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SNPs were identified between the subspecies using the Perl script used by Maughan et al. [ 28].
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Therefore, we used four nuclear molecular markers which reliably discriminate between the two subspecies using fixed polymorphisms (two autosomal and one from each sex chromosome [ 3]).
We summarized patterns of divergence between subspecies using the mean and variance of differences in allele frequencies between pairs of subspecies.
Due to the low levels of divergence within subspecies previously documented [ 22], we calculated pairwise estimates of variance in haplotype frequency (ΦST) between each presumed subspecies using haplotype frequencies in ARLEQUIN version 3.11 and we assessed significance using 10,000 permutations.
Molecular data were used to compute genetic distances (Nei and Li 1979) within and between V. unguiculata subspecies using TREECON (version 1.3b; Van de Peer and De Watcher 1994; http://bioc-www.uia.ac.be/u/yvdp/treeconw.html) and a dendrogram was generated using the unweighted paired group method with arithmetic averages (UPGMA).
Recently, molecular tests capable of distinguishing between the subspecies by using single nucleotide polymorphisms have been developed (2, 3 ).
The average DI between the subspecies was consistently low using different thresholds of minimum number of reads from each pool.
In contrast, however, Brown et al. [35] did document low but significant differentiation between the two subspecies when using contemporary samples.
Like most other QTL analyses in rice, this was performed using crosses between the subspecies indica and japonica where it is relatively easy to obtain molecular markers and construct saturated linkage maps.
The indica and japonica landraces from Kalimantan were highly differentiated, with an F st of 0.59 between the subspecies.
Differences between the subspecies are small and geographically gradual.
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