WP2

DELIVERABLE 2.2

Genomic Resources for the selected non-food crops

OIL CROPS

Oilseed rape (Brassica napus)

Oilseed rape (B. napus) is an allotetraploid species consisting of two genomes, derived from B. rapa (A genome) and B. oleracea (C genome).It is the closest major crop relative of A. thaliana and the world's second most important oilseed crop. The oil derived from crushingharvested seed is a major provider of calorific value to thehuman food chain, with variations in fatty acid profileincluding combinations of erucic acid, oleic and linolenic acidthat are of industrial value as oils, lubricants, surfactants andhigh-value plastics.

The Multinational Brassica rapa Genome Sequencing Project (BrGSP) has developed valuable genomic resources, including BAC libraries, BAC-end sequences, genetic and physical maps, and seed BAC sequences for Brassica rapa( integrated genetic linkage map for the A genome of Brassica napus using SSR markers derived from sequenced BACs in B. rapahas been constructed, facilitating the rapid transfer of valuable genomic resources from B. rapa to B. napus (Xu et al., 2010).A total of 604 SNPs that can be used for genetic analysis, were identified in oilseed rape (Durstewitz et al., 2010).Sun et al. (2007) constructed an ultradense genetic map containing 13,351 SRAP markers in B. napus, which is the most saturated map in Brassica species that has ever been constructed. Moreover, SRAP is an effective method for map-based gene cloning and molecular marker assisted selection (MAS) and very effective in studying genetic diversity since it does not need genome sequence information.Additionally, SRAP was adequate to perform QTL analysis, which was demonstrated in B. napus (Fu et al., 2007).The B. napus genetic map is being used to align other five genetic maps that are used to perform QTL mapping of Sclerotinia tolerance in B. napus (Li et al., 2011).

Quantitative trait locus (QTL) mapping has been employed to gain better understanding of the genetic factors controlling silique-traits and gain insights into the gene networks affecting erucic acid and oil content in seeds, plant height and flowering time, resistance to biotic and abiotic stresses (Cao et al., 2010; Kaur et al., 2009; Mei et al., 2009; Zhang et al., 2010; Zhao et al., 2010).

The total number of ESTs from Brassica species deposited in public databases has risen dramatically to more than 800,000 entries with about 280,000 from seed developmental stages. Microarrays have become a widely-used tool for transcriptome analysis in plants. Oligonucleotide microarrays constructed for A. thaliana have been used in the past for expression profiling in B. napus, but have not provided optimal signal intensity and reproducibility (Hudson et al., 2007; Li et al., 2005). 67,000 ESTs from seed developmental stages have been used to develop a B. napus cDNA microarray for analysis of seed gene expression patterns (Xiang et al., 2008). An endosperm EST collection of over 30,000 entries and a microarray dataset have provided a basic genomic resource for dissecting metabolic and developmental events important for oilseed improvement (Huang et al., 2009).A Brassica community microarray resource has been successfully developed and validated (Trick et al., 2009).

An alternative for accurate, quantitative global expression profiling is serial analysis of gene expression (SAGE). The LongSAGE approach was used for analysis of global gene expression in B. napusby matching B. napus tags via Brassica ESTs to annotated A. thaliana gene loci, including detection of tags matching in sense and antisense orientation (Obermeier et al., 2009).The cDNA amplified fragment length polymorphism (cDNA-AFLP) approach was employed for association of gene expression profiles with intersubgenomic heterosis in Brassica napus (Chen et al., 2008).

TILLING permits the rapid discovery of induced point mutations in populations of chemically mutagenized individuals. The application of TILLING to the generation and identification of a novel low erucic acid (LEA) genetic resource for rapeseed improvement has already been reported (Wang et al., 2008). EcoTILLING, a powerful genotyping method, was employed to assess FAE1 (fatty acid elongase1) polymorphisms in three Brassica species and their association with differences in seed erucic acid contents (Wang et al., 2010).

