Agarose-resolvable InDel markers based on whole genome re-sequencing in cucumber.

Insertion and Deletion (InDel) are common features in genomes and are associated with genetic variation. The whole-genome re-sequencing data from two parents (X1 and X2) of the elite cucumber (Cucumis sativus) hybrid variety Lvmei No.1 was used for genome-wide InDel polymorphisms analysis. Obtained sequence reads were mapped to the genome reference sequence of Chinese fresh market type inbred line '9930' and gaps conforming to InDel were pinpointed. Further, the level of cross-parents polymorphism among five pairs of cucumber breeding parents and their corresponding hybrid varieties were used for evaluating hybrid seeds purity test efficiency of InDel markers. A panel of 48 cucumber breeding lines was utilized for PCR amplification versatility and phylogenetic analysis of these markers. In total, 10,470 candidate InDel markers were identified for X1 and X2. Among these, 385 markers with more than 30 nucleotide difference were arbitrary chosen. These markers were selected for experimental resolvability through electrophoresis on an Agarose gel. Two hundred and eleven (211) accounting for 54.81% of markers could be validated as single and clear polymorphic pattern while 174 (45.19%) showed unclear or monomorphic genetic bands between X1 and X2. Cross-parents polymorphism evaluation recorded 68 (32.23%) of these markers, which were designated as cross-parents transferable (CPT) InDel markers. Interestingly, the marker InDel114 presented experimental transferability between cucumber and melon. A panel of 48 cucumber breeding lines including parents of Lvmei No. 1 subjected to PCR amplification versatility using CPT InDel markers successfully clustered them into fruit and common cucumber varieties based on phylogenetic analysis. It is worth noting that 16 of these markers were predominately associated to enzymatic activities in cucumber. These agarose-based InDel markers could constitute a valuable resource for hybrid seeds purity testing, germplasm classification and marker-assisted breeding in cucumber.

Identification of high-efficiency SSR markers for assessing watermelon genetic purity.

Genomic simple sequence repeat (SSR) markers were used to fingerprint and determine genetic similarity (GS) of the watermelon breeding lines, as well as the purity of their hybrid derivatives. Cluster analysis and Jaccard's distance coefficients using the unweighted pair group method with arithmetic mean (UPGMA) have classified these lines into three major groups. Notwithstanding,the genetic background of these lines is narrow as revealed by the restricted GS coefficients. Fifty-five sets of SSR markers were employed in this study. Fourteen of these markers were polymorphic between the breeding lines and were used for assessing hybrid purity. Cross-checking assay validated nine SSR markers as informative SSR markers for purity detection of these hybrids. To confirm the accuracy and efficiency of these markers, their derived PCR products were further sequenced, and ClSSR09643, ClSSR18153 and ClSSR01623 were selected as high-efficiency SSR markers. Interestingly, SSR markers ClSSR09643 and ClSSR18153 were broadly applied for purity detection of more than two different hybrids, while SSR marker ClSSR01623 behaved as a specific marker forpurity detection in this study. Genetic purity of six commercial watermelon hybrids was definitely evaluated using these SSR markers. Genetic purity of all tested hybrids exceeded 96% while the field purity was above 98%. Genetic purity test was an emergency for identifying off-types and selfed female in a lot of hybrid seeds. Here, we elucidated the potential of nine SSR markers including threewith higher breeding selection efficiency. We recommended them to seed company for purity improvement of watermelon commercial hybrid varieties.

A putative positive feedback regulation mechanism in CsACS2 expression suggests a modified model for sex determination in cucumber (Cucumis sativus L.).

It is well established that the plant hormone ethylene plays a key role in cucumber sex determination. Since the unisexual control gene M was cloned and shown to encode an ethylene synthase, instead of an ethylene receptor, the 'one-hormone hypothesis', which was used to explain the cucumber sex phenotype, has been challenged. Here, the physiological function of CsACS2 (the gene encoded by the M locus) was studied using the transgenic tobacco system. The results indicated that overexpression of CsACS2 increased ethylene production in the tobacco plant, and the native cucumber promoter had no activity in transgenic tobacco (PM). However, when PM plants were treated with exogenous ethylene, CsACS2 expression could be detected. In cucumber, ethylene treatment could also induce transcription of CsACS2, while inhibition of ethylene action reduced the expression level. These findings suggest a positive feedback regulation mechanism for CsACS2, and a modified 'one-hormone hypothesis' for sex determination in cucumber is proposed.

Molecular isolation of the M gene suggests that a conserved-residue conversion induces the formation of bisexual flowers in cucumber plants.

Sex determination in plants involves a variety of mechanisms. Here, we report the map-based cloning and characterization of the unisexual-flower-controlling gene M. M was identified as a previously characterized putative 1-aminocyclopropane-1-carboxylic acid synthase gene, while the m allele that mutated at a conserved site (Gly33Cys) lost activity in the original enzymatically active allele.

Development and fine mapping of three co-dominant SCAR markers linked to the M/m gene in the cucumber plant (Cucumis sativus L.).

Owing to its diverse sex types, the cucumber plant has been studied widely as a model for sex determination. In addition to environmental factors and plant hormones, three major genes-F/f, M/m, and A/a-regulate the sex types in the cucumber plant. By combining the bulked segregant analysis (BSA) and the sequence-related amplified polymorphism (SRAP) technology, we identified eight markers linking to the M/m locus. Among them, the two closely linked SRAP markers flanking the M/m locus were the co-dominant marker ME1EM26 and the dominant marker ME1EM23. Further, the co-dominant marker ME8SA7 co-segregated with the M/m locus. With the chromosome walking method using the cucumber genomic bacterial artificial chromosome (BAC) library, we successfully developed a co-dominant SCAR marker S_ME1EM23 from the ME1EM23 sequence. Along with the other two co-dominant SCAR markers S_ME1EM26 and S_ME8SA7 (developed from ME1EM26 and ME8SA7, respectively) in a larger segregating population (900 individuals), the M/m locus was mapped between S_ME1EM26 (5.4 cM) and S_ME1EM23 (0.7 cM), and S_ME8SA7 co-segregated with it.