Alien chromosome transmission and somatic elimination in monosomic addition lines of Gossypium australe F. Muell in G. hirsutum L

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ArticleTitle _{Alien chromosome transmission and somatic elimination in monosomic addition lines of}Gossypium

australe F. Muell in G. hirsutum L. Article Sub-Title

Article CopyRight Springer Science+Business Media B.V.

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Journal Name Euphytica

Corresponding Author Family Name Sarr

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Given Name Djibril

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Organization Minist_{ère de l’Enseignement Technique et de la Formation Professionnelle}

Address Bloc 23 rue Calmette, Dakar, S_énégal

Email djibrils@hotmail.com

Corresponding Author Family Name Mergeai

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Given Name Guy

Suffix Division

Organization Universit_{é de Liège, Gembloux Agro-Bio Tech, Unité de Phytotechnie}

Tropicale et Horticulture

Address Passage des d_{éportés, 2, 5030, Gembloux, Belgium}

Email gmergeai@ulg.ac.be

Author Family Name Lacape

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Given Name Jean-Marc

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Organization Centre de Coop_{ération Internationale en Recherche Agronomique pour le}

D_{éveloppement (CIRAD)}

Address Avenue Agropolis, Montpellier Cedex 5, France

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Author Family Name Jacquemin

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Given Name Jean-Marie

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Division Département de Biotechnologie

Organization Centre Wallon de Recherches Agronomiques

Address Chauss_{ée de Charleroi, 234, 5030, Gembloux, Belgium}

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Particle

Given Name Halima

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Author Family Name Toussaint

Particle

Given Name André

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Author Family Name Baudoin

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Given Name Jean-Pierre

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Received 13 February 2011

Revised

Accepted 11 June 2011

Abstract _{The efficiency of using monosomic alien addition lines (MAALs) to introgress agronomical traits of interest}

carried by wild diploid Gossypium species into the main cultivated cotton species G. hirsutum depends on

the opportunities of confronting the alien chromosome and the recipient background genome at each generation and on the occurrence of translocations and homoeologous recombinations. The selfed-progeny

of five MAALs of G. australe in G. hirsutum was screened with SSR markers to determine the transmission

frequency of the alien chromosome and monitor its integrity. Three MAALs revealed a transmission frequency significantly lower than the expected ratio and one MAAL presented an exclusive transmission of the additional chromosome. In these four MAAL the alien chromosome was transmitted almost unaltered. With the fifth MAAL the alien chromosome was normally transmitted but was altered in half of the plants containing it. In one MAAL normally carrying brown fiber, the emergence of some plants carrying white and brown fiber revealed the somatic elimination of the additional chromosome. The loss of this chromosome seems to be triggered by its deletion.

Keywords (separated by '-') Chromosome elimination - Cotton - Gossypium - Monosomic alien addition line

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1 2

3

Alien chromosome transmission and somatic elimination

4

in monosomic addition lines of Gossypium australe F. Muell

5

in G. hirsutum L.

6 Djibril Sarr •Jean-Marc Lacape•Jean-Marie Jacquemin•

7 Halima Benbouza•Andre´ Toussaint•Jean-Pierre Baudoin•

8 Guy Mergeai

9 Received: 13 February 2011 / Accepted: 11 June 2011

10 Springer Science+Business Media B.V. 2011

11 Abstract The efficiency of using monosomic alien 12 addition lines (MAALs) to introgress agronomical 13 traits of interest carried by wild diploid Gossypium 14 species into the main cultivated cotton species 15 G. hirsutum depends on the opportunities of con-16 fronting the alien chromosome and the recipient 17 background genome at each generation and on the 18 occurrence of translocations and homoeologous 19 recombinations. The selfed-progeny of five MAALs 20 of G. australe in G. hirsutum was screened with SSR 21 markers to determine the transmission frequency of 22 the alien chromosome and monitor its integrity.

23 Three MAALs revealed a transmission frequency

24 significantly lower than the expected ratio and one

25 MAAL presented an exclusive transmission of the

26 additional chromosome. In these four MAAL the

27 alien chromosome was transmitted almost unaltered.

28 With the fifth MAAL the alien chromosome was

29 normally transmitted but was altered in half of the

30 plants containing it. In one MAAL normally carrying

31 brown fiber, the emergence of some plants carrying

32 white and brown fiber revealed the somatic

elimina-33 tion of the additional chromosome. The loss of this

34 chromosome seems to be triggered by its deletion.

