For heaths and vitality Cordyceps sinensis has been widely used as an important medicinal mushroom. C. militaris is popular for its substitute and can be cultivated artificially. Its new strains have been improved by crossing single spore strains. A bipolar heterothallic fungus, C. militaris has two strains of compatible mating types and they can be differentiated by crossing, fruiting body formation ability and the production of perithecia. Each isolate was determined to be monokaryotic or dikaryotic on the basis of perithecial formation by fruiting tests. This process was also carried out by molecular markers for the detection of two opposite mating types or F1 hybrids using two sets of primers specific for C. militaris amplifying a 191-bp fragment for MAT1-2 and 233-bp fragment for MAT1-1. Therefore the crossing of two compatible mating types resulted in well-developed perithecial fruiting bodies and their crossings were confirmed by the PCR assays for the rapid and specific detection of both MAT1-1 and MAT1-2. This results showed that molecular markers provide an efficient alternative for identification of hybridal status or mating types in the breeding program for artificial cultivation and industrial-scale production of C. militaris.

Lentinula edodes shows atypical mating behaviors deviated from tetrapolar mating in outcrossing. To better understand the mating behavior, we investigated variations in the mating type genes, residing in A and B mating loci, of 129 dikaryotic strains from East Asia. Through sequence analysis of A locus, we found that hypervariable region spanning N-term of HD2-intergenic region-N-term of HD1 could represent A mating type of L. edodes. Mating and hypervariable region analyses revealed 50 unique A mating types: 27 from 98 cultivated strains, 33 from 31 wild strains, and 10 commonly found. It was also revealed that only a few A mating type alleles such as A1, A4, A5, and A7 were prevalent in cultivated strains. The A mating type in wild strains were highly diverse: 23 unique A alleles were discovered in small mountainous area in Korean peninsula. The B locus was assessed by allelic variations in pheromone (PHB) and pheromone receptor (RCB) pairs. Sequence analyses revealed 5 alleles of RCB1 with 9 associated PHBs in B sublocus and 3 alleles of RCB2 with 5 associated PHBs in B sublocus. Each RCB was primarily associated with two PHBs. This allowed us to propose 15 B mating types via combinations of five B and three B subloci. Thus, the total number of mating types in L. edodes became 750 through a combination of 50 A and 15 B. This number will further increase because of rapid diversification of A mating type and possible emergence of variants of RCBs and PHB-RCB combinations.

The mushroom species Cordyceps militaris has been studied and cultivated as a medicinal mushroom due to its multiple valuable biological and pharmaceutical activities. For breeding new strains of C. militaris, multiplex PCR assays were performed using primers specific for its mating type genes, MAT1-1 and MAT1-2. Mating types and mating status were confirmed, as evidenced by DNA bands at 233-bp and 191-bp for MAT1-1 and MAT1-2 respectively. The novel 'Dowonhongcho 2ho’ was developed through mating; they were found to possess high-quality fruiting bodies when grown in artificial media. The stromata of the new strain were club-shaped, with a bright orange-red color, and measured 7.1 cm in length. They had an average cordycepin content of 0.33%. Compared to 'Dowonhongcho,' the new strain had a 7% higher yield, as well as firm fruiting bodies. The optimum temperature for mycelial growth was 20~25°C, and the optimum temperature for stroma development was 18~22 °C. The fruiting bodies developed after 49.1 days from inoculation. The use of mating type molecular markers improved the breeding efficiency of the new strain 'Dowonhongcho 2ho.’ Thus, they may be valuable for artificial cultivation and industrial-scale production of C. militaris with excellent characteristics.

Cordyceps species are important mushrooms traditionally used for heaths and vitality. C. sinensis has been widely used as a medicinal mushroom and C. militaris is popular for its substitute. C. militaris can be cultivated artificially and new strains has been improved by crossing single spore strains. As a bipolar heterothallic fungus C. militaris has two strains of compatible mating types and they can be differentiated by crossing, fruiting body formation ability and the production of perithecia. However this process is very laborious and time consuming to carry out. In this study, molecular markers were designed and used for the detection of two opposite mating types. Two mating types were assayed using two sets of primers specific for C. militaris, which were amplified a 191-bp fragment for MAT1-2 and 233-bp fragment for MAT1-1. After crossing of two compatible mating types F1 hybrids resulted in well-developed perithecial fruiting bodies and their crossings were confirmed by the multiplex PCR assays for the rapid and specific detection of both MAT1-1 and MAT1-2. This results may serve as a efficient process in the breeding program for artificial cultivation and industrial-scale production of C. militaris.

