Artocarpus hypargyreus Hance (Moraceae), a tall evergreen tree endemic to southern China, is valued for its milky latex for making stiff rubber and for its wood for making furniture (Zhou and Gilbert, 2003). Its natural populations have declined because of overexploitation and habitat loss, and it was listed as a vulnerable species in the IUCN Red List of Threatened Species in 1997 (IUCN, 2015). Therefore, genetic information, such as genetic diversity and population structure, will be important for the conservation of this species.

Here, we developed 34 novel simple sequence repeat (SSR) markers for A. hypargyreus, of which 10 were polymorphic in A. hypargyreus and the additional 24 successfully amplified loci were monomorphic. These 10 polymorphic markers were tested on 67 individuals from four populations of A. hypargyreus, and their transferability was tested in two other Artocarpus species.

METHODS AND RESUTS

The transcriptome of A. hypargyreus was sequenced with Illumina pairedend sequencing for the development of expressed sequence tag (EST)–SSR markers. The total RNA was extracted from the fresh leaves of A. hypargyreus (Appendix 1) using the modified cetyltrimethylammonium bromide (CTAB) method (Fu et al., 2004). Normalized cDNA libraries were constructed and sequenced using the HiSeq 2000 system (Illumina, San Diego, California, USA). The raw reads were cleaned by removing reads containing unknown “N” bases or more than 10% bases with a Q value < 20 using custom Perl scripts. A total of 25.34 million cleaned 100-bp paired-end reads were de novo assembled into 121,556 contigs (N50 = 906 bp) using Trinity version 2.1.1 (Grabherr et al., 2011) with default parameters.

The software QDD version 3.1 (Meglécz et al., 2014) was used to search SSR motifs containing two to six nucleotides with the minimum number of repeats as follows: six for dinucleotide and five for trinucleotide, tetranucleotide, pentanucleotide, and hexanucleotide. A total of 14,143 SSR loci were detected in 12,013 contigs. Among them, dinucleotide repeats account for the largest proportion for 54.7%, trinucleotide repeats account for 40.5%, and tetranucleotide repeats account for 1.1%. Subsequently, using Primer3 (Rozen and Skaletsky, 1999) implemented in the QDD program, primer pairs were successfully designed for 3693 SSR loci, which were further subjected to an “all-against-all” BLAST with an E-value of 1E^-40 to remove redundancy. Finally, we obtained 2084 unique SSR loci based on which primer pairs were successfully designed.

Field investigations indicated that the individuals of A. hypargyreus showed a scattered distribution in their natural environments, causing difficulties in collecting large samples for each population. A total of 67 individuals were collected from four populations of A. hypargyreus (16–18 individuals for each population, see Appendix 1) to evaluate the polymorphisms of these SSR loci. In addition, five individuals of A. nitidus Trécul and nine individuals of A. heterophyllus Lam. were sampled to test the transferability of these primers. Genomic DNA was extracted from silica gel–dried leaves with the CTAB method (Doyle and Doyle, 1986).

Amplification and polymorphism tests were performed for 63 randomly selected primer pairs using two individuals from each population of A. hypargyreus. PCR amplification was performed according to Fan et al. (2013) with an appropriate annealing temperature, and PCR products were detected on 1% agarose gels. A total of 34 primer pairs were successfully amplified, generating legible products of the expected fragment size. Sequences of these SSR loci have been deposited in GenBank (Table 1, Appendix 2). The products were inspected with the Fragment Analyzer Automated CE system (Advanced Analytical Technologies [AATI], Ames, Iowa, USA) with the Quant-iT PicoGreen dsDNA reagent kit, 35–500 bp (Invitrogen, Carlsbad, California, USA). The raw data were analyzed by using PROSize version 2.0 software (AATI). Ten loci were polymorphic among the populations, and 24 loci were monomorphic.

The allelic polymorphisms of the 10 loci were further tested in 67 individuals from four populations of A. hypargyreus, and the efficiency of these markers in cross-species amplification was detected in A. nitidus and A. heterophyllus. GenAlEx version 6.5 (Peakall and Smouse, 2012) was used to calculate the average number of alleles per locus, the observed heterozygosity, the expected heterozygosity, and deviation from Hardy-Weinberg equilibrium (HWE). The results showed that the number of alleles per locus ranged from three to 10 (Table 1). The observed and expected heterozygosity ranged from 0.00 to 0.706 and from 0.328 to 0.807, respectively, and all loci showed significant deviation from HWE (Table 2). The scattered distribution of A. hypargyreus may cause difficulties in the long-distance dispersal of pollen and eventually lead to a decrease in the observed heterozygosity values and the significant deviations from HWE. Of the 10 SSR markers tested, all successfully amplified in A. nitidus and nine successfully amplified in A. heterophyllus (Table 3).

CONCLUSIONS

Ten novel polymorphic SSR markers were developed for A. hypargyreus, which are likely to be useful for evaluating the genetic diversity and population structure of A. hypargyreus, and for facilitating the development of a conservation strategy for this species. The cross-amplification of these microsatellite loci in A. nitidus and A. heterophyllus suggests that they will also be useful in studies of other species within Artocarpus.

Table 1.

Characteristics of the 10 polymorphic microsatellite loci developed for Artocarpus hypargyreus.

Table 2.

Polymorphism of the 10 EST-SSRs in four populations of Artocapus hypargyreus.^a

Table 3.

Cross-amplification of the 10 polymorphic EST-SSR markers developed for Artocarpus hypargyreus in A. nitidus and A. heterophyllus.