| Barley(Hordeum vulgare L.) is the fourth largest crop in China, and also one of the most important crops worldwide. It is mainly used for food, feed, beer and heath care industries. With the development of economy and the people's living standards rising quickly, the production of barley is imposed to higher request. It has become an important topic for the breeders to develop and utilize the rich germplasm resources of barley effectively, and cultivate barley varieties with high yield, high quality, and being riched in functional components. The molecular biology research of the specific germplasm resources of barley will not only mine excellent gene resource, but also lay the foundation for the molecular marker assisted breeding, molecular pyramiding breeding and map-based cloning of the main effect QTL, and has important theoretical and practical significance for the cultivation of barley varieties with high yield and quality.In the present study, two sets of specific germplasm materials were adopted. The first set is a Fi-derived doubled haploid (DH) population of 148 lines, which was generated from the cross between two six-rowed barley varieties Steptoe and Morex. There are significant differences between the parents in terms of agronomic and quality traits. A molecular genetic linkage map of barley was constructed using the DH population. Based on it, QTLs for 7 agronomic traits including plant height, main spike length, grain number of main spike, tiller number and heading date, maturity, thousand grain weight and 1 quality trait (flavone contents of grain) were analyzed. The second set of research materials are maintainer line (6602B) and seven cytoplasmic male sterile (CMS) lines (6602A/6602B,6605A/6602B, N-5A/6602B, N-9A/6602B, N-10/6602B, N-11A/6602B, US-A/6602B). Three related genes of cytoplasmic male sterility(orf256, orf300, orf25) was cloned and analysed in the eight materials.The results collected and analyzed from the first set of materials are following:1.1 The DH population of 148 lines derived from the cross between Steptoe and Morex was used for the construction of the genetic map and QTL mapping. A total of 366 markers (312 SSR markers and 54 SRAP markers) covering the whole barley genome were screened for polymorphism between parents of the DH pupulation. Of these,98 SSR markers and 21 SRAP markers revealed polymorphism between the parents and generated 107 and 52 polymorphic loci, respectively. Most loci met 1:1 Mendelian segregation ratio in the distribution of population. The result showed that the DH population was suitable for construction of genetic linkage map.1.2 A molecular genetic linkage map of barley containing 155 polymorphic loci (105 SSR and 50 SRAP loci) was constructed using MAPMAKER/Exp 3.0. The map covered a total length of 960.7 cM with an average intervals of 6.2 cM between adjacent markers.155 loci were located in the 9 linkage groups with a mean of 17.22 loci per linkage group, and mapped on the 7 chromosomes. Some gene-rich regions were found in the genetic linkage map of barley.1.3 Among all the 159 polymorphic markers,28 (17.6%) markers showed segregation distortion at the 0.05 level of significance, and 4 (2.5%) markers of them showed extremely significantly segregation distortion (P<0.01). About 67.9% of these 28 markers skewed towards the female parent Steptoe, whereas the left markers (32.1%) deviated towards the male parent Morex. This study further showed that SRAP markers revealed higher segregation distortion ratio than SSR markers, and all the 4 markers displayed the extremely significantly segregation distortion were SRAP markers. In this study, we also investigated the distribution of marker segregation distortion on genetic linkage map and found 3 segregation distortion regions.1.4 The main agronomic traits were analysed using the method of Composite Interval Mapping of Windows QTL Cartographer 2.5 in barley. A total of 25 QTLs for agronomic traits were detected (LOD>2.5), and 14 of them had higher relative genetic contribution, the range was 11.06%-48.13%, were major genes, the relative genetic contribution of the other 11 QTLs was 4.3%-9.44%.6 QTLs for plant height were located on 2H (1),3H (2), 4H (1) and 7H (2), respectively, and which could explain about 4.88%-20.04% of phenotypic variation; 6 main spike length QTLs were located on 2H (1),3H (3) and 7H (2), respectively, which could explain about 6.37%-48.13% of phenotypic variation, and the QTL with highest contribution located on 3H (48.13%), was a major QTL; 4 QTLs for grain number of main spike were located on 2H and 3H, respectively, and the 3 QTLs located on 3H had higher relative genetic contribution, the range was 15.63%-17.13%; Only 1 tiller number QTL was detected, was located on 5H, which could explained about 8.65% of phenotypic variation; 4 QTLs for heading date were located on 1H and 2H, respectively, and there were 3 QTLs located on 2H, the highest relative genetic contribution was 21.37%; For maturity, only one major effect QTL was detected, and located on 2H, the relative genetic contribution was 21.51%, the value of the additive effects was -1.83, and showed that the additive gene came from male parent Morex; 3 thousand seed weight QTLs were located on 5H (1) and 7H (2), respectively, which could explain about 6.44%,8.63% and 11.95% of phenotypic variation.1.5 Flavone is one of the important functional components of barley, which plays important roles during human health. In this study,3 QTLs controlling the contents of flavone were detected on 2H,3H and 6H, respectively, which could explained 7.72%, 6.85% and 7.36% of the phenotypic variance. The additive effect was negative, which showed alleles of increasing flavone contents came from male parent Morex. The results collected and analyzed from the secondary set of materials are following:2.1 orf256 is one of important genes related to cytoplasm male sterile, which loss of function can confer to partial or complete cytoplasm male sterile in planta. Degenerate primers developed efficiently from conserved-region of orthologous 'or/256' in wheat (Triticum aestivum L.), rye (Secale cereal L.) as well as A.tauschii were employed to amplify 1 maintenance line and 7 sterile lines, respectively. The 937 bp fragment of or/256 included 783 bp open read frame.100% AA-identity displayed no mutants, deletion, insertion as well as ORFs shift between seven sterile lines and maintainer line. The results also represented high-similarity in the four cereals, arranged from 97% up to 99%. A low-molecular-weight protein (79.8 kDa) encoded by or/256 showed the isoelectric points with the point of 5.08 through bioinformatic approach.2.2 The ortholog of orf300 leading to cytoplasm male sterile in sugar beet (Beta vulgaris L.) and Silene gallica L., etc. was amplified and isolated in barley. The fragment containing 933 bp element as well as 594 bp open read frame inside also represented complete AA-similarity in the materials. or/300 encodes a polypeptide with a molecular mass of 78.1 kDa and a pI of 5.07. 2.3 The resources of orf25 in other species, like wheat, tobacco(Nicotiana tabacum L.) and rape(Brassica napus L.), etc. were used for marker design.100% identify of 622 bp fragment (containing 474 bp ORF) was detected between maintainer line and seven sterile lines with the program of BlastN. orf25 encodes a polypeptide with a molecular mass of 51.5 kDa and a pI of 5.18.
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