Chinese fir [Cunninghamia lanceolate (Lamb.) Hook] is one of the most important indigenous timber tree species in China. The aim of the present work is to characterize simple sequence repeat (SSR) loci derived from the specific length amplified fragment sequencing (SLAF) data of the genome and to investigate the number of pollen donors for per cone was with novel SSR markers of low-frequency null alleles. A total of 58855 SLAF-SSR with frequency of 42.04 SSR/Mbp were identified in about 1.40 Gb Chinese fir genome. Dinucleotide repeat SSR contributed to 66.4% of the total SSR from SLAF data. The AT/AT and ATG/CAT motifs were predominant in the category of din-and trinucleotide repeat SSR. Low frequencies of null alleles (<5%) were detected at the nine novel SSR markers with average expected heterozygosity of 0.513 and polymorphism information content score of 0.508. The number of tested progeny of a cone was from 4 to 13. It could be 67 pollinizers for 15 cones and the average number of pollen donors per cone was 4.5. The study points out, for the first time, that there are multiple pollen donors for single cone in gymnosperm.
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