| Ormosia hosiei is a wild species in the second class of the State protection list.O.hosiei scatters in discontinuous or small patches and its natural habitat is within broadleaved forests near stream and in valleys in northern Shanxi and Jiangsu,Zhejiang,Jiangxi,Fujian,Hubei,Sichuan,Chongqing,Guizhou province,O.hosiei has been extensively used for craft and ornamental carving due to the high quality,color and luster of its heartwood.The natural resources have been seriously destroyed because of its high economic and cultural value.Due to difficult natural regeneration,,the existing natural populations of O.hosiei are usually small and becoming endangered.However,little is known about its genetic background.Based on previous researches,firstly,SSR markers were developed which were used to the genetic diversity analysis of O.hosiei;genetic diversity and genetic differentiation were systematically studied on the natural populations in different small watersheds;Secondly,the mating system of the natural population and individuals were illustrated,and the genetic differences between parents and offspring were compared to elucidate the maintenance mechanism of high genetic diversity.Finally,we carried out the progeny seedling test of plus tree to research the family variation of the growth and morphological traits of seedlings,which were also analyzed for the genetic control,and the effect of the genetic diversity on the growth of progeny.The results of this research would provide theoretical basis for the formulation of its genetic conservation strategies and effective utilization of resources for O.hosiei.The main results were summarized as follows.1.Specific-locus amplified fragment sequencing(SLAF-seq)was used to sequence the genome of O.hosiei.In total,6,426,462 reads and 16,653 polymorphic SLAF tags were obtained,with a total of 17,868 SSR locus.A SSR loci would be distributed per 6.98 kb on the average.The number of repeated elements types in different nucleotides and SSR loci had a big difference.In addition to the single nucleotide,the number of dinucleotide and trinucleotide repeated types were much more,accounting for 17.15% and 15.02% of the total SSR loci,respectively;among these the elements of AT/TA and the GGA/TCC had the most repeates,reaching 1 090(43.1%)and 349(15.2%),respectively.With the information got in O.hosiei,2718 pairs of SSR markers were finally designed out,with the types of di-nucleotide and tri-nucleotide dominate,of which the proportions reached 94.8%.Out of the 70 pairs of primers isolated from O.hosiei and its relative species,12 pairs were screened out with rich polymorphism and specific amplified bands.The percentage of polymorphic loci was 17.14%.The development of SSR primers laid foundation for genetic diversity,mating system and molecular marker assisted breeding of O.hosiei.2.Based on the developed SSR,genetic diversity and genetic differentiation of O.hosiei from three watersheds,LXH(Luxihe),XYX(Xiyangxi)and OJ(Oujiang)in Jiangxi,Fujian and Zhejiang provinces were analyzed,respectively.It was found that the populations in different small watersheds maintained high genetic diversity(HE>0.720).The order of genetic diversity from high to low was OJ(HE=0.835),XYX(HE=0.829),LXH(HE=0.796);In the Xixi river,the genetic diversity of FJFX in the middle reach was higher than that of in the upper reach(FJDY)and in the lower reach(FJZZ).Most of the genetic variation was observed in the watershed(92.73%)and within the population(89.66%),and the genetic differentiation among watersheds and populations was moderate.Genetic distance clustering and structure analysis clasified nine natural populations of O.hosiei from three small watersheds into two groups,among which LXH and XYX were classified as the first group,and OJ watershed for the second group.There was no significant linear correlation between genetic distance and geographic distance.Based on the above results,it was inferred that the fragmentation time was still quite short,therefore the genetic diversity was not significantly affected.3.Based on the analysis of genetic diversity of natural populations,three natural populations of O.hosiei from JXMTS,ZJBD and ZJJX-1 were selected for further analysis of its mating system The results revealed high levels of genetic diversity in both adults and offspring;slight difference existed among populations and their offspring in genetic diversity;the genetic diversity of JXMTS and ZJBD was higher than that of ZJJX-1.In addition,the fixed index(F)of offspring was significantly lower than that of adults,which indicated that the proportion of homozygous plants was higher than that of adults.The multilocus outcrossing rate(tm=0.884)was high.A small number ofbiparental inbreeding(tm – ts =0.078)was detected.We found a small number of effective pollen donors(Nep=2.096).The multilocus outcrossing rate of JXMTS(tm =1.000)was higher than that of ZJBD(tm =0.824),and less pollen donors were found in a bigger population.The tm of eight familes ranged from 0.772 to 1.000,most of which had a certain degree of biparental inbreeding and few of which had a small amout selfing.These results showed that it is important to create conditions to expand size of population,and to prevent the diaruption of gene flow caused by the destruction of marginal individuals or isolated individuals.4.The study with 76 open-pollinated families from plus-tree of O.hosiei,selected from Zhejiang,Fujian,Jiangxi and Sichuan province,were conducted to investigate the genetic variation of seed and seedling growth traits.The result of variance analysis showed that there existed significant or extremely significant differences among families for seed traits(seed width,seed thickness)and offspring growth(height and ground diameter),leaf shape traits and root characteristics etc,and the high growth rhythm parameters among families also reached a significant level.The correlation analysis showed that seedling height and ground diameter were positively correlated with seed thickness and hundred seed weight,respectively;the ground diameter has a higher positive correlation level with the characteristics of leaf and root than that with height of seedling;Meanwhile,the seedling height had extremely significant positive correlations with early stage of linear growth(t1)and growth parameters(MGR,LGR,TLG),and a weak negative correlation with final stage of linear growth,indicating that the family with a faster growth rate can preferentially occupy the growth space to obtain a high growth capacity.The family heritability of all growth traits was high,and all of the traits was under moderate or high genetic controll(hf2=0.479~0.854).20 superior families were selected from 76 families by taking seedling height as the main trait and taking ground diameter into account as well.In general,the effects of seed traits,leaf and growth rhythm parameters on seedling growth were significant and could provide a reference for future selection of superior family.5.We selected 26 representative families from open-pollinated families of O.hosiei to study the genetic diversity and its influence on the growth of seedlings.The results indicated that the genetic diversity of offspring was rich(NE= 7.766,HO=0.469,HE=0.865).Except for the loci of SSR8,the observed heterozygosity(HO)of the other locus were less than the expected heterozygosity(HE),indicating that the majority locus of the offspring was in heterozygous deletion state.The genetic diversity of different families was also different,the highest was the 12 th family,and the lowest was the 8th family.By comparison,it was found that the genetic diversity of the population progeny was significantly higher than that of the isolates.Both the F statistic and analysis of molecular variance(AMOVA)showed that the genetic variation offspring population mainly existed in the family,and the genetic differentiation among the families was small.The correlation analysis indicated that the genetic diversity of the offspring was positively correlated with the seed traits and the seedling height at the 7th month after sowing(r=0.378~0.527).It was showed that the natural populations of O.hosiei played an important role in the maintenance of genetic diversity,and the genetic diversity of offspring significantly affected the later growth of seedlings.These could provide theoretical basis for the selection of superior family. |
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