After crosses, the identification of true hybrids is not only the most important step in the initiation of a breeding program but also plays a crucial role in the improvement of hybrid varieties. However, current morphological or molecular-based hybrid identification methods are time-consuming and costly approaches that require knowledge and skill, as well as specific lab equipment. In the current study, xenia, direct or immediate effect of pollen on seeds was used to identify true hybrids in the genus Pisum L. for the first time without growing F₁ plants. The current study was therefore aimed to (i) elucidate the xenia effect on seeds in intra- and interspecific crosses between P. sativum L. subsp. sativum var. sativum or var. arvense L. Poir. and its wild relatives, including P. sativum subsp. elatius (M. Bieb.) Aschers & Graebn. and P. fulvum Sibth. & Sm., and (ii) illuminate the beneficialness of the xenia effect in a practical improvement of the genus Pisum L. The pea cultivars, including P. sativum subsp. sativum var. sativum and P. sativum subsp. sativum var. arvense, were therefore crossed with P. sativum subsp. elatius and P. fulvum, and the occurrence of the xenia effect was studied on the seeds of fertilized female plants immediately after the crosses. It was concluded that using the xenia effect for the early detection of true hybrid immediately after crossing was not only the fastest, most reliable, and least expensive option as early selection criteria, but that xenia also provided information about dominant seed and pod traits after double fertilization.

Many biological processes require cell fusion, therefore defects in cell fusion result in many diseases. The unicellular green alga Chlamydomonas reinhardtii is an excellent model organism to study cell-cell fusion. The objective of this study was to identify genes that are involved in C. reinhardtii mating type minus (MT_) gamete fusion. A forward genetics approach was taken in our work. We created several MT_ fusion defective mutants using DNA insertional mutagenesis. These mutants were normal in the early stages of mating; they agglutinated with mating type plus (MT+), removed their walls, adhered to their mating partner through their mating structures, but the cells did not fuse, indicating that the DNA insertional mutants were defective in the latest stages of fusion. The number of insertions was confirmed by Southern blots. Mutant J1 had one insertion and the flanking genomic DNA was cloned by TAIL-PCR and RESDA-PCR. The insertion is in a gene predicted to be involved in 5-deoxystrigol biosynthesis.

With the availability of the complete genome, Chlamydomonas BACs are being used extensively in functional genomics analysis. The following aspects of their construction, however, make them less than optimal for some types of analysis. (1) These BACs do not contain a gene to allow direct selection of transformants in complementation analysis. (2) Co-transformation using the BAC and an independent vector with a selectable marker has a low efficiency. (3) Most BACs have more than one gene, necessitating sub-cloning of each gene into a different vector (relying on the use of restriction enzymes). To simplify this process, we modified Chlamydomonas BACs by inserting 2 selectable marker cassettes, using the RED-ET system. We ligated a eukaryotic and a prokaryotic selectable marker cassette and used it in a one-step modification instead of a two-step counter selection protocol and showed the expression of both cassettes. This method will decrease the time needed for use of BACs in functional genomics analysis in Chlamydomonas.

Sulfate is an essential macroelement for all living organisms. However, sulfate can be limited in agricultural settings. Microorganisms change their gene expression to acclimate to sulfate deficient conditions. Green microalga Chlamydomonas reinhardtii expresses and secretes extracellular arylsulfatase (ARS) under sulfate deficient conditions. Our results show that C. reinhardtii ARS can hydrolyze both sulfate monoester (5-bromo-4-chloro-3 indolyl sulfate; X-SO42-) and phosphate monoester (5-bromo-4-chloro-3 indolyl phosphate; X-PO43-) providing evidence that ARS enzyme has promiscuous activity. C. reinhardtii is found in soil and fresh water habitats in nature. This promiscuous activity can be beneficial in making both sulfate and phosphate bioavailable for uptake by soil organisms and plant roots.

C. reinhardtii has two putative SEC23 genes, CrSEC23A and CrSEC23B. The encoded polypeptides are only ~18.9% identical, suggesting that they might have different functions. It is not clear whether SEC23 paralogs have same or different functions in diverse organisms. Interestingly, our alignment and homology modeling showed that CrSEC23B does not have the conserved SEC24 binding motif (VFR), but instead appears to have an LPA motif in the same position. While LPA might be part of a novel SEC24 binding motif, CrSEC23B might have an alternate function that is either associated with or independent of COPII. Our results also show SEC23 orthologs in various organisms have variations in the putative SEC24 binding motif. Phylogenetic analyses place the SEC23 orthologs into two clusters that we designated group A (conventional; CrSEC23A-like orthologs) and group B (unconventional; CrSEC23B-like orthologs). Our results suggest that many photosynthetic organisms have a divergent SEC23 paralog. This divergence is not seen in animals. We hypothesize that divergent (unconventional) SEC23 paralogs might be the result of gene duplication and divergence that may facilitate specific aspects of trafficking. Since we only identified the B-like proteins in photosynthetic lineages, we hypothesize that B-like proteins may not have been present in the common ancestor involved in the primary endosymbiotic event. Key words: Chlamydomonas reinhardtii, SEC23 paralogs, gene duplication, secretory pathway, COPII