Cytogenetic evidence of mixed disomic and - Annals of Botany

Cytogenetic evidence of mixed disomic and polysomic inheritance in anallotetraploid (AABB) Musa genotype

Mouna Jeridi1,2, Xavier Perrier1, Marguerite Rodier-Goud1, Ali Ferchichi2, Angelique D’Hont1,and Frederic Bakry1,*

1Centre de Cooperation Internationale en Recherche Agronomique pour le Developpement (CIRAD), UMR AGAP, AvenueAgropolis, F-34398 Montpellier, France and 2Institut des Regions Arides (IRA), 4119 Medenine, Tunisia

* For correspondence. E-mail [email protected]

Received: 7 June 2012 Returned for revision: 27 July 2012 Accepted: 10 September 2012 Published electronically: 18 October 2012

† Background and Aims Edible bananas originated mainly from two wild species, Musa acuminata Colla (AA)and Musa balbisiana Colla (BB), and triploid cultivars with an AAA, AAB or ABB genome are the most widelyused. In the present study, chromosome pairing affinities are investigated in a sterile AB Indian variety and in itsfertile colchicine-induced allotetraploid (AABB) derivative to determine the inheritance pattern of the tetraploidgenotype. The potential implications of interspecific recombination and chromosomal composition of diploidgametes for Musa improvement are presented.† Methods The pairing of different chromosome sets at diploid and tetraploid levels was investigated through acombination of conventional cytogenetic and genomic in-situ hybridization (GISH) analyses of meiotic chromo-somes, leading to a likelihood model of the pairing behaviour. GISH analysis of mitotic chromosomes was alsoconducted to reveal the chromosome constitution of hybrids derived from crosses involving the allotetraploidgenotype.† Key Results Analysis of chromosome associations at both ploidy levels suggested that the newly formed allote-traploid behaves as a ‘segmental allotetraploid’ with three chromosome sets in a tetrasomic pattern, three sets in alikely disomic pattern and the five remaining sets in an intermediate pattern. Balanced and unbalanced diploidgametes were detected in progenies, with the chromosome constitution appearing to be more homogenous inpollen than in ovules.† Conclusions Colchicine-induced allotetraploids in Musa provide access to the genetic background of natural ABvarieties. The segmental inheritance pattern exhibited by the AABB allotetraploid genotype implies chromosomeexchanges between M. acuminata and M. balbisiana species and opens new horizons for reciprocal transfer ofvaluable alleles.

Key words: Musa, banana, polyploidy, homoeologous pairing, inheritance pattern, gamete selection, GISH.

INTRODUCTION

Polyploidy is highly frequent in domesticated plants (Lewis,1980; Leitch and Leitch, 2008), including very economicallyimportant vegetatively propagated crops such as banana,potato, strawberry, sugarcane, sweet potato and yams. Thesevegetatively propagated crops, which are generally heterozy-gous, are vulnerable to inbreeding depression. Investigatinghow to maximize heterozygosity in hybrids is thus a key breed-ing objective for these species (Sanford, 1983).

There are two recognized polyploid types, i.e. allopolyploidsand autopolyploids. In autopolyploids, all chromosome setsare identical or very closely related while allotetraploidsresulting from interspecific hybridization have divergentchromosome sets (Leicht and Leicht, 2008; ZhiHui et al.,2011). Allopolyploidy is considered to be the most commonmode of polyploid speciation and is frequently reported inangiosperms (Grant, 1981). Allopolyploids potentially benefitfrom ‘hybrid vigour’, by combining traits from the two parentalspecies (Otto and Whitton, 2000).

Heterozygosity transmitted to progenies by polyploidgametes is directed by the inheritance patterns of the polyploid

parents. Considering an AABB allotetraploid derived fromchromosome doubling, strict autosyndesis would lead to theformation of homologous bivalents only (Sybenga, 1969).All gametes would be heterozygous AB, with diploid hetero-zygosity being entirely transmitted to progenies throughdiploid gametes. In contrast, in autotetraploids, multivalentand bivalent associations could randomly occur duringmeiosis (Sved, 1966; Sybenga, 1975). If they derive fromsomatic doubling, the proportion of diploid parental heterozy-gosity transmitted through gametes will be 66.6 % (Sanford,1983). An intermediate ‘segmental pattern’ of genetic inherit-ance may involve homologous and homoeologous chromo-some pairing in allotetraploids (Stebbins, 1947; Sybenga,1996). Transmission of diploid heterozygosity to diploidgametes ranges somewhere between 66.6 and 100 % and is ac-companied by high gamete variability (for details seeAppendix).

Meiotic instabilities are common in newly formed interspe-cific hybrids and polyploids (Navashin, 1934; Stebbins, 1971;Grant, 1975; Sybenga, 1975; McClintock, 1984; Ramsey andSchemske, 1998; Levin, 2002; Souza-Kaneshima et al.,2010). Recombinations among homoeologous chromosomes

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with distinct structural organization could contribute to manyof the small and large genetic changes detected in newlyformed allopolyploids. In addition, the formation of univalentsand multivalents randomly dividing during anaphase I ofpolyploid meiosis complicates the orderly separation ofhomologous/homoeologous partners and can result in ex-change and compensation between chromosomes (Ramseyand Schemske, 2002). Inheritance patterns are thus directlyinfluenced by the degree of pairing affinity and subsequentchiasmata formation between different chromosome sets(Stebbins, 1971; Soltis and Soltis, 1993; Wendel, 2000;Catalan et al., 2006).

Edible bananas originated mainly from two wild species,Musa acuminata Colla (AA) and Musa balbisiana Colla(BB), and triploid cultivars with an AAA, AAB or ABBgenome constitution (Simmonds and Shepherd, 1955;Simmonds, 1966) are the most widely used. These triploidsare generally more vigorous and productive than the diploids.New triploid genotypes should therefore be sought for geneticimprovement programmes geared towards increased productiv-ity and disease and pest resistance. Seedling production is byfar the most serious operational problem in banana breeding;too much effort is given to producing seedlings rather thanscreening them (Simmonds, 1962). In this regard, erratic fertil-ity of AB genotypes was considered unsuitable for breedingpurposes. Colchicine-induced allotetraploids have otherwiseproven to be fertile, and new breeding pathways have beendesigned for the synthesis of new AAB and ABB varietiesvia crosses between M. acuminata and M. balbisiana diploidsand these allotetraploids (Bakry et al., 2009). However, thisstrategy has raised new questions on the constitution of poly-ploid gametes in Musa.

