The statistics data of Archidendron clypearia transcriptome sequencing are listed in Supplementary Table S1. After assembly redundancy reduction, a final number of 173,172 transcripts were retained with an average transcript length of 1597.3 bp and an N50 length of 2427 bp. These length values were remarkably higher than those of other medicinal leguminous plants, e.g. an average transcript length of 626 bp and an N50 length of 987 bp in Mucuna pruriens (L.) DC. [19].
A total of 34,701 SSR loci were identified from 26,716 (15.4%) transcripts. Of the di- to penta-nucleotide repeat motifs, the most frequent was tri-nucleotide (17,751, 51.2% of the 26,716 transcripts), followed by di- (14,760, 42.5%), tetra- (1557, 4.5%), hexa- (378, 1.1%), and penta-nucleotide (254, 0.7%; Supplementary Table S2). The three most abundant repeat motifs were AG/CT (6908, 19.9%), AAG/CTT (5614, 16.2%), and AC/GT (3933, 11.3%; Supplementary Table S2). The prevalence of AG/CT motif is consistent with most plant species [20].
Out of the 718 primer pairs designed, 644 (89.7%) were of effective amplification. After excluding 70 effective primer pairs each with an amplicon larger than 500 bp, 456 polymorphic (Supplementary Table S3), 99 monomorphic, and 19 no-fluorescent-signal SSRs were identified. For all the polymorphic markers, Na, Ho, He, and PIC ranged between 2–16 (mean 3.0), 0.0–1.0 (mean 0.776), 0.056–0.944 (mean 0.511), and 0.053–0.912 (mean 0.409), respectively (Supplementary Table S4). The polymorphism estimates are generally higher than those reported in other legumes, e.g. PIC and He being 0.24 and 0.41 in M. pruriens [19] and 0.1956 and 0.1081 in Cyamopsis tetragonoloba (L.) Taub. [21].
Of the 456 polymorphic markers, 391 (85.7%) and 402 (88.1%) were transferable to A. lucidum and A. multifoliolatum, respectively, with 376 shared by both species (Supplementary Table S4). The cross-species transferability levels are higher than those reported for 89 EST-SSR markers from C. tetragonoloba in two Cyamopsis species (82% and 69%) [21].
The 15 polymorphic EST-SSR markers resulted in a total of 146 alleles over the two populations, with 6–16 alleles per marker (mean 9.7; Table 1). Only two markers showed significant deviation from HWE expectation after Bonferroni correction (Table 1). Locus Ho and He ranged from 0.500 to 1.000 (mean 0.825) and from 0.494 to 0.902 (mean 0.772), respectively (Table 1). Population Ho and He were 0.815 and 0.696 for EEP and 0.838 to 0.719 for JNFP (mean 0.826 and 0.707), respectively (Table 2). The genetic diversity is relatively high as compared to other legumes, e.g., He being 0.41 in M. pruriens [19] and 0.1081 in C. tetragonoloba [21]. This result demonstrates that the two populations are clearly needed to conserve the genetic diversity. Additionally, the higher Ho than He estimates indicate certain magnitude of heterozygote excess.
Table 2
Genetic diversity estimates for two A. clypearia populations
Population | N | Na | Ne | Ho | He | F |
EEP | 18 | 113 | 57.4 | 0.815 | 0.696 | −0.171 |
JNFP | 16 | 106 | 69.2 | 0.838 | 0.719 | −0.164 |
Total | 34 | 146 | 79.9 | – | – | – |
Mean | – | – | – | 0.826 | 0.707 | −0.168 |
Na, number of alleles; Ne, number of effective alleles; Ho, observed heterozygosity; He, expected heterozygosity; F, Wright’s fixation (inbreeding) coefficient; EEP, Erlongshan Ecological Park; JNFP, Jianfengling National Forest Park |
The indices F and Fis suggested certain degree of inbreeding, with general mean of F and Fis being − 0.168 (Table 2) and − 0.171 (Table 1). On the other hand, there were 73 (50.0%) private and 17 (11.6%) diagnostic alleles over EEP and JNFP populations (Table 1), indicating some difference between the two populations. An overall Fst of 8.7% (0.087; Table 1) was detected, suggesting relatively low level of population differentiation despite the long distance (about 700 km) separation of the two populations. This also indicates that A. clypearia has mainly an outcrossing mating system as population differentiation coefficient is usually less than 19% for outcrossing species [22]. In addition, significant bottleneck effect and allelic frequency distribution mode shift were not detected for EEP and JNFP populations under TPM and SMM models (Supplementary Table S5), indicating no recent bottleneck experienced by both populations in spite of certain magnitude of heterozygote excess.