Cytogenetics and Genetics of Pearl Millet

CYTOGENETICS AND GENETICS OF PEARLLET Prem P. Jauhar' and Wayne W. Hanna* I USDA-Agricultural

Research Service Northern Crop Science Laboratory State University Station, Fargo, North Dakota 58105 WSDA-Agricultural Research Service Coastal Plain Experiment Station Tifton, Georgia 31793

I. Introduction

II. Origin

111. Taxonomic Treatment

IV

V. VI. VII. VIII. M. X.

XI.

A. Taxonomic Placement of Pearl Millet B. Wild Annual Relatives of Pearl Millet C. Perennial Relatives of Pearl Millet Chromosomes, Karyotype, and Meiosis A. Chromosomes as Multiples of 5 , 7 , 8 , and 9 and Size Differences B. Chromosomes of Pearl Millet and Other Penicillarias C. Evolution of the Chromosome Complement of Pearl Millet Genome Relationships Aneuploidy and Gene Mapping Molecular Markers and Gene Mapping Wide Hybridization with Pearl Millet Wide Hybridization and Genetic Enrichment for Fodder Traits A. Interspecific Hybrids B. Intergeneric Hybrids Hybridization and Exploitation of Hybrid Vigor A. Grain Hybrids B. Forage Hybrids C. Germplasm D. Types of Hybrids Apomixis A. Incidence of Apomixis in Pennisentm Species B. Genetics of Apomixis C. Harnessing Apomixis for Exploitation of Heterosis

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PREM P. JAUHAR AND WAYNE W. HANNA XI. Genetics of QualitativeTraits XIII. Genetics of Quantitative Traits xn! Conclusion and Perspectives References

I. INTRODUCTION Pennisetum is one of the most important genera of the family Poaceae. It includes such important species as pearl millet, Pennisetum glaucum (L.) R. Brown [ =Pennisetum typhoides (Bum.) Stapf et Hubb., Pennisetum americanum (L.) Schumann ex Leeke] (2n = 14),a valuable grain and forage crop; and its tetraploid relative Napier grass (I? purpureum Schum.) (2n = 4x = 28), prized for its fodder grown throughout the wet tropics of the world. Pearl millet is widely cultivated in different parts of the world. It is a multipurpose cereal grown for grain, stover, and green fodder on about 27 million hectares, primarily in Asia and Africa (ICRISAT, 1996). In terms of annual production, pearl millet is the sixth most important cereal crop in the world, following wheat, rice, maize, barley, and sorghum. Among the millets, it is second only to sorghum. Pearl millet is the only cereal that reliably provides both grain and fodder on poor, sandy soils under hot, dry conditions. It is remarkable that it produces nourishment from the poorest soils in the driest regions in the hottest climates. In the drier regions of Africa and Asia, the crop is a staple food grain. In more favored areas, however, pearl millet grain is fed to bullocks, milch animals, and poultry. In areas where other types of feed are not available, stover provides feed for cattle (ICRISAT, 1996). Pearl millet is also grown in several other countries. It was planted to almost 1 million hectares in Brazil in 1996. In the United States, it is grown as a forage crop on an estimated half a million hectares. It is also grown as a forage crop in tropical and warm-temperate regions of Australia and several other countries (Jauhar, 198la). Pearl millet is an ideal organism for cytogenetic and breeding research. Several favorable features of its chromosome complement--e.g., the small number and large size of chromosomes with distinctive nucleolar organizers-make pearl millet a highly suitable organism for cytogenetic studies. Because of its low chromosome number, pearl millet offers a particularly favorable material for aneuploid analysis and thereby elucidation of its cytogenetic architecture. Moreover, its protogynous flowers and outbreeding system make it ideal for interspecific hybridization and breeding work, particularly heterosis breeding. Pearl millet has also been found suitable for molecular studies. Although pearl millet has great agricultural importance and is a favorable organism for cytogenetic and molecular studies, it has not received the attention it deserves. Consequently, the information available on its genetics and cytogenetics is far less than that available for other agricultural crops. In a comprehensive

CYTOGENETICSAND GENETICS OF PEARL MILLET

3

review, Jauhar (1981a) compiled the available literature on the cytogenetics and breeding of pearl millet and related species. The purpose of this article is to summarize the information on cytogenetics and genetics of pearl millet mostly since the publication of Jauhar’s book (198 la).

II. ORIGIN Pearl millet originated in West Africa, where it grows in chronically droughtprone areas. Selection exercised by early cultivators within a variety of cultural contexts resulted in a multitude of morphologically diverse forms. The protogynous flowers of pearl millet facilitated the introgression of characters from related wild species to cultivated annual species. Although researchers generally agree that pearl millet is of African origin, pinpointing its specific region of origination has been controversial. Vavilov (1949-1950) placed pearl millet in the Ethiopian Center of Origin (particularly Abyssinia and Sudan), considering this the region of maximum diversity. However, the center of diversity is not always the center of origin (Harlan, 1971). In light of the great morphological diversity present in introductions from Central Africa, Burton and Powell (1968) inferred that pearl millet originated there. Another method used to pinpoint its center of origin is the occurrence of B chromosomes. Because B chromosomes frequently occur in primitive varieties but not in commercially bred cultivars, Muntzing ( 1958) suggested that their occurrence might indicate a crop’s center of origin. Therefore, based on the occurrence of B chromosomes in pearl millet collections, some researchers consider Sudan (Pantulu, 1960) and Nigeria (Powell and Burton, 1966; Burton and Powell, 1968) to be the crop’s centers of origin. However, drawing conclusions on the basis of occurrence of B chromosomes may not be scientifically sound (Jauhar, 1981 a), because several ecological and edaphic factors influence the occurrence of B chromosomes. In rye (Secafecereale), for example, the frequency of Bs is higher in rnaterial growing on acidic soils than on basic soils (Lee, 1966). Working on clonal plants of rye grown under different regimes of soil, temperature, and humidity, Kishikawa (1970) found that the frequency of Bs was lower in progeny derived from plants grown under high temperatures or dry soil conditions. Considering that the greatest morphological diversity of pearl millet occurs in West Africa, south of the Sahara Desert and north of the forest zone, and that the wild progenitor also occurs in the drier, northern portions of this zone, Harlan (197 1 ) suggested that the center of origin lies in a belt stretching from western Sudan to Senegal. Based on present-day distributions, the Sahel region of West Africa appears to be the original home of pearl millet (Brunken et al., 1977). The cultivated types show the highest level of morphological variability in this region (Clegg et al., 1984).

