| Lentinula edodes (shiitake or Xianggu), well-known for the high nutritional and medical value, is one of the most cultivated edible mushroom worldwide. Breeding fine varieties is crucial for the sustainable development of shiitake industry. Most of the important agronomic traits of shiitake are quantitative traits controlled by multiple genes or quantitative trait loci (QTLs). Traditional breeding methods, such as selected based on the phenotypes, are difficult to improve these agronomic traits. Markers associated with phenotype may be integrated into regular breeding schemes, namely marker-assisted selection (MAS), to improve the breeding efficiency. Owing to lack of high-quality genetic map, as well as the inconsistency between the linkage mapping population and the phenotypic segregation population, QTLs associated with the features of fruiting body, yield-related traits has been rarely reported in L. edodes. In the current study, using an improved linkage map, QTLs controlling major agronomic traits were mapped, thus laying a solid foundation for marker assisted selection in L. edodes.1. Genetic linkage maps are powerful tools for detecting candidate loci corresponding to important traits of interest. In this study, we constructed an improved shiitake genetic linkage map (M map) by integrating insertion/deletion (InDels) markers into our previously published map. The monokaryotic mapping population (M population), including 146 F1 single spore isolates (SSIs), was used for InDels genotyping and linkage mapping. In the map M, a total of 572 markers were aligned to 12 linkage groups (LGs), with a total map length of 983.7 cM, and an average marker spacing of 1.8 cM. Owing to a set of new added InDel markers, comparative mapping between the M map and the other shiitake map S was performed. The results showed that 2 of 12 LGs in M map actually belonged to the same linkage group, therefore M map contained 11 LGs. Furthermore, four QTLs for three traits related with monokaryons growth were detected using map M. The QTL on LG4 (mgr-IV-1) for monokaryons growth rate was found to be in close proximity to the region containing the MAT-A and S278R/F loci.2. Evaluation and analysis of agronomic traits in the segregation populations provide the prerequisites for QTL mapping and the subsequent genetic improvement. Two testcross monokaryons,741-15 and 741-64, respectively paired with the 146 SSIs to construct two testcross dikaryon populations (LQ-15 and LQ-64). Through fruiting trials, phenotypic evaluation and analysis of twelve important agronomic traits of shiitake strains in LQ-15 and LQ-64 were performed using multivariate statistical methods. Results indicated that yield was positively correlated with the number of fruiting bodies, but negatively correlated with the weight of single fruiting body; significantly positive correlations were found between pairs of agronomic traits related to the basidiocarp; there were no significant correlations between growth performance in synthetic media and agronomic traits performance in fruiting trials. Factor analysis uncovered the three major factors (single fruiting body, yield and vegetative growth); path analysis revealed a mutual restriction between shiitake yield component traits (the number of fruiting bodies and the weight of single fruiting body), and the number of fruiting bodies was the key component influencing yield.3. The dissection of the agronomic traits in individualized loci by QTL mapping would greatly facilitate their effective genetic manipulation in breeding program. Composite interval mapping (CIM) and inclusive composite interval mapping (ICIM) were utilized to uncover the loci regulating and controlling important agronomic traits of shiitake in LQ-15 and LQ-64. In total,38 and 66 QTLs related to twelve agronomic traits were respectively detected in LQ-15 and LQ-64. The QTL (sdgr-Ⅱ-Ⅰ) for dikaryons growth rate was both identified in LQ-15 and LQ-64. The 103 QTLs were located on seven LGs,50 of them were both detected by CIM and ICIM methods. Seven QTLs were identified with high percentages of phenotypic variation explained (over 20%). Most of mapped QTLs uncovered here were clustered, implying the presence of main genomic areas responsible for investigated agronomic traits. For traits related to single fruiting body, QTLs detected in LQ-15 and LQ-64 were both clustered on LG2 (45-51 cM), as a QTLs-hotspot region. Three InDel markers (S48-ID1, S113-ID1 and S306-ID1) were located on this region. The candidate genes (e.g. para-aminobenzoate synthetase gene, MAP kinase gene, laccase gene, cyclopropane-fatty-acyl-phospholipid synthase gene, P450 monooxygenase genes) controlling the agronomic traits of shiitake were also presented. In LQ-15 and LQ-64, QTL analysis identified genomic regions controlling two or more related traits in a manner that was consistent with correlation among traits, suggesting either pleiotropy or tight linkage among QTLs. The co-localization of QTLs could be the hereditary basis for phenotype correlation of traits in L. edodes.4. In L. edodes, it is difficult to carry out QTL mapping due to the inconsistency between the linkage mapping population and the phenotypic segregation population. In this study, the dikaryon population (F2 population) consisted of 171 dikaryons, was generated by random mating between the 146 F1single spore isolates (SSIs) in M population. Based on the F2 population, we described an easy method to construct a dikaryotic linkage map (F2 map) using genotype data deduced from the two mating monokaryons. This first shiitake dikaryotic map included 440 markers assigned to 15 LGs, and covered a total genetic length of 970.8 cM with an average marker spacing of 2.3 cM. Comparative mapping between the M map and F2 map revealed a high level of synteny between the consensus markers in order and positions; 15 LGs in F2 map were matched with the 12 LGs in M map. Four QTLs for dikaryons growth rate were mapped in the F2 population, and showed dominant or over-dominant effect. Among them, fdgr-Ⅳ-2 and fdgr-Ⅷ-1 was respectively adjacent to sdgr-Ⅳ-2 (LQ-15) and dgr-Ⅷ-1 (LQ-64). The utilization of F2 population provides a new possible way to solve the inconsistency between the linkage mapping population and the phenotypic segregating population. The combination of F2 and testcross dikaryon populations would facilitate to uncover the genetic architecture of complex agronomic traits.In L. edodes, this is the first report of using sequence-based InDel markers for genetic mapping, and could facilitate the integration of genetic map and genome sequences. It is also the first time to identify the candidate genes responsible for shiitake agronomic traits using genome sequences.The QTL-marker associations highlighted here, are also useful tools to establish MAS breeding strategy in L. edodes. Furthermore, the utilization of F2 population enrichs the method of genetic mapping and QTL analysis of edible mushrooms. In summary, it is an important step to establish the associations among agronomic traits, molecular markers, genetic map, as well as genome sequence in L. edodes. |
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