Characterization Of Trititrigia Germplasm SN304 And QTL Analysis Of Its Important Traits

Thinopyrum intermedium(2n=6x=42)is one of the important wild relative species of wheat,which has the characteristics of luxuriant growth,large spike,multiple flowers,strong stress resistance,and wide adaptability,it also has good resistance to various diseases and insect pests.So it is a valuable gene pool for the genetic improvement of wheat.Many Trititrigia Germplasm lines were created by distant hybridization between Th.intermedium and common wheat,which retained many excellent characters of Th.intermedium,and is a bridge material to transferring excellent genes from Th.intermedium to wheat.More attention has been paid to the transfer of Th.intermedium to wheat for its excellent disease resistance and insect resistance genes,but there few studies are on the influence of yield-related characters from Th.intermedium.In this study,molecular cytogenetics and re-sequencing techniques were used to identify the genetic composition of the wheat-Th.intermedium germplasm line Shannong304(SN304);a RIL population was constructed using SN304 and common wheat Yannong 15(YN15),and specific locus amplified fragment sequencing was used to construct a high-quality genetic map.QTL analysis of yield-related traits in different environments was carried out,QTL from yield-related traits of Th.intermedium was located,and new Trititrigia germplasms with excellent comprehensive traits were screened.The main results are as follows:1.Th.intermedium genomic DNA was used as the probe to hybrid with SN304 by genomic in situ hybridization(GISH)analysis,which showed no Th.intermedium hybridization signal in SN304.Fluorescence in situ hybridization(FISH)analysis was performed on SN304 and YN15 using oligonucleotide AFA-3,AFA-4,p As1-1,p As1-3,p As1-4,p As1-6,p Sc119.2-1,and(GAA)₁₀ as probes,showed that there were significant differences in hybridization signals between SN304 and YN15 on chromosomes 2A,7A,2B,3B,6B and 7B.Many pairs of molecular markers were amplified specific bands of Th.intermedium in SN304.The above results indicate that SN304 is a wheat-Th.intermedium invisible introgression line.2.Constructing a genetic linkage map using the RIL population of SN340 and YN15 based on PCR markers and polymorphic markers developed by SLAF-seq.The genetic linkage map containing 3053 markers,which was located on 18 linkage groups,the total length was1401.44cM,and the average genetic distance between markers was 0.46cM.3.Under different cultivation environment,24 essential traits of SN304,YN15,and RIL populations were investigated and statistically analyzed.Except for some traits,such as the growth period,the essential traits of SN304 were significantly higher than YN15.Most of the important traits showed continuous variation in different environments,and the coefficient of variation was within the normal range and reached a significant level between genotypes and environments.4.Under different fertility conditions,QTL analysis of 24 essential traits was carried out by linkage analysis and genome-wide association analysis(GWAS).A total of 385 QTL of important traits were detected by linkage analysis,which distributed on 17 chromosomes of wheat.A single QTL can explain 1.52%-45.84%of phenotypic variation,and the maximum LOD value is 75.34.Among them,104 QTL could be detected in more than two environments,and 59 QTL were the main QTL stably expressed between environments.Through GWAS,a total of 3709 SNP were detected,2214 SNP sites of which accounted for more than 10%of the phenotypic contribution,of which 1444 SNP could be detected in more than five environments,which were SNP sites stably expressed in multiple environments.By combining linkage analysis and GWAS,159 QTL were co-located,including 82 QTL stably expressed among environments.Among them,including 50 QTL additive effects from SN304,and 48 QTL were explicitly expressed in low fertilizer environments,of which 18 were significant QTL.5.Under an arid environment,102 QTL of 24 essential traits were detected using linkage analysis and GWAS.Linkage analysis showed that all the QTL were located on 15chromosomes of wheat.A single QTL could explain 1.99%-40.85%of phenotypic variation,and the maximum LOD value was 42.88.Among them,8 QTL could be detected in two arid environments,and the phenotypic variation rate of 3 LOD was more than 10%,which were stably expressed QTL in the arid environment.A total of 2033 SNP were detected through association analysis,of which 494 could be detected in both environments,and 1312 SNP contributed more than 10%.Linkage analysis and association analysis showed that there were11 co-located QTL intervals.Compared with traditional irrigated land condition,10 QTL were co-located in the same intervals,and only one QTL was explicitly expressed in the arid environment.6.The relationship between QTL interval of yield-related traits and Th.intermedium was verified by developing double-terminal anchored primers and re-sequencing analysis.For 82pairs of stably expressed major QTL intervals,double-terminal anchored primers were developed.Three pairs of primers located on chromosomes 2B,6B and 7B,which amplified the bands of Th.intermedium in SN304.The resequencing sequences of SN304 and YN15 were compared with the Chinese Spring reference genome,and the differential sequences with large fragments of 2BL,6BS,and 7BL chromosomes were found.Furthermore,comparing the differential sequences with the Th.intermedium genome sequence,it was found that the specific sequences on chromosome 2B of SN304 has good homology with a sequence on the second homologous group of Th.intermedium.The changes of the above results on chromosome 2B were consistent with the differential signals of SN304 and YN15 in FISH.Moreover,chromosome 2B included 47 QTL of yield-related traits that formed 6 QTL clusters.It was speculated that the QTL interval of important traits on chromosome 2B might be related to the infiltration of Th.intermedium chromosome fragments.7.Based on the differences of spike-related traits between SN304 and YN15,transcriptome analysis was carried out on the young panicles of SN304,YN15,R104 and R223,which are representative lines of large and small spikes.There were 557 and 509 differentially expressed genes between SN304 and YN15 in the two stages.Through hierarchical clustering screening,216 and 106 genes were highly expressed in SN304 and R104.Through database annotation,nine candidate genes located on chromosome 4B were screened,and they were located in the QTL interval that controlled spike-related traits on chromosome 4B.The functions of these genes require further analysis and verification.8.We selected and identified 20 Trititrigia germplasm lines with good comprehensive characters and early growth period from the RIL population and 14 Trititrigia germplasm lines which the pollen of SN304 irradiated with ⁶⁰Co-?ray.In situ hybridization analyzed the genetic composition of these Trititrigia germplasm lines and laid the foundation for its further utilization.