| Psathyrostachys huashanica Keng （2n=2x=14, NsNs） is characterized by earlymaturity, dwarf, high quality and high resistance to scab, stripe rust, take-all, and powderymildew. While it is also noted for its resistance to cold, salinity, drought, and barrenconditions, which is a useful gene pool for wheat improvement. The production of alienaddition lines and substitution lines is an important step for succesfully transferring excellentgenes to wheat. Those exotic lines will be useful for molecular cytogenetic study and genetictraits evaluation in breeding programs. In order to identify a complete set of wheat-P.huashanica alien addition lines, cytology, genomic in situ hybridization （GISH）, fluorescentin situ hybridization（FISH）, SDS-PAGE, A-PAGE, SSR, EST-SSR and EST-STS were usedto determine the homoeologous group and genome affinity of the added or substitutedchromosomes and then agronomic traits evaluation were combined. The complete set ofwheat-P. huashanica alien addition lines （1Ns–7Ns） and partial substitution line will bedeveloped accompanied with stable cytology composition, desirable genes and variousmorphology, which could lay a solid foundation for wheat improvement via introducingoutstanding genes from P. huashanica. In addition, the P. huashanica genomic specific SCARmarkers were converted from RAPD and SSR of wheat. These markers were used to detectNs genome chromatin in wheat background via testing the complete set of wheat-P.huashanica alien addition lines. This diagnostic PCR assay for screening the target geneticmaterial may play a key role in marker-assisted selective breeding programs. The main resultsare as follows:1. A complete set of wheat-P. huashanica disomic addition lines （1Ns–7Ns,2n=44=22Ⅱ）, which were derived from common wheat cv.7182and Psathyrostachys huashanicaKeng via embryo culture. They were characterized by cytology, genomic in situ hybridization（GISH）, SDS-PAGE, A-PAGE, EST-SSR, EST-STS, disease resistance screening andagronomic traits evaluation. The mitotic and meiotic cytogenetic investigation and GISHanalyses indicated that the chromosome number and configuration of （1Ns–7Ns） disomicaddition lines were2n=44=22Ⅱ, which contained42wheat chromosomes and one pair of P. huashanica chromosomes. The genomic affinities of the introduced P. huashanicachromosomes were determined by SDS-PAGE, A-PAGE analyses and using835EST-SSR,EST-STS multipleloci markers from seven wheat homoeologous groups between the parentsand addition line. The results indicated that there were seven different wheat-P. huashanicadisomic addition line in accord with seven homoeologous groups of wheat. Based on diseasereaction to mixed races （CYR31, CYR32, and Su11-14） of stripe rust and （FHTT, PHST,and FHST）of leaf rust in the adult stages. The agronomic traits of a complete set of wheat-P.huashanica disomic addition lines were avaluated via field observation and lab identification.The results indicated that1Ns disomic addition line12-3carried the HMW-GS,LMW-GS and gliadin genes of P. huashanica, and high resistance to leaf rust.2Ns disomicaddition line3-6-4-1had higher thousand-grain weight, and high resistance to strip rust.3Nsdisomic addition line22-2had the lowest plant height in seven disomic addition lines, andhigh resistance to strip rust.4Ns disomic addition line24-6-3has been transmitted superiortraits from its exotic parent that enhanced the tiller number and improved stripe rust resistance.5Ns disomic addition line3-8-10-2had plant-type compact and high resistance strip rust;yield traits were higher than its wheat parent7182.6Ns disomic addition line59-11wasintroduced α-gliadin genes from P. huashanica, and produced twin spikelets per spike inwheat.7Ns disomic addition line2-1-6-3had higher setting percentage, and high resistance toleaf rust. The studies indicate that a complete set of wheat-P. huashanica disomic additionlines （1Ns–7Ns,2n=44=22Ⅱ）were isolated and characterized using cytology, genomic insitu hybridization (GISH), SDS-PAGE, A-PAGE, EST-SSR, and EST-STS. Agronomic traitsevaluation showed that the （1Ns-7Ns） disomic addition lines were introduced differentgenes from P. huashanica, they exhibited excellent agronomic traits than wheat parent7182.The complete set of wheat-P. huashanica disomic addition lines lay a solid fundation fortransfering favorable genes efficently and definitely during breeding programs.2. A wheat-P. huashanica Keng substitution line16-6has the characteristics of superiornumbers of florets and kernels per spike, which was also developed from the samehybridization between common wheat cv.7182and P. huashanica. It was also characterizedby cytology, genomic in situ hybridization （GISH）, SSR, fluorescent in situ hybridization（FISH） and EST-STS. The line16-6contained21bivalents during meiosis, while GISHstudies revealed one pair of alien chromosomes existed. Sixteen microsatellite markers forchromosome arms2DS and2DL of hexaploid wheat and FISH applying probe pAs1pointedto a missing chromosome2D. Then,780EST-STS multiple-loci markers covering all sevenhomoeologous groups and14EST-STS markers particularly associated with homoeologousgroup2of wheat was utilized to identify the homoelogous relationship between the exotic P. huashanica chromosome and wheat. The results revealed that16-6was one2Ns（2D）wheat-P. huashanica substitution line. After it was inoculated using mixed races （CYR31,CYR32, and Su11-14） of stripe rust at the adult stages, line16-6showed highly resistancewhich might derive from the donor species. Compared to the control spike length, the numberof spikelets per spike and kernel number per spike of16-6is significantly increased whichwas a desired donor for wheat yield improvment.3. P. huashanica genomic specific SCAR markers were used to test the complete set ofwheat-P. huashanica disomic addition lines （1Ns–7Ns,2n=44=22Ⅱ） and its parentscommon wheat cv.7182and P. huashanica to validate the practical applicability. Theamplification results indicated that the target band of two different SCAR markers（RHS23and RHS141）were present in P. huashanica and all of the addition lines （1Ns,2Ns,3Ns,4Ns,5Ns,6Ns, and7Ns）; two markers （RHS153and SHS10） were present in P.huashanica and1Ns disomic addition line; three markers （RHS7, RHS14, and RHS103）were present in P. huashanica and5Ns disomic addition line, but they all absent from thematernal parent7182. Therefore, RHS23and RHS141could be used as genomic specificSCAR markers for tracking P. huashanica chromatin; RHS153and SHS10can be used as1Ns chromosome specific SCAR; RHS7, RHS14, and RHS103could be used for detectingthe P. huashanica5Ns chromosome. These SCAR markers could be used powerful tool forthe marker-assisted selection of P. huashanica chromatins in a large genetic pool.Molecular cytogenetics research and excellent genes digging of a complete set ofwheat-P. huashanica disomic addition lines （1Ns–7Ns,2n=44=22Ⅱ） and a2Ns（2D）substitution line is important significance in both theory and practice for studying on geneticrelationship between P. huashanica chromosome and wheat. P. huashanica specific SCARmarkers will be used as useful tool for rapidly screening numerous samples because they areaccurate, rapid, as well as for the improvement of identification efficiency. It is importantsignificance in both theory and practice for transferring excellent genes designedly anddeveloping translocation lines directly. |
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