Construction Of Genetic Map And QTL Mapping Of Seedling Growth Traits Of Eucalyptus Cloeziana F. Muell

Eucalyptus cloeziana is an important high-value solid wood tree species,known as"Australian scentedrosewood",which is native to Queensland,Australia.Genetic improvement of E.cloeziana is just beginning,and how to improve genetic gain quickly and effectively are extremely urgent.Molecular breeding is selection using markers associated with traits,for trees with complex genetic background,high heterozygosity and easy to be affected by environment,it can improve the accuracy of early selection and accelerate the process of breeding.Genetic map,as an important tool for genetic improvement,provides important help for analyzing genetic structure,analyzing gene evolution and assisting genome splicing and assembly.QTL mapping analysis is the most effective way to study the genetic relationships between traits and markers,and plays an important role in molecular assisted breeding.In this study,the breeding population of E.cloeziana was used as the experimental material,based on SSR molecular marker technology,through the paternal identification,the mapping mother parent of E.cloeziana was determined,and the genetic characteristics and rules of breeding population of E.cloeziana were studied,and the full-sib offspring of the mapping population were screened.Based on GBS sequencing,SNP markers were developed to construct the genetic map of E.cloeziana,and QTL mapping was performed for seedling growth traits（Internode number,Ground diameter,0.5 year old Seedling Height,1.5 year old seedling height,Leaf length and Leaf width）.The main contents and conclusions are as follows:（1）The female parent of mapping population was selected and determined,and the genetic characteristics and rules of breeding population of E.cloeziana were analyzed.In this study,six E.cloeziana trees were selected as the mother trees.The paternal parent of 433progeny populations of 6 female E.cloeziana trees were identified based on ABI3130 XL sequencing platform by using the combination of previous multiple fluorescence SSR analysis system.The results show that,within the 80%confidence interval,84.6%（366 strains）of progeny could be identified as the paternal parent,and the highest identification rate was in 5-018 families（98.8%）.The number of outcrossing progeny from the six parent trees ranged from 20 to 62,among which 5-018 produced the most outcrossing progeny（62）.Nine male parent trees provided the main pollen for 5-018 female parent trees,and the maximum number of full sib progeny was 22.In particular,the progeny populations of these six parent trees all have different degrees of inbreeding.The results of the paternal identification showed that the self-crossing rate of E.cloeziana was low,and outcrossing was the main propagation method of E.cloeziana.In the end,the parent tree 5-018 with the highest proportion of paternal parent identification,the largest number of progeny produced by outcrossing,the least number of potential candidate paternal parents,good growth and abundant seed yield was selected as the parent tree of the mapping population,providing material for the subsequent map construction.In addition,the effective pollen dispersal range of E.cloeziana mainly concentrated within 100m.The multi-locus outcrossing rate（t_m）of E.cloeziana was 0.661,which indicated that E.cloeziana produced seeds mainly through outcrossing.The single-locus outcrossing rate（t_s）value of unit point is 0.309,and the biparental inbreeding（t_m-t_s=0.352）is greater than 0,indicating that there is a certain degree of inbreeding phenomenon.The multi-locus correlation of paternity(r_p（m）)and the single-locus correlation of paternity(r_p（s）)were 0.857 and 0.640,respectively.In the 6 families,the t_m values of each family ranged from 0.847 to 0.962,and the t_s values ranged from 0.221 to 0.603,and the biparental inbreeding of each family were all greater than 0,which also indicated that these families all had inbreeding phenomenon of different degrees.SSR genotyping was performed on 433 progeny populations,the average number of alleles（N_a）and effective number of alleles（N_e）were 6.3 and 1.8,respectively.The average observed heterozygosity（H_o）and expected heterozygosity（H_e）of progeny population were 0.403,0.659,Shannon index（I）and polymorphism information content（PIC）were 0.676and 0.605,respectively.The average fixed index（F）was 0.025,indicating a high level of genetic diversity.In addition,for breeding populations of E.cloeziana,the average N_aand N_ewere 10.5 and 1.9,respectively.H_owith an average of 0.431,H_e was 0.681,the average I and PIC were 0.673 and 0.654,respectively.The average of F was 0.046.This indicates that the level of genetic diversity is also high.Compared with the offspring population genetic parameters,although there are some differences,but no significant level,the results showed that the genetic diversity of E.cloeziana offspring population did not decrease.The analysis of population structure by NJ method showed that there was major genetic differentiation between the southern provenances and the northern provenances,but the genetic differentiation between individuals was not obvious and the genetic structure was weak.These conclusions can also provide technical guidance for the cultivation and thinning of E.cloeziana,and provide important theoretical basis for the prevention of inbreeding decline,rational planning and design,breeding strategies and effective protection of genetic resources.