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PREM P. JAUHAR AND WAYNE W. HANNA

Traditionally, characterization of genetic resources of crop plants has been accomplished through a combination of morphological and agronomic traits, e.g., growth habitat, earliness, and disease and pest resistance. Biochemical and molecular markers have also been used to obtain additional information on a crop plant’s center of domestication, the effect of domestication on genetic diversity, and potential gene flow between wild and cultivated types (Gepts and Clegg, 1989). However, using restriction fragment length polymorphisms (RFLPs) among chloroplast, nuclear ribosomal RNA, and alcohol dehydrogenase (ADH) sequences in a group of 25 wild and 54 cultivated accessions of pearl millet, Gepts and Clegg (1989) could not identify the precise pattern of its domestication. Brunken et al. (1977) hypothesized the existence of several independent domestications of pearl millet in the southern fringe of the Sahara. Based on polymorphisms in 12 genes coding for 8 enzymes in 74 cultivated samples and 8 wild samples from West Africa, the 82 samples were classified into three groups: (1) wild types, (2) early maturing cultivars, and (3)late cultivars (Tostain et al., 1987). The early maturing cultivars were found to have the highest enzyme diversity, whereas cultivars from Niger showed the most diversity. The high diversity of the early maturing group and its extensive divergence from West African wild millets further suggest multiple domestications.

III. TAXONOMIC TREATMENT A. TAXONOMIC PLACEMENT OF PEARL MILLET Pearl millet is the most important member of the genus Pennisetum in the tribe Paniceae. It has received a variety of taxonomic treatments, and its scientific binomials have been frequently shuffled by a variety of taxonomists. Consequently, it has had many Latin names, perhaps more than any other grass. In the post-Linnaean period from 1753 to 1809, pearl millet was treated as a member of at least six different genera, namely, Panicum, Holcus, Alopecuros, Cenchrus, Penicillaria, and Pennisetum (see Jauhar, 1981a,c). At the beginning of this century, pearl millet was commonly referred to as Pennisetum typhoideum, Penicillaria spicata, Panicum spicatum, and Pennisetum alopecuroides (Chase, 1921). By the mid-19th century, however, pearl millet was generally called Pennisetum typhoideum L. C. Rich, but this nomenclature was not widely accepted. The Latin name Pennisetum americanum given by K. Schumann (1895)-apparently based on the first name “Panicum americanum L.” used by Linnaeus (1753bwas accepted by Terrell (1976) and hence used by several American workers. However, this name is inappropriate and misleading because it inadvertently implies the American origin of pearl millet (Jauhar, 1981a,c).

CYTOGENETICSAND GENETICS OF PEARL MILLET

Stapf and Hubbard (1933, 1934) gave the name Pennisetum fyphoides (Bum.) Stapf et Hubb., which was accepted by several modem taxonomists, including Bor (1960), and used by most pearl millet workers outside the United States. In the 1960s, American workers joined the rest of the world in calling pearl millet Pennisetum ophoides (Burton and Powell, 1968).The name Pennisetum glaucum (L.) R. Br., based on Panicum glaucum (L.) R. Br., was adopted by Hitchcock and Chase (195 1) in Manual of the Grasses of the United States. Consequently, American scientists currently engaged in research on pearl millet use this name. All annual and perennial members of the section Penicillaria fall under the x = 7 group. They have typically penicillate anther tips. Whereas most penicillarias are diploid with 2n = 14 chromosomes, one, viz., Napier grass, is a perennial tetraploid.

B. WILDANNUAL RELATIVES OF PEARL MILLET Of the 32 species described by Stapf and Hubbard (1934) in the section Penicillaria of the genus Pennisetum, only two have been found outside Africa. There is considerable variation in seed and other characters both between and within different cultivars or races. Such variation could be attributed to independent domestications and migrational events resulting in geographical isolations. The protogynous nature of pearl millet and its intercrossabilitywith its wild relatives must have generated much of the existing genetic diversity. Meredith (1955) described four taxa, which he called “allied species,” closely related to pearl millet: Pennisetum americanum, I? nigritarum, I! echinurus, and I? albicauda. Since these are interfertile with pearl millet, they were merged into a single species with pearl millet (Brunken et al., 1977). However, for the sake of convenience, Brunken subdivided the morphologically heterogeneous pearl millet species he called “Pennisetum americanum” into three subdivisions: ( 1) ssp. americanum encompasses the wide array of cultivated pearl millets; (2) ssp. monodii includes all the wild and semiwild diploid races that are fully fertile with pearl millet and therefore form a single reproductive unit with it; and (3) ssp. stenostachyum is morphologically intermediate between the two preceding species. Amoukou and Marchais (1993) found some evidence of a partial reproductive bamer between wild and cultivated pearl millets. Crosses between 16 cultivated accessions (f? glaucum ssp. glaucum) (as female parents) and 11 wild accessions (f? glaucum ssp. monodii), from the whole range of diversity of the species, showed certain degrees of seed malformation and reduced 1000-grain-weightand germination ability. These are manifestations of a genetic imbalance between the cultivated and the wild groups, probably resulting from reproductive barriers that developed during the domestication process.

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C. PERENNIAL RELATIVESOF PEARL MILLET Elephant or Napier grass, Pennisetum purpureum (2n = 4x = 28), is a perennial relative of pearl millet (see Section V). It has typically penicillate anthers. Native to Africa, it is a robust perennial with creeping rhizomes. It was introduced into the United States in 1913. It is extensively grown in the humid tropics throughout the world.

N.CHROMOSOMES, KARYOTYPE, AND MEIOSIS A. CHROMOSOMES AS MULTIPLES OF 5,7,8, AND 9 AND SIZEDIFFERENCES The genus Pennisetum is a heterogeneous assemblage of species with chromosome numbers as multiples of 5 , 7, 8, and 9, for example, P. ramosum (2n = lo), P. ryphoides (2n = 14) and P. purpureum (2n = 28), P. massaicum (2n = 16,32), and P. orientale (2n = 18, 36, 54). The chromosome morphology is diverse and substantial size differences exist. A notable feature is that species with lower chromosome numbers have larger chromosomes. Thus, pearl millet (2n = 14) and P. ramosum (2n = 10) have relatively large chromosomes, larger than those of other members of the tribe Paniceae. In contrast, species with higher chromosome numbers, e.g., I? orientale (2n = 18), have strikingly smaller chromosomes than those of pearl millet (2n = 14) (Fig. 2C). A characteristicfeature of perennial species of Pennisetum is the occurrence of chromosomal races or cytotypes, e.g., P. orientale L. C . Rich. (2n = 18, 27, 36, 45, 54) and F! pedicellatum Tin. (2n = 36,45,54). However, no such cytotypes occur in the annual cultivated or wild pearl millets, all of which have 2n = 14 chromosomes.