35 Keywords _{Chromosome elimination Cotton}

36 Gossypium Monosomic alien addition line

37 38 39 Introduction

40 Upland cotton (Gossypium hirsutum L.), the world’s

41 leading natural fiber crop, is one of the five

allotet-42 raploı¨d species (2n = 49 = 52) of a genus which

43 also includes approximately 45 wild diploid species

44 (2n = 29 = 26) (Wendel et al. 1992). The use of

45 monosomic alien addition lines (MAALs) is one of

46 the strategy developed to transfer important

agro-47 nomic traits carried by these wild diploid species into

48 the main cultivated species (Stewart1995; Brubaker

49 et al. 1999; Ahoton et al. 2003; Mergeai 2006).

A1 D. Sarr (&)

A2 Ministe`re de l’Enseignement Technique et de la A3 Formation Professionnelle, Bloc 23 rue Calmette, Dakar, A4 Se´ne´gal

A5 e-mail: djibrils@hotmail.com

A6 H. Benbouza A. Toussaint J.-P. Baudoin A7 G. Mergeai (&)

A8 Universite´ de Lie`ge, Gembloux Agro-Bio Tech, Unite´ de A9 Phytotechnie Tropicale et Horticulture, Passage des A10 de´porte´s, 2, 5030 Gembloux, Belgium

A11 e-mail: gmergeai@ulg.ac.be A12 J.-M. Lacape

A13 Centre de Coope´ration Internationale en Recherche A14 Agronomique pour le De´veloppement (CIRAD), Avenue A15 Agropolis, Montpellier Cedex 5, France

A16 J.-M. Jacquemin

A17 De´partement de Biotechnologie, Centre Wallon de A18 Recherches Agronomiques, Chausse´e de Charleroi, 234, A19 5030 Gembloux, Belgium

Euphytica

DOI 10.1007/s10681-011-0479-x

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50 By limiting the donor genome to a single chromo-51 some, MAALs provide an effective way to identify 52 interesting genes carried by the wild species and to 53 achieve their accurate transfer to produce introgres-54 sed plants in a relatively short time (Hau 1981). 55 Comparable to a dissection of a diploid genome in the 56 same genetic background, MAALs constitute as well 57 a very useful genetic material to investigate genome 58 structure and phylogenetic relationships (Brubaker 59 and Brown 2003).

60 Introgression of desirable genes from the alien 61 chromosome to the cultivated species can be achieved 62 by use of ionizing radiation, tissue culture or chemical 63 mutagens to induce heterogenetic translocations (Jiang 64 et al.1994; Sears1993; Lapitan et al.1984). However, 65 these techniques may also cause great rearrangements 66 and aberrations in the chromosomes of the genetic 67 background resulting in production of genetically 68 non-compensated individuals (Konan et al. 2009). 69 Spontaneous translocations arising from a centromeric 70 or non-centromeric breakage and reunion is theoreti-71 cally another mechanism allowing the transfer of alien 72 chromosome fragments (Walters1950). But as trans-73 locations induced by irradiation they most likely 74 involve non-homeologous chromosomes and are 75 therefore associated with genetic imbalance due to 76 duplication of the genes brought by the alien segment 77 and deficiencies of the genes lost by the recipient 78 genome. Contrary to spontaneous or induced translo-79 cations, homoeologous recombination results in an 80 exchange of a similar-size fragment between the alien 81 chromosome and a homeologous chromosome of the 82 recipient genome and is therefore genetically compen-83 sated. In a species like wheat that possesses a meiotic 84 pairing genetic system (Chen et al.1994; Jiang et al. 85 1994), chromosomes can be ‘‘engineered’’ to induce 86 homoeologous recombination and minimize the link-87 age drag. In the genus Gossypium, which lacks such a 88 system, the only available solution to induce hetero-89 genetic associations has been to proceed by repeated 90 selfing in order to multiply meiotic events confronting 91 the donor chromosome and the recipient genome 92 (Stewart 1995; Mergeai 2006). In this perspective, 93 identification of the rare recovered recombinants, with 94 a high-throughput procedure is of a major importance. 95 But until recently detection of the recombinants has 96 been based on the time-consuming and painstaking 97 classical cytogenetic techniques combined with anal-98 ysis of the plant morphology (Rooney et al. 1991).

99 Molecular markers such as SSR, owing to their

PCR-100 based technique, wide distribution in the genome and

101 cost-effectiveness are very useful in overcoming these

102 impediments.

