal time for flowering in target environments such as for low rainfall areas, via marker-assisted selection.
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http://dx.doi.org/10.1007/s00122-012-1966-8DOI Listing
January 2013

Identification and characterization of candidate Rlm4 blackleg resistance genes in Brassica napus using next-generation sequencing.

Plant Biotechnol J 2012 Aug 23;10(6):709-15. Epub 2012 Jun 23.

Centre for Integrative Legume Research and School of Agriculture and Food Sciences, University of Queensland, Brisbane, Qld, Australia.

A thorough understanding of the relationships between plants and pathogens is essential if we are to continue to meet the agricultural needs of the world's growing population. The identification of genes underlying important quantitative trait loci is extremely challenging in complex genomes such as Brassica napus (canola, oilseed rape or rapeseed). However, recent advances in next-generation sequencing (NGS) enable much quicker identification of candidate genes for traits of interest. Here, we demonstrate this with the identification of candidate disease resistance genes from B. napus for its most devastating fungal pathogen, Leptosphaeria maculans (blackleg fungus). These two species are locked in an evolutionary arms race whereby a gene-for-gene interaction confers either resistance or susceptibility in the plant depending on the genotype of the plant and pathogen. Preliminary analysis of the complete genome sequence of Brassica rapa, the diploid progenitor of B. napus, identified numerous candidate genes with disease resistance characteristics, several of which were clustered around a region syntenic with a major locus (Rlm4) for blackleg resistance on A7 of B. napus. Molecular analyses of the candidate genes using B. napus NGS data are presented, and the difficulties associated with identifying functional gene copies within the highly duplicated Brassica genome are discussed.
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http://dx.doi.org/10.1111/j.1467-7652.2012.00716.xDOI Listing
August 2012

Molecular mapping of qualitative and quantitative loci for resistance to Leptosphaeria maculans causing blackleg disease in canola (Brassica napus L.).

Theor Appl Genet 2012 Jul 28;125(2):405-18. Epub 2012 Mar 28.

EH Graham Centre for Agricultural Innovation, NSW Department of Primary Industries and Charles Sturt University, Wagga Wagga Agricultural Institute, PMB, Wagga Wagga, NSW 2650, Australia.

Blackleg, caused by Leptosphaeria maculans, is one of the most important diseases of oilseed and vegetable crucifiers worldwide. The present study describes (1) the construction of a genetic linkage map, comprising 255 markers, based upon simple sequence repeats (SSR), sequence-related amplified polymorphism, sequence tagged sites, and EST-SSRs and (2) the localization of qualitative (race-specific) and quantitative (race non-specific) trait loci controlling blackleg resistance in a doubled-haploid population derived from the Australian canola (Brassica napus L.) cultivars Skipton and Ag-Spectrum using the whole-genome average interval mapping approach. Marker regression analyses revealed that at least 14 genomic regions with LOD ≥ 2.0 were associated with qualitative and quantitative blackleg resistance, explaining 4.6-88.9 % of genotypic variation. A major qualitative locus, designated RlmSkipton (Rlm4), was mapped on chromosome A7, within 0.8 cM of the SSR marker Xbrms075. Alignment of the molecular markers underlying this QTL region with the genome sequence data of B. rapa L. suggests that RlmSkipton is located approximately 80 kb from the Xbrms075 locus. Molecular marker-RlmSkipton linkage was further validated in an F(2) population from Skipton/Ag-Spectrum. Our results show that SSR markers linked to consistent genomic regions are suitable for enrichment of favourable alleles for blackleg resistance in canola breeding programs.
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http://dx.doi.org/10.1007/s00122-012-1842-6DOI Listing
July 2012
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