Buffelgrass [Pennisetum ciliare (L.) Link (syn. Cenchrus ciliaris L.)], an important pasture and range grass in the arid semi‐tropics and tropics, has excellent drought‐tolerance but lacks winter hardiness. It is a polymorphic, apomictic species and its most common chromosome number is 2n = 4x = 36. Eighty‐six pentaploid (2n = 5x = 45) accessions from South Africa were deposited into the USDA National Germplasm System (NPGS) in 1976 and these were more winter‐hardy than all tetraploid accessions. Prior to 1976, only 31 pentaploids were in the NPGS but they lacked cold tolerance. Since 1976, ten pentaploid accessions with unknown cold tolerance have been added to the NPGS and they were included. The genetic diversity among all these pentaploid accessions was investigated to determine if the winter‐hardy and non‐winter‐hardy genotypes were distinct. Amplified fragment length polymorphisms were used to determine the genetic diversity and phylogenetic relationships among the accessions. Ten primer combinations generated 862 polymorphic bands; the number of bands identified by each primer ranged from 74 to 117 (mean 93.4). Unweighted pair group method with arithmetic mean (UPGMA) cluster analysis and principal coordinate analysis revealed two groups with one separating into two sub‐groups and one sub‐group separating into two subdivisions. Genetic similarity coefficients ranged from 0.39 to 0.98 (mean 0.74). These findings show considerable genetic diversity among the pentaploid accessions. Most cold‐tolerant and non‐cold‐tolerant genotypes separated by subgroups but not by group. Nine tetraploid accessions, included for reference, clustered into several subgroups. The pentaploid genotypes appear to have polyphyletic origins.

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. × 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.

Sustainable cropping systems for leading candidate biofuel crops currently focus predominantly on perennial grasses for which assessments of invasiveness potential remain incomplete. Perennial C4 grasses have significant capacity for biomass accumulation across diverse environments, providing intrinsic value towards protection and restoration of underutilized, marginal, and degraded lands. Varied seed and vegetative reproduction mechanisms, however, contribute to their invasive potential. The development of feedstocks possessing the minimum vegetative propagules required for perennial life habit, combined with seed sterility, would therefore greatly reduce the risk of perennial biofuel crops becoming biological invaders. Pearl millet-napiergrass ("PMN"; Pennisetum glaucum [L.] R. Br. × Pennisetum purpureum Schumach.) and king-grass (P. purpureum × P. glaucum) are examples of such feedstocks, being "seeded-yet-sterile" crops in which fertile parents allow seeded production of hybrids that are subsequently both seed-sterile and devoid of rhizomes in biomass production fields. The use of genomics tools provide further tools suitable for both characterizing genetic mechanisms governing weediness and deploying marker-assisted breeding programs for biofuel crops with reduced risk of negative environmental impacts.

Napiergrass [Pennisetum purpureum (Schum.) Morrone] is a robust, perennial, warm‐season grass that grows throughout the tropical and subtropical regions of the world. Some genotypes have sufficient winter hardiness to survive winters in the Gulf Coast region of the United States. However, germplasm with increased cold tolerance is needed to expand where the grass can be grown as a forage and biofuel crop in the southern United States. A male‐sterile napiergrass accession was pollinated with several genotypes of flaccidgrass (P. flaccidum (Griseb.) Morrone], P. orientale (Rich.) Morrone, and buffelgrass [Cenchrus ciliaris L. syn. P. ciliare (L.) Link] to transfer cold tolerance to napiergrass. More than 38,500 florets were pollinated but only two weak, short‐lived napiergrass × P. orientale seedlings were recovered. Controlled pollinations were made to determine what prevented hybridization, and pollen germination and tube growth in the pollinated pistils were observed using fluorescent microscopy. Most did not exhibit barriers to hybridization; however, pollen tubes of one P. orientale accession did not grow into the napiergrass styles. Pollen tubes became disoriented in the ovary and grew in an irregular manner in most crosses, but some tubes entered the micropyle. Since pollen–pistil incompatibilities were not the primary cause for failure of hybridization, post‐fertilization events in the embryo sacs were observed. The egg cell and polar nuclei were not fertilized in many sacs, and when they were, the embryo and endosperm began deteriorating within 3 to 5 d following pollination. Thus, hybrids were not recovered from crosses between these species because of post‐fertilization barriers, suggesting alternative strategies to wide hybridization may be required for the development of cold‐tolerant napiergrass.