References

  • Cao Z, Tian F, Wang N, Jiang C, Lin B, Xia W, Shi J, Long Y, Zhang C and Meng J (2010) Analysis of QTLs for erucic acid and oil content in seeds on A8 chromosome and the linkage drag between the alleles for the two traits in Brassica napus. J Genet Genomics 37:231-40.
  • Chen X, Li M, Shi J, Fu D, Qian W, Zou J, Zhang C and Meng J (2008) Gene expression profiles associated with intersubgenomic heterosis in Brassica napus. Theor Appl Genet 117:1031-40.
  • Durstewitz G, Polley A, Plieske J, Luerssen H, Graner EM, Wieseke R and Ganal MW (2010) SNP discovery by amplicon sequencing and multiplex SNP genotyping in the allopolyploid species Brassica napus. Genome 53:948-56.
  • Fu FY, Liu LZ, Chai YR, Chen L, Yang T, Jin MY, Ma AF, Yan XY, Zhang ZS and Li JN (2007) Localization of QTLs for seed color using recombinant inbred lines of Brassica napus in different environments. Genome 50:840-854.
  • Huang Y , Chen L , Wang L , Vijayan K , Phan S , Liu Z , WanL et al. (2009) Probing the endosperm gene expression landscape in Brassica napus. BMC Genomics 10:256.
  • Hudson ME, Brugginkd T, Changa SH, Yuc W, Hana B, Wanga X, Toornd P and Zhua T (2007) Analysis of Gene Expression during Brassica Seed Germination Using a Cross-Species Microarray Platform. Crop Sci 47: 96-112.
  • Kaur S, Cogan NO, Ye G, Baillie RC, Hand ML, Ling AE, McGearey AK et al. (2009) Genetic map construction and QTL mapping of resistance to blackleg (Leptosphaeria maculans) disease in Australian canola (Brassica napus L.) cultivars. Theor Appl Genet 120:71-83.
  • Li F, Wu X, Tsang E and Cutler AJ (2005): Transcriptional profiling of imbibed Brassica napus seed. Genomics 86: 718-730.
  • Li W, Zhang J, Mou Y, Geng J, McVetty PB, Hu S and Li G (2011) Integration of Solexa sequences on an ultradense genetic map in Brassica rapa L. BMC Genomics 12:249.
  • Mei DS, WangHZ, Hu Q, Li YD, Xu YS and Li YC (2009) QTL analysis on plant height and flowering time in Brassica napus. Plant Breed 128: 458–465.
  • Obermeier C , Hosseini B, Friedt W and Rod Snowdon (2009) Gene expression profiling via LongSAGE in a non-model plant species: a case study in seeds of Brassica napus. BMC Genomics 10: 295.
  • Sun Z, Wang Z, Tu J, Zhang J, Yu F, McVetty PB and Li G (2007) An ultradense genetic recombination map for Brassica napus, consisting of 13551 SRAP markers. Theor Appl Genet 114:1305-17.
  • Trick M , Cheung F , Drou1 N , Fraser F , Lobenhofer EK, Hurban P et al. (2009) A newly-developed community microarray resource for transcriptome profiling in Brassica species enables the confirmation of Brassica-specific expressed sequences. BMC Plant Biology 29:50.
  • Wang N, Shi L, Tian F, Ning H, Wu X, Long Y and Meng J (2010) Assessment of FAE1 polymorphisms in three Brassica species using EcoTILLING and their association with differences in seed erucic acid contents. BMC Plant Biol 10: 137.
  • Wang N, Wang Y, Tian F, King GJ, Zhang C, Long Y, Shi L and Meng J (2008) A functional genomics resource for Brassica napus: development of an EMS mutagenized population and discovery of FAE1 point mutations by TILLING. New Phytol. 180: 751-765.
  • Xiang D, Datla R, Li F, Cutler A, Malik MR, Krochko JE, Sharma N, Fobert P et al. (2008) Development of a Brassica seed cDNA microarray. Genome 51: 236-42.
  • Xu J, Qian X, Wang X, Li R, Cheng X, Yang Y, Fu J, Zhang S, King GJ, Wu J and Liu K (2010) Construction of an integrated genetic linkage map for the A genome of Brassica napus using SSR markers derived from sequenced BACs in B. rapa. BMC Genomics 11:594.
  • Zhang L, Yang G, Liu P, Hong D, Li S and He Q (2010) Genetic and correlation analysis of silique-traits in Brassica napus L. by quantitative trait locus mapping. Theor Appl Genet 122:21-31.
  • Zhao H, Liu J, Shi L, Xu F and Wang Y (2010) Development of boron-efficient near isogenic lines of Brassica napus and their response to low boron stress at seedling stage. Genetika 46:66-72.

