Molecular Cytogenetics Of Studies On Wheat-Leymus Mollis Derivative Lines And Ns Genome Array Development

Leymus mollis（Trin.）Pilger（Ns Ns Xm Xm,2n=28）,a tetraploid perennial of the Leymus genus,which possesses numerous desirable traits and serves as a crucial tertiary source for genetic enhancement in wheat.In the 1980s,Chinese researchers successfully developed wheat-L.mollis hybrids using remote hybridization and chromosome engineering techniques,which led to extensive research on the topic.In this study,wheat-L.mollis derived lines were systematically identified and analyzed by using various techniques,such as cytology,in situ hybridization（GISH,FISH）,molecular markers（SSR,EST and PLUG）,array analysis（wheat55K SNP array,wheat-Psathyrostachys huashanica 45K liquid array）,resistance identification of stripe rust and FHB,and agronomic trait investigation.The main research findings were outlined as below:（1）Identification of the wheat-L.mollis 4Ns（4D）disomic substitution line M862.The wheat-L.mollis 4Ns（4D）disomic substitution line M862 was identified through the selection of a derivative line from the BC₁F₄ progeny,which was obtained by crossing common wheat7182 with wheat-L.mollis partial amphiploid M47,followed by backcrossing and selfing.Molecular cytogenetic analysis and GISH indicate that the chromosome configuration and composition of M862 was 2n=42=21II=40T.a+2L.m.Furthermore,sequential FISH-GISH,array and molecular markers confirms that M862 was a wheat-L.mollis 4Ns（4D）alien disomic substitution line.Moreover,the karyotype construction and comparative analysis of M862 and its parent 7182 using FISH indicated that chromosomes 1A,1D,2B,and 5A exhibited mutations in M862.The introduction of the 4Ns chromosome had a more significant impact on gene expression in the 4D and 2B chromosomes compared to the other chromosomes.This suggests that the substituted 4Ns chromosome provided better compensation for the 4D chromosome.Specifically,449 L.mollis 4Ns-specific sequences were obtained through RNA-seq,and 50 of these were selected for validation.A total of 16specific molecular markers of L.mollis 4Ns were screened.Among them,9 were incomplete specific markers and 7 were completely specific markers located on chromosomes.The markers provided can be utilized to trace and identify genetic material of chromosome 4Ns in a wheat background.On the other hand,M862 was renowned for its remarkable resistance to both stripe rust and FHB.Additionally,it had desirable traits such as dwarfism and large grain,making it an invaluable resource in enhancing wheat genetics.（2）Identification of wheat-L.mollis 4Ns L translocation lines and preliminary fragment investigation of stripe rust resistance.M892 and M956 were obtained from the F₄ progeny of a cross between common wheat Norin 26+3C（gametocidal chromosomes）and wheat-L.mollis 4Ns disomic addition line M852.The results indicate that M892 was highly susceptible to FHB and stripe rust,whereas M956 displayed high resistance to stripe rust and FHB.In situ hybridization and 45K liquid chip analysis of wheat-P.huashanica showed that M892 was a wheat-L.mollis T5DL-4Ns L-5DL·5DS translocation line that had been inserted,while M956was a terminal wheat-L.mollis T4Ns L·5DL-5DS translocation line.What’s more,the stripe rust resistance genes of chromosome 4Ns were narrowed down to the 648-746Mb segment in M956.Through a comparison of differentially expressed unigenes in M892 and M956,11,368unigenes were identified in M956,48 of which were linked to disease resistance.On this basis,eight 4Ns L chromosome-specific molecular markers were successfully developed,two of which were also associated with disease resistance gene（s）.The development of specific markers would accelerate the identification and screening of 4Ns L genetic material in the wheat breeding process,and lay the foundation for the mining and utilization of 4Ns L genes for resistance against stripe rust and FHB.