B. CHROMOSOMES OF PEARL MILLET AND &HER

PENICILLARTAS

Rau (1929) was the first to determine the somatic chromosome number of pearl millet as 2n = 14, and he mentioned these chromosomes as being large. The chromosomes have median to submedian centromeres; the shortest chromosome pair is satellited, and during meiosis the shortest bivalent is associated with the nucleolus. The chromosomes of diploid taxa of the section Penicillaria are similar to those of pearl millet. Thus, I? ancylochaete, P. gambiense, I! maiwa, and I? nigritarum have 2n = 14 chromosomes, and their chromosome morphology is similar to one another and to chromosomes of pearl millet (Veyret, 1957). Not surpris-

CYTOGENETICS AND GENETICS OF PEARL MILLET

7

ingly, therefore, these taxa are interfertile with pearl millet, and there is no barrier to gene flow across these taxa. Pennisetum violaceum and R mollissimum, the two close wild relatives that form a primary gene pool with pearl millet, and I? schweinfurthii (a representative species of tertiary gene pool) were assessed for their genomic organization, using in situ hybridization with rDNA probes on somatic metaphase spreads and interphase nuclei (Martel et al., 1996). These studies showed chromosomal similarity of rDNA sequence locations in the three taxa in the primary gene pool. Pearl millet regularly forms seven bivalents at meiotic metaphase I. A characteristic feature is the rapid terminalization of chiasmata, such that at diakinesis mostly loose ring bivalents with two terminalized chiasmata each are observed. The annual, semiwild taxa also have regular meiosis with 7 11. They all have the genomic constitution AA. Recently, Reader et al. (1996) used fluorescence in situ hybridization (FISH) to characterize the somatic complement of pearl millet. A metaphase spread was hybridized with Fluorored-labeled rDNA (derived from plasmic clone pTa71; Gerlach and Bedbrook, 1979) and then stained with DAPI. In that double exposure. two large and two small NOR loci were observed. Napier grass is a perennial relative of pearl millet. Burton (1 942) determined its somatic chromosome number as 2n = 28 chromosomes. It is an allotetraploid (2n = 4x = 28) with diploidlike meiosis (see Jauhar, 1981a). It is genomically represented as AABB, the A genome being largely homologous to the A genome of pearl millet (see Section V).

C. EVOLUTION OF THECHROMOSOME COMPLEMENT OF PEARL MILLET Researchers generally believe that several crop species have evolved from species with lower basic chromosome numbers, with increase in chromosome number occurring by means other than straight polyploidy. Evidence supporting this view has been found by RFLP studies of maize (Helentjaris et al., 1986; Whitkus et al., 1992), brassicas (Slocum et al., 1990; Kianian and Quiros, 1992), and sorghum (Hulbert et al., 1990; Whitkus et al., 1992; Chittenden et ul., 1994). Based on cytogenetic evidence, Jauhar (1968, 1970a, 1981a) hypothesized that x = 5 may be the original basic number in Pennisetum and that pearl millet (2n = 14) may be a secondary balanced species as a result of ancestral duplication of chromosomes. If duplication of a part of the original genome occurred during the evolution of pearl millet, some duplicate loci should be observed in the present genome. Liu et al. (1 994) indeed detected several duplicate loci in their RFLP linkage map of the pearl millet genome. However, further studies are needed to fully characterize the duplicated regions of the genome.

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PREM P. JAUHAR AND WAYNE W. HANNA

V. GENOME RELATIONSHIPS Knowledge of genome relationships between plant species is very useful in planning effective breeding strategies designed to transfer desirable genes or gene clusters from one species into another, thereby producing fruitful genomic reconstructions. Traditionally, the principal method of assessing the genomic affinities among species has been the study of chromosome pairing in their hybrids (Jauhar and Joppa, 1996). Genomic relationships are inferred from the degree of pairing between parental chromosomes.However, pairing in the hybrids may be due to allosyndesis (Le., pairing between chromosomes of the parental species) andor autosyndesis (i.e., pairing within a parental complement).Therefore, information on the nature of chromosome pairing is important for assessing the genomic relationships. The chromosomes of pearl millet are much larger than those of other species of Pennisetum (e.g., see Fig. 1). This size difference makes it possible to study intergenomic chromosome pairing relationships. A clearly distinguishable size difference between chromosomes of pearl millet (2n = 14 large chromosomes; AA genome) and those of Napier grass (2n = 28 relatively small chromosomes; AABB) makes it possible to study, in their hybrids (e.g., see Figs. 2A, 2B), the degree of allosyndetic and autosyndetic pairing (Jauhar, 1968). Based on pairing in triploid hybrids (2n = 3x = 21; AAB), it was inferred that the two species basically share a genome (A and A being very similar). However, the source of B genome remains unknown.

Figure 1 Somatic chromosomesof a hybrid between pearl millet and fountain grass, Penniseturn setaceurn (Forsk.) Chiov. Note the 7 large pearl millet chromosomes and 18 much smaller fountain grass chromosomes.

CYTOGENETICSAND GENETICS OF PEARL MILLET

L

;*

'I)

.;

9

P

* I .

A

C

D

Figure 2 Chromosome pairing in interspecific hybrids (2n = 3x = 21;AAB) between pearl millet (2n = 2x = 14;AA) and Napier grass (2n = 4x = 28;AAB). (A) Metaphase I showing 21 univalents-7 large ones from pearl millet (arrows) and 14 small ones from Napier grass. (B) Metaphase I with 7 11 (2 11 overlapping) + 7 I; the bivalents comprise 2 large, symmetrical bivalents within the A genome (hollow arrows), 1 heteromorphic intergenomic bivalent between chromosomes of A and A genomes (solidarrow),and 4 intragenomic bivalents within A and B genomes. Note 2 large univalents of the A genome. (C, D)Chromosome pairing in interspecific hybrids (2n = 16) between pearl millet (2n = 14) and P. orienrule (2n = 18). (C) Diakinesis with 16 univalents-7 large ones (arrows) from pearl millet and 9 small ones from orientale. Note the striking size differences among the parental chromosomes. (D) Metaphase I with 2 heteromorphic bivalents between pearl millet chromosomes and orientale chromosomes (solidarrows),and 1 autosyndetic bivalent within the orienrule complement (hollow arrow). (Reprinted from Jauhar, 1981a. by permission of the publisher.)