It has been demonstrated clearly that interspecific recombi-nations between M. acuminata and M. balbisiana chromo-somes do occur and may be frequent in triploid hybrids(Jeridi et al., 2011). In the present study, we first determinedhow A and B chromosomes pair and recombine duringmeiosis in an interspecific diploid AB accession and in itscolchicine-induced allotetraploid derivative (AABB), in acontext of choice and non-choice, respectively, for pairingpartners. To address this issue, conventional cytogenetic andgenomic in-situ hybridization (GISH) analyses were focusedon meiotic chromosomes of both genotypes. A likelihood-model approach was used to investigate potential chromosomeaffinity variations among the basic set of chromosomesbetween the two genomes and to deduce the inheritancepattern of the allotetraploid accession. By applying GISH onmitotic chromosomes, we also examined the impacts of this in-heritance pattern on the genomic chromosome constitution ofsome progenies. The implications of these results for bananagenetic improvement programmes are discussed.

MATERIALS AND METHODS

Plant material

Four diploid and one tetraploid Musa accessions were analysedin this study. ‘Malaccensis’ (AAw genome, internal CIRADcode II-04001001505) is a wild Musa acuminata subsp. malac-censis accession that originates from Malaysia. The three other

diploids were deposited at the International Transit Center(ITC, Katholieke Universiteit Leuven, Belgium). ‘Cameroun’(BBw genome, ITC0246) is a wild M. balbisiana accessionof undetermined origin. ‘IDN110/AAcvRose’ (AAcv genome,ITC0712) is a dessert M. acuminata cultivar that originatesfrom Indonesia. ‘Kunnan’ (ABcv genome, ITC1034) is adessert diploid interspecific variety native to India. ‘Kunnan4x’ (AABB genome) is a synthetic allotetraploid obtainedfrom colchicine-induced chromosome doubling of ‘Kunnan’(Bakry et al., 2007).

In addition, progenies from crosses involving these genotypeswere produced for chromosome constitution investigations.They consisted of eight hybrids from the cross ‘Malaccensis’(AA – C) × ‘Kunnan 4x’ (AABB – F), eight hybrids fromthe cross ‘Kunnan 4x’ (AABB – C) × ‘IDN110/AAcvRose’(AA – F) and three hybrids from the cross ‘Kunnan 4x’(AABB – C) × M. balbisiana ‘Cameroun’ (BB – F).

Crossing was conducted at the CIRAD research station inGuadeloupe (French West Indies). Hybridization was carriedout according to Menendez and Shepherd (1975). The progen-ies were transferred from Guadeloupe to metropolitan Franceas in vitro plants; multiplication, weaning and acclimatizationwere carried out in the laboratories and greenhouses at CIRADMontpellier (France).

Meiotic chromosome preparation

The meiotic chromosome preparation protocol based onacetocarmine staining used in this study was described byDessauw (1987) and Shepherd (1999) and modified by Faureet al. (1993). The meiotic chromosome preparation procedurefor GISH was described in Jeridi et al. (2011).

Mitotic chromosome preparation

Root tips were harvested from plants cultivated in pots in thegreenhouse. They were treated with 0.04 % hydroxyquinolinefor 4 h and fixed for 12 h in ethanol/acetic acid (3 : 1) atroom temperature, then for 24 h in fresh ethanol/acetic acid(3 : 1) at 4 8C, and stored in 70 % ethanol at 4 8C. Themitotic chromosome preparations were performed as describedin D’Hont et al. (2000) and modified as follows: the enzymemixture comprised 1 % (w/v) cytohelicase (Duchefa,www.duchefa.com), 1 % (w/v) pectolyase Y-23 (Duchefa)and 1 % (w/v) cellulase ‘Onozuka’ RS (Duchefa).

Fluorescence in situ hybridization (FISH) and GISH

FISH and GISH were performed on mitotic and meioticchromosomes according to D’Hont et al. (2000) and Jeridiet al. (2011) for mitotic and meiotic chromosomes, respective-ly. The 45S rDNA from wheat (Gerlach and Bedbrook, 1979)was used as probe for FISH.

For GISH, we used total genomic DNA from ‘Pahang’(M. acuminata, AA genome) and ‘Pisang Klutuk Wulung’(M. balbisiana, BB genome), and 6 ng mL21 of each parentaltotal genomic DNA probe was taken. Probes were labelled byrandom priming with biotin-14-dUTP (Invitrogen LifeTechnology, www.invitrogen.com) and digoxigenin-11-dUTP(High Prime DNA Labelling Kit, Roche, www.roche.com).

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Fluorescence images were captured separately using a cooledhigh-resolution black and white CCD camera (ORCAHamamatsu, www.hamamatsucameras.com) and a LeicaDMRXA2 fluorescence microscope (www.leicamicrosystems.com). The camera was interfaced to a PC running Volocity soft-ware (Perkin Elmer, www.perkinelmer.com).

Statistical analysis

Possible differential pairing affinities between the differentsets of chromosomes from the two species were investigatedvia pairing configuration statistics.

Pairing probability. Statistically, the pairing between hom-ologous/homoeologous chromosomes can be viewed as abinary random event (pairing or not pairing) with an asso-ciated pc probability measuring their degree of affinity. Ifthe two chromosomes are always associated, like standardhomologous chromosomes, then pc ¼ 1; if the two chromo-somes have largely diverged and are never associated, thenpc ¼ 0; intermediate pc values indicate that the two chro-mosomes are not strictly similar but still have a sufficientlevel of homology to occasionally match according to theconditions during the meiosis. From these pc probabilitiesfor all chromosomes, under some sound hypotheses, thetheoretical frequency distributions of the different pairingconfigurations can be deduced in both diploid and tetra-ploid situations.

