Identification Of Common Wheat-agropyron Cristatum 2P And 6P Disomic Addition Lines And Locating Desirable Genes

Agropyron cristatum(L.)Gaertn.(2n=4x=28,PPPP)is a significant wild relative of wheat.Due to grown in harsh environment,it has many desirable genes to broaden the genetic resources for wheat improvement.such as tolerant genes of cold,drought and salt;resistant genes of fungal diseases;high grain number per spike;ideal plant architecture;high thousand grain weight and multiple fertile tiller number per plant.In order to study the homeologous relationship between Agropyron cristatum chromosomes and wheat chromosomes,locate the desirable genes on different P chromosomes,and produce the translocation/introgression lines with useful agronomic genes to improve wheat,we created a complete series of wheat-Agropyron cristatum disomic addition lines.In this study,using Expressed sequences tags(EST)markers,specific-locus amplified fragment sequencing(SLAF-seq)PCR markers and genome in situ hybridization(GISH),we identified and detected the homoeologous relationships between P chromosomes and wheat chromosomes,located desirable agronomic genes in different P chromosomes.The main results were as follows:(1)Molecular cytogenetic identification of wheat-Agropyron cristatum 2P disomic addition lines: we developed 4 wheat-Agropyron cristatum 2P disomic addition lines from a cross between common wheat cv.Fukuhokomugi(Fukuho)and Agropyron cristatum cv.Z559.Cytological observations and genomic in situ hybridization(GISH)identifications demonstrated that 4 wheat-Agropyron cristatum 2P disomic addition lines had different chromosome constitutions.According to the number of chromosomes contained,4 wheat-Agropyron cristatum derivatives lines were classified into three categories: a,42 wheat + Agropyron cristatum chromosomes: II-9-3 and II-29-1;b,42 wheat chromosomes + Agropyron cristatum II PS chromosomes: II-8-1;c,42 wheat chromosomes + Agropyron cristatum I PS chromosomes: II-7-1.In addition,we used the STS markers developed by the transcriptome sequencing of Agropyron cristatum and SLAF-seq markers to identified the homeologous relationships between Agropyron cristatum chromosomes and wheat chromosomes by comparative genomics which the SLAF-sequences were aligned with barley genomic sequences,Brachypodium distachyon genomic sequences,wheat A genomic sequence and D genomic sequences.II-9-3,II-29-1,II-7-1 and II-8-1 were assigned to wheat homoeologous group 2.(2)We identified and located the disomic addition line II-9-3 with resistance to powdery mildew: Cytological studies showed that II-9-3 contained 44 chromosomes.GISH using Agropyron cristatum genomic DNA as a probe and the common wheat Fukuho DNA as the blocker demonstrated that a pair of P chromosomes with strong hybridization signals had been introduced into II-9-3.The disomic addition line II-9-3 and the common wheat Fukuho were evaluated for adult-plant resistance to powdery mildew in greenhouse trials,which were inoculated with a mixture of different races of Blumeria graminis f.sp.tritici that contained the highly toxic strain E09.The results showed that the II-9-3 was resistant to powdery mildew,while the common wheat Fukuho was susceptible.To determine is indeed the P-genome chromosome harbored the powdery mildew resistance gene in II-9-3,the parents,F1 and F2 plants were selected randomly and identified by GISH and molecular markers.The result showed that plants containing P chromosome which had chromosome configuration of 21 II + 1 I and 22 II,respectively,were highly resistant or immune to powdery mildew.However,plants with no P chromosomes were susceptible to powdery mildew.The phenotype of resistance and susceptibility to powdery mildew was consistent with cytological identification.Genetic analysis of powdery mildew resistance in parents,F1 and F2 populations indicated that the resistant gene which came from the P chromosomes,was controlled by a single dominant gene.We screened 24 SLAF-seq markers to trace and locate the powdery mildew resistant gene.The SLAF-seq markers which were located on 2P chromosome,amplified unique band of Agropyron cristatum in II-9-3.II-9-3 can be used as a donor source for transferring novel powdery mildew resistant gene to wheat with the assistance of molecular markers and cytological identification.The disomic addition line II-9-3 could be exploited as an important bridge for wheat improvement.(3)Chromosomal localization of the genes conferring desirable agronomical traits from wheat-Agropyron cristatum disomic addition line 5113: Wheat-Agropyron cristatum derivative line 5113 was proved to be a novel wheat-Agropyron cristatum disomic 6P addition line by GISH and SLAF-seq technology.5113 displayed multiple elite agronomical traits,including higher uppermost internode/plant height ratio,larger flag leaf,longer spike length,higher grains number per spike,more spikelet number per spike,more kernel number in the middle spikelet,more fertile number per plant,and high resistance to powdery mildew and leaf rust.The genes conferring these elite traits were located on the Agropyron cristatum 6P chromosome by using the SLAF-seq markers,and the genetic populations(F1,BC1F1 and BC1F2 populations)produced from the produced from crosses between Fukuho and 5113.Therefore,chromosomal localization of the desirable genes are the fundamental prerequisites for the utilization of 5113,which will facilitate transferring high-yield and high-resistance genes from Agropyron cristatum into common wheat.(4)II-30-5,which derived from hybridization between wheat cv.Fukuho and Agropyron cristatum(L.)Gaertn.(2n = 4x = 28,PPPP),was proved to be a novel wheat-Agropyron cristatum disomic 6P addition line by GISH and SLAF-seq technology.II-30-5 possessed several elite agronomical traits,such as ideal plant architecture,erect leaf position,short flag leaf,high thousand-grain weight,and high resistance to powdery mildew and leaf rust.Genes conferring those useful agronomic traits were located on Agropyron cristatum 6P chromosome by genetic analysis of F1,BC1F1 and BC1F2 populations.Chromosomal rearrangements of 6Pchromosomes lead to generate more than one 6P addition lines,providing increased genetic variation for wheat genetic improvement.Taken together,we could make a conclusion that II-30-5 was a novel wheat-Agropyron cristatum disomic 6P addition line different from other 6P addition lines reported before,and could be used for genetic improvement for common wheat.