Construction And Characterization Of Four Bac Genomic Libraries Made For Maize And Its Wild Relatives

Maize is one of the most important crops in the world, not only as a food crop, a nutritious fodder, but also as a widely used industrial material, such as alcohol, starch etc. Breeders also breed multiple specialized maize species and expant the use of maize widely. Many phenomenons in genetics and genomics were found first in maize, such as transposable element, epigenetics and heterosis etc. The genome of maize is complicated and diverse and maize becomes a model plant for the research of genome sequencing of large plant genomes and for nuclear genomic evolution. There are plenty of germplasm of maize in the world, their wild relatives such as Tripsacum and teosinte are gene treasure for many useful characters and biotic or abiotic resistance. The first step for exploring and reading the genome resource of maize and its wild relatives is the construction of the large DNA genomic bacterial artificial chromosome library (bacterial artificial chromosome library, BAC Library), construction of physical map with high quality and obtain the whole genome sequence. We can also transfer the large DNA fragment containing genes or gene families of important agrinomic traits into plant directly in order to research gene functions. Considering the importance of exploring maize and its wild relatives and the role of BAC library in genomic research, we constructed four BAC libraries for maize and its wild relatives and charicterized these BAC libraries. We analyze the two wild maize BAC libraries preliminarily.The four materials of maize and its wild relatives is inbred line Zheng58and Chang7-2, teosinte Parviglumis (Zea mays ssp:Parviglumis), Tripsacum (Tripsacum dactyloides). Zheng58and Chang7-2are the parents of Zheng dan958which is the most widely planted hybrid cultivar in China and they contain plenty of useflul agrinomic genes. Parviglumis is the progenitor of modern cultivated maize, Tripsacum L. is the closest genus to Zea L. in trib maydeae and is also associated with maize origin. Both Parviglumis and Tripsacum are huge gene treasure and represent untapped genetic resource for crop improvement. The results for the four materials above in this study are shown as follows: 1. Constructed two BAC libraries for two maize inbred lines (Zheng58and Chang7-2) and two BAC libraries for two wild maize materials (Parviglumis and Tripsacum). The BAC library of Zheng58contains148,992clones, the average insert size is144kb, the organellar DNA contamination is0.55%and the genome equvilent is8.4plus. The BAC library of Chang7-2contains145,920clones, the average insert size is139kb, the organellar DNA contamination is0.58%and the genome equvilent is8.0plus. The BAC library of Parviglumis contains100,608clones, the average insert size is148kb, the organellar DNA contamination is0.31%and the genome equvilent is5.5plus. The BAC library of Tripsacum contains128,256clones, the average insert size is139kb, the organellar DNA contamination is0.26%and the genome equvilent is4.7plus.2. Choose12single copy genes of maize B73distributing on the ten chromosomes of maize. Use these12genes as probes to screen the fliters of Parviglumis and Tripsacum BAC libraries. All the12genes got positive clones after screened the two wild maize BAC libraries. These results indicated that the two wild maize libraries were ready for target gene screening. The two maize inbred line BAC libraries of Zheng58and Chang7-2have much more possibility for target gene screening since they reprisent much higher genome equvilent.3. Amplify the upstream and downstream genes of four loci, adhl, adh2, tbl, bal, by PCR. Streen the Parviglumis and Tripsacum BAC libraries with these gene probes.120clones (Parviglumis) and159clones (Tripsacum) were streened. All these clones were contig assembled by fingerprinting for further sequencing analysis.4. All the442BAC clones screened from two wild maize BAC libraries were sequenced both ends and88%of the BESs were sequenced sucessfully. All the positive clones screened with12gene probes were assembled into contigs by fingerprinting. There are20contigs from64clones in Parviglumis library and there are27contigs from85clones in Tripsacum library. Estimate the genome equvilent by contigs since one contig represents one loci, the genome equvilent of Parviglumis BAC library is3.2plus and the genome equvilent of Tripsacum BAC library is3.1plus.5. We tried to anchor the positive clones screened from Parvglumis BAC library onto maize B73reference sequence by BAC end sequences.102clones in Parviglumis had hits on B73reference sequence by BESs.202BESs were sequenced successfully and196BESs had hits on B73reference sequence.70clones could be anchored on reference sequence and18of the clones could be assembled into contigs, anchoring on the expected site.9contigs could be anchored on the reference sequence.6. We tried to anchor the positive clones screened from Tripsacum BAC library onto maize B73reference sequence by BAC end sequences.104clones in Tripsacum had hits on B73reference sequence by BESs.229BESs were sequenced successfully and150BESs had hits on B73reference sequence.20clones could be anchored on reference sequence and5of the clones could be assembled into contigs, anchoring on the expected site.3contigs could be anchored on the reference sequence.7. We analyzed the adhl contig which could be anchored onto the B73reference sequence. Check the clones’ real insert size of these two contigs by digesting the BAC clones with I-Scel and seperated on CHEF gel. Compare the real size with the anchored size on the maize B73reference and we found that the anchored size is twice as longer as the real insert size of Tripsacum adhl clones. Amplify the genes on both sides of maize B73adhl from adh1contigs of Tripsacum and we found that all the clones from this Tripsacum adhl contig could get the first adhl gene while the anchored sequence on B73does not contain this gene.