Current methods in retrotransposon based marker production and utilization

Doc. Ruslan Kalendar

Visit Program to Egypt: FCRI-Institute of Field Crops Research, ARC, Egypt; 30.03 - 04.04.2014

RAPD (Random Amplified Polymorphic DNA)

Theoretically any primers: from 10 to 18 bases, 40-80% GC single or several primer(s) per reaction Simple reaction set-up Comfortable for study many species But: The reaction is sensitive to reaction conditions

ISSR (Inter-Simple Sequence Repeat)

Specialized RAPD primers: simple repeat with selective base: 5-САСАСАСАСАСАСАG Simple reaction set-up Comfortable for study many species

Marker methods

SSRs

RAPDs

SNPs

TKW Association genetics

Plant species display remarkable variations in genome size

Fritillaria davisii 12.9 x 1010 bp

largest diploid Pinus edulis, 3 x 1010 bp

Zonneveld BJM 2010. J. Bot. http://www.hindawi.com/journals/jb/2010/527357.html

Plant species display remarkable variations in genome size

Brachypodium distachyon 272 Mbp Arabidopsis thaliana, 170 Mbp

Genome size varies even within plant genera

Drosophila 15%

Human 45%

Arabidopsis 14%

Lilium 99%

Maize 50-60%

Retrotransposons form the greatest proportion of the genome

The C-value paradox

How much DNA do all the genes occupy? 30,000 genes x 4800 bp = 1.44 x 108 bp for genic portion of “average”

genome

• 90 % of the Arabidopsis genome • 1.3 x 108 bp, 25498 genes

• 51 % of the rice genome

• 42 653 genes

• 4.8 % of the barley genome •50 000 genes?

• 0.27 % of the Fritillaria genome !

What is the rest of the genome if not genes?

•Microsatellites and other simple repeats •DNA transposons (Class II transposable elements)

•Retrotransposons (Class I transposable elements)

•Unidentified sequence

Retrotransposons form the greatest proportion of the genome by number and by bulk!

Two groups of RNA derived retrotransposons

DNA transposons (Class II Elements)

Retrotransposons (Class I Elements)

DNA

AAAA n

RNA

rev. transcription

DNA

Retrotransposition is inherently replicative

Sukkula Sabrina LINE

BAGY-2 Nikita MITE

BARE-1 En/Spm gene

Retrotransposons often form nested insertions in large cereal genomes

Retrotransposon-based molecular marker methods

Kalendar R, Flavell A, Ellis THN, Sjakste T, Moisy C, Schulman AH 2011. Analysis of plant diversity with retrotransposon-based molecular markers. Heredity, 106: 520-530. Kalendar R 2011. The use of retrotransposon-based molecular markers to analyze genetic diversity. Field and Vegetable Crops Research, 48(2): 261-274.

Multiplex products of various lengths from different loci are indicated by the bars beneath the diagrams of each reaction. (a) The SSAP method. The geometry of amplification from a DNA

fragment cut with two restriction enzymes (El, E2), containing a retrotransposon LTR (shaded, labeled) and ligated to an adapter (stippled) is shown. Primers used for amplification match the adapter (PA) and retrotransposon (PT).

(b) The IRAP method. Amplification takes place between retrotransposons (internal regions hatched) near each other in the genome (open bar), using retrotransposon primers (PT). The elements are shown oriented head-to-head, using a single primer.

(c) The REMAP method. Amplification takes place between a microsatellite domain (vertical bars) and a retrotransposon, using a primer anchored to the proximal side of the microsatellite (PM) and a retrotransposon primer (PT).

(d) RBIP. Full sites, depicted left, are scored by amplification between a primer in the flanking genomic DNA (here, primer PFL matches the left flank) and a retrotransposon primer (PT). The single product is shown as one bar beneath the diagram. The alternative reaction between the primers for the left and right flanks, PFL, and PFR, respectively, is inhibited in the full site by the length of the retrotransposon. The product that is not amplified is indicated by the grey bar beneath the diagram. The flanking primers are able to amplify the empty site, right, depicted as a bar beneath the diagram.

Retrotransposon-Based Marker Assays

IRAP (Inter-Retrotransposon Amplified Polymorphism)

Retrotransposon-Based Marker Assays

REMAP (REtrotransposon-Microsatellite Amplified Polymorphism)

IRAP - REMAP

Specialized primers from conserved regions in LTR Single or combination of primers per reaction Simple reaction set-up Comfortable for study only closely related species Highest polymorphism and reliable amplification But : Cloned sequences necessary for new retrotransposons for new species

Kalendar R, Schulman AH 2006. IRAP and REMAP for retrotransposon-based genotyping and fingerprinting. Nature Protocols, 1(5): 2478 - 2484. Kalendar R, Grob T, Regina MT, Suoniemi A, Schulman AH 1999. IRAP and REMAP: Two new retrotransposon-based DNA fingerprinting techniques. Theoretical and Applied Genetics, 98: 704-711.

