Transcriptome Analysis And KASP Marker Development Of Psathyrostachys Huashanica

Wheat(Triticum aestivum L.,2n = 6x = 42,AABBDD)as one of the most widely cultivated crops in the world,is the main food crops for human beings.At present,the global wheat genetic improvement is facing the bottleneck problem for abundance of genetic resources.A great number of wild relative species are widely excavated and utilized in order to improve the yield,resistance and quality of common wheat.As a unique species in China,Psathyrostachys huashanica Keng ex Kuo(2n = 2x = 14,Ns Ns)contains many superior characters and genes,such as high tolerance to cold,drought and barren,early maturity and high resistance to wheat stripe rust,take-all,powdery mildew,etc.Consequently,it is an important gene resource for wheat genetic improvement.Since 1980s,many wheat-P.huashanica deritived lines have been produced,such as common wheat-P.huashanica amphidiploid,alien addition,substitution and translocation lines.Therefore,it is an important and substantial work for wheat genetic improvement to accurately analyze the genetic information of P.huashanica.In this study,RNA-seq was applied to analyze the transcriptome of P.huashanica.Some homologous genes and SNP were detected via the wheat reference genome sequence as well as some known genomic species.Based on the SNP obtained,a series of specific SNP and KASP markers of P.huashanica were obtained,which laid a foundation for the identification of wheat-P.huashanica introgression lines and breeding lines.The main findings are as follows:1.RNA-seq was applied to P.huashanica,178,501,084 clean reads were generated after filtering.A total of 456,056 transcripts with a mean length of 813 bp were obtained via De novo assemble.The size of N50 was 1325 bp.After hierarchical cluster,248,835 unigenes were obtained and applied for functional annotated.Altogether,159,988(64.29%)transcripts had at least one hit in databases.2.16 putative orthologous gene clusters were identified through comparing P.huashanica and CDS of other genomes of the tribe Triticeae.Phylogenetic relationships among P.huashanica and other genomes were constructed on the basis of orthologous gene clusters.A clear division between P.huashanica and other Triticeae species was observed,indicating that Ns genome expressed abundant genetic variations.3.The average sequence similarity is 95.36% between P.huashanica and wheat.717,743 variants were identified by mapping the transcriptome sequencing data to wheat reference sequences to discover the variations between P.huashanica and wheat.The number of variants in each chromosome was not in prpportion to the chromosome length and gene number.The B genome contained the most variants(250,438) and expressed the highest variant density(53.74 SNPs/Mb),followed by D genome(237,225 variants and53.56 SNPs/Mb variant density)and A genome(230,080 variants and 49.23 SNPs/Mb variant density).These results suggested that P.huashanica was most distantly related to the wheat B genome among wheat genomes.674 genes possess high diversity and harbored more than 50 SNPs.4.Specific markers of Ns genome were developed according to SNPs and verified the polymorphisms via KASP genotyping assays among diploid species containing A~b,A~m,S,D,R,H,E~b,E~e,V,P,St genomes and polyploid species containing Ns Xm genome.A total of 43 pairs of Ns genome-specific markers were developed.24 of them were specific to Ns genome of P.huashanica,and 19 were specific to Ns genome of P.huashanica and P.juncea.43 Ns genome-specific markers were then used to amplify the wheat-P.huashanica derived line,5 markers were heterozygous in wheat-P.huashanica translocation line K13-835,4 markers were heterozygous in wheat-P.huashanica addition line 18-3-3,2markers were heterozygous in wheat-P.huashanica addition line 18-3-6.