Characterization of different pairing models. The simplest andthus easily tractable model is to assign an identical p valueto the 11 chromosomes. Of course, this minimal model isunable to detect any differential pairing and the best modelfor that would assign a pairing probability to each chromo-some. However, this full model requires the estimation of11 parameters, which is clearly over-parameterized regardingthe number of available data and cannot generate accurateparameter estimations. A pragmatic intermediate option isthus to consider that the degrees of affinity of each homoelo-gous pair, measured by pc, are not uniformly spread over thewhole 0–1 interval but can be grouped in subsets of roughlysimilar probability. Here we adopted the model family withtwo subsets P and Q, for the different combinations of Npand Nq ¼ 11 – Np chromosomes (by convention Np . Nq),assuming that the chromosomes are divided into twogroups of clearly differentiated levels of affinity, and withthe advantage of requiring the estimation of only two para-meters. Note that, for the tetraploid case, a third subset ofNs chromosomes (Np + Nq + Ns ¼ 11), with a pairing prob-ability s set at 0, was also considered to test a specific modelpresented in the Results.

Assuming that the chromosomes are independent, thepairing distribution among N chromosomes with identicalprobability can be approximated by a binomial distribution.In the diploid case, for example, the probability of a value xfor the number X of bivalents in the subset P is B(x, Np, p).Then the detection of xi bivalents in a cell ci may resultfrom the presence of xi bivalents in subset P and 0 in subsetQ, or xi – 1 in P and 1 in Q, and so on. Thus, the probability

distribution to observe xi bivalents in a cell is a sum ofproducts:

Pr(X = xi)= B(xi,N p, p) × B(0,Nq, q) + B(xi − 1,N p, p)

× B(1,Nq, q) + . . .

Model selection and pairing probability estimations. The selec-tion of the best model among the different combinations ofNp and Nq (11–0, 10–1, 9–2, and so on) and the estimationof p and q in each case were based on a likelihood maximiza-tion. The likelihood, i.e. the probability of obtaining the valueobserved in a cell under this model, is given by the above prob-ability distribution. As the cell observations are independent,the overall likelihood is the product of the likelihoods foreach cell, or their sum after logarithmic transformation:

LH = SiLog[P(X = xi)].

For a given Np/Nq model, the form of the likelihood in sums ofproducts did not allow analytical estimation of the parametersp and q and numerical estimation was required. The iterativeapproximation procedure was stopped when an accuracy of1023 was reached. Models corresponding to the best combina-tions of Np and Nq were selected on the basis of their LH andmodified likelihood accounting for the number of estimatedparameters, i.e. Akaike information criterion (AIC) andBayesian information criterion (BIC), which also accountsfor the number of useful observations.

A likelihood ratio test, which would concern only nestedmodels with the full model as reference, was not applicablehere. To facilitate model selection, we thus also calculated ax2 statistic to evaluate the goodness-of-fit of the observedpairing configuration distribution relative to the theoreticalone. This statistic tests the null hypothesis that observed andtheoretical distributions are identical, with the degree offreedom being k – 1 – l, where k is the number of classes(after grouping under-represented classes at both ends) and lis the number of estimated parameters. A statistic greaterthan the threshold for a given probability means that the hy-pothesis of identity between the estimated and observed distri-butions is rejected. In the opposite case, the hypothesis ofidentity is confirmed and thus the model correctly fits the data.

RESULTS

Genomic constitution of parental genotypes

Chromosome counts on mitotic cells of M. acuminata‘Malaccensis’ (AAw) and M. balbisiana ‘Cameroun’ (BBw)accessions confirmed their diploid status with 2n ¼ 2x ¼ 22.The ‘IDN110/AAcvRose’ accession was already confirmedas being diploid (Faure et al., 1993).

Chromosomes of ‘Kunnan’ and ‘Kunnan 4x’ were first ana-lysed by GISH to identify the genomic origin of each chromo-some, and secondly by FISH to label the rDNA sites. Thesesites, in satellite-position on chromosomes, are a major

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source of error for the chromosome counts. Indeed, rDNA sitesin banana often appear to be associated with secondary nucle-olar constrictions that delimit satellites. These satellites oftenbreak away from the main part of the chromosomes. Theymay then be confused with small whole chromosomes(D’Hont et al., 2000). The GISH and FISH results in thisstudy showed that the ‘Kunnan’ accession contained 11 Achromosomes and 11 B chromosomes, confirming its interspe-cific origin previously deduced from agromorphological char-acters. ‘Kunnan 4x’ displayed 22 A chromosomes and 22 Bchromosomes.

Chromosome pairing in the ‘Kunnan’ accession

Chromosome pairing in ‘Kunnan’ was assessed by acetocar-min staining on 77 pollen mother cells (PMCs) at metaphase I(Fig. 1). The number of observed bivalents per cell rangedfrom six to ten (Table 1), with 8.8 on average. Plates witheight, nine and ten bivalents respectively represented 33.8,42.9 and 20.8 % of the total number of observed cells, butcells with 11 bivalents were never observed. Moreover, thenumber of monovalents ranged from two to ten in metaphaseI, with a mean of 4.4 (or 2.2 pairs) per cell. No multivalentswere observed.

Chiasma frequency was evaluated over 49 well spreadPMCs. The proportion of ring bivalents (15.8 %), indicatingchiasmata on both chromosome arms, was significantlylower in comparison with rod bivalents (84.2 %), indicatingchiasma on only one arm. The mean chiasma frequency percell was 10.81, while 1.23 chiasma per bivalent was observed.

Several models of homoeologous chromosome affinity weretested for ‘Kunnan’. A first minimal model was assumed withthe same pairing probability for all chromosomes (Np ¼ 11).The other models were designed with two groups of Np andNq chromosome pairs, with respectively a probability of pand q of being associated in bivalents. Parameters p and qfor the minimal model and for the different combinations ofNp and Nq (Np + Nq ¼ 11) were estimated to maximize thelikelihood (Table 2).

The minimal model with identical pairing probability wasimmediately found to be the worst model, unable to correctlyadjust the observed data. The model likelihood was 105-foldhigher (LH increased from –47.35 to –42.59) when twogroups of chromosomes with contrasting probabilities wereassumed. The highest likelihood was obtained for Np ¼ 8and Nq ¼ 3 (Fig. 2). Fit with the observed data was excellent(Fig. 3) and this was the only model for which the x2 test didnot reject the hypothesis of identity between the observed andtheoretical distributions. Models for Nq ¼ 1 or 2 showed thelowest likelihoods, but they were still close to that of theNq ¼ 3 model, whereas they decreased rapidly for Nq valuesgreater than 3.

These results suggested that chromosome pairs in ‘Kunnan’do not form bivalents with the same frequency. The data muchmore strongly support a model with two subsets: one of eightchromosome pairs showing high affinity (probability p ¼0.939 of forming bivalents) and a second of three chromosomepairs with markedly lower pairing affinity (probability q ¼0.312 of forming bivalents).

