Development of Novel Polymorphic EST-SSR Markers in Bailinggu (Pleurotus tuoliensis) for Crossbreeding

Abstract

Identification of monokaryons and their mating types and discrimination of hybrid offspring are key steps for the crossbreeding of Pleurotus tuoliensis (Bailinggu). However, conventional crossbreeding methods are troublesome and time consuming. Using RNA-seq technology, we developed new expressed sequence tag-simple sequence repeat (EST-SSR) markers for Bailinggu to easily and rapidly identify monokaryons and their mating types, genetic diversity and hybrid offspring. We identified 1110 potential EST-based SSR loci from a newly-sequenced Bailinggu transcriptome and then randomly selected 100 EST-SSRs for further validation. Results showed that 39, 43 and 34 novel EST-SSR markers successfully identified monokaryons from their parent dikaryons, differentiated two different mating types and discriminated F₁ and F₂ hybrid offspring, respectively. Furthermore, a total of 86 alleles were detected in 37 monokaryons using 18 highly informative EST-SSRs. The observed number of alleles per locus ranged from three to seven. Cluster analysis revealed that these monokaryons have a relatively high level of genetic diversity. Transfer rates of the EST-SSRs in the monokaryons of closely-related species Pleurotuseryngii var. ferulae and Pleurotus ostreatus were 72% and 64%, respectively. Therefore, our study provides new SSR markers and an efficient method to enhance the crossbreeding of Bailinggu and closely-related species.

Keywords: Pleurotus tuoliensis; expressed sequence tag-simple sequence repeat; genetic diversity; monokaryon; transferability.

Figure 1

The distribution of SSR motif types in Bailinggu transcriptome.

Figure 2

Examples of expressed sequence tag-simple sequence repeat markers applied in Bailinggu. (A) Polymorphic bands between monokaryons and their parent dikaryons using blgSSR24, blgSSR13, blgSSR25 and blgSSR15 (1, 6, 11, 16: parent dikaryons CCMJ973 (D10); 2–3, 7–8, 12–13, 17–18: monokaryon M29; 4–5, 9–10, 14–15, 19–20: monokaryon M30; the dikaryons CCMJ973 (D10) had two bands, while monokaryons M29 and M30 only had one band at the corresponding position); (B) polymorphic bands between the monokaryotic strains and their F₁ hybrid using blgSSR12, blgSSR22 and blgSSR21 (1–2, 7–8 and 13–14: monokaryon M28; 3–4, 9–10 and 15–16: monokaryon M33; 5–6, 11–12 and 17–18: F₁ hybrid; monokaryon M28 and monokaryon M33 only had one band, while their F₁ hybrid was the combination of M28 and M33); (C) the monokaryotic strains and their F₂ hybrid using blgSSR13 (1: monokaryon M28; 2: monokaryon M33; 3–5: F₂ homozygous-like monokaryon M28; 6–8: F₂ homozygous-like monokaryon M33; 9–14: F₂ heterozygous).

Figure 3

Unweighted pair group method with arithmetic mean (UPGMA) dendrograms of Bailinggu based on 18 novel EST-SSR markers. (A) UPGMA dendrogram of 37 monokaryons of Bailinggu based on 18 novel EST-SSR markers. M1–M37 represented monokaryons obtained from parent dikaryons. D1–11 represented the parent dikaryotic strains: D1-CCMJ814, D2-CCMJ1077, D3-CCMJ967, D4-CCMJ974, D5-CCMJ968, D6-CCMJ1044, D7-CCMJ980, D8-CCMJ1001, D9-CCMJ1002, D10-CCMJ973, D11-CCMJ1123. (B) UPGMA dendrogram of 11 parent dikaryotic strains of Bailinggu based on the same 18 novel EST-SSR markers.