Jianwu Li

Comparison of genomic SSR and EST-SSR markers for estimating genetic diversity in cucumber

Thirteen genomic microsatellite (gSSR) and sixteen expressed sequence tag (EST)-SSR (eSSR) markers were compared to estimate genetic diversity among 29 cucumber (Cucumis sativus L.) accessions. gSSR markers detected mean 4.46 alleles with a mean polymorphic information content (PIC) of 0.664, against eSSR markers with mean 3.38 alleles and a mean PIC of 0.397. gSSRs amplified more null alleles than eSSRs. Genetic diversity within the accession set was estimated by construction of dendrograms using gSSR or eSSR data. There was a clear consistency between gSSR and eSSR trees in terms of positioning of most cucumber germplasms. gSSR markers could separate various types of cucumber germplasms on the whole, although clustering of some accessions was not based on their geographical origins in eSSR tree. eSSR markers identified an independent sub-cluster containing five accessions resistant to downy mildew, suggesting a probable relationship between eSSRs and disease-resistance trait in cucumber. The Mantel test between gSSR and eSSR matrices revealed a good fit correlation (r = 0.836). The general dendrogram constructed using the combined data of gSSRs and eSSRs was similar to those obtained separately with each marker.

Utilization of a set of high-polymorphism DAMD markers for genetic analysis of a cucumber germplasm collection

Eighteen minisatellite core sequences, derived from rice, human and phage M13, were used as primers in a PCR technique, known as directed amplification of minisatellite-region DNA (DAMD), to genotype 19 cucumber (Cucumis sativus L.) accessions from a wide collection. All the primers amplified polymorphic bands across the accessions. Out of 165 bands scored, 129 were polymorphic with 78.2% polymorphism. The average of polymorphism information content of the primers was 0.844, revealing a high discrimination power in cucumber. Based on Jaccard’s similarity indices and matrix generated by the DAMD markers, a dendrogram was constructed using the unweighted pair group method using arithmetic averages and allowed for separation of the 19 accessions into four distinct groups which demonstrated genetic relationship among the different types of germplasms. Sequencing of six polymorphic amplicons resulted in the identification of only one minisatellite locus, which indicated that variation in minisatellite number was not always the factor underlying DAMD polymorphism.

Genetic relationship of a cucumber germplasm collection revealed by newly developed EST-SSR markers

Cucumber (Cucumis sativus L.) is one of the most widely cultivated vegetables, ranked fourth after tomato, onion and cabbage (Pitrat et al. 1999). It originates from India, and China is widely regarded as its secondary origin centre (Staub et al. 1997). There are a large number of cucumber germplasms spread around China and they are generally classified into six ecotypes based on the geographical distribution and ecological characters, i.e. south China ecotype, north China ecotype, Europe greenhouse ecotype, Europe openfield ecotype, occident processing ecotype and wild ecotype (Xu 1994). Although various ecotypes have been identified in cucumber, the fact that cucumber has a narrow genetic base is widely acknowledged (Staub et al. 2005), and this limits the possibilities for trait improvement using cross breeding. Genetic diversity is desirable for long-term crop improvement and reduction of vulnerability to important crop stresses. Many cucumber cultivars have been developed from closely related parents, but limited improvement in certain agricultural traits in recent years demands a more diverse range of germplasms. Molecular markers have extensively been used for studying genetic diversity and genetic relationship in cucumber, especially PCR-based markers, such as random amplified polymorphic DNA (RAPD) (Horejsi and Staub 1999), amplification fragment length polymorphism (AFLP) (Li et al. 2004), inter-simple sequence repeat (ISSR) (Wang et al. 2007) and simple sequence repeat (SSR) (Danin-Poleg et al. 2001). From all these studies, different estimates for the degree of genetic variation were obtained, reflecting the differences in the selected sets of genotypes or marker systems.

Thidiazuron and silver nitrate enhanced gynogenesis of unfertilized ovule cultures of Cucumis sativus

Gynogenesis of Chinese long cucumber (Cucumis sativus L.) was obtained from unpollinated ovules cultured on cucumber basal medium (CBM) supplemented with thidiazuron (TDZ) and in some experiments AgNO3. High induction frequencies (7.85–12.14 %) were induced from unpollinated ovules at the time of anthesis at 0.03–0.07 mg dm−3 TDZ. Histological analysis indicated that embryo sacs developed completely at the time of anthesis. Further, the highest plant regeneration rate was achieved at CBM supplemented with 0.05 mg dm−3a-naphthaleneacetic acid, 0.2 mg dm−3 6-benzyladenine and 5–10 mg dm−3 AgNO3. Flow cytometry analysis showed that 80 % of the regenerated plants were haploid. Histological micrographs and ploidy level analyses clearly revealed initiation, development, and germination of embryos from the unpollinated ovules.

Development of novel chloroplast microsatellite markers for Cucumis from sequence database

The development of chloroplast microsatellite (cpSSR) markers in Cucumis species and analysis of their polymorphism and transferability were reported. Fifteen microsatellite markers, represented by mononucleotide repeats, were developed from the complete sequence of Cucumis sativus chloroplast genome. Intraspecific variation was successfully detected in C. sativus and C. melo and revealed mean 1.6 and 1.9 alleles per cpSSR locus, respectively. With the exception of two exon region-located cpSSR markers being monomorphic, each of the others amplified polymorphic fragments in C. sativus or C. melo. A total of 34 polymorphic loci were detected with these cpSSR markers in the two species. Transferability of the newly developed cpSSR markers was checked on an additional set of 41 Cucurbitaceae accessions (belonging to 12 different species), and except for two markers with no amplification in Cucurbita maxima, the others could be transferable to all the accessions tested. Of the 15 cpSSR markers, 14 markers generated fragments with expected band sizes and 13 markers detected interspecific polymorphism among the accessions. Intraspecific polymorphism was also observed within four Cucurbitaceae species excluding C. sativus and C. melo.

Analysis of cytoplasmic variation in a cucumber germplasm collection using chloroplast microsatellite markers

Nine PCR-based markers were developed from the microsatellites in non-coding regions of chloroplast genome of Cucumis sativus and used to detect chloroplast DNA variation. These markers successfully detected intraspecific polymorphism among 37 cucumber accessions containing Chinese native germplasms (CNGs) and non-Chinese germplasms (NCGs). Each marker detected between two and four alleles and the diversity value of the makers ranged from 0.105 to 0.528. Based on the data from allele size variation, a total of 17 distinct haplotypes were identified from the 35 accessions (excluding the two accessions possessing null genes). Three haplotypes were prevalent among CNGs but most NCGs had unique haplotype. No identical haplotype was found between CNGs and NCGs, reflecting lack of exchange of CNGs with others in the 60–80s of last century. A wild species (C. hystrix Chakr.) tested herein shared a haplotype with some CNGs, suggesting that it could be the ancestry of C. sativus or at least had a common ancestral lineage. The genetic relationship among the 37 cucumber accessions was further analyzed through construction of dendrogram based on Jaccard coefficient of similarity obtained from the allele sizes. All the CNGs were clustered into a group (containing the wild accession) that distinctly differed from the other four groups containing NCGs. This result agreed with the findings above obtained from haplotype analysis. Our research documented here will offer useful information for cucumber breeding.