Chromosome pairing in ‘Kunnan 4x’

GISH analysis. GISH analysis of ‘Kunnan 4x’ metaphase I pre-parations clearly highlighted A and B chromosomes (Fig. 4),

I

II

II

II

II

II II II

II II II

I5 µm

A B

FI G. 1. (A). Chromosome pairing in ‘Kunnan’ (genome AB) at metaphase I assessed by conventional cytogenetic acetocarmine staining: pollen mother cell(PMC) showing 10II and 2I. Photograph taken under natural light, magnification: ×1250. (B) Schematic interpretation of (A).

TABLE 1. Cell distribution (number and proportion) of thedifferent pairing configuration classes in ‘Kunnan’ observed on

77 pollen mother cells (PMCs) at metaphase I

Classes of pairing configuration

No. of monovalents per cell 0 2 4 6 8 10 . 10No. of bivalents per cell 11 10 9 8 7 6 , 6No. of observed cells 0 16 33 26 0 2 0Percentage* 0.0 20.8 42.9 33.8 0.0 2.6 0.0

*Percentage of cells presenting each chromosome distribution under the 77studied PMCs.


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although only centromeric and pericentromeric regions werelabelled by probes, in accordance with previous observations(D’Hont et al., 2000; Jeridi et al., 2011).

Chromosomes were superimposed throughout most of thepreparation, thus hampering complete analysis of chromosomepairing. Nevertheless, eight readable cells were observed withvarious pairing configurations.

Bivalent pairing was most frequently observed (Table 3,Fig. 4A–E). The total number of recorded bivalents rangedfrom seven to 17 per cell. Most of the bivalents resultedfrom homologous pairing, with about 42 and 48 % of AAand BB homologous pairs, respectively. Homoeologous biva-lents represented about 10 % of the observed associations.Multivalents were also observed, always associating A and Bchromosomes. Trivalents were found in different forms, e.g.V-shaped, Y-shaped and ‘frying pan’-shaped forms (Fig. 4B,G, H). These configurations included chromosomes belongingto both species organized in A/A/B or A/B/B associations(Fig. 4). Likewise, all tetravalents involved non-alternate A/A/B/B chromosome associations (Fig. 4F, I, J). Some univa-lents were also noted.

Conventional cytogenetic analysis. As for the GISH analysis, itwas hard to assess complete pairing configurations in ‘Kunnan4x’ by acetocarmin staining because of the greater number ofsuperimposed paired chromosomes. Nevertheless, 42 PMCswere observed clearly enough at metaphase I to determinethe number of mono-, bi-, tri- and tetravalents in cells (Fig. 5).

The observed proportion of bivalents ranged from aminimum of ten to a maximum of 20, with an average of15.64 bivalents per cell (Table 4). The proportion of ring biva-lents (47 % of the total number of bivalents) was higher thanobserved in the diploid ‘Kunnan’ accession (16 %). Thisresult, indicative of high similarities between associated chro-mosomes, confirmed a high number of homologous bivalentsin ‘Kunnan 4x’. A few tetravalents (1–4 per cell and 1.55on average) and trivalents (1–4 per cell and 1.14 onaverage) were observed. In addition, 1–9 monovalents percell were observed (3.1 on average) (Table 4).

With 44 chromosomes, the expected number of multiva-lents would be 7.3 per cell if all chromosomes have anequal opportunity of pairing at meiosis, in agreement withtetrasomic inheritance (Sybenga, 1975). The low multivalentfrequency observed in ‘Kunnan 4x’ suggested a mixedpairing model in which not all chromosomes have the samepairing behaviour.

Pairing models were developed in which the proportion ofhomoeologous bivalents was considered to be statistically neg-ligible in comparison with homologous bivalents. This assump-tion was supported by the low frequency of homoeologousbivalents observed in the GISH results. Tetravalent and

TABLE 2. Models of bivalent pairing while assuming two groups of Np and Nq chromosomes: maximum-likelihood estimations ofthe pairing probabilities p and q

Np 11 10 9 8 7 6Nq 0 1 2 3 4 5p 0.799 0.879 0.939 0.982 0.984 1.000q 0.000 0.171 0.312 0.476 0.558LH –47.35 –42.59 –42.19 –42.14 –44.02 –44.57param 1 2 2 2 2 2AIC 96.71 89.19 88.39 88.28 92.05 93.14BIC 96.60 88.96 88.16 88.05 91.82 92.91x2 16.27** 7.67* 3.99* 0.65 n.s. 5.53* 7.80*

Maximum log-likelihood (LH), number of estimated parameters (param), Akaike information criterion (AIC), Bayesian information criterion (BIC),x2 statistic between observed and predicted distributions. n.s.: no significant difference between estimated and observed distributions; *5 % and **1 %significant differences between estimated and observed distributions.

–390 1 2 3 4 5

–40

–41

–42

–43

Max

imum

log-

likel

ihoo

d (L

H)

Nq

–44

–45

–46

–47

–48

FI G. 2. Maximum log-likelihood of bivalents/cell in ‘Kunnan’ for models as-suming two groups of Np and Nq chromosomes (Np + Nq ¼ 11).

0

5

10

15

20

25

30

Fre

quen

cy (

%) 35

40

45

50Observed

Np = 8, Nq = 3

11109876543210Bivalents per cell

FI G. 3. Distribution of bivalents per cell for ‘Kunnan’: observed (white) andunder the model Np ¼ 8 and Nq ¼ 3 (black).


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trivalent data were grouped under the generic term ‘multiva-lent’ (more than two chromosomes involved) as a trivalent ne-cessarily resulted from interspecific chromosome pairing and

could be considered as an ‘incomplete tetravalent’ in whichone chromosome is disjoined as monovalent by the absenceof chiasma on its two arms (Sybenga, 1975). Likewise,

5 µm 5 µm

5 µm

5 µm

A B

C

F

D

E

G

H

I

J

FI G. 4. GISH study of chromosome pairing in ‘Kunnan 4x’ (genotype AABB). (A, B, C, F) Meiotic plates of ‘Kunnan 4x’. Superimposition of the three colours:DAPI staining chromosomes in blue, A genomic DNA revealed in green (FITC), B genomic DNA revealed in red (Texas red). (D) Higher magnification of part of(C): two homoeologous bivalents and one homologous balbisiana bivalent. (E) Schematic interpretation of (D). (G) Higher magnification of part of (B): homo-eologous ‘frying pan’ trivalent (two chromosomes A + 1 chromosome B). (H) Schematic interpretation of (G). (I) Higher magnification of part of (F): homo-

eologous B/B/A/A/ tetravalent. (J) Schematic interpretation of (I). Arrows indicate bivalents and circles indicate multivalents. Magnification: ×1000.


