In this study, we determined and analyzed the cp genome of ZJGS (a hybrid plant) and compared it with the Moraceae species for structural comparison and sequence alignment. From the structure composition of the cp genome, it can be seen that the seven Broussonetia species and the six Morus species are grouped independently and each of them have similar compositions. Among the seven Broussonetia species, total GC content of ZJGS cp genome sequence is the closest to the maternal B. kazinoki, shows high species affinity [41], which is also reflected in the comparison of sequence diversity and SSRs. In addition, the sequence comparison analysis of ZJGS cp genome and other 12 Moraceae plants at the genomic level reflects the high sequence conservation of the IRs regions, so the IRs regions changes can be used as a marker of species evolution [46, 47]. At the boundary of the IRs regions, the changes in the length of the rpl2, rps19, trnN and ycf1 reflect the contraction and expansion of the IRs regions. In the evolution of higher plants, all kinds of repetitive elements have a common feature, which tends to show “co-evolution”, which plays a crucial role in the genome sequence diversity and gene rearrangement [48, 49]. Studies have shown that the number of repeats is related to the degree of genome rearrangement [50–52]. Here, long repeat sequences in ZJGS cp genome are the largest, and the number of 20–30 bp repeats may promote the rearrangement of ZJGS [53], these repeats can serve as good molecular markers for species evolution, and play an important role in the variation of cp genome sequence.
The breeding process of ZJGS is long and complicated. It has undergone many cross-breeding and space mutation to form excellent plants with stable inheritance. And its maternal B. kazinoki has experienced controversy [15, 54], therefore, it is necessary to compare ZJGS and its hybrid parent systematically. Unlike the previous study [36], the cp genome of ZJGS was analyzed in detail. Through comparison, it is found that the total length and total GC content of ZJGS cp genome sequence (160,903 bp and 35.74%, respectively) are closest to its maternal B. kazinoki (160,841 bp and 35.73%, respectively), while its paternal B. papyrifera is 160,239 bp and 35.83%, respectively. This reflects a high degree of species affinity in the history of system evolution [41, 55]. Similarly, in the sequence diversity comparison, we also found that the cp genome sequence of ZJGS and maternal B. kazinoki remains highly consistence. This is related to sequence conservation and provides a basis for the species evolution of ZJGS [40, 56]. The construction of the phylogenetic tree shows us the most intuitive results, closely related species are grouped into the same branch. And ZJGS is closest to maternal B. kazinoki, which strongly supports the characteristics of cp maternal inheritance [57, 58].
As we have known before, when Ka/Ks > 1, the gene is strongly positive selection [42, 43]. We can screen some genes according to Ka/Ks ratio and then carry out functional studies, which have been commonly applied to the field of molecular evolution [59, 60]. In our research, three genes of atpF, ndhB and rpoC1 show a complete positive selection. In previous studies, some genes have been reported with a faster evolution rate, including ycf1, ycf2, accD, clpP, ndhA, rbcL, matK, ccsA, and cemA [22, 61]. Compared with two cross parents, the same genes (atpF, ndhA, ndhB, ndhD, petB and rpoC1) with positive selection were screened out. Most of the NADH genes (ndhA-ndhK) have a higher Ka/Ks ratio than photosynthesis genes (Photosystem I: psaA, psaB, psaC, psaI, psaJ; Photosystem II: psbA, psbB, psbC, psbD, psbE, psbF, psbH, psbI, psbJ, psbK, psbM, psbN, psbT, psbZ), which is similar to the previous research [55, 62], shows that photosynthesis genes have strong purification options. NADH genes have higher activity during cellular senescence and oxidative stress [63, 64], and early hybrid selection experiments showed that most proteins synthesized by chloroplasts in the early stages of aging are NDH polypeptides [65]. It can be seen that these genes under positive selection belong to the advantageous genes of hybrid plants, and may be the key to speciation.