103 Previous analyses of alien addition stocks issued

104 from the backcrossing to G. hirsutum of the [2

105 (G. hirsutum 9 G. australe) 9 G. hirsutum] pentaploid

106 allowed the isolation of 11 MAALs of G. australe in

107 G. hirsutumout of the possible 13 MAALs (Ahoton

108 et al.2003; Sarr et al.2011). The primary objective of

109 this study was to investigate with SSR markers the

110 transmission frequency and integrity of the alien

111 supernumerary chromosome carried by five of the

112 isolated MAALs.

113 Materials and methods

114 Plant material

115 The self-progeny of five MAALs of G. australe in

116 G. hirsutumwas grown in pots in the tropical

green-117 houses of Gembloux Agro-Bio Tech. They carried

118 the G. australe chromosomes homoeologous to the

119 G. hirsutumchromosome pairs c2–c14, c3–c17, c6–c25,

120 c10–c20 and c12–c26. (Ahoton et al.2003; Sarr et al.

121

2011). The MAALs have been identified from the 122 derived backcross progenies of the allohexaploid

123 between G. hirsutum and G. australe by cytological

124 (genomic in situ hybridization) and molecular markers

125 (SSR) analyses (Sarr et al.2011). In this paper these

126 five MAALs will be designated by the combination of

127 the prefix MAAL with the chromosome number of the

128 G. australechromosome (e.g., the MAAL carrying the

129 G. australec10 homoelogous to G. hirsutum

chromo-130 some pair c10–c20 will be designated MAAL-10) (Sarr

131 et al.2011). Each of the MAALs was characterized by a

132 more or less marked altered morphology when

com-133 pared to G. hirsutum. MAAL-6 was particularly easily

134 identified: the alien chromosome turned the white-fiber

135 of the G. hirsutum cultivar into brown color (Sarr et al.

136

2011). The self-progeny of each MAAL came from a 137 unique plant. Self-pollination was forced by clipping the

138 flower bud at candle stage.

139 SSR genotyping

140 The self-progenies of the five MAALs were

investi-141 gated with SSR markers specific to the involved

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142 G. australe chromosome. The number of plants 143 analyzed for each MAAL varied between twenty and 144 ninety-eight. DNA extraction was carried out on 145 sampled leaves of 30-day-old seedlings according to 146 Benbouza et al. (2006a). PCR amplification, gel 147 electrophoresis and silver staining detection were 148 performed according to Benbouza et al. (2006b). 149 The SSR linkage groups characterizing each of the 150 G. australe chromosome were established from the 151 progeny analysis of the [2(G. hirsutum 9 G. aust-152 rale) 9 G. hirsutum] pentaploid (Sarr et al.2011) and 153 are given in Table1. Sequences of all these markers are 154 available on the websitehttp://www.cottonmarker.org. 155 Out of the ten markers characterizing G. australe 156 chromosome c6 only seven have been used in this study 157 to screen the self-progeny of MAAL-6 (Table1). 158 Statistical tests

159 v2Tests were performed to detect bias from the 3:1 160 expected ratio in the alien chromosome transmission. 161 Results

162 Alien chromosome transmission in the five 163 self-pollinated MAALs

164 Taking into account all plants carrying a supernu-165 merary G. australe chromosome, whether altered or 166 not, the alien chromosome transmission frequency of 167 all the MAALs but MAAL-2 (v2=0.6; P = 0.438) 168 were significantly different from the 3:1 expected

169 ratio. But while MAAL-10 showed exclusive

trans-170 mission of the supernumerary chromosome,

MAAL-171 3, MAAL-6, and MAAL-12 presented an alien

172 chromosome transmission frequency lower than the

173 expected ratio (Table2). Considering that each SSR

174 linkage group defines a complete G. australe

chro-175 mosome, only seventeen plants of MAAL-2 (38%)

176 and two plants of MAAL-6 (2%) carried altered

177 G. australechromosomes (Table2).

178 Somatic elimination of the supernumerary

179 chromosome in MAAL-6

180 The self-progeny plants of MAAL-6 that did not

181 carry any of the alien chromosome markers produced

182 only bolls with white-fiber. Except for one plant

183 (#13), all the plants carrying the entire markers

184 specific to the alien chromosome produced only bolls

185 with brown fiber. The plant #13 gave brown-fiber

186 bolls and white-fiber bolls (Fig. 1). Of the two plants

187 (#37 and #39) carrying altered G. autrale

chromo-188 somes (plants not carrying all the markers

character-189 istic of the G. australe chromosome), one plant (#37)

190 produced only white-fiber bolls while the other (#39)

191 produced white-fiber bolls and brown-fiber bolls.