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bivalents and the remaining pairs of monovalents not complet-ing a trivalent were grouped under the generic term ‘bivalent’(two or fewer chromosomes involved). These data are summar-ized in Table 5.

As for the diploid study, several chromosome associationmodels were compared according to their likelihood. Three hy-potheses were tested: (1) a first model where all chromosomesets have the same pairing affinity; (2) a second class of

TABLE 3. Chromosome configurations at (a) metaphase I and (b) anaphase I in ‘Kunnan 4x’ (AABB) revealed by GISH

(a) Metaphase I

Readablechromosomes/44

IIHomeologous

AB

IIHomologous

AA

IIHomologous

BB

Total ofbivalents/

cell III IVI chromosome

BI chromosome

A

Cell1

38/44 2 8 6 16 1(2A + 1B) 0 3 0

Cell2

24/44 2 3 5 10 0 1(2B + 2A) 0 0

Cell3

34/44 5 5 7 17 0 0 0 0

Cell4

27/44 2 6 4 12 1(2B + 1A) 0 0 0

Cell5

18/44 0 3 4 7 0 1(2B + 2A) 0 0

Cell6

23/44 0 5 5 10 1(2A + 1B) 0 0 0

Cell7

16/44K 0 3 5 8 0 0 0 0

Cell8

37/44 2 7 8 17 1(2A + 1B) 0 0 0

(b) Anaphase I

Pole 1 Pole 2

Anaphase I (1) 10B + 11A 12B + 11AAnaphase I (2) 11B + 11A 11B + 11A

Bivalents were divided into homologous and homoeologous bivalent classes. The chromosome composition of the complex configurations is also presented.A, acuminata (chromosome); B, balbisiana (chromosome); I, monovalent; II, bivalent; III, trivalent; IV, tetravalent. Pole 1 and Pole 2 indicate the twoopposing poles of the cells during anaphase I.

5 µm IIII

IV

II

II

II

II

II

I

II

III

IIII

III

II

II

II

II

II

II

II

I

A B

FI G. 5. (A). Chromosome pairing in ‘Kunnan 4x’ (genome AABB) at metaphase I assessed by conventional cytogenetic acetocarmine staining. (B) Schematicinterpretation of (A). Photograph taken under natural light; magnification: ×1250.


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models with two subsets of Np and Nq chromosomes havingdifferent homoeologous pairing probabilities; and (3) a thirdcomplementary class of models with three subsets (of Np,Nq and Ns chromosomes) adding a third level of homoeolo-gous pairing probability. This third model family was testedaccording to the results obtained at the diploid level suggestingthat several chromosome sets (probably three) were poorlypaired in interspecific associations. They would logically notbe associated in tetravalents in ‘Kunnan 4x’. The third prob-ability was therefore set at 0, and only p and q were left freefor variation. In addition, as the tetravalent formation probabil-ity cannot be greater than 2/3 (Sved, 1966; Sybenga, 1975), theupper limit for p and q was set at 2/3.

The likelihood of the first minimal model, with 11 equiprob-able chromosomes (Np ¼ 11), was markedly lower than formodels based on several groups, even after correction of thelikelihood with respect to the number of estimated parameters.In the second class of Np (from 10 to 6) and Nq (from 1 to 5)models (Table 6), the highest likelihood was recorded formodels with Np ¼ 8 and Nq ¼ 3, i.e. eight chromosome setsforming tetravalents at low frequency ( p ¼ 0.101) and threechromosome sets pairing freely in tetravalents in a tetrasomicpattern, with q reaching its upper limit of 0.66. Np ¼ 9 andNq ¼ 2 was an acceptable alternative model, with a likelihoodjust slightly lower and with close x2 values. Models with Nq ¼1, 4 or 5 clearly had a lower likelihood.

The last family of models included a third subset of Ns chro-mosomes never pairing (s ¼ 0), with Ns varying from 1 to 5,and the remaining chromosomes dispatched in Np and Nq

TABLE 4. Distribution of the different pairing configurations in‘Kunnan 4x’ (AABB) assessed on 42 PMCs at metaphase I byacetocarmin staining: I, monovalent; II, bivalent; III, trivalent;

IV, tetravalent.

Chromosome configuration

I II III IV

Range per cell 1–9 10–20 1–4 1–4Total no. of observations 130 657 48 65Mean per cell 3.10 15.64 1.14 1.55Average of the implied chromosomes on the44

3.10 31.29 3.43 6.19

TABLE 5. Cell distribution of the different multivalent andbivalent classes of metaphase I of 42 PMCs in ‘Kunnan 4x’

(AABB)

Classes of pairing configurations

Multivalents 0 1 2 3 4 5 6 .7Bivalents 22 20 18 16 14 12 10 ,8No. of observedcells

0 6 13 15 6 0 2 0

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subsets (data not shown except for Ns ¼ 3, according to thediploid results). The highest likelihoods were observed forNs ¼ 1–3 models (Table 6), but at a level close to that ofNs ¼ 0 model (Fig. 6). The likelihoods decreased rapidly forNs greater than 3. For these optimal models (Ns ¼ 1–3), asfor the second model family (equivalent to Ns ¼ 0), Nqalways consisted of at least two but more likely three chromo-some sets pairing freely in tetrasomic inheritance in ‘Kunnan4x’. According to the second model family, the Np ¼ 8 or 9other chromosome sets showed lower interspecific affinityand preferentially paired in homologous bivalents. The thirdmodel family indicated that among these eight or nine chromo-some sets, setting-up some of them (no more than three) tohomologous pairing only, as suggested from diploid results,did not reduce the resulting likelihoods, making the results atthe diploid and tetraploid levels compatible and consistent.

Chromosome constitution of ‘Kunnan 4x’ gametes and progenies

Chromosome distribution in anaphase I. Cells in anaphase I werevery rare and hard to assess by GISH. Nevertheless, two readablecells were clearly observed – one showed a balanced distribu-tion of A and B chromosomes in each daughter cell while the

second showed 10 B and 11 A at one pole and 12 B and 11 Aat the other (Table 3). These few plates demonstrated that bothbalanced and unbalanced A and B chromosome distributionsmay occur in daughter cells.

