| Cucurbitaceae are mostly vine herbaceous plants,which are widely distributed in subtropical and temperate regions of the world.It includes Cucumis,Citrullus and so on.The genus Cucumis contains more than 50 species.Among them,cucumber（Cucumis sativus L.）,melon（C.melo L.）are important plants with economic value.C.hystrix is a wild species found in Yunnan.It has many excellent disease resistance and stress resistance genes.It is an important material for the improvement of cultivated cucumber varieties.Interspecific hybridization is an important breeding method.Then foster Cucumis sativus-hystrix alien addition lines,which can transfer some excellent genes from wild species to cultivated species,broaden the genetic background of cucumber,and realize the improvement of cultivated cucumber varieties.Alien addition lines are of great significance for excavate high-quality wild germplasm resources and for in-depth study of donor and recipient genomes.Studying the plant chromosome preparation technology is the basis for carrying out relevant research on plant chromosomes.Plant chromosome preparation technology usually uses root tip as the test material,and mostly adopts the wall-removing,hypotonic and flame drying method established by predecessors.Cut the apical meristem of root tips.After carry out cellulase and pectinase dewall treatment to tissue,use hypotonic operation to expanded cells and disperse chromosome distribution.At last,use fire to dry the slides.We use C.hystrix and C.sativus for interspecific hybridization,the allotetraploid formed by doubling was backcrossed twice in succession to obtain candidate addition line plants.In order to better apply the previous methods to the chromosome research of Cucurbitaceae,we propose an optimized chromosome prepare procedure.Because the ovaries of melon crops are mostly inferior and easy to obtain,this study proposes to use young ovary walls as materials to establish a chromosome preparation procedure suitable for Cucurbitaceous plants.And then the established chromosome preparation procedure is used to assist the identification of Cucumis sativus-hystrix alien addition lines materials.The results are as follows:1.Study on the method of chromosome preparation of ovary wall in Cucurbitaceae plants.Plant chromosome preparation usually uses seeds or plant root tips with cells in the vigorous division period as test materials,but this sampling method is likely to cause waste of seeds and damage to plants,and not applicable to precious and rare materials.Therefore,it is necessary to find a new production method.In this study,the young ovary walls of cucumbers,melons,watermelons,and West Indian cucumbers were used as materials for chromosome preparation.The effects of sample size,pretreatment time and enzymolysis time on chromosome preparation were compared and analyzed,and a method for preparing ovary materials was established.Then use the resulting slides for chromosome counting and fluorescence in situ hybridization analysis.It is found that the cell division of the young ovary of melons with a length of 0.2-1 cm is more vigorous,and more division phases can be observed in the slides.The best pretreatment time is 1h 30 min for cucumber,1h for melon,55 min for watermelon,and 45 min for West Indian cucumber.When cut the young ovary wall material into small pieces with a side length of 1-1.5 mm,the appropriate enzymatic hydrolysis time is 1h 10 min-1h 20 min.Using this method,the chromosomes of cucumber,melon,watermelon and West Indian cucumber can be clearly identified as 14,24,22 and 24,respectively.Clear and bright hybridization signals can be obtained after fluorescence in situ hybridization.It is proved that the established method is generally applicable to Cucurbitaceous plants and meets the requirements of fluorescence in situ hybridization.2.The creation of Cucumis sativus-hystrix alien addition lines.We produced BC2 generation by continuously backcrossing allotetraploids produced by interspecific crosses between wild hystrix cucumber and cultivated diploid cucumber.A total of 299 plants were obtained by planting BC2 seeds.Through the combination of GISH technology and hystrix-specific sequence markers,118 alien addition lines were identified,accounting for 55.40%of the total number of plants.Among them,there are materials with additional 1 to 3 foreign chromosomes,and the largest proportion is the MAALs.Accurate identification of the three heterogeneous addition line materials by hystrix-oligo-FISH,clear signals can be observed on the foreign chromosomes,and the identification results are consistent with those of specific molecular markers.Preliminary observations were made on the phenotypic traits of the Cucumis sativus-hystrix alien addition lines,and it was found that the overall phenotypic characteristics of most additional lines were biased towards cultivated cucumber.However,there are some differences between some traits of addition lines and diploid cultivated cucumbers.CC+H06 plants showed wavy leaf edges,and CC+H08 plants showed deep corolla nicks.3.Analysis of the transfer characteristics of foreign chromosomes in alien addition linesThrough statistical results,we found that the transmission rate of different foreign chromosomes is very different.Chromosomes 6 and 10 of hystrix have the highest probability of appearing in different addition lines,which are 46.3%and 44.9%,respectively.the ALs exist certain fertility,and many plump seeds can be produced through selfing.After carry out molecular marker identification and cytological identification of selfed progeny,we found that the transmission rate of No.6 hystrix chromosome in the selfing process reached 28.57%,indicating that selfing of heterogeneous addition lines is a feasible method to preserve foreign chromosomes.After self-crossing the two double-monomer addition lines,two MAAL materials were obtained,which provided a basis for simplifying the genetic background of the addition line and carrying out more targeted and in-depth research. |