（2）Identification of full-sib seedlings in E.cloeziana to improve the efficiency of conventional breeding.Based on the previously established multiple fluorescent SSR marker analysis system,A total of 2589 progenies of E.cloeziana open pollination were selected for paternal identification,pollen propagation distance detection,separation ratio of 12 markers（χ²）in the whole sib population,and correlation of corresponding genetic parameters with seedling height growth were estimated.The results showed that all the markers could be amplified and detected effectively.A total of 1,986（76.6%）progenies were identified with clear paternal parents.There were 6 main paternal parents with more than 140 progenies,and the number of progenies of a single paternal parent ranged from 142 to 456.The pollen dispersal distance can be up to 160 m,but the effective pollen dispersal distance is mainly within 100 m.For 7full-sib families with more than 10 progenies,12 markers showed different degrees of deviation from the expected Mendelian separation ratio.Diversity parameters such as H_o and H_e were relatively low,which had no significant correlation with seedling height and variation coefficient,and no self-crossing was found in this experiment.In this study,paternal identification was carried out on the progeny of free pollination of the female parent to obtain full-sib progeny,which is a relatively fast and effective method compared with conventional breeding strategy.These results can provide material basis for further studies on genetic determination,genetic map construction and quantitative trait locus localization based on the full-sib family.（3）The genetic linkage map of E.cloeziana was constructed based on GBS technique.One female parent（5-018）,four male parents and 441 progeny were used as experimental materials,the genetic map of the mother was constructed.In this study,the SNP markers were obtained by using simplified genome sequencing technology（GBS）.A total of 617 GB of original sequencing data was obtained.After screening,clean data with an average reads of 16599161was obtained,with an average sequencing coverage of 29.94%and an average sequencing depth of 4.23×for all samples,Compared with the reference genome,a total of 78289 highly reliable SNP data were obtained,and the average distance of the whole genome was 6.80Kb/reads,the genetic linkage map of E.cloeziana was divided into 11 linkage groups,and there were 1783 markers in the figure above.The total length of the map was 1135.300 c M,the average coverage distance of each linkage group was 103.209 c M,and the average genetic distance between the two markers was 0.69 c M,and the genetic spacing Gap of most of the markers was less than 5 c M,indicating good quality of the map.These results may be helpful for comparative genomics research among Eucalyptus and play an important role in genome assembly and splicing.In addition,the drawing of genetic map also provides the premise for QTL mapping of the traits of interest.（4）QTL mapping of growth traits in E.cloeziana seedling stage.Based on the genetic linkage map of E.cloeziana,the seedling Internode number,Ground diameter,0.5 year old Seedling Height,1.5 year old seedling height,Leaf length and Leaf width determination of six main growth traits such as evaluation,the results are accord with normal distribution.A total of29 growth-related QTL were detected,mainly distributed on all linkage groups except lg6,but the distribution is not uniform on each linkage group.There were 6 QTLs related to internode number,namely IN-lg2-1,IN-lg3-1,IN-lg9-1,IN-lg10-1,IN-lg11-1 and IN-lg11-2,and the phenotypic variation rates that could be explained by a single QTLs ranged from 2.2%to5.5%;There were 7 QTLs related to ground diameter,namely IN-lg3-1,IN-lg5-1,IN-lg5-2,IN-lg7-1,IN-lg7-2,IN-lg9-1,and IN-lg9-2,and the phenotypic variation rates that could be explained by a single QTLs ranged from 2.2%to 12.2%;There were 5 QTLs related to 0.5year old seedling height,namely 0.5SH-lg1-1,0.5SH-lg3-1,0.5SH-lg7-1,0.5SH-lg7-2 and 0.5SH-lg11-1,and the phenotypic variation rates that could be explained by a single QTLs ranged from 2.4%to 4.3%;There were 4 QTLs related to 1.5 year old seedling height,namely1.5SH-lg2-1,1.5SH-lg3-1,1.5SH-lg7-1 and 1.5SH-lg10-1,and the phenotypic variation rates that could be explained by a single QTLs ranged from 2.3%to 3.7%;There were 3 QTLs related to Leaf length,namely LF-lg3-1,LF-lg4-1 and LF-lg11-1,and the resolvable phenotypic variation rates were 3.7%,4.5%and 2.6%,respectively;There were 4 QTLs related to leaf width,namely LW-lg3-1,LW-lg8-1,LW-lg8-2,and LW-lg11-1,and the phenotypic variation rates that could be explained by a single QTLs ranged from 2.4%to4.9%.These results can lay a foundation for the fine mapping of QTL,the discovery of candidate genes and the identification of markers and auxiliary breeding,thus contributing to the genetic improvement of E.cloeziana.