Retrotransposon-Based Marker Assays

IRAP (Inter-Retrotransposon Amplified Polymorphism)

H.spontaneum lines

IRAP with Sukkula LTR primer (9900) with species of tribe Triticeae. 1 – H.perica, 2 – T.aestivum; 3 - T.durum, 4 - Ag.taushii, 5 - T.dicoccoides, 6 - S.cereale, 7 - S.strictum, 8 - H.

erectifolium, 9 - H.pussilum, 10 – H.marinum, 11 – H.murinum, 12 - H.spontaneum, 13 - H.patagonicum, 14 - H.muticum, 15 - H.roshevitzii, 16 - H. euclaston, 17 - H.brachyantherum, 18 – El.repens, 19 - Er.distans, 20 - Er.triticeum, 21 - L.elongatum, 22 - T.caput-medusa, 23 - Ps.spicata, 24 - H.piliferum, 25 - Am.muticum, 26 - C.comosum, 27 - S.speltoides, 28 - D.villosum, 29 – Cr.delileana, 30 – A. retrofractum, 31 – Th.bessarabicum

IRAP with single primer

1-5: Brachypodium distachyon lines; 6: Triticum eastivum (ABD); 7. T.durum (AB); 8. Aegilops tauschii (1704) (D); 9. Aegilops tauschii (1691) (D); 10. Triticum dicoccoides (AB); 11. T.dicoccoides (138); 12. T.dicoccoides (156); 13. Aegilops peregrina (S); 14. Phleum pratense; 15. Avena sativa; 16. Secale strictum (H4342).

M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 1. Sabrina LTR (489) 2. Wham LTR (515)

Single IRAP and two primer mixing

Nikita Nikita+Sukkula Sukkula

Retrotransposon-Based Marker Assays

18 hours 1.7% agarose gel Electrophoresis 70V

IRAP (original) +TaqI +TaiI

IRAP- PCR bands: restriction digestion

Implemented IRAP & REMAP marker systems Poaceae: Barley, Wheat, Rye, Maize, Oat, Spartina, Phleum, Brachypodium, etc.

Arecaceae: Oil Palm (Elaeis) Musaceae: Banana Brassicaceae: Brassica spp. Rosaceae: Apple, Rose, Plum, Quince, Rubus spp. Asteraceae: Sunflower Fabaceae: Pisum sativum L., Lotus corniculatus, Medicago truncatula, etc. Solanaceae: Tobacco, Tomato, Potato Linaceae: Flax Vaccinium: Blueberry Vitaceae: Grape Cucurbitaceae: Melon Clusiaceae: Mangosteen (Garcinia mangostana) Lithocarpus: Oak Ranunculaceae: Adonis sp. Campanulaceae: Adenophora liliifolia Plantaginaceae: Digitalis grandiflora Paeoniaceae: Paeonia anomala Pinaceae: Pine, Spruce Ferns: Nephrolepis exaltata, Sphaeropteris cooperi, Didymochlaena truncatula

Retrotransposon-based insertional polymorphism (RBIP)

Using three primers, RBIP yields co-dominant marker scores, which are particularly useful for phylogenetic studies because retrotransposon insertions are irreversible. In the case of a retrotransposon, a primer designed in the LTR is used together with a primer designed in the flanking region to allow the amplification of an insertion site, whereas primers specific to both the 5′ and 3′ flanking regions are used to score the corresponding empty site. TE insertions are usually more than thousands of bases long, and hence the flanking primers do not generate an amplicon from the occupied site.

Tagged microarray marker (TAM) TAM fingerprinting of two RBIP markers The basic RBIP method has been developed for high-throughput applications by replacing gel electrophoresis with array hybridization to a filter (Flavell et al., 1998; Jing et al., 2007). Initially, PCR reactions detecting the occupied sites and unoccupied sites carried out together were spotted onto membranes, and probed with a locus-specific probe. TAM is an extension of this to a microarray format (Jing et al., 2007). TAM based on the PDR1, Cyclops and Tpv LTR retrotransposons of pea has been developed for scoring thousands of DNAs for a co-dominant molecular marker on a glass microarray slide.

a

b

A total of 3263 Pisum lines were scored for the RBIP markers (a) Birte-B1 (b) 1794-2 by the TAM approach (Flavell et al., 2003; Jing et al., 2007, 2010).