Genomic constitution of ‘Kunnan 4x’ progenies. The chromo-some constitution of several hybrids derived from three differ-ent crosses involving ‘Kunnan 4x’ was assessed by GISH. Theresults are summarized in Table 7.

Eight individuals from the cross ‘Kunnan 4x’ (AABB –C) × ‘IDN110 (AA – F)’ were analysed. Two individuals(A18 and B18) displayed 22 A and 11 B chromosomes.Three individuals (M9, I9 and D18) displayed 23A + 11Bchromosomes, having a supernumerary chromosome and atotal of 34 chromosomes (Fig. 7, Table 7). Two individuals(J9 and C18) showed a deficit of A chromosomes, with 20A + 12 B chromosomes, and a total of 32 chromosomes.One individual (E18) displayed 21 A + 12 B chromosomesand presented a deficiency on A-chromosomes and an excessof B-chromosomes.

Eight individuals from the cross ‘Malaccensis’ (AAw –C) × ‘Kunnan 4x’ (AABB – F) were analysed and all dis-played 22 A chromosomes and 11 B chromosomes.

Three individuals from the cross ‘Kunnan 4x’ (AABB –C) × M. balbisiana ‘Cameroun’ (BB – F) were studied.Two individuals (R17 and G19) displayed 11 A + 22 B chro-mosomes. One individual (E19) displayed a 10 A + 23 Bchromosome constitution. Interestingly, this E19 individualpresented three rDNA sites derived from the B-genome(Fig. 8, Table 7).

DISCUSSION

Homoeologous chromosome pairing between A and B genomes

Analytical data on pairing configurations in interspecific Musaaccessions are abundant, suggesting that partner exchanges arepossible between A and B genomes. Bivalents were frequentlyobserved in diploid AB hybrids (Agarwal, 1988). In triploids,trivalent chromosome associations were also frequentlyobserved and no marked differences in meiotic associationwere noted between auto- and allotriploids, while multivalentsin AAB and ABB were on average less frequent than in AAA(Shepherd, 1999). Likewise, multivalent formation has also

–29·5

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Ns ¼ 11) for Ns ¼ 0 (plain line) or Ns ¼ 3 (dotted line) values.

TABLE 7. Genomic constitution and chromosome number based on GISH analysis of the hybrid progenies

Cross Chromosome constitution

Female Male Genotype class A chromosomes B chromosomes Total chromosome number No. of hybrids

Cross no. 1Kunnan 4x, AABB IDN110, AAcv AAB 22 11 33 2

23 11 34 320 13 33 221 12 33 1

Cross no. 2Malaccencis, AAw Kunnan 4x, AABB AAB 22 11 33 8Cross no. 3Kunnan 4x, AABB Cameroun, BBw ABB 11 22 33 2

10 23 33 1Total number of hybrids 19


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been reported in some cross-derived AABB hybrids (Dessauw,1987; Shepherd, 1999). Evidence of homoeologous chromo-some pairing between A and B chromosomes in triploidswas obtained using GISH (Jeridi et al., 2011).

Applying GISH on the AABB ‘Kunnan 4x’ genotyperevealed that homoeologous bivalents also occur in a pairingpartner choice context, but in a lower proportion than homolo-gous bivalents. As in most allopolyploids (Stift et al., 2008;Gaeta and Pires, 2010), ‘Kunnan 4x’ was shown to fall some-where between disomic and tetrasomic inheritance, with somechromosomes pairing in homologous bivalents only, whileothers paired in homoeologous bivalents and multivalents(see Appendix). Further observations on acetocarmin-stained

cells were thus used to quantify pairing configurations and tohighlight intergenomic affinities in the AB accession and itsAABB derivative.

Pairing behaviour in the AB diploid ‘Kunnan’

The chiasma frequency observed in ‘Kunnan’ was equiva-lent to frequencies found in wild AB hybrids (1.10 % chiasmasper bivalent) and slightly superior to that reported in ‘Guindy’,which is a natural AB cultivar (0.63 % chiasma per bivalent –Shepherd, 1999). These values are all low in comparison withmean chiasma frequencies in wild M. acuminata andM. balbisiana genotypes (1.71 and 1.75 % chiasmas per

5 µm 5 µm

A B

FI G. 7. GISH analysis at mitotic metaphase in interspecific triploid Musa progeny: I9 [genotype AAB, derived from the cross ‘Kunnan 4x’ (AABB) × ‘IDN110’(AAcv)] with 23 A + 11 B. (A) After superimposition of the three colours: B genomic DNA revealed in red (Texas red) and A genomic DNA revealed in green(FITC) and DAPI counter-stained chromosomes in blue. (B) Superimposition of DAPI (blue), used as DNA marker, and 45S rDNA probe revealed in green

(FITC). Arrows indicate chromosomes with 45S rDNA. Magnification: ×1000.

A B

5 µm 5 µm

FI G. 8. GISH analysis at mitotic metaphase in interspecific triploid Musa progeny: E19 (genotype ABB) with 10 A + 23 B. (A) After superimposition of thethree colours: B genomic DNA revealed in red (Texas red) and A genomic DNA revealed in green (FITC) and DAPI counter-stained chromosomes in blue. (B)

Superimposition of DAPI and 45S rDNA probe revealed in green (FITC). Arrows indicate chromosomes with 45S rDNA. Magnification: ×1000.


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bivalent, respectively – Dodds and Pittendrigh, 1946). It islikely that the low chiasma frequency observed in ‘Kunnan’reflects a low pairing affinity for some A and B chromosomepairs, or a low chiasma frequency between already paired chro-mosomes, or a combination of the two mechanisms.

In artificial AB hybrids, previous studies reported a generalrun of cells showing from eight bivalents to complete pairing,with averages ranging from nine to 10.2 bivalents per cell(Dodds and Pittendrigh, 1946; Dodds and Simmonds, 1946).Various authors have shown a similar behaviour in naturalAB varieties (Dodds, 1943; Agarwal, 1983, 1988; Shepherd,1999). Chromosomes were usually associated with fromthree to ten bivalents per cell, with marked variations accord-ing to the authors and plant material. Nevertheless, cells with11 bivalents have been frequently found in original AB geno-types from India (Agarwal, 1983, 1988).