버섯은 세계적으로 양송이, 표고 느타리버섯류 재배가 주류를 이루고 있으며, 한국의 시중에서 재배하고 있는 버섯품종이 1969년 이후 꾸준히 증가하여 약 24품목 224품종이 신고되거나 품종보호등록되어 농가에 보급되었다. 그 중에서 느타리와 큰느타리가 전체 생산 및 소비가 약 50%를 차지하고 있다. 이러한 상황에 비춰 하나의 품종이 다른 이름으로 신고되거나 등록되는 경우가 존재하여 먹는 버섯만 해도 구별하기가 쉽지 않은 상황이다. 과거에는 일반인이 외국에서 품종을 수집하여 그대로 판매신고를 하여 상품으로 판매하는 사례도 있는 실정이다. 지금은 품종등록을 한 종균을 허가없이 사용할 경우 로열티를 지불하거나 소송이 진행될 수 있음을 간과해서는 안된다. 일례로 새송이버섯 사건이 대표적인 예라 할 수 있다. 이 사건의 품종판별 방법으로 유전자 식별법을 사용하였다고 한다. 이것은 형태적 형질만으로는 품종구분 방식에 한계가 있음을 보여주고 있다. 우리는 미토콘드리아 유전자를 이용하여 품종구분을 하였는데 느타리에서 원형느타리, 수한1호, 춘추2호 등이 뚜렸한 차이를 보여주어 품종을 구분할 수 있는 마커로서의 가능성을 보여주었다. 그리고 교배육종된 교잡주의 모본유래를 확인함으로서 육종 마커로 사용할 수 있으리라 판단된다. 또한 육종효율 증진을 위해 mating type을 결정하는 homeodomain 및 pheromone receptor 유전자를 이용하여 교배형을 결정함으로서 교배육종 시 시간과 경비를 절감할 수 있으리라 사료된다.

The mating-type genes control formation of the dikaryon from two haploid strains. These genes are now used in mating-type-assisted breeding programs for economically important mushrooms, especially the oyster mushroom, Pleurotus ostreatus, aiming at high-yield and high-quality standard mushroom production. However, it improves the breeding program when the breeder is able to quickly identify compatible strains in a given set of progeny. The two mating factors with their mating-type loci are used as markers for breeding and have been incorporated in a chromosome mapping investigation. The linkage maps include not only genetic markers such as the mating types that can be cored, but also molecular markers such as PCR-assisted approaches, e.g. RAPD analyses, or RFLP markers. Once mating-type genes within progeny may be more easily identified by the use of PCR-directed cloning of partial mating-type genes. We analyzed homeodomain (HD1 and HD2) and pheromone receptor(rcb1, 2 and 3) genes as molecular markers for breeding using mating type A and B of Pleurotus eryngii and Pleurotus ferulae by direct PCR.

Lentinula edodes is an important cultivated mushroom in China. The development of Lentinula edodes production promotes more studies on it. In our previous work, degenerate PCR and chromosome walking technologies were used to obtain one pheromone receptor gene and one pheromone precursor gene from Lentinula edodes. In this study, four pairs of specific primers were designed according to the whole genome sequencing of the protoplast monokaryon of Lentinula edodes strain 135, to amplify STE3-like pheromone receptor gene and its flanking conserved genes in the protoplast monokaryon strain SUP2 derived from Lentinula edodes strain Suxiang and 33655bp DNA sequence was obtained. By BlastX search, seven putative genes were identified, and three of them are pheromone receptor encoded genes. Furthermore, near to two pheromone receptor genes, four genes encoding proteins with conserved motifs of pheromone precursors were found. This study firstly reveals the molecular organization of the B mating type locus of Lentinula edodes.

