Study On Genetic Mechanism Underlying The Reproductive Isolation Between Pinus Massoniana And P. Huangshanesis

Speciation is one of the most interesting processes in evolution, and reproductive isolationis essential for the process of speciation to be complete. Thus, understanding the geneticmechanism triggering reproductive isolation is a key goal in the study of speciation. To date, theunderlying causes of reproductive isolation are only partially understood in a few species.Hybridization between different species often has negative consequences that result in reducedfertility or viability, which leads to transmission ratio distortion (TRD) of the parental gametes.Occasionally, hybridization can raise hybrid vigor in which a hybrid offspring performs well.Yet, recombination and allelic segregation may render hybrid breakdown in the next generation.Genetic mapping methods provide a unique opportunity to study the interactions ofdifferentiated genes and genomes in a hybrid genetic background. The degree of TRD isthought to be positively correlated with the level of genome divergence between taxa, mappingthe SDLs that control the TRD marker clusters can be used to infer the genome regions relatingto reproductive barriers in distant crosses. To date, the majority evidences of TRD occurring inhybrid genetic backgrounds are based on studies employing experimental hybrids. By contrast,map-based studies of natural hybrids are much rarer.Pinus hwangshanensis and P. massoniana are native conifers in south of China. In theareas to the south of Yangzi River, P hwangshanensis is mainly found in the elevation range of800-1800m, and P. massoniana distributes below elevation of800-900m. The major differencein the ecological niches of the two species is elevation. With an increase in elevation,environmental factors, such as oxygen partial pressure, air temperature and moisture regime,soil temperature and water regime, sunray and ultraviolet light intensity, will change. Theseenvironmental factors are closely related to plant growth and fitness. They are environmentalstresses to cause differentiation in plant phenology and fitness, subsequently, to maintain thespecies-specific characteristics of the alternate species. The distribution ranges of P.hwangshanensis and P. massoniana are frequently found to be adjacent to each other, andoverlapped with a narrow contact zone. This distribution pattern leads them to be the desirableresearch materials to uncover the genetic mechanism triggering the parapatric speciationprocess. In my thesis, studies were carried out in the following aspects: Variation in conemorphology and seed characteristics for P.massoniana, P. Hwangshanensis, and their hybridzone; Variation trends of physiological and biochemical indexes of P. hwangshanensis withincrease in elevation; AFLP protocol optimization and primer combination screening for Pinusspp.; Potential chromosomal introgression barriers revealed by linkage analysis in a hybrid of P. massoniana and P. hwangshanensis; Genome-wide detection of genetic loci triggering unevendescending of gametes from a natural hybrid pine. The main context include:1. Variation in cone morphology and seed characteristics for P. massoniana, P.hwangshanensis and their hybrid zone. The cones and seeds were collected from differentelevations of Mt. Huang in Anhui Province. Six characteristics of cones and seeds, includingcone length, cone width, ratio of cone length to width, seed plumpness, germinationratio and weight per thousand seeds were analyzed to reveal the variation trends with thechanging of elevations. The results indicated that: Cone length, cone width, ratio of conelength to width decreased with increase in elevation; Seed plumpness, germination ratioand weight per thousand seeds were lower for plants in hybrid zone than that of theother zones. We propose it may relate to the reproduction barrier between P. massonianaand P. hwangshanensis. It was noticeable that the majority of variation for cones andseeds arose among individual trees, and traits were commonly diverged amongindividuals at different elevations.2. Variation trends of physiological and biochemical indexes of P. hwangshanensiswith increase in elevation. P. hwangshanensis seeds collected from different elevations ofHuanggang Mountain in Wuyi Nature Reserve were cultivated in a container at25℃. Aftersprouting,the seedlings were stored in a refrigerator within a temperature range of2-5℃for24hours. Five physiological and biochemical indices of P. hwangshanensis seedlings weredetermined before and after the treatment,and the variation trends of these indexes withincreasing elevation were also studied. Three of the tested indices,including relative electricalconductivity of extract fluid,SOD activity,and MDA content of the seedlings after lowtemperature treatment decreased with increasing elevation. Therefore,the above three indicesare related to the adaptability of P. hwangshanensis to elevation. These indices could be appliedto explore the mechanism of parapatric speciation.3. AFLP protocol optimization and primer combinations screening for Pinus spp.Pines possess gigantic genomes (10-20Gb), which are about10fold that of the human genomeand about40fold that of the poplar genome, leading them out of the genome range suitable forthe common AFLP(Amplified Fragment Length Polymorphism) protocol. This paper speciallyoptimized an economic and reliable AFLP analysis protocol for pines. The number of selectivenucleotides added to the core primers and the primer combinations suitable for AFLP analysisin such species were tested. Some AFLP primer combinations that were screened their utilityacross different species were tested. The results could also be used as a reference for allowingbetter amplification in complex templates isolated from plants species with large genomes4. Potential chromosomal introgression barriers revealed by linkage analysis in ahybrid of P. massoniana and P. hwangshanensis. Distortion of marker transmission ratio isfrequently ascribed to selection against alleles that cause hybrid incompatibility. The naturalintrogression between P. massoniana and P. hwangshanensis and their distribution ranges lead to the emergence of the two species as desirable organisms to study the genetic mechanisms forspeciation. Using seeds sampled from trees at different elevations, we consistently detectedsharp decreases in seed germination rates of trees in the hybrid zone, which might be duelargely to the hybrid incompatibility. A genetic map was established using192megagametophytes from a single tree in the hybrid zone of the two species. Segregationdistortion analysis revealed that the percentage of significant-segregation-distortion (SSD)markers was extremely high, accounting for more than25%of the segregating markers. Theextension range, the distortion direction, and the distortion intensity of SSD markers also varieddramatically on different linkage groups. This study displays the potential chromosomalintrogression barriers between P. massoniana and P. hwangshanensis. The established mapprovides a valuable platform for conducting genome-wide association of hybrid incompatibleQTLs and/or candidate genes with marker transmission ratio distortion in the hybrid.5. Genome-wide detection of genetic loci triggering uneven descending of gametesfrom a natural hybrid pine. Marker transmission ratio distortion (TRD) revealed in geneticmapping studies of distant crosses can be used to infer the genetic basis relating to reproductivebarriers between species. Unlike measure the degree of TRD by the overall number ofsegregation distorted markers in the affected genome regions, mapping the segregationdistorting loci (SDL) provides reliable statistic parameters that help to confine the targetgenomic region for further characterization at molecular level. Using the linkage mapconstructed for a natural hybrid of P. hwangshanensis and P. massoniana, we performed SDLanalyses and aligned the established map to the pine consensus map. Altogether,6SDLs withrelatively strong LOD supports were detected on4linkage groups of the established map. Sincegametes inheriting different alternate chromatid blocks from the SDL affecting genome regionshad uneven chance to descend to the offsprings, the corresponding genome regions weresupposed to play more significant role in rendering the speciation of P. hwangshanensis and P.massoniana. This paper presents a case study on a crucial step for uncovering the geneticfactors that triggered the uneven descending of gametes in a natural distant cross, and it maydraw broad interest for the evolutionary biologists.