Rosy Raman

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

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.

Genetic and in silico comparative mapping of the polyphenol oxidase gene in bread wheat (Triticum aestivum L.)

Polyphenol oxidases (PPOs) are involved in the time-dependent darkening and discolouration of Asian noodles and other wheat end products. In this study, a doubled haploid (DH) population derived from Chara (moderately high PPO activity)/WW2449 (low PPO activity) was screened for PPO activity based on l-DOPA and l-tyrosine assays using whole seeds. Both these assays were significantly genetically correlated (r=0.91) in measuring the PPO activity in this DH population. Quantitative trait loci (QTLs) analysis utilising a skeleton map enabled us to identify a major QTL controlling PPO activity based on l-DOPA and l-tyrosine on the long arm of chromosome 2A. The simple sequence repeat (SSR) marker GWM294b explained over 82% of the line mean phenotypic variation from samples collected in both 2000 and 2003. Four SSR markers were validated for PPO linkage in genetically diverse backgrounds and proven to correctly predict the PPO activity in more than 92% of wheat lines. Physical mapping using deletion lines of Chinese Spring has confirmed the location of the GWM294b, GWM312 and WMC170 on chromosome 2AL, between deletion breakpoints 2AL-C to 0.85. In order to identify functional gene markers, data searches for alignments between rice BAC/PAC clones assembled on chromosome 1 and 4, chromosome 7, and (1) the wheat expressed sequence tags mapped in deletion bin (2AL-C to 0.85) and (2) the coding sequence of a previously cloned wheat PPO gene were made and found significant sequence similarities with the PPO gene or common central domain of tyrosinase. Available PPO gene sequences in the National Centre for Biotechnology Information (NCBI) database have revealed that there is a significant molecular diversity at the nucleotide and amino acid level in the wheat PPO genes.

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

BackgroundDense 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.ResultsThe 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.ConclusionsThe 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.

Genome-wide association analyses of common wheat (Triticum aestivum L.) germplasm identifies multiple loci for aluminium resistance.

Aluminium (Al3+) toxicity restricts productivity and profitability of wheat (Triticum aestivum L.) crops grown on acid soils worldwide. Continued gains will be obtained by identifying superior alleles and novel Al3+ resistance loci that can be incorporated into breeding programs. We used association mapping to identify genomic regions associated with Al3+ resistance using 1055 accessions of common wheat from different geographic regions of the world and 178 polymorphic diversity arrays technology (DArT) markers. Bayesian analyses based on genetic distance matrices classified these accessions into 12 subgroups. Genome-wide association analyses detected markers that were significantly associated with Al3+ resistance on chromosomes 1A, 1B, 2A, 2B, 2D, 3A, 3B, 4A, 4B, 4D, 5B, 6A, 6B, 7A, and 7B. Some of these genomic regions correspond to previously identified loci for Al3+ resistance, whereas others appear to be novel. Among the markers targeting TaALMT1 (the major Al3+-resistance gene located on chromosome 4D), those that detected alleles in the promoter explained most of the phenotypic variance for Al3+ resistance, which is consistent with this region controlling the level of TaALMT1 expression. These results demonstrate that genome-wide association mapping cannot only confirm known Al3+-resistance loci, such as those on chromosomes 4D and 4B, but they also highlight the utility of this technique in identifying novel resistance loci.

Diversity array technology markers: Genetic diversity analyses and linkage map construction in rapeseed (Brassica napus L.)

We developed Diversity Array Technology (DArT) markers for application in genetic studies of Brassica napus and other Brassica species with A or C genomes. Genomic representation from 107 diverse genotypes of B. napus L. var. oleifera (rapeseed, AACC genomes) and B. rapa (AA genome) was used to develop a DArT array comprising 11 520 clones generated using PstI/BanII and PstI/BstN1 complexity reduction methods. In total, 1547 polymorphic DArT markers of high technical quality were identified and used to assess molecular diversity among 89 accessions of B. napus, B. rapa, B. juncea, and B. carinata collected from different parts of the world. Hierarchical cluster and principal component analyses based on genetic distance matrices identified distinct populations clustering mainly according to their origin/pedigrees. DArT markers were also mapped in a new doubled haploid population comprising 131 lines from a cross between spring rapeseed lines ‘Lynx-037DH’ and ‘Monty-028DH’. Linkage groups were assigned on the basis of previously mapped simple sequence repeat (SSRs), intron polymorphism (IP), and gene-based markers. The map consisted of 437 DArT, 135 SSR, 6 IP, and 6 gene-based markers and spanned 2288 cM. Our results demonstrate that DArT markers are suitable for genetic diversity analysis and linkage map construction in rapeseed.

