| Hermaphrodite is the most common type in angiosperms, and a lot of evidence shows that the hermaphrodite can evolve into the dioecy through multiple ways. Monoecy, gynodioecy and androdioecy are all likely to be the intermediate stage from hermaphrodite to dioecy. Addtionally, androdioecy is the rarest breeding system. The androdioecy populations consist of male and bisexual individuals. The evolutionary stable strategy (ESS) theory holds that male individuals in order to maintain in the population of hermaphrodites, male fitness of male individuals need more than twice higher than hermaphrodites. If hermaphroditic flowers selling fertile, males will need a higher fitness. Because of the harsh conditions to maintain androdioecious breeding system, it was once believed that the functional androdioecy do not exist. Studies have proven that most morphological androdioecy are functional dioecy, namely hermaphroditic individuals in the populations do not have male function. However, recent studies have confirmed that more than ten types of angiosperms are androdioecy; in these populations, hermaphroditic individuals can bear fruit as well as produce fertile pollen.Tapiscia sinensis, the tertiary ancient tropical relict plant of China, is a perennial tall tree that belongs to Tapisciaceae family Tapiscia genus. Due to habitat fragmentation, scarce number of individuals within populations, poor natural regeneration ability and habitat destruction, T. sinensis has been listed on the endangered species red list by the International Union for Conservation of Nature (IUCN). The study found that male and hermaphroditic individuals coexist in T. sinensis populations, showing androdioecious characteristics. Even though it is demonstrated in the artificial pollination experiment that both the bisexual and male pollens can give rise to fruit, it is still unknown about whether the wild type of T. sinensis is functional androdioecy plants in the natural environment. What this breeding system involves from? Does the androdioecy is the transition phase of the development in which, the hermaphrodite involve into dioecy plants? Or is it a situation that female individual recover the male function in dioecy plants results from the limited pollens existence? In this study, we conducted the transcriptomes sequencing of T. sinensis flower, found a number of homologous genes related to flower development, developed a large number of SSR molecular markers; analyzed breeding system of T. sinensis by using the method of paternity analysis, analyzed genetic diversity of T. sinensis populations, and discussed it's endangered mechanism and protection strategy. The main results are as follows:(1) By means of assembly,52,169 unigenes with an average length of 1116 bp were obtained from T. sinensis floral transcriptome sequencing data. A total of 36,662 unigenes (70.27%) were annotated,10,002 (19.17%) unigenes were assigned to 124 KEGG pathway. Among them,224 unigenes were assigned to "Starch and sucrose metabolism" pathway which is associated with pollen development; 260 unigenes were assigned to "plant hormone signal transduction" pathway that related to the regulation of sex differentiation. A number of transcription factors and candidate genes with function related to sex, flower development and flowering time monitoring were obtained.(2) A total of 10,371 loci were identified from the 52,169 unigenes, and 16,317 pairs of SSR primers were designed. A set of 150 pairs of primers was synthetized and assessed,117 pairs (78%) successfully amplified target fragments for SSR-PCR, including 68 pairs that were able to produce clear polymorphic bands (45.5%). By screening, we also got a pair of SSR primer that can identify the gender of T. sinensis. Six polymorphic SSR markers can be used to distinguish the population genetic differences based on different geographical locations.(3) Twelve pairs of polymorphic SSR primers were used to conduct paternity analysis for 91 male candidates and 556 progenies that from 8 sample groups in four natural populations "Shaanxi Ningshan (NS), Hunan Yongshun (YS), Hunan Yanling (YL), and Hubei Shennongjia (SN)" of T. sinensis. A total of 367 offspring (66%) was determined belong to the only male parent under 80% confidence level. Among all the offspring that have determined male parent, those from male or hermaphroditic male parent were respectively 226 and 141, accounts for 61.58%,38.42% respectively. Experiments show that the natural population of T. sinensis is functional androdioecy, and self compatibility.(4) In the eight groups of samples, except NS101S and YS26S, the other six groups own the ratio of the average male sire rate between male and hermaphrodite individuals (F) are equal to or greater than two. This result consists of the ESS theoretical model about androdioecy. T. sinensis hermaphroditic individuals with extremely long reproductive cycle and their flowers and fruits develop synchronously; pollens of male and hermaphroditic individuals have different shapes; anemophilous combined with insect-borne pollination; setting rate of artificial pollination was not higher than that of open pollination, all of these features are pointing to that the androdioecy of T. sinensis might evolve from hermaphroditic ancestors.(5) The four of T. sinensis populations above mentioned coupled with Guizhou Xifeng (XF) populations, the total of five populations were conducted genetic diversity analysis. The results show that natural populations of T. sinensis have rich genetic diversity(PPL = 100%, He = 0.6904,I = 1.4368). The high Shannon's index shows that the T. sinensis populations present at a relatively stable stage. Analysis of genetic relationships among populations indicates that the genetic distance between HunanYanling (YL) and Guizhou Xifeng (XF) populations was the smallest (0.4829), genetic distance between Shaanxi Ningshan (NS) and Guizhou Xifeng (XF) populations was the largest (0.9821). Mantel Test shows that there is no correlation between geographic distance and genetic distance. AMOVA suggested that 33.3% of the variation happened among populations,66.7% of the variation happened in the populations. Moderate gene flow among populations(Nm = 0.7274) is not sufficient to counteract the genetic drift within the populations and result in significant differentiation among populations (Fst = 0.2987).(6) In this study, Hunan Yongshun (YS) and Hubei Shennongjia (SN) populations that with abundant genetic diversity are the focus of genetic conservation. It can be further to increasing the number of individuals, and expanding the scope of the populations to maintain their stability. Hunan Yanling (YL) population existing only 12 individuals should be strictly protected to avoid the danger of extinction. Furthermore, it is necessary to stabilize the population structure through applying artificial pollination, reintroduction and other manual intervention methods. In addition, the habitat's conservation of the T. sinensis is needed to pay more attention. |
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