Molecular Cytogenetic Identification Of Wheat-Leymus Mollis Derivatives M13063A-1 And M13086A

The long-term artificial selection and domestication narrows the genetic diversity of modern common wheat cultivars,as well as limits improving and breeding of common wheat cultivars.Previous researches have shown that distant hybridization is one of the main approach to broaden wheat genetic diversity.Leymus mollis is a tertiary gene source for common wheat,the superior gene of which can be transferred into common wheat through distant hybridization.In this study,the molecular cytogenetic identification was used to identify wheat-L.mollis derivatives M13063A-1 and M13086 A.(1)Identification and analysis of M13063A-1The results of cytological observation of pollen mother cells and root tip cells showed that M13063A-1 contained 44 chromosomes at mitotic metaphase ?,with 22 bivalents at meiotic metaphase I.Moreover,homologous chromosomes were equally separated at meiotic anaphase ?.Genomic in situ hybridization using genomic DNA of L.mollis and Psathyrostachyshuashanica as probes revealed that M13063A-1 contains 42 common wheat chromosomes and a pair of translocation chromosomes,the long arm of which is from the Ns genome of L.mollis.Sequential fluorescence in situ hybridization-genomic in situ hybridization was performed with L.mollis genomic DNA probe as well as oligonucleotides Oligo-p Ta535 and Oligo-p Sc119.2,the results showed that 42 common wheat chromosomes were cytologically intact set of wheat genomes.Multi-color genomic in situ hybridization using genomic DNA of Triticum urartu,Aegilops tauschii and L.mollis indicating that the short arms of the translocation chromosomes are from the B subgenome of common wheat.Genomic in situ hybridization using L.mollis genomic DNA probe with three oligonucleotides Oligo-p Ta71-2,Oligo-119 and Oligo-60,respectively,showed that 6BS is involved in the formation of translocation chromosomes.Molecular marker analysis revealed that 1 PLUG primer(TNAC1677),3 EST primers(BQ159615,CD454198,CD454353)and 2 SSR primers(WMC672,CWM532)amplified unique fragment of L.mollis and M13063A-1,all 6 markers are located in the of wheat chromosomes.Among the 6 markers,TNAC1677,BQ159615,CD454198 and CD454353 are located in the short arms of the homoeologous groups 6 of comman wheat.Combined together,the results of molecular marker analysis and genomic in situ hybridization using Psathyrostachyshuashanica genomic DNA probe,indicated that the chromosome fragment transferred into M13063A-1 was 6Ns S.In terms of agronomic traits,the plant height of M13063A-1 was significantly lower than parental wheat 7182,and the spike length of M13063A-1 was significantly shorter than 7182.There was no significant difference in other traits.At the adult stage,with regard to the resistance to stripe rust,M13063A-1 showed good resistance to Puccinia striiformis f.sp.tritici strains,CYR31 and CYR32.Therefore,M13063A-1 is common wheat-L.mollis 6BS·6Ns S additional translocation line,which is potential usefulness for creating new wheat germplasm as well as breeding and improving common wheat cultivars.(2)Identification and analysis of double disomic addition line M13086AThe results of cytological observation of pollen mother cells and root tip cells showed that M13086 A contained 46 chromosomes at mitotic metaphase ?,with 23 bivalents at meiotic metaphase I.Moreover,homologous chromosomes were equally separated at meiotic anaphase ?.,indicating that M13086 A was cytologically stable.Genomic in situ hybridization was performed at mitotic metaphase ? with genomic DNA of L.mollis,the results showed that M13086 A contained 44 common wheat chromosomes and 4 alien chromosomes.Genomic in situ hybridization performed at meiotic metaphase I with genomic DNA of L.mollis showed that four alien chromosomes formed two bivalents,which further verified the genetic stability of M13086 A.Fluorescence in situ hybridization using oligonucleotides Oligo-p Ta535 and Oligo-p Sc119.2 revealed that 42 common wheat chromosomes were cytologically intact set of wheat genome;the signal patterns of two pairs of alien chromosomes were compared with the results of previous studies.By comparison,the two pairs of alien chromosomes were confirmed as 6Ns and 7Ns.The results of disease resistance identification showed that M13086 A was resistant to Puccinia striiformis f.sp.tritici strains,CYR31 and CYR32 at adult stage,and was resistant to powdery mildew.Therefore,M13086 A is a double disomic addition line with chromosomes 6Ns and 7Ns of L.mollis,with application values in breeding and improving common wheat cultivars.