AFLP and SSR polymorphism evidence levels of introgression

1

• AFLP and SSR polymorphism evidence levels of
  introgression
• from Manihot glaziovii into traditional
  varieties of cassava
• in their area of origin
• Morillo E 1., Fuenmayor F., Carvalho P.C. and
  Second G. 2
• 1 INIAP, National Department of Phytogenetic
  Ressources, DENAREF (Quito, Ecuador)

2
Manihot glaziovii
?
M.carthaginensis s.l. complex
Manioc (Manihot esculenta C.)
M.esculenta ssp.flabellifolia
M.carthaginensis
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Objectives

• To study the molecular structure of Manihot
  carthaginensis species complex

• Based on diagnostics molecular markers, to
  suggest spontaneous introgressions into the
  cassava crop from M.carthaginensis especies
  complex

• To demonstrate introgression trough comparative
  sequencing of candidate introgressed markers

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PLANT MATERIAL

• 76 accessions
• Cassava crop Mostly sweet var. (35)
• (Artif.Int.)

• ancestral sp M.e.ssp.flabellifolia (6)

Tree cassava (12) M.carth s.l
complexe M.glaziovii (10) M.carthaginensis
(6)

• Other sp. M.brachyloba (3)
• M.quinquepartita (2)

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APPROACHES

• 1. AFLPs Three primer combinations E-AGA/M-CAG
• E-AGT/M-CTC
• E-AAT/M-CCC

2. 27 SSRs (21 mapped loci) (Chavarrriaga et al.,
1998 Mba et al., 2001) Analyse per linkage
group Focus on linkage group D -
QTL for CBB resistance (Jorge et al., 2000) -
Cross resistance to CBB and CMD