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PREM P. JAUHAR AND WAYNE W. HANNA

Even more striking size differences exist between the chromosomes of pearl millet and those of oriental grass (Penniseturn orientale; 2n = 18) (Fig. 2C). The nature of chromosome pairing was analyzed in hybrids between these species (Patil and Singh, 1964;Jauhar, 1973,1981a,b). Association between chromosomes of the parental species resulted in the formation of conspicuously heteromorphic bivalents (Fig. 2D), suggesting an ancestral relationship between the two species. In addition to intergenomic pairing, intracomplement associations within the glaucum and the orientale complements were also observed.

VI. ANEUPLOIDY AND GENE MAPPING The establishment of a complete series of aneuploids is very useful in elucidating the cytogenetic architecture of a crop plant. Jauhar initiated work on the isolation of aneuploids of pearl millet. From the progeny of triploid X diploid crosses, he isolated two primary trisomics (2n + I = 15) (Jauhar, 1970b). Jauhar (198 la) summarized research on aneuploids in pearl millet. Over the years, there have been numerous reports on double trisomics, triple trisomics, double telotrisomics, ditertiary compensating trisomics, multiple interchange trisomics, and so on. Minocha et al. (1980a) described a set of primary trisomics and used them to assign genes to five of the seven chromosomes. Vari and Bhowal(1985) reported a set of primary trisomics distinguishable by morphological characteristics. Using trisomic analyses, Sidhu and Minocha (1984) located genes controlling peroxidase isozyme production on all seven chromosomes. Minocha et al. (1 982) described a translocation tester set of five translocation stocks, each of which involved two nonhomologous chromosomes. Rao et al. (1988) described various types of trisomics, some involving interchanges, and also reviewed some of the earlier work on aneuploids in pearl millet. However, it appears that little use has been made of these aneuploids and translocation stocks in genetic and breeding studies.

VII. MOLECULAR MARKERS AND GENE MAPPING An important aspect of genetic research is creating genetic maps that are useful to geneticists and plant breeders. DNA markers can be employed in the construction of genetic maps, which help determine the chromosomal location of genes affecting either simple or complex traits (Paterson et al., 1991). With these molecular methods, genetic maps of diploid plants can be developed more rapidly than those of polyploids. Pearl millet has a haploid (1C) DNA content of about 2.5 pg (Bennett, 1976).

CYTOGENETICS AND GENETICS OF PEARL MILLET

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Using RFLP, Liu et al. (1994) constructed a linkage map of pearl millet. The RFLP map so generated is relatively dense, with a 2 cM distance between markers. However, specific chromosome regions with tightly linked markers are still evident. Using molecular markers, Jones ef al. (1995) assigned part of the genes controlling quantitatively inherited resistance to downy mildew to linkage group 1, 2,4, 6, and 7 of pearl millet. Busso et al. (1995) used RFLP markers to study the effect of sex on recombination in pearl millet. They found no differences in recombination distances at the whole-genome level; only a few individual linkage intervals differed, but all were in favor of increased recombination through the male. These results are contrary to those obtained with tomato. Using RFLP markers to compare male and female recombination in two backcross populations of tomato, De Vicente and Tanksley (199 1) reported a significantly higher recombination rate in female meiosis.

Vm. WIDE HYBRIDIZATION WITH PEARL MILLET In recent years, experimental hybridization has been effected between taxonomically distant taxa. Using pearl millet as a pollen parent in crosses with barley, Zenkteler and Nitzsche ( 1984) obtained globular embryos. In crosses between hexaploid spring wheat cv. Chinese Spring and the pearl millet genotype Tift 23 BE, Laurie (1989) observed fertilization in 28.6% of the 220 florets pollinated. Chromosome counts in zygotes confirmed the hybrid origin of the embryos; three embryos had the expected 21 wheat and 7 pearl millet chromosomes and a fourth had 21 wheat and 14 pearl millet chromosomes. However, the hybrid embryos were cytologically unstable and probably lost all of the pearl millet chromosomes in the first four cell division cycles. The elimination of pearl millet chromosomes at an early stage will limit the chances of gene transfer from pearl millet into wheat. In crosses between five cultivars of oat with pearl millet (as pollinator), Matzk (1996) obtained a hybrid frequency of 9.8%. However, the pearl millet chromosmes were lost during embryo or plant development. In one hybrid, 5 pearl millet chromosomes were retained with 21 of oat. Hybrids like this could offer an opportunity for transfer of pearl millet genes into oat or vice versa. Such hybrids could also help produce alien addition or substitution lines in the two crop plants.

M.WIDE HYBRIDIZATION AND GENETIC ENRICHMENT FOR FODDER TRAITS

The potential for producing and using hybrids for forage production is greater in Pennisetum than in many other genera. A number of the species can be inter-

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crossed with various degrees of ease. Bridging species can be used to increase success of wide crosses. Pearl millet usually contributes vigor and high forage quality to wide hybrids, whereas the wild species contributes perennial growth habit and short-day sensitivity to extend the vegetative growing period. Successful propagation of hybrids will depend on commercial production of hybrid seed (usually in a frost-free or tropical area) (Osgood et al., 1997), vegetative propagation, andor apomictic seed production.