Sunflower (Helianthus annus)

The genus Helianthus is a member of the Asteraceae family. This cosmopolitan family comprises of 1600–1700 genera, 24000–30000 species, and several agronomically, horticulturally, and medically importantspecies (Jansen et al. 1991; Funk et al. 2005).

Sunflower is one of the most importantoilseed crops cultivated in the world.It is the preferredsource of oil for domestic consumption andcooking in much of central and eastern Europe.Sunflower oil contains less than 11% totalsaturated fat and does not contain any trans fat.The inexpensive production of biofuel fromplant vegetable oils such as sunfloweroil has been achieved. Furthermore, sunflower canproduce latex naturally. It is an ideal plantforproducinghigh quality rubber in its leaves and stemsand some of the taller perennial specieshave potential for high latex yields (Wood, 2002).

The multiple usages of sunflower products in food,feed, and industry are stimulating the discovery of new sources of biodiversity for sunflower molecular breeding programs in combination with the application of high throughput approaches and genetic manipulation.

A rich and diverse germplasm collection is the backboneof every successful crop improvement program.Assessing genetic diversity within a genetic pool of novelbreedinggermplasm could make crop improvement moreefficient by the directed accumulation of desired alleles (Darvishzadeh et al., 2010).

The sunflower, Helianthus annuus, genome is diploid with a base chromosome number of n=17 and an estimated genome size of about 3.500 Mb. Dominant markers such as random amplified polymorphic DNA (RAPDs), amplified fragment length polymorphism (AFLPs),restriction fragment length polymorphisms (RFLPs),simple sequence repeats (SSRs) have been employed for sunflower genome mapping and genetic variability studies. Conservatively,18 genetic maps of varying density and completenesshave been constructed in wild and cultivated sunflowers. The constructed sunflower genetic maps consisted of 17 to 23 stable linkage groups and differed in their genetic length from 1,423 cMin the SSR map to 2,916 cM AFLP map.The target region amplification polymorphism (TRAP) marker approach was used to define Helianthus annuuslinkage group endsand to expand the publishedsunflower simple sequence repeat (SSR) linkage map (Hu et al., 2007).

Several BAC libraries have been constructed for sunflower(Gentzbittel et al., 2002; Özdemir et al., 2004; Feng et al., 2006). The librariesare equivalent to approximately 8 haploid genomes of sunflower and provide a greater than 99% probability of obtaining a clone of interest and they have been employed for isolating and physical mapping ofloci such as the FAD2-1 locus (Tang et al. 2007), or the fertility restorer Rf1locus (Hamrit et al., 2008).In situ hybridization techniques involving GISH, FISH and BAC-FISH are being optimized for diversity and evolutionary studies between species of the genus Helianthus and development of a physical sunflower map allowing a cross reference to the genetic map (Paniego et al. 2006).

VariousEST sequencing programs have been carried out in sunflower, including the

Compositae Genome Project, and other programs reported by Fernandez et al.(2003), Tamborindeguy et al. (2004), and Ben et al. (2005). More than 261,699 sunflower ESTs have been developed for sunflower primarily by the Compositae GenomeProgram (CGP; and 306090 sunflower ESTs have been deposited in GenBank (Heesacker et al., 2008). Forsunflower, 93428ESTs were derived from H. annuus L representing approximately 31605 unigenes (

Dex.ph). Interesting associations have been detected between ESTs and QTLs for salt tolerance and for domestication traits (Lai et al., 2005).

Sequencing the genome of cultivated sunflower will dramatically enhance Compositae genomic resources. As of January 2010, a$10.5 million research project titled“Genomics of Sunflower” use next-generation genotyping and sequencing technologies to sequence, assemble and annotate the sunflower genomeand to locate the genes that are responsible for agriculturally important traits such as seed-oil content, flowering, seed-dormancy, and wood producing-capacity. A total of 304.2 Gbp of Illumina sequence and a total of 8.5X coverage with 454 sequence have been obtained. However, even the most complete assemblies of the sunflower genome generated to date include several hundred thousand scaffolds and cover approximately 60% of the genome (

These genomic resources are valuable tools that can be used to obtain direct access to some genes of interest by map-based cloning or candidate gene approaches for physicalmapping or for the development of markers.