In our study, we obtained similar chromosome pairingresults in ‘Kunnan’. However, we observed no formation of11 bivalents despite the high number of recorded cells. Thetheoretical meiotic distribution resulting from our statisticalanalysis does not exclude possible cells with 11 bivalents,but the low probability could explain their absence in particu-lar samples. As there is strong evidence of environmentaleffects and the impact of inflorescence age on chromosomepairing (Shepherd, 1999), we consider that the meiotic behav-iour of the ‘Kunnan’ accession in the present study is similar tothose previously reported by Agarwal (1983, 1988) on prob-ably closed varieties.

In such accessions, our likelihood model approach clearlydistinguished two subgroups of pairing chromosome behav-iour. One subgroup of eight chromosome sets displayed high(but not complete) pairing affinity, suggesting a high degreeof homology between A and B chromosomes. A second sub-group of three chromosome sets presented low pairing affinity,indicating a lower degree of homology.

Pairing behaviour in the AABB tetraploid‘Kunnan 4x’

The results presented previously were obtained in a diploidcontext, where A and B chromosomes were ‘forced’ to pair.This pairing context differs from what would occur in thederivative allotetraploid in which each chromosome canassociate with either its homologous chromosome or its homo-eologous pairing partner.

Our likelihood-model approach first rejected a full tetraso-mic inheritance pattern for the overall genome in favour of amodel that divided the 11 chromosomes into two groups.One group (Group 3) consisted of three sets of chromosomespairing freely in a tetrasomic pattern (as in autotetraploids),while the pairing frequency was significantly lower for theeight other chromosome sets. The pairing affinity in Group 3thus led to numerous exchange possibilities via crossing-overor chromosome substitution between the A and B genomes.However, the higher frequency of chain tetravalents in com-parison with ring tetravalents along with the presence of multi-valents composed of non-alternate A/B chromosomeassociations indicated that chiasmata was easier between chro-mosomes belonging to the same species than between chromo-somes of the two species.

All of these observations at tetraploid level suggest globalhomoeologous relationships between the chromosomes of thetwo genomes. Nevertheless, the likelihood of the model wasnot reduced when the second subset of eight chromosomeswas split into a group of five sets of chromosomes ofmedium interspecific pairing affinity (Group 2) and anothergroup of three sets of chromosomes with very low to nopairing affinity exclusively associated in homologous biva-lents, as identified at the diploid level (Group 1). Group 2 con-sisted of chromosomes able to form multivalents at lowfrequency. They could pair in homoeologous associations, asconfirmed by GISH. However, in a pairing partner choicecontext in ‘Kunnan 4x’, they would preferentially pair in hom-ologous bivalents (as suggested by the higher frequency ofring bivalents – an indicator of high chromosome affinity onboth chromosome arms) than of rod bivalents, when comparedwith the diploid genotype. Conversely, Group 1 consisted ofthree sets of chromosomes that were assumed to pair exclu-sively in homologous bivalents according to strict disomicinheritance.

Initially, full disomic inheritance was hypothesized for‘Kunnan 4x’ because of its high gamete fertility as comparedwith the complete sterility of ‘Kunnan’. Based on the presentstudy, a mixed inheritance pattern is now proposed for‘Kunnan 4x’, with three sets of chromosomes segregating intetrasomic inheritance, three sets in disomic inheritance andfive sets in an intermediary pattern. ‘Kunnan 4x’ is consideredhere as being a ‘3/5/3’ segmental allotetraploid (Stebbins,1950, 1971; Sybenga, 1996), with chromosome associationsresulting in various configurations, including cross-structures,rings and chains between homoeologous and homologouschromosomes (Sybenga, 1975).

Divergent genome evolution

Comparisons of a few genomic regions of M. acuminata andM. balbisiana revealed a high level of microsynteny conserva-tion and colinearity between the two species that were esti-mated to have diverged approx 4.6 Mya (Lescot et al.,2008). Pairing configurations in ‘Kunnan’ and ‘Kunnan 4x’here suggested some marked differential pairing affinitiesand/or chiasma frequencies between the different A and Bchromosomes. These pairing affinity variations may thereforebe explained by large structural rearrangements involvingsome chromosomes that could have occurred during the diver-gence period. Low multivalent frequency in tetraploids isusually expected when the pairing affinity is reduced as a con-sequence of evolutionary differentiation between genomes.However, in some species, homoeologous pairing can beunder genetic control (Cifuentes et al., 2010), notably inwheat (Griffiths et al., 2006; Qi et al., 2007) and Brassica(Jenczewski et al., 2003; Nicolas et al., 2007). In Musa, no in-formation is available on a possible genetic control of chromo-some pairing. Nevertheless, we can assume that thishypothesis is improbable because, if that were the case, suchgenetic control would probably equally affect all chromo-somes. Regardless, further information on interspecieschromosome homology can be expected in the near future.The M. acuminata ‘Malaccensis’ genome A is now fullysequenced (D’Hont et al., 2012) and the B genome will


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soon be sequenced. It will thus be interesting to compare se-quencing data for the two genomes with the meiotic observa-tion data we obtained here.

Genomic composition of gametes

GISH analyses of anaphase I demonstrated the presence ofbalanced and unbalanced gametes produced by ‘Kunnan 4x’.The presence of univalents, multivalents and homoeologousbivalents may lead to such unequal chromosome distributionsduring anaphase I and, by the same token, generate aneuploidand pseudoeuploid gametes (Ramsey and Schemske, 2002). Inour study, we noted that the excess of B chromosomes coin-cided with a deficit in A chromosomes for the two crossesbetween ‘Kunnan 4x’ as female parent and the two diploidaccessions ‘IDN110’ and ‘Cameroun’. In addition, chromo-some homoeologous compensations were observed in twohybrids (E18, E19). However, despite these chromosomeexchanges, most of the hybrids were found to be triploidwith 33 to 34 chromosomes, indicating a trend in favour of aeuploid chromosome constitution.

The selection of efficient equilibrated gametes is suspectedfor pollen. Indeed, no variation in chromosome compositionwas observed for ‘Kunnan 4x’ as male parent. Conversely,the gamete chromosome composition was variable when‘Kunnan 4x’ was used as female parent. As noted byTakahashi et al. (1997), such genetic imbalances generallyresult in gamete abortion. However, some studies havepointed out that there may be lesser selection for euploidovules than for pollen cytotypes (Riley and Kimber, 1961;Ramsey and Schemske, 2002). This latter assumptionappears to be confirmed for ‘Kunnan 4x’ because our findingsindicated higher gamete equilibrium through pollen than viaovules.

