Genetic Linkage Map Construction And QTLs Analyses For Gardenia Jasminoides Ellis

Gardenia（Gardenia jasminoides Ellis）is an evergreen shrub of the Rubiaceae（Rubiaceae）genus in Gardenia（Gardenia）family.As early as the Tang Dynasty,gardenia had become an important ornamental object.Its dried and mature fruits are commonly used bulk Chinese medicinal materials.In recent years,the demand for gardenias in domestic and foreign markets has increased rapidly,and the blind large-scale artificial planting of gardenias has significantly increased the pests and diseases of gardenia,which has seriously affected the industrialization of gardenias.As a medicinal plant,gardenia has a weak research foundation,and there is no report on constructing a genetic map,and it is difficult to conduct in-depth research such as gene discovery.In this paper,the F₁population of the hybrid combination of"high-branched,large-fruited,mid-leaf and wide-crown gardenia"（coded as GD1,♀）and"short-branched,small fruit,fine-leaf and dwarf-crown gardenia"（coded as AX5,♂）was used as the test material.We dedicate to develop SSR molecular markers,use simplified genome sequencing technology（Genotyping by sequencing,GBS）to obtain SNP markers,and construct a high-density gardenia genetic map based on SNP molecular markers.The traits of the F₁ generation populations were investigated for two years,and QTL mapping was performed.The main results are as follows:1.Full-length transcriptome sequencing and SSR primer design.Gardenia was used as the test material to perform the three-generation transcriptome sequencing,and SSR primers were designed based on the sequencing results to screen primer polymorphisms and identify hybrids.After completing the transcriptome sequencing,a total of 25.5Gb of Clean Data was obtained,and 387,265 CCS（Circular consensus）reads were obtained.The average CCS sequence length was 2,709 bp,and the insert size was 1 to 6 kb.Among them,there were 315,271 non-chimeric（FLNC,Full length reads non-chimeric）full-length sequences,and the full-length non-chimeric percentage of CCS sequences was 81.41%.The Iso Seq module of SMRT Analysis software was used to cluster the similar sequences in the full-length non-chimeric sequence,and a total of 32,933 identical transcripts with an average length of 2,493bp were obtained.After the consensus sequence was polished,32,704 high-quality transcript sequences were obtained,accounting for 99.30%of the consensus sequence.Using CD-HIT software to remove redundancy,18,957 non-redundant transcript sequences were finally obtained.Combining the transcript data obtained from sequencing,using Msat Commander,in GAP,Micro Family and SSRTool software to develop SSR molecular markers,finally 447 pairs of primers were obtained.2.Screen effective SSR amplification primers and identify hybrids.In addition to using software to design primers,the experiment also used 47 pairs of primers in published articles,and currently a total of 494 pairs of primers.In order to obtain stable and reliable amplification results,494 pairs of SSR primers were screened.Due to the large number of primers,we chose to conduct preliminary screening in the parents GD1 and AX5 to determine the primer amplification conditions.After the initial screening,amplification was carried out in the F₁ generation mapping population,and the primers that were polymorphic,had few miscellaneous bands,and the target bands were clearly distinguishable,and met the gene coding rules were selected.In the end,39 pairs of primers were successfully retained,and 200 plants were identified as true hybrids with SSR primers.3.Develop SNP molecular markers and construct gardenia genetic maps containing SNP molecular markers.From the F₁hybrid population of"GD1"and"AX5",200 identified hybrids were selected as mapping materials,and simplified genome sequencing was performed on the sequencing platform Illumina Nova 6000 to develop SNP molecular markers.A high-density gardenia genetic map was constructed based on SNP molecular markers.This map contained 11 linkage groups,and 4,249 SNP markers.The total covered genetic distance was 1956.28c M,and the average genetic distance was 0.46c M.Among them,there are 120 molecular markers on the ninth linkage group,which is the smallest number of markers among the 11linkage groups,and there are 648 molecular markers on the second linkage group,which is the largest number of markers in the linkage group.4.Gene mapping of quantitative traits.A two-year phenotypic data survey was conducted on 188 hybrids.The investigated yield-related traits included crown width,number of branches at the base of a single plant,main stem tilt angle,plant height（plant height）,and main stem length.,The thickness of the base of the main stem,the number of leaves on the main stem,the number of leaves per plant,the length and width of the longest leaf on the main stem,and the length and width of the shortest leaf on the main stem,a total of 12 traits.According to the constructed gardenia genetic map,using interval mapping method,combined with two-year phenotypic survey data,QTL（Quantitative trait locus）positioning was carried out for 12 traits.When the LOD threshold is 2 to 4.34,a total of 55 QTLs were detected in the two test years of 2020 and 2021,of which 16 QTLs controlling the number of leaves per plant,8 QTLs controlling plant height,and controlling the main stem There are 6 QTLs for the shortest leaf length,4 QTLs for controlling the number of branches at the base of a single plant,main stem inclination angle and main stem length,and 3 QTLs for the crown width and the shortest leaf width on the main stem.There are 2 QTLs controlling the thickness of the base of the main stem,the number of leaves on the main stem,and the longest leaf width on the main stem.There is 1 QTL controlling the longest leaf length on the main stem.Among them,the crown width,the number of branches at the base of a single plant,the inclination angle of the main stem,the height of the plant,and the number of leaves per plant can be detected within the same QTL interval of the same chromosome in two years of determination.