References

  • Darvishzadeh R, Azizi M, Hatami-Maleki H, Bernousi I, Mandoulakani BA, Jafari M and Sarrafi A (2010) Molecular characterization and similarity relationships among sunflower (Helianthus annuus L.) inbred lines using some mapped simple sequence repeats.African J Biotech 9: 7280-7288.
  • Feng J, Vick BA, Lee M-K, Zhang H-B and Jan CC (2006) Hong-BinConstruction of BAC and BIBAC libraries from sunflower and identification of linkage group-specific clones by overgo hybridization. Theor Appl Genet 1:23-32.
  • Funk VA, Bayer RJ, Keeley S, Chan R, Watson L, Gemeinholzer B, Schilling E, Panero JL, Baldwin BG, Garcia-Jagas N, Susanna A, Jansen RK (2005) Everywhere but Antarctica: using a supertree to understand the diversity and distribution of the Compositae. Biol Skr 55:343–374.
  • Gentzbittel L, Abbott A, Galaud JP, Georgi L, Fabre F, Liboz T, and Alibert G (2002) A bacterial artificial chromosome (BAC) library for sunflower, and identification of clones containing genes for putative transmembrane receptors. Mol Genet Gen 266: 979-987.
  • Hamrit S, Kusterer B, Friedt W, Horn R (2008) Verification of positive BAC clones near the Rf1 gene restoring pollen fertility in the presence of the PET1 cytoplasm in sunflower (Helianthus annuus L.) and direct isolation of BAC ends. In: Proc 17th Int Sunflower Conf. vol.2, Córdoba, Spain, International Sunflower Association, Paris, pp 623-628.
  • Heesacker A, Kishore VK, Gao W, Tang S, Kolkman JM, Gingle A, Matvienko M, Kozik A, MichelmoreRM, Lai Z, RiesebergLH, Knapp SJ (2008) SSRs and INDELs mined from the sunflower EST database: abundance, polymorphisms, and cross-taxa utility. Theor Appl Genet 117:1021–1029.
  • Hu J, Yue B, Vick BA (2007) Integration of TRAP markers onto a sunflower SSR marker linkage map constructed from 92 recombinant inbred lines. Helia 30: 25-36.
  • Jansen RK, Michaels HJ, Palmer JD (1991) Phylogeny and character evolution in the Asteraceae based on chloroplast DNA restriction site mapping. Syst Bot 16: 98–115.
  • Jan CC, Vick BA, Miller JF, Kahler AL, Butler ET (1998) Construction of an RFLP linkage map for cultivated sunflower. Theor Appl Genet 96:15–22.
  • Lai Z, Livingstone K, Zou Y, Church SA, Knapp SJ, Andrews J, Rieseberg LH (2005) Identification and mapping of SNPs from ESTs in sunflower. Theor Appl Genet 111: 1532-1544.
  • Özdemir N, Horn R and Friedt W (2004) Construction and characterization of a BAC library for sunflower (Helianthus annuus L.). Euphytica 138: 177-183.
  • Wood, M. (2002) Sunflower rubber? Agriculture Research Magazine 50: 22.
  • Ishitani M, Rao I, Wenzl P, Beebe S, Tohme J (2004) Integration of genomics approach with traditional breeding towards improving abiotic stress adaptation: drought and aluminum toxicity as case studies. Field Crops Research 90:35–45

FIBRE CROPS

Flax (Linum usitatissimumL)

Flax (Linum usitatissimum L.) is a globally important agricultural crop producing edible and industrial oils as well as fibres. It belongs tothe Linaceae family and is one of about 200 species in the genus Linum.Despite renewed interest in flax as a source of phloem (bast) fibres, relatively few genomic resources have been established for this crop.

Total Utilization Flax GENomics (TUFGEN) project was initiated, aiming to develop genomic tools needed for molecular breeding. The first genome-wide physical map of flax and the generation and analysis ofBAC-end sequences (BES) from 43,776 clones, providing initial insights into the genome was recently reported.As a genomic resource, this map will be useful for fine mappingof target genomic regions and map-based cloning of genes/QTLs.Flax genome size was estimated to range from 370 Mb to 675 Mb(Ragupathy et al., 2011).