Flammulina velutipes, amongst others known as Winter Mushroom or Enokitake is an important economic crop in Asia. The tetrapolarity (having four mating types) of this mushroom obligates mating and results in self-sterile progeny that carries unique genetic traits, making understanding of the genetic base desirable for breeding. Moreover, mating type genes are significant for evolutionary studies as their high polymorphism benefits phylogenetic comparisons. This polymorphism further makes mating type genes interesting candidates for genetic markers that allow identification of specific strains. Mating type loci in Agaricomycotina are classically termed A and B and control two different developmental pathways [for a review see 1]. They consist of tightly linked subloci that encode multiallelic genes. MatA loci contain two groups of divergently transcribed homeodomain proteins (HD1 and HD2) and heterodimerization of HD1 with a non-self HD2 protein forms a functional transcription factor that activates the A pathway. MatB loci hold pheromones and pheromone receptors. Pheromone genes encode small precursor proteins that are characterized by a C-terminal CAAX motif. Pheromone receptors typically contain 7 membrane spanning regions and are coupled to G-proteins. Binding of a pheromone to a receptor, triggers splicing of the (trimeric) G-protein, which activates the B pathway. New genetic data from recent genome sequences is challenging the strict concepts of old mating type models in fungi. MatB loci turn out to be rather diverse and contain considerable varying numbers of pheromone receptors and associated pheromones. To this, pheromone receptors which are not linked to matB loci have now been reported for C. cincerea, S. commune and L. bicolor [2, 3]. Also the organization of the matA locus is less strictly conserved than anticipated. Though far more tightly maintained than the matB locus, substantial differences in HD gene numbers and overall organization are reported [2, 3]. These differences stress the importance of determination of the individual mating type systems from industrially important mushrooms to assist breeding. Our analysis of F. velutipes strain 4019-20 uncovered 7 pheromone receptors together with 3 pheromones. The matB-3 locus of this strain however, is defined by only a single pheromone receptor and pheromone gene and our data strongly indicates that a 2nd pheromone receptor recently lost its function. The other receptor genes are non mating type specific. Finally, we detected three homeodomain genes distributed over two distant subloci. These subloci have been separated by two large inversions. Strikingly the distant matA subloci in S. commune seem to be separated by inversions as well. Synthenic mapping of a large regions from Coprinus cinerea, Laccaria bicolor, S. commune and F. velutipes shows that the matA loci originate from a single locus in a common ancestor of S. commune and F. velutipes that is represented by L. bicolor and C. cinerea. [1] U Kües. Micr Mol Biol Rev 64, 316 (2000) [2] H Niculita-Hirzel, J Labbé, A Kohler, F le Tacon, F Martin, IR Sanders and U Kües. New Phytol 180, 329 (2008). [3] RA Ohm, JF de Jong, LG Lugones, A aerts, E Kothe, JE Stajich, RP de Vries, E Record, A Levasseur, SE Baker, KA Bartholomew, PM Couthino, S Erdmann, TJ Fowler, AC Gathman, V Lombard, B Henrissat, N Knabe, U Kües, WW Lilly, E Lindquist, S Lucas, JK Magnuson, F Piumi, M Rausdaskoski, A Salamov, J Schmutz, FWMr Schwarze, PA van Kuyk, JS Horton, IV Grigoriev and HAB Wösten. Nat. Biotechnol ISSN: 1087-0156 (2010).

버섯의 품종육성을 위해서는 종간 및 종내의 다른 두 개의 단포지를 분리하여 교배한 후 우량형질을 선발하여 신품종으로 등록된다. 전세계적으로 25%를 재배생산되고 있는 느타리버섯은 4극성교배시스템을 가지고 있어서 이러한 교배 불화합성을 이용하면 육종 시간 및 노력을 단축할 수 있다. 느타리버섯의 교배형은 4개의 딸핵으로 분열되는 세포분열에 A형, 클램프 연결체의 형성에 관여하는 B형으로 나눈다. 최근에는 게놈프로젝트에 의해 교배기작에 관련된 유전자가 밝혀짐에 따라 이것을 이용한 육종효율 제고를 위해 많은 연구자들이 교배형 결정인자의 동정에 심혈을 기울이고 있다. 최근에는 분홍느타리(Pleurotus djamor)에서 CLA4(MAP kinase) 유전자의 CAPS 분석에 의해 교배형 B가 동정되었다. 따라서 본 실험에서는 제한효소를 사용하지 않고 바로 PCR로 동정할 수 있는 방법으로 교배형 A에 해당하는 homeodomain 유전자를 이용하여 교배형 A를 동정하였다.

본 연구는 1999년부터 2001년까지 경남에 있는 K 종돈장의 Yorkshire(Y), Landrace(L), Duroc(D), L×Y and Y×L 5품종 종빈돈 3,893복의 분만성적인 복당 총산자수, 복당 포유개시두수, 복당 사산두수, 복당 미이라두수, 복당 포유개시체중 및 1회 종부 후 분만율에 영향하는 교배조합, 분만년도, 분만계절 및 분만 산차의 효과를 추정하였다. 교배조합의 효과는 각 형질에 대하여 교배조합별로 순종은 복당 총산자수, 복당 포유개시두수와 복당 미이라두수에서 다소 유의적(p<0.05)인 차이를 보였으며, 교잡종에 있어서는 유의적인 차이가 없었다(p<0.05). 분만년도의 효과는 여러 연구자의 보고와 상이한 결과를 보였는데, 복당 총산자수는 1999년 및 2001년이 2000년도에 비해 유의하게(p<0.05) 높았다. 분만 계절의 효과는 복당 총산자수를 제외한 각 형질에 대하여 유의하게(p<0.05) 영향하는 것으로 나타났다. 복당 총산자수는 4～7산차에서 유의하게(p<0.05) 높게 나타났으며, 7산차 성적이 가장 우수하고, 1산차 성적이 가장 저조했다. 이는 조사대상 집단에 대한 점진적인 관리 환경 개선과 모돈 자체에 대한 체계적인 사양관리의 개선 때문이라고 사료된다.