192 Table 3 gives the relationships between the fiber

193 color and the number of the G. australe-specific loci

194 markers in the 98 self-progeny plants of MAAL-6.

195 The production of fiber of different colors by

196 plants #39 and #13 was surprising as well as the

197 exclusive production of white-fiber bolls by plant

198 #37. The hypothesis of the elimination of the alien

199 chromosome was tested. The three plants #13, #37

Table 1 SSR linkage groups characterizing the G. australe chromosomes (Sarr et al.2011)

MAAL-2 MAAL-3 MAAL-6 MAAL-10 MAAL-12 CIR202 (195)a MUSS073 (210) BNL1440 (210) NAU1182 BNL1679 (137) BNL3989 (245) MUCS620 (237) BNL1061 (155) CIR187 (290) BNL3537-2 (170) JESPR231 CIR180 BNL1047 (155) CIR171 (218) BNL1673 (185) BNL1897 (118) BNL2443 (124) BNL3436 (190) BNL3563 (210) BNL3599 (175) CIR228 (192) BNL3413 (112) BNL3359 (187) BNL119 BNL3261(190) CIR210 (102) BNL2569 (152) CIR292 (202) BNL3103 (180) BNL3259 (198) BNL3594 CIR407 (157) BNL2884 (175)

a _{Number in parenthesis gives the G. australe-specific fragment size in base pairs}

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200 and #39 were ratooned to produce new fresh leaves 201 that were sampled on different branches for DNA 202 extraction and SSR analysis. The results of this 203 analysis combined with the fiber color of the bolls are 204 given in Table4.

205 All the samples from plants #13 and #37 gave 206 results identical to the initial analysis before ratoon-207 ing while some branches of plant #39 lost the 208 G. australe-specific marker (Fig.2).

209 After ratooning the plants #13 and #37 produced

210 only bolls containing white-fiber as the branches of

211 plant #39 that lost all the G. australe markers. The

212 two branches of plant #39 that retained the

213 G. australe markers carried white-fiber and

brown-214 fiber bolls (Table4).

215 The systematic absence of brown-fiber bolls on

216 branches of plant #39 without any marker revealed

217 clearly that the appearance of the white-fiber

charac-218 ter was due to the elimination of the alien

chromo-219 some. The exclusive production of white-fiber by

220 plants #13 and #37 despite the presence of all the

221 markers is comprehensible if one keeps in mind the

222 fact that, due to the very late chromosome

elimina-223 tion, the genotype of the leaves does not necessarily

224 reflect the genotype of the fibers which are epidermal

225 cells of the seed-coat developing later after leaves

226 have been sampled (see discussion below).

227 Since the low number of bolls carried by the plants

228 grown in pots (1–3 bolls per branch) (Table 4) did not

229 allow a real understanding of the pattern of the

230 supernumerary chromosome elimination, a total of

231 104 plants of MAAL-6 self-progeny were grown in

232 the field during the summer in Senegal (West-Africa).

233 Out of the 104 plants, only two revealed mosaicism.

234 With branches much longer and carrying more bolls

235 than branches of the plants grown in pots, we

236 observed that some branches carried white-fiber and

237 brown-fiber bolls while other branches carried

white-238 fiber bolls or brown-fiber bolls only.

239 Considering two consecutive branches the

first-240 produced could carry white-fiber bolls while the

241 later-produced branches carried brown-fiber bolls.

242 Considering also two successive bolls within a

243 branch, the first-produced could carry white-fiber

244 bolls whereas the second carried brown-fiber bolls.

