Asjad Ali

Identification and Functional Analysis of Pheromone and Receptor Genes in the B3 Mating Locus of Pleurotus eryngii

Pleurotus eryngii has recently become a major cultivated mushroom; it uses tetrapolar heterothallism as a part of its reproductive process. Sexual development progresses only when the A and B mating types are compatible. Such mating incompatibility occasionally limits the efficiency of breeding programs in which crossing within loci-shared strains or backcrossing strategies are employed. Therefore, understanding the mating system in edible mushroom fungi will help provide a short cut in the development of new strains. We isolated and identified pheromone and receptor genes in the B3 locus of P. eryngii and performed a functional analysis of the genes in the mating process by transformation. A genomic DNA library was constructed to map the entire mating-type locus. The B3 locus was found to contain four pheromone precursor genes and four receptor genes. Remarkably, receptor PESTE3.3.1 has just 34 amino acid residues in its C-terminal cytoplasmic region; therefore, it seems likely to be a receptor-like gene. Real-time quantitative RT-PCR (real-time qRT-PCR) revealed that most pheromone and receptor genes showed significantly higher expression in monokaryotic cells than dikaryotic cells. The pheromone genes PEphb3.1 and PEphb3.3 and the receptor gene PESTE3.3.1 were transformed into P5 (A3B4). The transformants were mated with a tester strain (A4B4), and the progeny showed clamp connections and a normal fruiting body, which indicates the proposed role of these genes in mating and fruiting processes. This result also confirms that PESTE3.3.1 is a receptor gene. In this study, we identified pheromone and receptor genes in the B3 locus of P. eryngii and found that some of those genes appear to play a role in the mating and fruiting processes. These results might help elucidate the mechanism of fruiting differentiation and improve breeding efficiency.

Identification of Degenerate Nuclei and Development of a SCAR Marker for Flammulina velutipes

Flammulina velutipes is one of the major edible mushrooms in the world. Recently, abnormalities that have a negative impact on crop production have been reported in this mushroom. These symptoms include slow vegetative growth, a compact mycelial mat, and few or even no fruiting bodies. The morphologies and fruiting capabilities of monokaryons of wild-type and degenerate strains that arose through arthrospore formation were investigated through test crossing. Only one monokaryotic group of the degenerate strains and its hybrid strains showed abnormal phenotypes. Because the monokaryotic arthrospore has the same nucleus as the parent strain, these results indicated that only one aberrant nucleus of the two nuclei in the degenerate strain was responsible for the degeneracy. A sequence-characterized amplified region marker that is linked to the degenerate monokaryon was identified based on a polymorphic sequence that was generated using random primers. Comparative analyses revealed the presence of a degenerate-specific genomic region in a telomere, which arose via the transfer of a genomic fragment harboring a putative helicase gene. Our findings have narrowed down the potential molecular targets responsible for this phenotype for future studies and have provided a marker for the detection of degenerate strains.

Multiplex Simple Sequence Repeat (SSR) Markers Discriminating Pleurotus eryngii Cultivar

For development of a method for differentiation of Pleurotus eryngii cultivars, simple sequence repeats (SSR) from whole genomic DNA sequence analysis was used for genotyping and two multiplex-SSR primer sets were developed. These SSR primer sets were employed to distinguish 12 cultivars and strains. Five polymorphic markers were selected based on the genotyping results. PCR using each primer produced one to four distinct bands ranging in size from 200 to 300 bp. Polymorphism information content (PIC) values of the five markers were in the range of 0.6627 to 0.6848 with an average of 0.6775. Unweighted pairgroup method with arithmetic mean clustering analysis based on genetic distances using five SSR markers classified 12 cultivars into two clusters. Cluster I and II were comprised of four and eight cultivars, respectively. Two multiplex sets, Multi-1 (SSR312 and SSR366) and Multi-2 (SSR178 and SSR277) completely discriminated 12 cultivars and strains with 21 alleles and a PIC value of 0.9090. These results might be useful in providing an efficient method for the identification of P. eryngii cultivars with separate PCR reactions.

Breeding of Pleurotus eryngii with a high temperature tolerance trait

In order to breed a new P. eryngii cultivar with high temperature tolerance trait to cope with climate change, strains and cultivars were characterized at 20oC that is 5oC higher than normal condition followed by screening for the characteristics such as required days to harvest, quality and yield. Monokaryons from the selected strains were crossed. Da-32×KNR2322-15 derived from the crosses between KNR2322 having characteristics of short growing day and Da(Ga5Na5-4×KNR2312-7) having charateristics of high guality and yield at 20oC, showed 14.9 days for harvest, 120.6 g yield, and 7.0 quality in the first trial. The strains were named as Taeyangsongi and cultivated on a large scale to compare with Kenneutari No. 2 at a mushroom farm. Yield of Taeyangsongi (109 g) was significantly different(P=0.001) from Kenneutari No.2. Quality of the new (6.6) and the reference cultivar (3.5) was also statistically different (P=0.001) The brightness of pileus of Taeyangsongi (59.5) was 10 points less than the reference cultivar due to which it has an inability to bear high temperature stress. Thus, PCR reactions with URP2 discriminated between Taeyansongi and reference cultivars (Keneutari No. 2 and Aeryni).

Plant Transformation via Pollen Tube-Mediated Gene Transfer

Genetic transformation using foreign genes and the subsequent development of transgenic plants has been employed to develop enhanced elite germplasm. Although some skepticism exits regarding pollen tube-mediated gene transfer (PTT), reports demonstrating improved transformation efficiency with PTT systems are increasing and encouraging and the adoption of increasingly refined pollen-mediated methodologies may lead to species-dependent improvements in breeding. Here, we highlight PTT technology as an alternative to genetic transformation.

The complete chloroplast genome sequence of Coix lacryma-jobi L. (Poaceae), a cereal and medicinal crop

Abstract Coix lacryma-jobi is a cereal and medicinal crop belonging to the Poaceae family. This study characterized complete chloroplast genome sequence of a Korean cultivar Johyun of C. lacryma-jobi var. ma-yuen through the de novo hybrid assembly with Illumina and PacBio genomic reads. The chloroplast genome is 140,863 bp long and composed of large single copy (82,827 bp), small single copy (12,522 bp), and a pair of inverted repeats (each 22,757 bp). A total of 123 genes including 87 protein-coding genes, 32 tRNA genes, and four rRNA genes were predicted in the genome. Phylogenetic analysis confirmed a close relationship of C. lacryma-jobi with species in the Panicoideae subfamily of the Poaceae family.