Implications for Musa breeding

As seedling production is far the most serious operationalproblem in banana improvement, we proposed a breedingpathway which takes advantage of the genetic background ofnatural AB varieties by crossing their fertile colchicine-induced allotetraploid derivatives to create high quantities ofnew AAB and ABB hybrids (Bakry et al., 2009).

Heterosis is a common feature of these triploid hybrids inthe field. For most characters, the mean values obtained forthe hybrids were significantly higher than the average parentalvalues, and in some cases hybrid values exceeded that of thebest parent (Jenny et al., 2012). In allotetraploids, heterozy-gosity transmission depends on the inheritance patterns. Ourpresent study suggests that about 80–85 % of the ‘Kunnan’heterozygosity, corresponding to a figure midway between66 % (for full tetrasomic inheritance) and 100 % (for strictdisomic inheritance), would be transmitted to triploid progen-ies via the diploid gametes. This rough estimate is in line withthe hybrid vigour observed in the field.

The segmental inheritance pattern of ‘Kunnan 4x’ alsoopens new horizons as it implies a much wider range ofgametic types as compared with the strict disomic inheritancepattern. Furthermore, as the efficient female and male gametepopulations did not seem to have the same structure, we could

expect lower variability in progenies involving ‘Kunnan 4x’ asmale parent and wider variability when ‘Kunnan 4x’ serves asfemale progenitor. In addition, possibilities of chromosomeexchanges between M. acuminata and M. balbisiana opennew avenues for the reciprocal use of valuable sources ofalleles in the two species, e.g. elimination of e-BSV sequences(Iskra-Caruana et al., 2010), improvement in fruit productivityand quality for the B genome and, conversely, introgression ofdrought and salinity tolerance and disease resistance from theB genome into the A genome.

In conclusion, the segmental inheritance pattern of a duplexallotetraploid Musa genotype was highlighted through a com-bination of conventional cytogenetics, GISH and a likelihood-model approach for data processing. Further research is stillneeded to complete this pioneering study. Development ofBAC-FISH on meiotic chromosomes, as well as the use ofmany A- and B-molecular markers well spread on thegenomes and anchored to specific chromosomes, will bringsignificant added value for the study of larger progenies.Clearly, the results of the Musa genome sequencing (D’Hontet al., 2012) will facilitate implementation of these newprojects.

ACKNOWLEDGEMENTS

We thank Prof. Edmond De Langhe for his critical commentsand helpful assistance on an earlier version of this manuscript.We also thank Christophe Jenny for producing hybrids andproviding plant material. This work was supported by theTunisian Ministere de l’Enseignement Superieur de laRecherche Scientifique et de la Technologie, while M.J. bene-fited from a 23-month (2010–2011) scholarship awarded bythe European AVERROES programme and from CIRADfunding.

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APPENDIX: INHERITANCE PATTERNS ANDTRANSMISSION OF PARENTAL

HETEROZYGOSITY IN TETRAPLOIDS

Transmission of heterozygosity to progenies via polyploidgametes is directed by the inheritance patterns of polyploids,which in turn are influenced by the degree of pairing affinityand subsequent chiasmata formation of different chromosomesets. Thus, segregation patterns range from strict disomic in-heritance in true diploidized allopolyploids to full polysomicinheritance in autopolyploids (Stebbins, 1971; Soltis &Soltis, 1993; Wendel, 2000; Catalan et al., 2006).

Allotetraploid genomes consist of two sets of homoeologouschromosomes (one derived from each ancestral lineage). At themeiotic level, this results in bivalent pairing configurations inwhich chromosomes from one ancestral lineage only pair withtheir homologues from the same ancestral lineage (Soltis andSoltis, 1993; Ramsey and Schemske, 2002). This gives no op-portunities for recombination between the diverged genomes,and strict autosyndesis leads to equal distribution of eachchromosome set in daughter cells. When considering a duplexallotetraploid (denoted A1A1B1B1), strict autosyndesis willlead to the formation of homologous AA or BB bivalents only,and all gametes will be heterozygous A1B1. If derived fromchromosome doubling, it is the entire nuclear genome of the ori-ginal AB diploid that will be transmitted to progenies without re-combination, i.e. 100 % parental heterozygosity to progeniesthrough diploid gametes (Ramsey and Schemske, 2002).

In autotetraploids, each chromosome has four homologousversions. Each chromosome may then pair randomly with anyof its homologues (Stift et al., 2008), chromosomes being


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http://dx.doi.org/10.1186/1471-2164-9-58

http://dx.doi.org/10.1186/1471-2164-9-58

statistically paired about one-third in bivalents and two-thirdsin tetravalents (Sved, 1966; Sybenga, 1975). If derived froman A1A2 diploid by somatic doubling, the doubled diploidwill be diallelic duplex (A1A1A2A2), at all loci which were ori-ginally heterozygous and the genetic consequence of crossingis determined by polysomic segregation. Thus, the gameticarray from such a tetraploid will be such that gametes will bedivided into 1/6 A1A1 gametes, 1/6 A2A2 gametes and 4/6A1A2 gametes, with the last accounting for 66.6 % of the ori-ginal diploid’s heterozygosity (Sanford, 1983).

An intermediate genetic inheritance class may involvehomoeologous chromosome pairing in allotetraploids, generat-ing so-called segmental allopolyploids (Stebbins, 1947). Asegmental inheritance pattern may be derived from doubled

diploid hybrids between closely related species (Sybenga,1996). There is considerable pairing in the diploid hybrid,but as a result of preferential pairing between entirely homolo-gous chromosomes over less fully homologous chromosomes,so bivalents are mainly formed in the tetraploid. However,preferential pairing is incomplete, and some pairing betweenhomoeologous chromosomes is possible, not only in thediploid but also at the polyploidy level, where homoeologouspairing must compete with homologous pairing. For breeding,heterozygosity transmission falls somewhere between 66.6 and100 % of the transmission accompanied by numerous chromo-some exchanges and possible chromosome compensation,leading to high variability in the progeny gametes (Ramseyand Schemske, 2002).


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