A. INTERSPECIFIC HYBRIDS The pearl millet (2n = 2.x = 14, AA genome) X Napier grass (2n = 4x = 28, AABB genomes) cross produces a vigorous, sterile triploid (PMN) hybrid (2n = 3x = 2 1, AAB). This hybrid can be produced by hand pollinations from which superior plants can be vegetatively propagated, or commercial hybrid seed can be produced on a cms (cytoplasmic-nuclear male sterile) pearl millet in a tropical area (Osgood er al., 1997). The interspecific hybrid needs to be produced in a frost-free area, because Napier grass is short-day sensitive and will not mature seed in the traditional pearl millet hybrid seed production areas. The PMN hybrids are perennial and extend the vegetative growing period into late fall. Muldoon and Pearson (1979) and Jauhar (1981a) published extensive reviews on most aspects of these hybrids. A 3-year study conducted by Hanna and Monson (1980) on 20 PMN hybrids showed that they can significantly out-yield the best pearl millet hybrids. Hanna and Monson also found that interspecific hybrids made with a tall cms pearl millet parent out-yielded those made with a dwarf parent. Napier grass genotypes varied in their combining ability with pearl millet to produce superior hybrids, and certain Napier grass pollinators produced varying amounts of seedling lethals in crosses with pearl millet. Schank and Hanna (1995) summarized reseal ch on the forage potential of derivatives of the PMN triploid hybrid. Doubling the chromosome number of the PMN triploid results in a seed fertile hexaploid (2n = 6x = 42, AAAABB) with excellent forage potential and which can be vegetatively or seed propagated. A vigorous leafy sterile tetraploid (2n = 4x = 28, AAAB) is produced when the fertile hexaploid is backcrossed to diploid pearl millet. This sterile tetraploid is perennial and can be vegetatively propagated. High-forage-yielding, leafy, perennial trispecific hybrids can be produced by pollinating the fertile hexaploid PMN hybrids with fertile apomictic hybrids from tetraploid pearl millet X apomictic I? squamulatum crosses (Hanna et al., 1989). Apomictic genotypes can be selected among the trispecific hybrids that combine germplasm from pearl millet, Napier grass, and P. squamulatum. Hussey er al. (1993) showed that 2n n hybrids from the P. jaccidum X P. mezianum cross have excellent forage potential. The pearl millet X P. squamularum hybrid has forage potential but does not appear to be as high-yielding as the preceding hybrids

+

CYTOGENETICS AND GENETICS OF PEARL MILLET

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(Patil and Singh, 1980a; Hanna et al., 1989). Several other interspecific hybrids and derivatives (Patil and Singh, 1980; Jauhar, 1981a; Hanna et al., 1992) have been produced, but more research is needed to establish their forage potential. More potential exists for producing vigorous 2n n hybrids among the apomictic Pennisetum species.

+

B. INTERGENERIC HYBRIDS Jauhar (1981a) and Patil and Singh (1980) summarized studies on various intergeneric hybrids involving Pennisetum species. Most intergeneric hybrids are weak, and/or more information is needed to establish their usefulness. Hussey et al. (1993) reported on a 2n n Cenchrus ciliaris X t?orientale intergeneric hybrid that had excellent forage potential. It appears that more potential exists for producing vigorous hybrids between Cenchrus and Pennisetum species by taking advantage of the relatedness of these genera, apomixis, and the potential for 2n + n fertilization.

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X. HYBRIDIZATION AND EXPLOITATION OF HYBRID VIGOR Heterosis is significant in pearl millet for both grain and forage production. Use of hybrids is increasing each year in all of the pearl millet growing areas except Africa and Pakistan. Most cultivars grown outside the major pearl millet growing areas of Africa, India, and Pakistan are hybrids and used for forage. However, there is an increased emphasis on production of grain hybrids in the United States. Researchers estimate that 40% of the cultivars in India are F, hybrids, but the areas planted to hybrids range from about 95% in Gujarat to about 10% in Rajasthan (Andrews, 1987; Dave, 1987). Reviews and summaries on the history and progress of inbred and hybrid development and breeding methods used to produce superior hybrids have been published by Andrews (1987), Andrews et al. (1989, Burton (1983), Jauhar (1981a), Anand Kumar and Andrews (1984), Rachie and Majmudar (1980), and Williams and Andrews (1983).

A. GRAINHYBRIDS Heterosis for grain yield in pearl millet was recognized in the mid-1940s. The first pearl millet hybrids released were X . 1 and X.2. These were single-cross grain hybrids produced by chance hybridization due to protogyny and yielded on average 45% more grain than the local types (Rao et al., 1951). It was recognized at

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that time that a commercial method for producing 100% hybrid seed was needed, because the varying amounts of selfed seed produced by the chance method did not allow maximum expression of hybrid vigor at the low seeding rate for a commercially planted grain hybrid. However, Burton (1948, 1989) showed that up to 50% selfed plants in forage chance hybrids would not decrease forage yields at recommended seeding rates, which are higher than those for grain hybrids. Anand Kumar and Andrews (1984) found that research in the 1950s demonstrated the large yield increases possible with F, hybrids and that a crns system was needed to produce hybrids on a commercial scale. Tift 23A, a crns inbred, was made available to Indian pearl millet breeders in 1962 (Burton, 1965).Indian pearl millet breeders pollinated Tift 23A with Bil-3B, an Indian inbred, to produce HB 1, the first released pearl millet single-cross grain hybrid using the crns system. Hybrids using Tift 23A and Tift 18A as female parents and Indian inbreds as pollinators averaged 102% more grain production than the best available varietal checks in India from 1964 to 1967 (Rachie and Majmudar, 1980). Hybrids such as HB 1, using Tift 23A as the seed parent, eventually became susceptible to downy mildew (Sclerospora graminicola Sacc. Schroet.) and ergot (Clavicepsfusiformis Loveless). This initiated a concentrated effort to develop inbreds resistant to these diseases for production of resistant hybrids. The research is ongoing today. Scientists at ICRISAT (International Crops Research Institute for the Semi-And Tropics), India, have been exploring new sources of cytoplasmic male sterility for hybrid production (Sujata er al., 1994; Rai, 1995). The first release in India of a top-cross hybrid was announced in 1996 by government authorities in Madhya Pradesh. The hybrid named “Jawahar Bajra Hybrid 1 (JBHl)” has high grain-yield potential, medium height, nonbristled compact ears, and medium bold, globular grains. Both the hybrid and its top-cross pollinator are highly resistant to downy mildew. Similarly, Gujarat State Fertilizers Company Limited has developed a hybrid “Sardar Hybrid Bajra (SHB I),” which has about 20% more yield, has better quality grain, and matures earlier than the existing hybrids (SATNews, 19961997). Interest in producing pearl millet for grain in the United States and Australia has increased. HGM 100 was the first commercial grain hybrid released in the United States in the early 1990s (Hanna el al., 1993). The area planted to the crop was increasing in the southeastern United States until a new race of rust attacked the crop in late plantings. Pearl millet’s high-quality grain, drought resistance, and flexibility in rotation and multiple cropping systems have caused interest in it as a grain crop outside its traditional growing areas.

