Location: Crop Germplasm ResearchTitle: Confirmation of Pearl Millet-Napiergrass Hybrids Using EST-derived Simple Sequence Repeat (SSR) Markers) Author
Submitted to: American Journal of Plant Sciences
Publication Type: Peer reviewed journal
Publication Acceptance Date: 4/9/2013
Publication Date: 7/24/2013
Citation: Dowling, C.D., Burson, B.L., Foster, J.L., Tarpley, L., Jessup, R.W. 2013. Confirmation of pearl millet-napiergrass hybrids using EST-derived Simple Sequence Repeat (SSR) markers. American Journal of Plant Sciences. 4:1004-1012. Interpretive Summary: Pearl millet was crossed with napiergrass to produce hybrids to be used as a bioenergy crop. The seed produced were either hybrid seed or self-pollinated pearl millet seed. The seed were germinated and the seedlings and resulting plants could not be identified as hybrids by their appearance because of similarities with the parents. The hybrids could be identified by counting their chromosomes, but this is a difficult, time consuming approach. A quicker approach is to determine the DNA content in the nuclei of the offspring and both parents using a flow cytometer. However, this approach did not work because the DNA content of the hybrids and both parents was very similar and could not be distinguished from one another. Seed were collected from the plants that were assumed to be hybrids and those seed were placed in soil to germinate. None of these seed germinated which indicates the seed were sterile and these plants are probably wide hybrids between the parental species. This is one way the hybrids can tentatively be identified but it is time consuming because the suspected hybrids have to grow to maturity before any seed are produced and then the seed have to be subjected to a germination test before any information is available regarding their hybrid nature. This delays the identification of true hybrids for 8 to 12 months, which significantly reduces the efficiency of the breeding program. An alternative approach was used to quickly identify the hybrids, and that was the development and use of molecular markers that are present in the male parent (napiergrass) but absent in the female parent (pearl millet). Using molecular techniques, tissue from the seedlings was analyzed to determine if male-specific markers were present, and those seedlings with the markers were true hybrids. This approach accurately and quickly identifies true hybrids and will significantly increase progress in developing 'Seeded-yet-Sterile' pearl millet x napiergrass hybrids that have potential as high biomass crops that pose no threat as an invasive weed because of seed sterility.
Technical Abstract: Prospects for deploying perennial grasses that are currently considered leading candidates for dedicated energy crops over large acreages are debatable because of several limitations, including vegetative propagation or small seed size, low biomass production during the first growing season, and incomplete assessments of crop invasiveness risk. Pearl Millet-Napiergrass hybrids ('PMN'; Pennisetum glaucum [L.] R. Br. x P. purpureum Schumach.), in contrast, are large-seeded, sterile feedstocks capable of high biomass production during establishment year. Novel methods are warranted for confirmation of PMN hybrids, as traditional morphological observations can be inconclusive and chromosome number determination using cytological methods is laborious and time consuming. Six putative PMN lines were produced in this study, and 10 progeny from each line were evaluated using morphological traits, seed fertility, flow cytometry, and expressed sequence tag-simple sequence repeat (EST-SSR) markers. All putative hybrid lines were sterile and failed to produce seed. The PMN hybrids could not be distinguished from either parent using flow cytometry due to highly similar nuclear genome DNA contents. A number of paternal napiergrass-specific EST-SSRs were identified for each PMN line, and four paternal-specific EST-SSRs conserved across all napiergrass accessions were selected to screen the putative PMN hybrids. These EST-SSRs confirmed that all F1 individuals analyzed were PMN hybrids. The use of paternal-specific markers therefore provides a valuable tool in the development of both 'Seeded-yet-Sterile' biofuel PMN feedstocks and additional PMN cultivar- and parental species-specific markers.