245 From these observations we could conclude that

246 the chromosome elimination, either inter-branch or

Table 2 Transmission frequency and integrity of the alien chromosome in the five self-pollinated MAALs

MAAL-2 MAAL-3 MAAL-6 MAAL-10 MAAL-12 Number of plants without the alien chromosome 9 (20)a _{19 (51)} _{52 (53)} _{0 (0)} _{57 (66)}

Number of plants with the complete alien chromosome 19 (42) 18 (49) 45 (45) 20 (100) 30 (34) Number of plants with an altered alien chromosomeb 17 (38) 0 (0) 2 (2) 0 (0) 0 (0) Total number of plants 45 37 98 20 87

a _{Number in parenthesis indicates the percentage}

b _{An altered alien chromosome does not carry all the markers characteristic of the G. australe chromosome}

Fig. 1 Self-progeny plant #13 of MAAL-6 carrying white-fiber and brown-white-fiber bolls

Table 3 Relationships between fiber color and presence of G. australe-specific loci markers in the 98 self-progeny plants of MAAL-6

Plants Number of markers Color of the fiber 52 Plants 0 Marker White

43 Plants All markers Brown

Plant #13 All markers White and browna

Plant #37 1 marker White

Plant #39 3 markers White and browna a _{All the seeds of a boll carry fibers of the same color}

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247 intra-branch, occurred completely at random.

More-248 over, one of the two mosaic plants observed in

249 Senegal produced one boll containing seeds with

250 white-fiber and seeds with brown fiber. However, all

251 the seeds in a loculus carried fiber of the same color

252 (Fig.3).

253 Discussion

254 Alien chromosome transmission in self-pollinated

255 MAALs

256 Alien chromosome transmission in the self-progeny

257 of MAAL-3, MAAL-6 and MAAL-12 was

signifi-258 cantly lower than the expected 3:1 ratio. Distortion in

259 the segregation ratio of MAALs in the genus

260 Gossypium is a rather common observation. Rooney

261 and Stelly (1991) found in the self-progeny of 3

262 MAALs of G. sturtianum in G. hirsutum an average

263 alien chromosome transmission of 23%. Mergeai

264 (1992) reported transmission frequencies of 52 and

265 53.8% in two MAALs involving the diploid species

266 G. areysianum. It is also well-known that

self-267 pollination in the genus Gossypium is likely to

268 overestimate the bias of the alien chromosome

269 transmission because of the differential ability to

270 transmit the alien chromosome between the male and

271 the female gametes on the one hand and because of

272 the strong selection against disomic alien addition

273 lines with the same chromosome in duplicate on the

274 other hand (Hau 1981). Female gametes are better

275 suited for transmission of the alien chromosome than

276 male gametes. Poisson (1970) reported transmission

277 frequencies by the female gamete ranging from 26.2

278 to 39.2% for six MAALs of G. anomalum in

Table 4 G. australe -specific loci markers and fiber color in three self-progeny plants of MAAL-6 Plant #13 b Plant #37 b Plant #39 b Branches of plant #13 c Branches of plant #37 c Branches of plant #39 c 1 234 561 2 3 12 3 45 6 7 Markers a BNL3436 ? ??? ??? BNL3359 ? ? ??? ??? BNL2569 ? ? ??? ??? BNL3103 ?? d ? ? ? ? ? ? ? ???? ? BNL3594 ? ? ? ? ? ? ? ? ? ???? ? Number of bolls with … White fiber 1 3 3 3 0 2 2 1 0 2203 1 0 1 1 0 0 Brown fiber 4 0 3 0 0 0 0 0 0 0001 0 0 2 0 0 0 a Only half of the 10 markers identifying G. australe c6 of MAAL-6 have been used to screen the branches of the plants #13, #37 and #39 b Constitution of the plant before ratooning c Constitution of the plant after ratooning d The sign ‘‘ ? ’’ indicates presence of the G. australe -specific fragment

Fig. 2 Loss of the G. australe-specific fragment produced by the marker BNL3103 in the self-progeny plant #39 of MAAL-6. Note: a = G. australe, h = G. hirsutum, H = Hexaploid, b =branch. The arrow indicates the G. australe-specific fragment

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279 G. hirsutumand from 0 to 5.89% by the male gamete 280 for the same MAALs. The four MAALs of 281 G. sturtianum in G. hirsutum studied by Rooney 282 and Stelly (1991) transmitted the alien chromosome 283 exclusively by the female gamete. The two MAALs 284 described by Mergeai (1992) transmitted the alien 285 chromosome through the male gamete at a rate of 2.6 286 and 6.2% only. The lower transmission of the alien 287 chromosome by the male gamete is usually attributed 288 to a lesser viability and/or competitiveness of male 289 gametophyte with an additional chromosome in the 290 pollen tube growth. On the contrary to the three 291 previous MAALs, MAAL-10 showed an exclusive 292 preferential transmission of the supernumerary chro-293 mosome. This preferential transmission was already 294 observed by Rooney and Stelly (1991) in one MAAL 295 of G. sturtianum in G. hirsutum with a transmission 296 rate through the male gamete of 90%. Becerra Lopez-297 lavalle and Brubaker (2007) also identified a 298 G. australe chromosome designated Aust-M (which 299 could be the same than the one carried by our 300 MAAL-10) transmitted at a rate of 100% by the 301 female gamete. Preferential transmission of a super-302 numerary chromosome in MAALs was also observed 303 in other species. Cameron and Moav (1957) 304 described a MAAL of Nicotiana plumbaginifolia in 305 N. tabacumwhose alien chromosome was transmitted 306 at 100% by the male gamete; alien chromosomes of 307 Aegilops sharonensis and of A. longissima in the 308 wheat background were reported to be selectively