Each spot represents a single sample (sample locations in the array are conserved between slides) and in these two cases a red spot indicates an occupied (retrotransposon insertion present) locus and the green spot an unoccupied locus. Yellow spots indicate an individual heterozygous for the retrotransposon insertion.

What is next for the developing molecular markers based on repeated DNA

The genome DNA contain huge amount of short and conservative for all

eukaryotes sequences: From ancient RNA world – Pol III promoters: 5S rRNA, tRNA and other small RNA; In silico search short and conservative sequences;

LTR retrotransposon structure: LTR and PBS sequence Two nested LTR retrotransposons in inverted orientations amplified from single primer or two different primers from primer binding sites. PCR product contains both LTRs and PBS sequences as PCR primers in the termini. In figure schematically showing general structure for PBS and LTR sequences, between 5’LTR(5’-..CA) and PBS (5’-TGG..3’) is spacer with several nucleotides (0-5 bases).

The inter PBS amplification (iPBS) scheme

Kalendar R, Antonius K, Smykal P, Schulman AH 2010. iPBS: A universal method for DNA fingerprinting and retrotransposon isolation. Theoretical and Applied Genetics, 121(8):1419-1430.

Ginkgo biloba Aegilops speltoides Equisetum arvense

The inter PBS amplification

Detection polymorphism maize callus lines

Detection polymorphism with animals Cow: P14 P70 P69 P34 P88

P167 P161 P212 P213 P134 P137 P16

Yak

Lama Chicken

Polymorphism between people

PCR with universal primers and detection transcription polymorphism in barley cDNA (stress and tissues)

1. CI9819, leaves 2 days old seedlings

2. CI9819, roots 2 days old seedlings 3. Adele, leaves 2 days old seedlings 4. Adele, roots 2 days old seedlings 5. Kristap, leaves 2 days old seedlings 6. Kristap, roots 2 days old seedlings 7. Rolfi, leaves 2 days old seedlings 8. Rolfi, roots 2 days old seedlings 9. Rolfi, spike 30 days, greenhouse 10. Rolfi, soil, greenhouse, leaves 11. Rolfi, cold(7 C), soil, leaves 12. Rolfi, cold(7 C), soil, leaves 13. Rolfi, cold(7 C), vermiculate, leaves 14. Rolfi, cold(7 C), vermiculate, leaves 15. Rolfi, sand, dark, 1 week old plant 16. Callus, K19 (barley Kymppi), old12 A1

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

QTL

mapping

fine

mapping

saturation

mapping

sequence

BAC

contigs

phenotype genotype

TILLING

micro-

arrays

GMOs

Prote-

omics

Meta-

balomics

B I O I N F O R M AT I C S

M A T R I X

Genomics toolbox for connecting traits to genes

crop

traits

crop

improvement

Implementation of the genomics toolbox

QTL

mapping

fine

mapping

saturation

mapping

sequence

BAC

contigs

phenotype genotype

TILLING

micro-

arrays

GMOs

Prote-

omics

Meta-

balomics

B I O I N F O R M AT I C S

M A T R I X

crop

traits

crop

improvement

Genomics decodes quantitative trait variation

9000 High throughput Illumina SNP markers with known map locations

SNPs for barley on Illumina

Hybridized Agilent chip: 44 000 barley genes

Track a gives the seven barley chromosomes. Green/grey colour depicts the agreement of anchored fingerprint (FPC) contigs with their chromosome arm assignment based on chromosome-arm-specific shotgun sequence reads. For 1H only whole-chromosome sequence assignment was available. Track b, distribution of high-confidence genes along the genetic map; track c, connectors relate gene positions between genetic and the integrated physical map given in track d. Position and distribution of track e class I LTR-retroelements and track f class II DNA transposons are given. Track g, distribution and positioning of sequenced BACs.

Landscape of the barley gene space

“A physical, genetic and functional sequence assembly of the barley genome”. The International Barley Genome Sequencing Consortium. Nature (2012) doi:10.1038/nature11543

Cereal genome synteny

15 X change, 4.9 x 109 bp (140 bp/ year)

Barley chromosomes indicated as inner circle of grey bars. Connector lines give the genetic/physical relationship in the barley genome. SNV frequency distribution displayed as five coloured circular histograms (scale, relative abundance of SNVs within accession; abundance, total number of SNVs in non-overlapping 50-kb intervals of concatenated ‘Morex’ genomic scaffold; range, zero to maximum number of SNVs per 50-kb interval). Selected patterns of SNV frequency indicated by coloured arrowheads. Colouring of arrowheads refers to cultivar with deviating SNV frequency for the respective region.

Single nucleotide variation (SNV) frequency in barley

“A physical, genetic and functional sequence assembly of the barley genome”. The International Barley Genome Sequencing Consortium. Nature (2012) doi:10.1038/nature11543