• 3. Sequencing AFLP fragments
• flanquing regions of SSRs alleles

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RESULTS1. MULTIVARIATE APPROACH (PCO) OF
THE MOLECULAR POLYMORPHISM OBSERVED
224 AFLPs
27 SSRs
Correlation index (r) 0.85
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b q M.cth M.glaz
Tree cassava M.esculenta
M.flab
M.glaziovii
.
Shared alleles genic flow or homoplasy
High alellic diversity good representativity
of samples
Hybrids diversity diversity of genotypes
involved in the crosses
High heterozygoty of cassava Outbreeding
over a broad genetic base
Specific alleles of M. carth. complex ancient
separation
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2. A group of introgressed cultivars with
heterogenous origins
Artificially introgressed
SRT1316
Col22
Introgressed candidates varieties
MULTIVARIATE ANALYSIS OF CASSAVA CULTIVARS (PCO)
BASED ON 224 AFLPs MARKERS
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EVIDENCE OF INTROGRESSION AMONG LOCI BELONGING TO
A SIMPLE LINKAGE GROUP (D)
M.glaziovii
Cassava
Artif. Int.
V.Venez
Res1
Ar7
Introgressed var.
30395
MULTIVARIATE ANALYSIS (PCO) BASED ON FIVE SSR
LOCI OF LINKAGE GROUP D
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EVIDENCE OF INTROGRESSION AMONG LOCI BELONGING TO
A SIMPLE LINKAGE GROUP (F)
Introgressed var.
Cassava
M.glaziovii
MULTIVARIATE ANALYSIS (PCO) BASED ON THREE SSR
LOCI OF LINKAGE GROUP F
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3.TESTING INTROGRESSION HYPOTHESIS BY SEQUENCING
Sequencing of six AFLP candidates bandes
Some bands (4) are not confirmed to be from
homologous loci
G E E E E F
New primers designed
Do not document an introgression hypothesis
PCR Amplification
E G F
Sequencing
AFLP profile
GLAZ Bre 5-CACTATTTCCTTCATATTTCCCAAACGTTCGGTTTTC
GCAGAAACAGAG-ATGGCCATA- GCTATGTCTCTCTTCTTCTTCTTCTT
CTTCTTCTTCTT----CTGCTACTACAGCAGTGGAAAAGGCTGACCAGTG
ATCTTCTAATCGCTCCCGATTGGACTATGAATATCGATATTTGTG-3' C
ol 22 5-CACTATTTCCTTCATATTTCCCAAACGTTCGGTT
TTCGCAGAAACAGAG-ATGGCCATAAGCTATGTCTTCTTCTTCTTCTTCT
TCTTCTTCTT----------- CTGCTACTATAGCAGTGGAAAAGGCTGA
CCAGTGATCTTCTAATCGCTCCCGATTGGACTATGAATATCGATATTTGT
G-3' FLA 5-89 5-CACTATTTCCTTCATATTTCCCAAACGTTCG
GTTTTCGCAGAAACAGAG-ATGGCCATA- GCTATGTCTCTCTCTTCTTC
TTCTTCTTCTTCTTCTTCTTCTGCTACTATAGCAGCGGAAAAGGCTGACC
AGTGATCTTCTAATCGCTCCCGATTGGACTATGAATATCGATATTTGTG-
3' FLA 5-88 5-CACTATTTCCTTCATATTTCCCAAACGTTCGGT
TTTCGCAGAAACAGAG-ATGGCCATA- GCTATGTCTCTCTCTTCTTCTT
CTTCTTCTTCTTCTTCTTCTGCTACTATAGCAGCGGAAAAGGCTGACCAG
TGATCTTCTAATCGCTCCCGATTGGACTATGAATATCGATATTTGTG-3'
SRT 1344 5-CACTATTTCCTTCATATTTCCCAAACGTTCGGTTTT
CGCAGAAACAGAG- ATGGCCATA-GCTATGTCTCTCTCTTCTTCTTCTT
CTTCTTCTTCTTCTTCTGCTACTATAGCAATGGAAAAGGCTGACCAGTGA
TCTTCTAATCGCTCCCGATTGGACTATGAATATCGATATTTGTG-3' GL
AZ Af 5-CACTATTTCCTTCATATTTCCCAAACGTTCGGTTTTCGCAG
AAACAGAG-ATGGCCATA-GCTATGTCTCTCTCTTCTTCTTCTTCTT---
------------------- CTGCTACTATAGCAGCGGAAAAGGCTGA
CCAGTGATCTTCTAATCGCTCCCGATTGGACTATGAATATCGATATTTGT
G-3' BRACH 5-CACTATTTCCTTCATATTTCCCAAACGTTCGG
TTTTCGCAGAAACAGAG- ATGGCCATA-GCTATGTCTCTCTCTTCTTCT
TCTTCTT----------------------CTGCTACTATAGCAGCGGAAA
AGGCTGACCAGTGATCTTCTAATCGCTCCCGATTGGACTATGAATATCGA
TATTTGTG-3' TC 5-CACTATTTCCTTCATATTTC
CCAAACGTTCGGTTTTCGCAGAAACAGAG- ATGGCCATA-GCTATGTCT
CTCTCTTCTTCTTCTTCTT----------------------
CTGCTACTATAGCAGCGGAAAAGGCTGACCAGTGATCTTCTAATCGCTCC
CGATTGGACTATGAATATCGATATTTGTG-3' BRA 110
5-CACTATTTCCTTCATATTTCCCAAACGTTCGGTTTTCGCAGAAACAG
AGTATGGCCATA-GCTATGTCTCTCTCTTCTTCTTCTTCTT---------
------------- CTGCTACTATAGCAGCGGAAAAGGCTGACCAGTGAT
CTTCTAATCGCTCCCGATTGGACTATGAATATCGATATTTGTG-3' SRT
1316 5-CACTATTTCCTTCATATTTCCCAAACGTTCGGTTTTCGCA
GAAACAGAG- ATGGCCATA-GCTACGTCTCTCTCTTCTTCTTCTTCTT-
--------------------- CTGCTACTATAGCAGCGGAAAAGGCTGA
CCAGTGATCTTCTAATCGCTCCCGATTGGACTATGAATATCGATATTTGT
G-3
One case favors but another one rejects an
introgression hypothesis
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PHYLOGRAMMES BASED ON THE COMPARATIVE SEQUENCES
OF SSRs FLANKING REGIONS
SSR-3 clon (520 p)
SSR-40 clon (600 p)
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Conclussions

• The genetic structure of the wild
  M.carthaginensis complex in relation to cassava
  and its direct ancestor has been described. It
  includes introgression from cassava.

• Confirmation that a chromosome fragment (D
  linkage group) was introgressed from M.glaziovii
  in one of the parents of the cassava genetic map
  and also with different allelic combinations in
  various traditional cultivars originated in South
  America

• Evidences of several introgressed fragments and
  differents levels of introgressions into
  M.esculenta genome.

• Sequencing work is at present the most efficient
  tool to confirm introgression.

• Study of introgression should be a good strategy
  to mine important genes of interest.

• Chromosome areas recalcitant to introgression
  should bear good phylogenetic markers in the
  genus Manihot.

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Perspectives

• Study of introgression including other wild
  species such as M. guaranitica, M. aesculifolia.

• Sequencing of flanking regions of mapped SSR
  distributed in the whole genome

• Analysis of repeated sequences by microarray
  technique to investigate whether introgression is
  a stimulus to the dynamics of mobile elements.