B. FORAGE HYBRIDS Gahi 1, the first commercial pearl millet forage hybrid-produced by harvesting all the seed from a field planted to a mixture of four inbreds that flowered at the same

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time and gave high-yielding hybrids in all combinations-yielded 52% more than Common and 35% more than Stan: Gahi 3 replaced Gahi 1 and was the first singlecross forage hybrid produced using crns (Burton, 1983).Subsequentsingle-crosshybrids, such as Tifleaf 1, and Tifleaf 2, and a three-way hybrid, Tifleaf 3, have increased animal gains because of improved forage yields, leafiness, quality, andor disease resistance (Burton, 1983; Hanna et al., 1988; Hanna et al., 1997).

C. GERMPLASM Over 20,000 accessions of cultivated pearl millet and its wild relatives are stored in India and the United States. These accessions include landraces, improved populations and breeding lines, and wild relatives from the primary, secondary, and tertiary gene pools that are available to plant breeders. Most germplasm is in the primary gene pool. Objectives need to be clearly defined to effectively select and use the best germplasm. Principal component and cluster analyses can be used to help identify the genetic and phenotypic diversity needed in a breeding and improvement program (Wilson et al., 1991). Weedy relatives in the primary gene pool (Hanna et al., 1988; Hanna, 1989) and wild relatives in the secondary (Hanna, 1990) and tertiary gene pools (Hanna et al., 1993) are also potential sources of valuable genes (Hanna, 1987).

D. TYPES OF HYBRIDS Hybrids usually out-yield open-pollinated cultivars (Andrews, 1987; Burton, 1983). However, since all cross combinations may not always produce superior hybrids, inbreds with good general combining ability (GCA) and/or specific combining ability (SCA) need to be identified (Anand Kumar et al., 1992). Hybrids maximize yields and can be most easily made using crns in the seed parent (Anand Kumar and Andrews, 1984),especially if pollen-fertility restorer genes are present in the pollinator of hybrids grown for grain. Lack of complete male fertility restoration can result in poor grain yields and a higher incidence of smut and ergot diseases. Restorer genes are not needed (and probably undesirable) in pollinators of forage hybrids. Most pearl millet hybrids are single crosses. A single cross between two elite inbreds with high SCA is probably the best way to maximize yield. In addition to using crns in one inbred to produce single-cross F, hybrids, single-cross hybrids can also be made between two elite male fertile inbreds by taking advantage of naturally occumng protogyny in pearl millet. Protogyny can be used to make hybrids in at least two ways: (1) equal quantities of seed of two or more inbreds, equal in height and maturity, can be mixed, planted, and allowed to interpollinate; and (2) elite male fertile inbreds can be planted in adjacent rows and seed harvested from

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only one inbred.The inbred from which seed is harvested should flower 3 or 4 days earlier than the other inbred used to produce the hybrid. The use of protogyny to produce hybrids will result in some selfed and sibbed seed. The effects of selfed and sibbed seed can be overcome to some extent in the hybrid production field by increasing the seeding rate to crowd out the weaker plants. Seed from selfing and sibbing in grain hybrids may be more objectionable, especially when the hybrid grain is mechanically harvested. Seed yields can be increased in the hybrid seed production fields by producing three-way hybrids. Two inbreds are used to produce a cms F, hybrid, which is used as the seed parent and pollinated by a third inbred in hybrid production fields. The commercial forage hybrid Tifleaf 3 is produced by pollinating crns F, Tift 8593 (Hanna, 1997) with inbred Tift 383 (Hanna et al., 1997). Twice as much hybrid seed is produced on Tift 8593 as on crns inbred Tift 85D,A,, the seed parent of Tifleaf 2. Forage yields of Tifleaf 2 and Tifleaf 3 are similar. Inbred (crns or male fertile) X landrace hybrids may not maximize hybrid vigor but should increase yields and provide more genetic diversity in a hybrid population. These hybrids would maintain some of the agronomic characteristics of landraces preferred by farmers and provide more genetic diversity for diverse environmental growing conditions. Mean grain yields of crns inbred X open-pollinated variety crosses have been equal to or superior to the open-pollinated variety (Mahalskshmi et al., 1992). Landrace X landrace crosses seem to have the most potential for improving yield and reliability in harsh, variable climates. Ouendeba er al. (1993) showed that the better-parent heterosis for hybrids among five West African landraces ranged from 25 to 81% for grain yield.

XI. APOMMIS Apomixis is a reproductive mechanism that bypasses the sexual process and allows a plant to clone itself through seed. In Pennisetum, a chromosomally unreduced egg cell develops into an embryo in an embryo sac derived from a vegetative nucellar cell. This type of apomixis is called apospory. In addition to the egg cell developing into an embryo without fertilization by a sperm, pseudogamy or fertilization of the central cell is needed for endosperm and seed development. Apospory is the only type of apomixis confirmed in Pennisetum.

A. INCIDENCE OF h o r n s m Pennisetum SPECIES Apomixis is relatively common in the polyploid species of Pennisetum, especially those in the tertiary gene pool. Apomixis has been reported in polyploids

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(triploid and higher) of both the x = 8 and x = 9 chromosome groups. Only x = 7 chromosome species have been reported in the primary and secondary gene pools, and all are sexual. Likewise, tertiary gene pool species with the x = 5 and x = 7 chromosome groups and diploids with x = 8 or x = 9 have been reported to be sexual. Jauhar (1981a) listed at least nine species that have been reported to reproduce by apomixis. Additionally, F! squamulatum, F! polystachyon, and t! macrourum have been reported to be apomictic (Dujardin and Hanna, 1984). Apomixis may have played a role in building and maintaining new genome combinations in Pennisetum. Hanna and Dujardin (1991) summarized some of their research, which showed how apomixis was used in crosses among two sexual and three apomictic species in the x = 7 and x = 9 chromosome groups from the primary, secondary, and tertiary gene pools to develop and maintain more than 20 new chromosome and/or genome combinations. These were developed from sexual X apomictic crosses, parthenogenesis of a reduced gametophyte, and fertilization of an unreduced egg. Hussey er al. (1 993) and Bashaw ef al. (1 992) showed that facultative apomictic F! fiaccidum hybridized with Cenchrus setigerus, P. massaicum, F! mezianum, and P. orientale, as n + n and/or 2n + n hybridizations, produced new genome combinations.

B. GENETICSOF APOMIXIS The genetics of apomixis is difficult to study because sexual and apomictic counterparts are usually not available within the same species. Therefore, crosses need to be made between sexual and apomictic plants from different species. Genetic studies on apomixis are made more complex by facultative apomixis, lack of F, segregatingpopulations, and the limitation of having to use the apomictic plant as pollen parent in crosses. Asker and Jerling (1992) summarized the current status of the genetics of apomixis. Most researchers agree that it is probably under relatively simple genetic control. Both dominant and recessive gene actions have been reported. Crosses between sexual and apomictic Penniserum species indicate a major dominant gene and some modifiers (Hanna et al., 1993).