309 retained in wheat by both male and female gametes

310 (Maan 1975; Miller et al. 1982). Maguire (1963)

311 reported a MAAL of Tripsacum in corn whose alien

312 chromosome was transmitted at 91% by the female

313 gamete.

314 If a low transmission of the alien chromosome can

315 be explained by prezygotic (meiotic irregularities

316 resulting in the loss of the alien chromosome,

317 unviability and lack of competitiveness of the male

318 gamete) or postzygotic phenomena (less viability of

319 zygotes or plants with an alien chromosome) (Rooney

320 and Stelly 1991), other mechanisms such as

apo-321 mixis, preferential segregation or gametocidal genes

322 are put forward to explain preferential transmission of

323 the alien chromosome (Rhoades1942; Maguire1963;

324 Miller et al.1982; Gao and Jung2002). Apomixis has

325 been reported to occasionally occur in hybrids of

326 Beta vulgaris and species of the section Corrolineae.

327 It has been assumed to cause the transmission of the

328 alien chromosome in a MAAL of B. corolliflora in

329 B. vulgaris at a rate of 60% through the female

330 gamete (Gao and Jung2002). Preferential segregation

331 was described by Rhoades (1942) as the passage of a

332 specific chromosome to a specific pole. He found that

333 the abnormal maize chromosome 10 with an extra

334 piece of chromatin attached to the end of the long

335 arm, observed in some strains grown in the

south-336 western of the USA, was transmitted at more than

337 70% in the ovules. He explained this distorted ratio

338 by the fact that the abnormal chromosome passed

339 preferentially to the lower pole of the spindle from

340 which the basal megaspore that gives the embryo sac

341 arises while the three other megaspores degenerate.

342 Gametocidal chromosomes carry genes that make

343 non-functional the gametes not carrying them

(Cam-344 eron and Moav1957; Jiang et al.1994; Nasuda et al.

345

1998). Numerous Aegilops species (A. cylindrica, 346 A. triuncialis, A. sharonensisetc.) are known to carry

347 these genes. The G. australe chromosome designated

348 Aust-M by Becerra Lopez-lavalle and Brubaker

349 (2007) is suspected to carry gametocidal genes as it

350 could also be the case with the alien chromosome

351 carried by MAAL-10. If that turned out to be true, a

352 powerful means will be at the disposal of cotton

353 breeders to harness G. australe for the improvement

354 of the main cultivated species. Gametocidal

chromo-355 somes from Aegilops species for example have been

356 used for mapping as an alternative to radiation

357 (Masoudi-Nedjad et al. 2005) and for introgression

Fig. 3 Boll with white-fiber and brown-fiber seed-cotton produced by a self-progeny plant of MAAL-6

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358 in wheat (Shi and Endo 1999; Shi and Endo 2000; 359 Masoudi-Nedjad et al.2002).

360 Determination of the factors causing the distorted 361 transmission in MAAL-3, MAAL-6, and MAAL-12 362 will require to test the progeny obtained by back-363 crossing to G. hirsutum as male and female parent 364 and to carry out cytogenetic studies.