Mapping of genomic regions associated with net form of netblotch resistance in barley

Quantitative trait loci (QTLs) associated with resistance to net blotch and their chromosomal locations were determined from analyses of doubled haploid progeny of Alexis/Sloop, Arapiles/Franklin, Sloop/Halcyon, and recombinant inbred lines of Sloop-sib/Alexis. Five QTLs on chromosomes 2H, 3H, and 4H were found to be associated with seedling resistance to the net form of net blotch. In Arapiles/Franklin and Alexis/Sloop populations, 4 significant QTLs explaining 9–17% of the variation in net blotch resistance were detected on 2H and 3H. A major locus, QRpts4L accounting for 64% of the variation in infection type, was detected on 4H in the Sloop/Halcyon population. In Sloop/Halcyon, 2 microsatellite markers, EBmac0906 and GMS089, and AFLP marker P13/M50-108, co-segregated and detected maximum variability for net blotch resistance as revealed by bootstrap analysis. EBmac0906 and Bmac0181 were validated in F2 progeny of an Ant29/Halcyon population and reliably predicted phenotypes of 93% of lines resistant and susceptible to net blotch. These markers may be used within breeding programs to select alleles favourable for net blotch resistance derived from Halcyon.

Repetitive Indel Markers within the ALMT1 Gene Conditioning Aluminium Tolerance in Wheat (Triticum aestivum L.)

ALMT1 gene encoding a membrane protein that facilitates an aluminium stimulated malate efflux has been characterised and mapped in wheat (Triticum aestivum L.). Here, we have identified molecular markers targeting insertion/deletion (indel) and SSR repeats within intron 3 region of the ALMT1 gene. Both the markers: ALMT1-SSR3a and ALMT1-SSR3b based on repetitive indels, exhibited complete cosegregation with Al tolerance, malate efflux, and a CAPS marker discriminating ALMT1-1 and ALMT1-2 alleles, in a doubled haploid population derived from Diamondbird (Al-tolerant)/Janz (Al-sensitive). A parental screen of 20 diverse wheat genotypes with repetitive indel markers indicated that six allele variants exist at the ALMT1SSR3 locus. Sequence analysis confirmed that these variations were due to indels, copy number of SSR repeats, and base substitution within SSR repeats. The higher level of variation in intron 3 suggests that this genomic region has been constrained by indels, SSR and single nucleotide polymorphisms. Results have proven that repetitive indel markers cosegregating with the Al tolerance locus will be useful for marker assisted selection and population and evolution studies.

Development and allele diversity of microsatellite markers linked to the aluminium tolerance gene Alp in barley

Aluminium (Al) toxicity is one of the main factors restricting barley production in acidic soils. The utilisation of barley cultivars tolerant to Al is one of the most economic strategies for expanding barley production in these soils. Among barley genotypes, the cultivar Dayton has been reported to exhibit the highest level of Al tolerance. The gene conferring Al tolerance in Dayton, Alp, has been mapped to the long arm of chromosome 4H using RFLP markers. However, such markers are not useful for routine marker-assisted selection in breeding programs due to the cost and labour associated with their use. To increase the ease by which marker-assisted selection can be conducted for Alp, we sought to identify microsatellite markers linked to this gene. Several such markers that flank Alp were identified in a mapping population from a cross between Dayton and Harlan Hybrid. The most tightly linked microsatellite markers, HVM68 and Bmag353, flank Alp and are 5.3 cM and 3.1 cM from this locus, respectively. The linkage between Bmag353 and Alp was validated in a separate F3 population derived from the cross between Dayton and F6ant28B48-16, where this microsatellite marker was found to predict the Al tolerance phenotype with over 95% accuracy. Allele diversity for the 3 most tightly linked microsatellite markers was evaluated among 40 barley genotypes currently used in Australian barley breeding programs. The high levels of polymorphism detected among the genotypes with the markers indicated that the microsatellite markers, especially Bmag353 and Bmac310, will be broadly useful for marker-assisted selection of Alp in breeding programs seeking to improve Al tolerance. AR M ic a d tance H. Ra m et al

Genome‐wide association analyses reveal complex genetic architecture underlying natural variation for flowering time in canola

Optimum flowering time is the key to maximize canola production in order to meet global demand of vegetable oil, biodiesel and canola-meal. We reveal extensive variation in flowering time across diverse genotypes of canola under field, glasshouse and controlled environmental conditions. We conduct a genome-wide association study and identify 69 single nucleotide polymorphism (SNP) markers associated with flowering time, which are repeatedly detected across experiments. Several associated SNPs occur in clusters across the canola genome; seven of them were detected within 20 Kb regions of a priori candidate genes; FLOWERING LOCUS T, FRUITFUL, FLOWERING LOCUS C, CONSTANS, FRIGIDA, PHYTOCHROME B and an additional five SNPs were localized within 14 Kb of a previously identified quantitative trait loci for flowering time. Expression analyses showed that among FLC paralogs, BnFLC.A2 accounts for ~23% of natural variation in diverse accessions. Genome-wide association analysis for FLC expression levels mapped not only BnFLC.C2 but also other loci that contribute to variation in FLC expression. In addition to revealing the complex genetic architecture of flowering time variation, we demonstrate that the identified SNPs can be modelled to predict flowering time in diverse canola germplasm accurately and hence are suitable for genomic selection of adaptative traits in canola improvement programmes.

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

Brassica carinata (BBCC) is an allotetraploid in Brassicas 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 B. carinata with genotyping by sequencing approach DArTseq, generating a total of 54,510 polymorphic markers. Two subpopulations were exhibited in the B. carinata accessions. The average distance of LD decay (r2 = 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 B. napus. These results would not only provide insights for genetic improvement of this species, but will also identify useful genetic variation hidden in the Cc subgenome of B. carinata to improve canola cultivars.