C. HARNESSING APOMIXIS FOR EXPLOITATION OF HETEROSIS Apomixis has tremendous potential for revolutionizingfood, feed, and fiber production around the world because it makes possible true-breeding hybrids through seeds. Apomixis not only would fix hybrid vigor but also could make possible commercial hybrids in seed-propagated crops lacking an effective male-sterility system for producing hybrids. The opportunities apomixis offers for developing

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superior hybrids and simplifying hybrid production have been previously discussed (Hanna and Bashaw, 1987; Hanna, 1995). Probably more progress has been made in transferring the apomictic mechanism from wild I! squamulatum to cultivated pearl millet than in any other grain crop. The mechanism has been transferred to the BC, generation where high levels of apomixis have been maintained (Hanna et al., 1993; and unpublished data). However, a problem encountered has been the loss of 80-90% of the seed set postanthesis. Efforts are under way to transfer apomixis from Tripsacum dactyloides (L.) L. to maize (Savidan er al., 1993; Kindiger et al., 1996) and from Elymus rectisetus (Nees in Lehm.) to wheat (Carman and Wang, 1992). The greatest impact of apomixis may be realized by cloning and inserting the gene(s) controlling apomictic reproduction into various sexual species by molecular methods. To be useful, a transferred gene must express itself and be stable in an alien genome. The gene(s) controlling apomixis needs to be mapped before it can be cloned and used in other species. Molecular markers linked to apomixis are being developed in Pennisetum (Ozias-Akins et al., 1993; Lubbers et al., 1994).

XII. GENETICS OF QUALITATIVE TRAITS Numerous qualitative traits have been reported for pearl millet. Comprehensive reviews on the genetics of qualitative traits in pearl millet have listed at least 145 mutants (Koduru and Krishna Rao, 1983; Anand Kumar and Andrews, 1993). These consisted of chlorophyll deficiencies (26%), plant pigmentation (1 8%), earhead characters (14%), pubescence and plant form (each 7%), seed characters and reproductive behavior (each 6%), foliage striping and sterility (each 4%), leaf characters and disease resistance (each 3%), and earliness (1%) (Anand Kumar and Andrews, 1993). Other mutants have been described and not included in the preceding reviews. Some of these include a naked flower mutant (Desai, 1959) and a “spreading” mutant (Goyal, 1962). Most mutants are controlled by one or two loci and dominant or recessive gene action. Recently described qualitative characters include phylloid (Wilson, 1996), narrow leaf (Appa Rao et al., 1995), brown midrib (Gupta, 1995), and xantha terminalis (Appa Rao et al., 1992) mutants controlled by the phm phm, In In, bm, bm,, and xt xt genes, respectively. Hanna and Burton (1992) showed that two plant-color mutants, red (Rp,)and purple (Rp,), are allelic; and RpI is dominant over Rp2 and normal green, whereas Rp, is dominant over normal green. Uma Devi et al. ( 1996) observed linkage of semidwarf phenotype to interchange homozygosity. Most of the mutants have potential for mapping and various genetic and physiological studies. Some appear to have direct application in commercial cultivars. Dwarf genes, especially the d , locus, has been widely used to produce high qual-

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ity shorter forage hybrids and dwarf grain hybrids that can be mechanically harvested. The early genes have been effectively used to produce early grain hybrids. Forage quality could be rapidly increased with the brown midrib bm,gene, which can reduce lignin by 20% in the plant (Cherney et al., 1988). The trichomeless or tr locus could potentially have an effect on improving drought resistance, disease and insect resistance, and palatability. Loci controlling disease resistance are being used in both commercial grain and forage hybrids. Linkage relationships have been established for only a few of these mutants (Minocha et al., 1980b; Hanna and Burton, 1992, and summarized by Koduru et al., 1983; and Anand Kumar and Andrews, 1993). Minocha et al. (1980a) used trisomics to map genes to chromosomes 1,2,4,5,and 6 . Liu er al. (1994) placed 181 RFLP markers on a molecular map. The length of the linkage map for seven linkage groups was 303 cM, with an average map distance of 2 cM between loci.

Xm. GENETICS OF QUANTITATIVE TRAITS Burton (195 1, 1959) conducted some of the first quantitative genetic studies on various plant characters and yields of pearl millet. Virk (1988) published a comprehensive review on quantitative studies conducted on pearl millet. Both additive and nonadditive genetic variances are important in pearl millet. However, the nonadditive component tends to be more important, indicating the opportunity to successfully take advantage of hybrid vigor for both grain and forage production. This, in fact, has been the case in pearl millet (see Section X). Efforts have been made to identify qualitative characters linked to quantitative characters affecting forage yield. Burton et al. (1980) showed that three recessive mutants, T13 orange node, T18 early, and T23 stubby head, increased forage yields 34, 38, and 22%, respectively, when heterozygous in an F, hybrid. In another study involving crosses between nonlethal genetic markers and exotic pearl millet lines, the Rp, gene was associated with 1861% heterotic chromosome block heterosis (HCB), and the tr was associated with 1 7 4 % HCB heterosis (Burton and Werner, 1991). A similar approach used to identify HCBs in Burkina Faso landraces identified up to 5 1% HCB heterosis associated with the R p , locus in certain crosses (Burton and Wilson, 1995).

