Cytogenetic Formation Mechanism And Genetic Characters Of Resistance To Fusarium Wilt In Cucumber Alien Translocation Line

Fusarium wilt is a serious destructive disease for cucumber in the world. However, theintraspecific genetic variation for disease resistance is very limited in cucumber. Alientranslocation line is usually cross- compatible to the receptor cucumber parent, thus it isvery important to the transferring of alien useful traits. In order tO exploiting cucumbergene resources resistant to fusarium wilt, the series progenies derived from the interspecifichybridization performed in our lab between Cucumis hystrix Chakr. and cucumber, i.e. theamphidiploid C.×hytivus Chen & Kirkbride, allotriploid, alien translocation line AT-04 andthe allied genernations derived from the crossing between AT-04 and suspectible cultivarCC₂, were adopted as primary researching materials in present paper. For alientranslocation plant AT-04, its agronomic and cytogenetic traits, cytogenetic formationmechanism were investigated in detail. And based on the isolation and comparison offusarium wilt fungi from cucumber and C. hystrix, and on the evaluation of severalinoculation methods, characters of genetics and heterogeneity of resistance gene to thefusarium wilt in cucumber alien translocation line were revealed in detail.1 Cytogenetic formation mechanism and characters of the cucumber alientranslocation lineIn order to improve the accuracy of chromosome analysis for cucumber alientranslocation line, mitotic chromosome behaviors at prophase, karyotype at metaphase andC-banding at prometaphase of cucumbe (C. sativus cv. Beijing Jietou, 2n=2x=14) werestudied using cycloheximide in pretreatment in vivo. Prophase chromosome distributedspatially in a circle form, while prometaphase chromosomes varied from 3 to 8μm in length.Twenty one stable and distinct C-bands in a haploid complement could be identified atprometaphase, including 12 telomeric-, 7 centromeric-, 1 intercary- and 1 satellite- bands.Length of metaphase chromosomes was from 1.65 to 2.30μm. Karyotype formula ofcucumber was 2n=14=12m (SAT)+2 sm with the satellite on the long arm ofchromosome 3.In order to reveal the formation mechanism of the alien transloctaion line (ATL) in Cucumis, genetics exchange between homologues through chromosome pairing in theamphidiploid and allotriploid was investigated in present paper. The multivalents wereobserved in～52ï¼…pollen mother cells (PMCs) and the configuration formula was 38=0.56â… +17.36â…¡+0.35â…¢+0.26â…£+0.046â…¤+0.056â…¥for the amphidiploid. Meanwhile,genome separation, i.e. chromosomes from cucumber and C. hystrix distributing at differetspace in PMC, was observed through prophaseâ… to anaphaseâ… in the amphidiploids. And attelophaseâ…¡, 7 chromosomes were observed to separate into one pole in about 1ï¼…PMCs. Inallotriploid, a low frequency of trivalents (0.22～0.24ï¼…)was observed existing at diakinesisand prophaseâ… . And 7 chromosomes were also detected to separated into one pole both atanaphaseâ… and telophaseâ…¡. Some backcrossed progenies from mating amphidiploid- andallotriploid- to cucumber was found to have 2n=14 chromosomes, which confirmed theviability of the gametes from the separation of 7 chromosomes to one pole. Furthermore,the alien DNA fragments from C. hystrix was detectedd in line HH_1-8-1-2 derived from themating amphidiploid to cucumber, which proved there is ATL in this population. Thus,genetic exchange between homeologous chromosomes and the subsequent genomeseparation might be an improtant mechanism for the formation of cucumber ATL.In morphology, the plant AT-04 was characterized to have small-sized leaf, short fruit,brown spine and multi-branching habit, which were closely similar to those of the originalparent wild species-C. hystrix, and had a fertility as high as that of cultivated cucumber.Cytological observation in AT-04 found that sister chromatids in at least one pair of somaticchromosomes were delayed obviously in separation at late metaphase. At diakinesis, 0.2tetravalents, 0.05 hexavalents and 0.05 octovalents per pollen mother cell (PMC) weredetected. In 30ï¼…PMCs, crossed chromosomes occurred at diakinesis. At metaphase I, 7bivalents were frequently detected to separate into three groups, with a form of 3+2+2,indicating that at least four translocation events once occurred. C-banding analysis of thefirst selfed progeny of AT-04 (AT-04S₁) revealed that morphology variation of chromosomeno 4 and 5 had occurred at mitotic prometaphase. Field investigation on plants alsoconfirmed a relative high resistance to fusarium wilt both exisiting in AT-04 and its firstselfed progeny (AT-04S₁).2 Genetic characters of resistance to fusarium wilt in cucumber alien translocationline AT-04To provide infectible fusarium fungi for cucumber disease resistance breeding, three types of fusarium wilt fungi were isolated from the diseased cucumber plants, including F.oxysporum, F. solani and F. equiseti. And Fusarium oxysporum is the major infectingpathogeny in the cucumber production. The Fusarium oxysporumcan form 3 differentcolonies. The big spores were hook-shape and most of them had 3 space components. Thesmall spore was elapis without space. The spore-producing cell was bottle neck-shaped andseparative. The purified Fusarium oxysporum strain Foc-W01 can infect cucumber.In order to screen the resistance in C. hystrix from the fungi respects, morphologicaltraits, POD isozyme patterns and pathogenicity of Fusarium oxysporum, isolated from wildspecies C. hystrix and cucumber, were compared. The results showed characters of the twofungi cultured in PSA medium differed from each other singificantly. And the twopathogenies had different number of peroxidase (POD) isozyme bands. A strongpathogenicity was found in fungi from C. hystrix when using cucumber seedlings asmaterias, while a weak one in fungi from cucumber to C. hystrix seedling was detected.Using cucumber cultivars Changchunmici, Jinyan no.4 (CC₄), Erzaozi (CC₂),interspecific hybrid progeny HH_1-8-1-2, and some hybrid F₂ plants derived from the matingHH_1-8-1-2 (R)×CC₂ (S) as materials, efficiencies of the identification methods, includingsuppression of seed germination, radicel inoculation, soaking roots and peroxidase (POD)enzyme electrophoresis analysis, were evaluated in present paper. The results showed thatthe suppression of radicel growth and seeds germination treated with crude toxin solutioncan partially reflex difference between the resistant and susceptible plants. Meanwhile,some theoretical disadvantages were also found existing in the wide used radicelinoculation method-screening the sprouted seedlings. Inoculation methods of soaking rootin fungi and filling fungi into the base of roots were both proved to be accurate in thediscrimination of resistant and susceptible cucumber germplasm, but with a long latentperiod. And POD enzyme electrophoresis analysis is proved to be an rapid and effectiveasistant selection method in the resistance evaluation at the begin of breeding program.In order to reveal the resistance genetic law of cucumber alien translocation line tofusarium wilt, the cucumber alien translocation line AT-04 (high resistant) and cucumbercultivar CC₂ (high susceptible) were used as parents to construct the allied generations.When resistant line AT-04 was used as maternal parent, the distribution of resistant gene inits progenies of F₁, F₂, BC₁P₁, BC₁P₂ fit the genetic model of one pair of domiant genes.But when the suspectible cultivar CC₂ as maternal parent, the distribution of resistant genein its progenies of F₁, F₂, BC₁P₁, BC₁P₂ didn't fit the model of one pair of dominatn genes anymore, and disease resistance of F₁, BC₁P₁, BC₁P₂ and F₂ population was weaker as awhole than that of the respective population derived from the crossing with AT-04 asmaternal parent. Meanwhile, in general, a higher disease resistance was detected in the BC₁generation than that in the F₁ generation. The POD isozyme analysis revealed that densityof isozyme bands is negatively related to the resistance to fusarium wilt, and thus can beused to distinguish the disease resistance in the same population.The heterogeneity of resistanc gene to fusarium wilt in line AT-04 was evaluated bytechniques of RAPD, SSR and AFLP. When amplified by primers for RAPD reportedlinked to resistance to fusarium wilt in cultivated cucmber, no expected DNA fragmentswas attained neither from resistance nor suspectible gene pool. And no expected DNAfragments linked to the resistance between the two gene pools were either obtained whenamplified by the the SSR (CSWCT06A) or by the 5 pairs of degenerate primers synthesizedaccording to the resistance gene analog (RGA). DNA bands with expected weight weredetected in the resistant gene pool of AT-04, wild species C. hystrix, reciprocal F₁ whenamplified by primers of AFLP (E25/M70), but in a dominant genetic model not in acodominat one. Meanwhile, when amplified by the RAPD primer AI-5, an additionalpolymorphic DNA fragment with a weight of～1600bp was detected in the gene pool of C.hystrix, AT-04 and AT-04×CC₂, but not in that of the CC₂×AT-04. In a word,heterogeneity of resistance gene to the fusarium wilt was found existing between thecucumber alien translocation line and those reported in the cultivated cucumbers.