365 Integrity of the alien chromosome 366 in the self-pollinated MAALs

367 Of the five MAALs, only MAAL-2 and MAAL-6 368 carried altered chromosomes that could be identified 369 using SSR markers. The fragments carried by 370 MAAL-6 self-progeny seemed not to be recombined 371 (see discussion on the somatic elimination). As for 372 the fragments carried by the MAAL-2 self-progeny, 373 one can suppose that the altered chromosomes 374 observed in 38% of the plants are not recombined 375 since G. australe belongs to the tertiary gene-pool of 376 the cultivated species which is characterized by the 377 lowest level of homoeology and pairing affinity with 378 G. hirsutum chromosomes (Stewart1995). Genomic 379 in situ hybridization (GISH) analysis of these plants 380 will be necessary to confirm this hypothesis. 381 Somatic elimination of the supernumerary 382 chromosome in plant #39 of MAAL-6

383 Fiber color mosaicism and loss of the G. australe-384 specific loci markers in some leaves in plant #39 385 of MAAL-6 clearly revealed an elimination of the 386 G. australe chromosome. Somatic elimination of 387 the supernumerary chromosome has been already 388 reported by Rooney et al. (1991) in a monosomic 389 addition line of Gossypium sturtianum in G. hirsutum. 390 Complete or partial uniparental chromosome elim-391 ination following an interspecific hybridization, 392 whether by sexual or somatic fusion hybridization, 393 is a frequently observed phenomenon which has even 394 been used to produce haploid or aneuploid plants 395 (Koba et al.1991; Riera-Lizarazu et al.1996; Kynast 396 et al.2001). But usually it is during the first zygotic 397 divisions of the hybrid embryo development, that the 398 chromosome elimination takes place. For example in 399 wheat and maize hybrids, all maize chromosomes are 400 eliminated during the first three-cell division cycles 401 in the embryo (Laurie and Bennett1989); in hybrids

402 between wheat and pearl millet, all pearl millet

403 chromosomes are eliminated between 6 and 23 days

404 after pollination (Gernand et al. 2005). When the

405 chromosomes are lost in the early stages of the

406 embryo development, the derived-plant can grow

407 with the same number of chromosomes in all its cells.

408 The very peculiarity of the chromosome elimination

409 reported here is its late occurrence, during the adult

410 plant growth.

411 After ratooning the fragment carrying the markers

412 BNL2569 and BNL3359 is lost in all analyzed

413 branches while the fragment with the markers

414 BNL3103, BNL3594, CIR407, and BNL2884 was

415 still retained in some branches. This observation

416 seems to reveal that the first fragment was less stable

417 than the second.

418 Production of some brown-fiber bolls on these

419 branches indicates that the gene responsible of the

420 brown color of the fiber is located on the retained

421 fragment carrying the markers BNL3103, BNL3594,

422 CIR407, and BNL2884.

423 The apparent inconsistency observed between the

424 results obtained with the SSR markers and the color

425 of the fiber in the plants #13 and #37 of MAAL-6 has

426 to be interpreted considering the results obtained

427 from plant #39. The important conclusion that can be

428 drawn from these results is that the chromosome

429 elimination can occur at any moment in the growing

430 plant.

431 Because the chromosome elimination can occur at

432 any moment a correlation may not exist between the

433 genotype of the leaves that gave the DNA analyzed

434 with the SSR markers and the other parts of the plant,

435 notably the fiber constituted by epidermal cells of the

436 seed-coat. This discrepancy is all the more likely

437 since fibers are among the latest produced cells.

438 The plant #37 carried only white-fiber bolls,

439 despite the fact that it carried the two same

440 G. australe-specific SSR loci as the branches of

441 the plant #39 that gave some brown-fiber bolls.

442 The number of four SSRs being too low to

charac-443 terize satisfactorily a chromosome fragment it is

444 likely that the fragments present in the two plants

445 carry the same two markers but are not identical. Two

446 hypotheses could be put forward : (i) the chromosome

447 elimination in plant #37 is more severe in the fibers

448 than in the leaves or (ii) the chromosome elimination

449 severity is the same whatever the type of cell, but the

450 fragment carried by the plant #37 has in fact lost the

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451 region carrying the gene. Considering the pattern of 452 the chromosome elimination in the plant #39, insta-453 bility in leaves being accompanied by instability in 454 the fibers, there is no reason to think that the 455 chromosome elimination varies according to the type 456 of cell. So the persistence of the four markers 457 BNL3103, BNL3594, CIR407, and BNL2884 in the 458 leaves of plant #37 combined with the regular 459 production of white-fiber favored the second hypoth-460 esis: although plant #37 and the two branches of plant 461 #39 carry the four same G. australe-specific loci, they 462 carry in fact two different fragments. The fragment 463 carried by plant #37 likely lost the gene responsible 464 of the fiber brown color and did not undergo somatic 465 elimination. As for the fragment carried by plant #39 466 it carried the gene responsible of the fiber brown 467 color and did undergo somatic elimination. Thus, 468 there seems to be a variation in the level of stability 469 according to the nature of the G. australe fragment. 470 Timing, origin and mechanisms

