| Larix principis-rupprechtii Mayr is an important afforestation tree species in North China,which has both economic and ecological values.The breeding goal of L.principis-rupprechtii is to develop new varieties with fast growth,high timber quality,and strong resistance.However,because of its long growth cycle,complicated genetic background,and quantitative genetic attributes of most economic traits,the efficiency of breeding new varieties by traditional methods is usually very low.Using DNA molecular markers for genetic map construction and QTL mapping,and then carrying out molecular marker-assisted selection can shorten the breeding cycle and improve breeding efficiency.However,due to the deficiency of genomic data and high-quality molecular markers,there has been no report on the genetic map construction and QTL mapping in this tree species thus far.In this study,polymorphic EST-SSR markers were developed using transcriptome sequencing data of L.principis-rupprechtii,and the utility of these markers was verified by conducting genetic diversity analysis.After using a part of the newly mined markers to perform hybrid identification on the F1 mapping population,the SLAF-seq technology was used to identify large-scale SNP markers and construct the first high-density genetic map of L.principis-rupprechtii.QTL analysis was performed on 8 growth and needle traits based on the phenotypic data measured for two successive years.It is of great theoretical and practical significance to carry out the above research with regard to 1)increase the number of high-quality molecular markers;2)analyze the genetic structure of important quantitative traits;and 3)promote ge netic improvement at the molecular level.The main results are as follows:(1)A new set of polymorphic EST-SSR markers across Larix species were developed by transcriptome sequencing.Based on 1300 Unigene sequences,1065 primer pairs were successfully designed.After screening 240 primer pairs randomly selected from these candidates,52 polymorphic primer pairs were obtained.Then 20 polymorphic EST-SSR markers with the clearest banding patterns was further selected to genotype 66 clones of L.principis-rupprechtii,a total of 77 alleles were detected,and the number of alleles per locus ranged from 2 to 7.Additionally,all of the 20 EST-SSR markers could amplify clear and stable bands across three related Larix species,with an efficient amplification ratio of 100%.(2)The newly developed EST-SSR markers were used to analyze the genetic diversity of 66 clones deployed in a seed orchard of L.principis-rupprechtii.The mean values of the number of alleles and polymorphism information content at 20 loci were 3.85 and 0.424,respectively,indicating that the seed orchard had a moderate level of genetic diversity.The genetic distance between 66 clones ranged from 0.012 to 0.585,with an average of 0.317.The relatively wide range of genetic distances indicated that the 66 clones studied were genetically diverse.Clustering analysis distributed these 66 clones into three main clusters,which was further validated by principal coordinate analysis(PCo A),but due to incomplete information and confusion in records,it is not clear whether the distribution of 66 clones follows their geographical origins.(3)An F1 mapping population composed of 145 individuals was constructed by intraspecific hybridization between two elite clones of L.principis-rupprechtii.After authenticity identification of hybrids,SLAF-seq technology was used to develop genome-wide SNP markers.SLAF-seq generated a total of 1501.22 M pair-end reads,approximately 300.20 Gb of raw data.After sequence alignment and clustering,6,323,943 SLAFs were generated.The sequence with the highest copy number for each SLAF was selected as the reference sequence and was used to mine SNP markers,and 122,785 of the 324,352 SNP markers detected were polymorphic,with a polymorphism rate of 37.86%.Finally,6931 valid SNP markers with parental sequence depths of >10-fold,integrities of >75%,and expected Mendelian ratio were obtained.(4)The first high-density genetic linkage map of L.principis-rupprechtii was constructed.6099 SNPs were organized into the genetic map containing 12 linkage groups,consistent with the haploid chromosome number of larch and most other species in the Pinaceae family.The linkage map spanned 2415.58 c M and covered 99.6% of the L.principis-rupprechtii genome with an average of 0.4 c M between adjacent markers.The average sequencing depth of the finally mapped SNPs in the parent and progeny was 65.84-and 7.73-fold,respectively,and a mean integrity of >99% for all samples.(5)According to the information from the genetic map,QTL mapping of 8 growth and needle traits measured for two successive years was performed using composite interval mapping.When the LOD(Logarithm of Odds)threshold was 2.5,a total of 36 putative QTLs were detected,including 7 QTLs for needle area,5 QTLs each for seedling height,basal diameter and needle width,4 QTLs each for needle length and needle thickness,and 3 QTLs each for length-width ratio of needle and number of stomata rows.These QTLs were assigned to 10 linkage groups except LG7 and LG10,and each QTL explained 4.2%-18.2% of the phenotypic variation.A total of 6 QTL cluster regions were detected in the two test years,of which 136.365-161.717 c M region on LG8 and 43.453-65.422 c M region on LG9 contained many QTLs controlling different traits,and the contribution of each QTL was high,which will be the target regions for future research.Based on transcriptome sequencing and SLAF-seq technology,a large number of high-quality SSR and SNP markers were developed at the genome-wide level and the first high-density genetic map of L.principis-rupprechtii was constructed in the current study.On this basis,QTL analysis of growth and needle traits of L.principis-rupprechtii was performed for the first time,and some QTLs controlling these important phenotypic traits were obtained.These results will provide powerful molecular tools and information support for accelerating the genetic improvement of L.principis-rupprechtii. |
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