Improvement of flax varieties through breeding for various traits can be assistedby development of molecular markers and by understanding the genetic andbiochemical bases of these characteristics. A comprehensive EST resource was developed for flaxrepresentingdevelopmental stages of specific seed tissues, some vegetative and reproductive tissues (Venglat et al., 2011).A queryable flax unigene database is also publicly available ( recently published flax-specific microarray based on EST sequencesobtained from a fiber focused study, provides a complimentary genomic tool for flax gene expression analysis.Initial studies have enabled the identification of specifically-expressed cellwall- and defence-related genes in 2 different flax varietiesshowing contrasting fibre quality and resistancetowards a fungal pathogen(Fenart et al., 2011).

Different research groups have developed reverse genetics and genomicapproaches to decipherfibre and seed formationin this economically-important species.EMS mutagenized populations of CDC Bethune,an elite linseed cultivar, have already been produced (10,000 individualM2 families).A proteomic approach has been employed to increase our understanding of the proteins that contribute to the unique properties of flax bast fibres (Hotte and Deyholos, 2008). The RNA interference approach was employed to study the impact of reduced expression ofb-galactosidase during fibre development in flax (Roach et al., 2011).

Molecular markers are highly useful to identify potentially novel genotypes among the many flax accessions, and to assess genetic diversity for both germplasm management and core collection assembly.A variety of marker systems, including random amplified polymorphic DNA (RAPD), inter-simple sequence repeat (ISSR), amplified fragment length polymorphism (AFLP), and simple sequence repeat (SSR), been used to analyze flax germplasm (Cloutier et al., 2009; Diederichsen and Fu, 2006; Everaert et al., 2001; Wiesnerova and Wiesner, 2004). Inter-retrotransposon amplified polymorphism (IRAP) markers were recently developed for cultivated flax and the genetic diversity among 708 accessions of cultivated flax comprising 143 landraces, 387 varieties, and 178 breeding lines was evaluated (Smýkal et al., 2011).

References

  • Cloutier S, Niu Z, Datla R and Duguid S (2009) Development and analysis of EST-SSRs for flax (Linum usitatissimum L.). Theor Appl Genet 119:53–63.
  • Diederichsen A and Fu YB (2006) Phenotypic and molecular (RAPD) differentiation of four infraspecific groups of cultivated flax (Linum usitatissimum L. subsp. usitatissimum). Genet Resour Crop Evol 53:77–90.
  • Everaert I, de Riek J, de Loose M, van Waes J and van Bockstaele E (2001) Most similar variety grouping for distinctness evaluation of flax and linseed (Linum usitatissimum L.) varieties by means of AFLP and morphological data. Plant Var Seeds 14:69–87.
  • Fenart S, Ndong Y-P, Duarte J, Riviere N, Wilmer J, van Wuytswinkel O, Lucau A, Cariou E, Neutelings G and Gutierrez L et al: Development and validation of a flax (Linum usitatissimum L.) gene expression oligo microarray. BMC Genomics 11: 592.
  • HotteNS and Deyholos MK (2008) A flax fibre proteome: identification of proteinsenriched in bast fibres. BMC Plant Biol 8:52.
  • Ragupathy R, Rathinavelu R and Cloutier S (2011) Physical mapping and BAC-end sequence analysis provide initial insights into the flax (Linum usitatissimum L.) genome. BMC Genomics 12: 217
  • Roach MJ, Mokshina NY, Snegireva AV, Badhan A, Hobson N, Deyholos MK and Gorshkova TA (2011) Development of cellulosic secondary walls in flax fibers requires {beta}-galactosidase. Plant Physiol. May 19. [Epub ahead of print].
  • Smýkal P, Bačová-Kerteszová N, Kalendar R, Corander J, Schulman AH and Pavelek M (2011) Genetic diversity of cultivated flax (Linum usitatissimum L.) germplasm assessed by retrotransposon-based markers. Theor Appl Genet 122: 1385-97.
  • Venglat P, Xiang D, Qiu S, Stone SL, Tibiche C, Cram D, Alting-Mees M et al. (2011) Gene Expression Analysis of Flax Seed Development. BMC Plant Biol 11: 74.
  • Wiesnerova D and Wiesner I (2004) ISSR-based clustering of cultivated flax germplasm is statistically correlated to thousand seed mass. Mol Biotechnol 26:207–214.

Hemp (Cannabis sativa)