471 of the chromosome elimination

472 The configuration of a mosaic formation due to 473 chromosome elimination allows the determination of 474 the timing of the chromosome loss. Since a sector is 475 constituted by a cell lineage originating from the 476 same cell, the size of a sector depends on the moment 477 when the chromosome is eliminated. The largest a 478 sector is, the earliest the chromosome has been 479 eliminated. Based on this principle, Rooney and 480 Stelly (1991), observing no intra-branch mosaicism, 481 concluded that somatic elimination of the supernu-482 merary chromosome happened at a precocious stage, 483 during the formation of the branches developed from 484 a single adventitious bud. In the case studied here, the 485 existence of inter-branch, intra-branch and even an 486 intra-boll mosaicism revealed clearly that the chro-487 mosome can be lost at every moment, even until 488 flowering, in the growing plant.

489 It is most likely that the G. australe chromosome 490 elimination has been triggered by its previous break-491 age. Correlation between chromosome breakage and 492 instability is a well-known fact. McClintock (1941) 493 described how breakage of a single chromatid results, 494 after division, in the fusion of the two sister halves of 495 the broken chromosome creating a dicentric bridge that 496 will break during poleward migration in the following 497 mitotic anaphase. This breakage-fusion-bridge (BFB)

498 sequence will continue in each successive nuclear

499 division until the broken chromosome end is healed.

500 This BFB cycle can occur at meiosis as well as in

501 mitosis of somatic cells. An important question to be

502 answered in the present study, because it would help

503 understanding the mechanisms of the somatic

elimi-504 nation, is to determine whether the breakage of the

505 chromosome undergoing elimination occurred after

506 fertilization in the sporophytic tissues (maybe even

507 beginning in the embryo) or before (during

gameto-508 genesis). In the first case, the chromosome is not

509 expected to be healed. Though healing of broken

510 chromosome ends in the sporophytic tissues is not

511 impossible, it is very rare and is thought to occur only in

512 certain physiological conditions (McClintock1941). It

513 is also known that breaks induced by mechanical

514 rupture, as it is the case here, and by X-ray, contrary to

515 those caused by ultraviolet radiation, are followed by

516 fusions of broken ends of the chromosomes

(McClin-517 tock1941). So in the present case the mosaicism could

518 be explained by instability due to the BFB cycle. But

519 this explanation should result in a high-intensity of

520 variegation (Moav1961) and for example more

intra-521 boll and even intra-loculus variegation should be

522 observed. In the second case the centric fragment of the

523 broken chromosome is not expected to undergo a BFB

524 cycle but should be transmitted normally like a

525 complete chromosome because the broken end of a

526 chromosome is known to be permanently healed after

527 its passage in the embryo. So if the broken

chromo-528 some is unstable in the sporophytic tissues causing

529 mosaicism, it is more likely because the chromosome

530 fragment undergo rare aberrant mitotic anaphase

(non-531 disjunction of sister chromatids and lagging or

migra-532 tion of both chromatids to one daughter cell) resulting

533 in the deficiency of the fragment in some cell lineages

534 expressed by the mosaicism. Gupta (1968) studying

535 different plants with a chromosome fragment of

536 Nicotiana plumbaginifoliain N. tabacum found out a

537 frequency of mitotic anaphase bridges formed by the

538 N. plumbaginifoliachromosome ranging from 2.6 to

539 20.9% positively correlated with the intensity of the

540 variegation.

541 Conclusion

542 Presence of a dominant marker gene in the

543 G. australechromosome c6 provided an opportunity

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544 to detect the somatic elimination of the alien 545 chromosome. The frequencies obtained in the green-546 house and in the field are consistent and are around 547 2% of a self-progeny. Because it reduces the meiotic 548 events confronting the donor chromosome and the 549 recipient genome, the chromosome elimination is an 550 obstacle for the achievement of homoeologous 551 recombination and above all can result in dramatic 552 consequences in terms of waste of time and resources 553 if it goes unnoticed.

554 So in the breeding programs involving MAALs 555 lacking a qualitative morphological marker much 556 attention will have to be paid to the possible 557 elimination of the alien chromosome.

558 Acknowledgment We thank the Belgian Technical

Coopera-559 tion for funding the PhD scholarships of the first author.

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