Germplasm Evaluation, Molecular Marker Development And Genetic Linkage Map Construction Of Switchgrass

Switchgrass (Panicum virgatum L.) belonging to the family Poaceae and genus Panicum, is a warm season C4 perennial grass, native to North American tall grass prairies. The high biomass production potential of switchgrass with low inputs makes it an excellent choice as a sustainable bioenergy crop. The genetic backgrounds of switchgrass germplasm were study by SRAP,SSR and flow cytometry analysis. The biomass production capacity, rust and anthracnose disease resistance of switchgrass germplasm were also assessed. Our phenotypic evaluation lead us to idenifytwo lines 'Alamo'and'Dacotah',which have significant difference in many agronomic traits including biomass yields, plant hight, flower date, feedstock quality, and rust disease resistance etc. Alamo'and'Dacotah'were chosen as parental lines to to construct the frame genetic linkage map. The EST-SSR and RGA-SNP markers were developed and using these markers along with SRAP markers, we constructed the frame genetic linkage map. The QTLs controliong the rust resistance were idenitified. The key results are as follows:1,To study the genetic diversity and relationships between switchgrass and other species in Panicum,91 accessions from USDA's germplasm collection representing 22 species of Panicum from six continents and 22 countries were investigated by using the Sequence-Related Amplified Polymorphism (SRAP) and Expressed Sequence Tags-Simple Sequence Repeat (EST-SSR) markers. High genetic diversity was detected in Panicum species (PPB=96.74%,I=0.463 based on SRAP markers; PPB= 94.73%,I=0.424 based on EST-SSR markers). Most genetic variations were present among the different species (70.02%revealed by SRAP,73.35%revealed by EST-SSR) and the cluster analysis indicated that all Panicum accessions could be distinguished by using the SRAP or EST-SSR markers. The dendrogram faithfully reflected the phylogenetic relationships of Panicum species but did not suggest a possible domestication process of the cultivated switchgrass in relationship to the geographical sources of the accessions. Panicum amarum was found to be the closest species of switchgrass. SRAP markers were considered to be more efficient than EST-SSR markers for screening large numbers of Panicum accessions.2,The SRAP markers was employed to detect the genetic diversity of 94 switchgrass accessions obtained from USDA germplasm center. Twenty primer pairs were used for the DNA amplification, and in total 209 distincnt DNA fragments were amplified, of which 167 DNA fragments were polymorphic. In average 8 DNA fragments could be amplified per SRAP primer pairs. The Shannon's Information index was 0.241. The nuclear DNA content and the Ploidy level of each switchgrass line were estimated by using the Flow cytometry analysis. Our results suggested that all lowland accessions were tetraploid and among the upland accessions, most of them were tetraploid (79.06%); only 8 accessions were octoploids (18.60%).3,122 germpasm of switchgrass were assessed based on their dry biomass per plant, the ratio of stem to leaf, rust resistance and anthracnose resistance. Our results suggested that most accessions were susceptible to the rust disease. In general, the lowland switchgrass accessions were more resistant to rust than that of upland accessions. Three best germplasms'Alamo','TEM-LoDorm', and'Kanlow'in terms of biomass yield and disease resistance were identified.4,We downloaded about 20,000 switcghrass EST sequences from Genbank database. By using the SSRIT program, we identified 587 ESTs that carry DNA fragments with SSR sequences that were longer than 18bp. The 587 microsatellites were consisted of 21.47%dinucleotide,56.22%trinucleotide,2.04%tetranucleotide,3.41% pentanucleotide and 16.87%Hexanucleotide, respectively. CGG/GCC (12.27%) and GCG/CGC (10.05%) were the most two rich motifs.587 SSR primer pairs were designed based on these ESTs. By screening all 587 SSR makers with the DNA of two parental lines'Alamo'and'Dacotah', we found that 430 pair of primers (73.25%) could give clear and strong PCR amplification, where 123 pair of primers (21.95%) detected polymorphism between the two parental lines.51 pair primers (8.69%) were detected polymorphism in the F1 segregation population. The SSR markers were also used to perform the UPGMA cluster analysis. Based on the genetic similarity, we were able to classify the accessions into two major groups that were corresponding to their ecotypes (lowland and upland). However, the octoploid germplasm did not cluster together within the upland group.5,By using the rice disease resistance gene analog (RGA) sequences as query, we BLAST searched against the switchgrass ESTs in Genbank batabase. One hundred ninty RGAs of switchgrass were idenified. One hundred ninty RGA-STS primer pairs were designed. By screening the 190 pairs of RGA-STS primers with the DNAs of two parental lines'Alamo'and'Dacotah',78 pairs of primers (41.06%) of gave clear and strong PCR amplification. Ninety DNA fragments (RGA-PCR production) (57.69%) were successfully sequenced. By comparing the seqeunces of RGA alleles from'Alamo'and'Dacotah', we idenitified 249 SNPs from the 18090bp sequence, we got an estimated frequency of 1 SNP per 61.5bp in the switchgrass ESTs. By using these RGA-SNPs, we developed 15 RGA-CAPS or RGA-dCAPS markers.6,EST-SSR,SRAP and RGA-SNP markers were employed for construction of a draft linkage map by using F1 population (165 plants) derived from a cross between 'Alamo'(lowland) and'Dacotah'(upland) In total,139 molecular marker with Mendelian segregation ratio (1:1) (P=0.01) were identified. Furthermore, the draft linkage maps of'Alamo'and'Dacotah'were constructed respectively by using the Map Manager QTX b20 program (link?). The map of female parental line included 9 linkage groups with 26 molecular markers, which covered a total map length of 452.9cM. The average genetic distance between markers was 17.42cM. The map of the male parental line included 16 linkage groups with 32 molecular markers, which covered a total map length of 541.43cM. The average genetic distance between markers was 16.92cM. Based on the composite interval mapping, one QTL were identified that contributes to the rust resistance in'Alamo'. The rust resistance QTL was located in genetic linkage Group 5, explaining 9.61%of rust resistance variance. We also found 6 markers which had a significant accociation with the rust resistance.