（3）Comparative analysis of the wheat-L.mollis and wheat-P.huashanica 7Ns alien lines.The chromosome 7Ns was successfully identified with both the genomic DNA probes of L.mollis and P.huashanica through GISH identification,including the wheat-L.mollis disomic alien addition line（M10）,wheat-L.mollis disomic alien substitution line（M8）and wheat-P.huashanica disomic alien addition line（H1）.These probes produced similar signals,presenting their effectiveness.However,there were differences observed between the L.mollis and P.huashanica 7Ns chromosomes in terms of FISH signal distribution and arm ratio.Moreover,the wheat-P.huashanica liquid array analysis showed that a large segment of the proximal part of the P.huashanica 7Ns L was missing in H1,whereas the L.mollis chromosome 7Ns was structurally intact in M10 and M8.All three 7Ns alien lines exhibited resistance to FHB,but showed variations in resistance to stripe rust.M10 and M8demonstrated broad-spectrum resistance to six of the most common Pst races of stripe rust at both the seedling and adult stages,H1 only displayed immunity to Pst races of CYR23,CYR31and CYR32.The agronomic trait assessment results revealed that the three materials shared similarities in spikelet number,thousand grain weight,and awnedness.Furthermore,all three subjects displayed an increase in grain weight compared to parent 7182.However,significant differences were observed in plant height,spike shape,spike length,seed size,and seed color.Based on transcriptome data,668 7Ns chromosome-specific unigenes were screened,and 437Ns chromosomal markers were developed and validated.Of these markers,27 markers were identified as 7Ns chromosomal universal markers,9 were specific to the L.mollis Ns chromosome,and 7 were specific to the P.huashanica 7Ns chromosome.The identification of these markers specific to each chromosome provides a foundation for further investigation into the composition of L.mollis chromosomes.（4）The development and application of the wheat-P.huashanica 45K liquid array,also known as Geno Baits（?）Wheatplus Ph.The P.huashanica genome was sequenced and assembled,resulting in a genome size of 7.02 Gb,with contig N50 and contig N90 of 53,727bp and 2,372,002 bp,respectively.The wheat-P.huashanica liquid array was developed using the genomes of common wheat and P.huashanica,and consists of 5,798 wheat-specific intervals and 47,887 P.huashanica-specific intervals that were evenly distributed across chromosomes.Each interval measured approximately 300 bp and was associated with 1-2targeting capture probes.The array capture efficiency test revealed that the specific intervals for common wheat,P.huashanica,L.mollis,Leymus racemosus,Agropyron cristatum,and Eremopyrum triticeum could be effectively captured.Obviously,the capture efficiency of P.huashanica,L.mollis,and L.racemosus was significantly higher than that of A.cristatum and E.triticeum.Geno Baits（?）Wheatplus Ph was successfully identified three wheat-P.huashanica and 14 wheat-L.mollis derived lines.This demonstrates that the microarray has potential for identifying wheat-P.huashanica and L.mollis lines of addition and substitution and translocation.Furthermore,the Oligo-p Ta535 probe was utilized to construct a preliminary FISH karyotype of the L.mollis Ns genome.This karyotype would be a valuable reference point for identifying L.mollis Ns chromosome-derived lines within in wheat backgrounds.This study identified six new germplasms,including the wheat-L.mollis 4Ns（4D）disomic substitution line,T4Ns L-5DL-5DS and T5DL-4Ns L-5DL-5DS translocated lines,two 7Ns disomic addition lines and 7Ns（7D）disomic substitution line.These lines possessed desirable traits,such as resistance to stripe rust and FHB,short straw and large grain,which make them potentially valuable for improving wheat genetics and breeding disease-resistant varieties.Furthermore,the development of wheat-P.huashanica liquid array could expedite the tracking and detection of the chromosomal genetic material of P.huashanica and L.mollis Ns in the wheat background.This advancement provided theoretical and technical support for the evolutionary relationships,exogenous superior gene mining and utilization of wheat,L.mollis and P.huashanica.