Electrophoretic karyotyping and construction of a bacterial artificial chromosome library of the winter mushroom Flammulina velutipes

Abstract

An electrophoretic karyotype of Korean Flammulina velutipes was obtained using contour-clamped homogeneous electric field (CHEF) gel electrophoresis. The monokaryotic progenies (4019-20 and 4019-18) and a dikaryotic strain (4019-2018) had 6-8 chromosomes, with sizes ranging from 1.6 to 5.8 megabase (Mb) pairs. Among the 3 strains that were examined, strain 4019-20 resolved into at least 7 chromosomes, with a total genome size of approximately 26.7Mb. We selected several chromosome-specific genes from the cDNA library of F. velutipes using Southern hybridization analysis. In order to determine the whole genome sequence, we constructed a bacterial artificial chromosome (BAC) library of the 4019-20 strain. The BAC library comprised 3840 clones, and the insert size ranged from 60 to 228kb, with an average size of 136kb.

β-mercaptoethanol assists efficient construction of sperm bacterial artificial chromosome library

“…The important point is that β-mercaptoethanol did not influence DNA purity and length. Only a few reports have described the use of β-mercaptoethanol for BAC library construction [15][16][17][18], and thus β-mercaptoethanol is uncommon chemical regent for large-insert library construction. The reason for use has not been described at all, and the usage, efficiency and side effects of β-mercaptoethanol have never been assessed in detail.…”

Assignment of RAPD marker probes designed from 12 linkage groups of Flammulina velutipes to CHEF-separated chromosomal DNAs

“…By this method, electrophoretic karyotypes have been revealed for many fungi including cultivated edible mushrooms such as A. bisporus (Royer et al 1991(Royer et al , 1992Sonnenberg et al 1991;Lodder et al 1993), L. edodes (Arima and Morinaga 1993), Pleurotus species (Sagawa and Nagata 1992;Tamai et al 1995), and F. velutipes (Kim et al 2000;Tanesaka et al 2003;Park et al 2010). Various reports have suggested that F. velutipes has at least six (Kim et al 2000), or six or seven (Park et al 2010), or seven or eight (Tanesaka et al 2003) haploid chromosomes. Chromosome-specific probes have also been constructed by assignment of DNA probes prepared from a cDNA library to CHEF-separated chromosomes (Kim et al 2000;Park et al 2010).…”

“…Various reports have suggested that F. velutipes has at least six (Kim et al 2000), or six or seven (Park et al 2010), or seven or eight (Tanesaka et al 2003) haploid chromosomes. Chromosome-specific probes have also been constructed by assignment of DNA probes prepared from a cDNA library to CHEF-separated chromosomes (Kim et al 2000;Park et al 2010). On the other hand, assignment using DNA probes prepared from a genetic linkage map is more effective to elucidate the karyotype involved in haploid chromosome number and genome size and for analysis of meiotic behavior, as demonstrated in A. bisporus (Royer et al 1991(Royer et al , 1992Kerrigan et al 1993;Sonnenberg et al 1996).…”

“…The electrophoretic karyotype of FSB presented here is slightly different from those previously reported for other cultivated varieties of F. velutipes; for example, Japanese variety ''Nakano-JA'' has seven or eight haploid chromosomes ranging from 1.4 to 4.9 Mb, with total sizes of 24.0-24.6 Mb (Tanesaka et al 2003), and the karyotype from a Korean commercial variety has six or seven haploid chromosomes ranging from 1.60 to 5.84 Mb, with total sizes of 23.98-26.98 Mb (Park et al 2010). The CLP between parental monokaryons, omFSB1 and omFSB2, presented here and also that observed in a Korean variety (Park et al 2010) is relatively large in comparison with the CLP among monokaryotic progeny generated from basidiospores (Tanesaka et al 2003). It is unclear whether the size differences among monokaryons are attributable to a property of individual dikaryons or to other effects, i.e., selection during the meiotic process or spore germination, or afterward to eliminate karyotype aberration among progeny.…”

“…No probes hybridized to the smallest two chromosomes, CB 10 and 11 (Table 1). Kim et al (2000) and Park et al (2010) assigned several DNA probes prepared from a cDNA library to CHEFseparated F. velutipes chromosomes using Southern hybridization analysis. This analysis was powerful enough to construct chromosome-specific probes and also enabled detection of weak bands on gels.…”