Development And Physical Localization Of Rye Genome-specific Markers Based On Transcriptome Sequences Of Secale Cereale

Chromosome engineering is an important method to broaden wheat genetic diversity and enhance biotic and abiotic resistance.Development of molecular markers provided new tools for chromosome engineering.Chinese rye cultivar Jingzhouheimai(Secale cereale L.cv.Jingzhouheimai,2n= 2x =14,JZHM)showed multiple resistance to powdery mildew,yellow mosaic and sharp eye spot of wheat,two major powdery mildew resistance alleles were allocated to the chromosomes 2R and 6R.Development of small segmental translocations was necessary to break the linkage drag and develop new wheat germplasm or cultivars.Idenfication of enough molecular markers specific for these chromosomes will facilitate this process.In this study,transferability of Thinopyrum bessarabicum transcriptome markers was firstly identified in JZHM,and then RNA-seq was used to obtain rye trancriptome sequences and used to develop markers specific for rye after blasted against D genomic sequences of wheat,the specific markers were finally chromosomally mapped in rye chromosomes particularly for 2R and 6R and some of them were used to identify rye chromosomes and its variations occured in a hybrid population.The major results were as following.Of 342 primer pairs of Th.bessarabicum,194 pairs(56.7%)produced stable amplification in HXH,JZHM,HXH-JZHM amphiploid and Th.bessarabicum,while only 50 pairs(14.6%)produced polymorphic loci of JZHM.Based on the transcritome sequences of JZHM,total 3602 rye specific sequences were found after blasted against the D genomic sequences of wheat progenitor Aegilops tauschii,233 pairs of primers were designed,except 4 pairs(1.7%)without amplification,104 pairs(44.6%)produced same amplicons in HXH and JZHM,while 125(53.6%)produced specific amplicons in JZHM and HXH-JZHM amphiploid,indicating higher efficiency of these markers than the former Th.bessarabicum markers(14.6%).Using HXH-JZHM alien chromosome lines,of 178 markers,53 have been located,which included 50 rye amplified by 47 primer pairs and 3 Th.bessarabicum markers,the remaining 125 pairs were not positioned which might be located in other rye chromosomes not involved in this study.Among 53 mapped markers,25 were chromosome specific including 7 in 1RL,8 in 2R including 2RS(4)and 2RL(2),1 in 5RS,and 9 in 6R including 6RS(5)and 6RL(3),while 28 were duplicated in more than two chromosomes of rye,indicating recombination occurred in these chromosomes.Using four markers specific for chromosome 6RS(Lfz2044 and Lfz2007)and 6RL(Lfc1999 and Lfz2065),a F2 population derived from MA6R/Yangmai 158 were analyzed,and found these markers could be effectively used to identify the alien chromosome,and six plants with modified chromosomes of 6R were detected,indicating potential of these markers in chromosome engineering.