XIV. CONCLUSION AND PERSPECTIVES With world population currently growing at the alarming rate of more than 2% per year, meeting the ever-expanding need for food will be difficult in the near fu-

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ture. The planet’s carrying capacity is not unlimited, and environmentalconstraints are ever increasing. Moreover, the balance of demographic power has shifted to the developing world, where about 78% of human beings live. Poverty is taking its toll, and more than 1 billion people today survive on less than a dollar a day. Immediate measures must be undertaken to provide quick and reasonable relief to this large segment of society. About one-sixth of the world’s population live in the semi-arid tropics encompassing parts of Asia, Africa, and Latin America-the regions typified by limited and erratic rainfall and poor soils. Pearl millet provides sustenance to a large proportion of poor people in these regions. It has the capacity to grow in some of the poorest soils in chronically drought-prone regions. The need for genetic improvement of pearl millet cannot, therefore, be overemphasized. Although its importance as a research tool in cytogenetics and breeding has been recognized, its potential as an economic crop has not been fully realized. This poses a challenge for cytogeneticists,breeders, agronomists, and biotechnologists. Pearl millet is endowed with an efficient C, photosynthetic pathway, and it responds well to fertilizers. Although it has a remarkable ability to grow on poor, depleted soils, nitrogen deficiency is a major factor limiting grain production. Therefore, genotypes with high-nitrogen-use efficiency should be produced. Fortunately, pearl millet responds extremely well to heterosis breeding. Utilization of hybrid vigor will, therefore, be the most efficient means of increasing both grain and forage production. If the vast pearl millet growing areas in Africa and Asia could be planted to improved hybrids, grain production would increase phenomenally. Apomixis provides a unique tool for reaping the fruits of heterosis over an extended period of time. If apomixis is transferred to hybrids with desired heterozygosity and superior gene combination, it can fix and help perpetuate heterosis, thereby obviating the need to produce hybrid seed year after year. Research in this area will be very rewarding. Developing a broad genetic base of hybrids is imperative to ensuring resistance to future diseases. With the availability of cytoplasmic-genic male-sterile lines in the mid- 1960s, several excellent hybrids were produced in India. Particularly promising among these was HB 3, which, because of its high yields, became widely accepted throughout India in the early 1970s. Soon afterward, however, the hybrid became vulnerable to downy mildew caused by the fungus Sclemsporu gruminicolu. The disease devastated the relatively genetically uniform hybrid crop. An effective solution to such an eventuality is to produce genetically broadbased male-sterile lines using disease-resistantgenetic resources. Recently, several male-sterile lines have been developed at ICRISAT, and thnx of these (ICMA 91113, ICMA 91114, and ICMA 91115) provide not only reasonable yields but also resistance to ergot, smut, and even downy mildew. Pearl millet is an important source of dietary protein for a sizable portion of those living in poverty in Africa and Asia. Therefore, the nutritional quality of the

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grain, particularly its protein content and amino acid balance, needs to be improved. With genetic enrichment of the quantity and quality of its proteins, pearl millet will be a more nutritional food source. Cytogenetic manipulations have no doubt been instrumental in producing superior cultivars of pearl millet. An exciting recent development is the availability of tools of modern biotechnology for crop improvement.The development and use of molecular markers-random amplified polymorphic DNA (RAPDs) and restriction fragment length polymorphism (RFLPs)-are beginning to revolutionize molecular mapping. For example, until recently, our knowledge of the inheritance of downy mildew resistance was limited. Resistance was generally believed to be monogenic dominant. However, molecular mapping has demonstrated that many genes contribute to downy mildew resistance and that these genes are scattered throughout the host genome. The use of DNA markers could help identify desired genotypes more precisely and hence assist in adopting appropriate breeding strategy for pearl millet. Pearl millet provides unlimited opportunities for both basic and applied research. With further cytogenetic manipulation and marker-assisted selection, combined with the exploitation of recent advances in biotechnological research, pearl millet may emerge as a leading economic crop that plays an ever-increasing role in the welfare of those living in poverty, particularly in the semi-arid tropics of the world.

REFERENCES Amoukou, A. I., and Marchais. L. (1993). Evidence of partial reproductive barrier between wild and cultivated pearl millets (Penniseturn gluucurn). Euphyrica 67, 19-26. Anand Kumar, K., and Andrews, D. J. (1984). Cytoplasmic male sterility in pearl millet [Penniserurn americunum (L.) Leekel-A review. Adv. Appl. Eiol. 10,113-143. Anand Kumar, K., and Andrews, D. J. (1993). Genetics of qualitative traits in pearl millet: A review. Crop Sci. 33, 1-20. Andrews, D. J. (1987). Breeding pearl millet grain hybrids. In “Hybrid Seed Production of Selected Cereal Oil and Vegetable Crops” (W. A. Feistzer and A. F. Kelly, eds.), pp. 83-109. FA0 Plant Production and Protection, Paper 82, Rome. Andrews, D. J., King, S. B., Witcomb, J. R., Singh, S. D., Rai, K. N., Thakur, R. P., Talukdar, B. S., Chavan, S. B., and Singh, P. (1985). Breeding for disease resistance and yield in pearl millet. Field Crops Res. 11,241-258. Appa Rao, S., Mengesha, M. H., and Rajagopal Reddy. C. (1992). Characteristics and inheritance of xantha terminalis in pearl millet. J. Hered. 83,6243. Appa Rao, S., Rai, K. N., Mengesha, M. H., and Rajagopal Reddy, C. (1995). Narrow leaf mutant: A new plant type in pearl millet. J . Hered. 86,299-301. Asker, S. E., and Jerling, L. (1992). “Apoxirnis in Plants.” CRC Press, Boca Raton, FL. Bashaw, E. C.. Hussey, M. A,, and Hignight, K. W. (1992). Hybridization (n + n and 2n + n) of facultative apomictic species in the Penniseturn agamic complex. fnf.J. Planr Sci. 15,466470. Bennett, M. D. (1976). DNA amount, latitude, and crop plant distribution. Environ. Exp. Bor. 16, 93- 108.

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Bor, N. L. (1960). “Grasses of Burma, Ceylon, India, and Pakistan (excluding Bambuseae).” Pergamon Press, London. Brunken, J. N., de Wet, J. M. J.. and Harlan, J. R. (1977). The morphology and domestication of pearl millet. Econ. Bot. 31, 163-174. Burton, G. W. (1942). A cytological study of some species in tribe Paniceae. Am. J. Bot. 29,355-359. Burton, G. W. (1948). The performance of various mixtures of hybrid and parent inbred pearl millet, Pennisetum glaucum (L.) R. Br. J . Am. SOC.Agron. 40,908-915. Burton, G. W. (1951). Quantitative inheritance in pearl millet (Pennisetum glaucum) indicated by genetic variance component studies. Agron. J. 51,47948 l . Burton, G. W. (1959). Breeding methods for pearl millet (Pennisetum glaucum). Agron. J. 43,409417. Burton, G. W. (1965). Pearl millet Tift 23A released. Crops Soils 17, 19. Burton, G. W. (1983). Breeding pearl millet. Plant Breeding Reviews 1, 162-182. Burton, G. W. (1989). Composition and forage yield of hybrid-inbred mixtures of pearl millet. Crop Sci. 29,252-255. Burton, G. W., and Powell, J. B. (1968). Pearl millet breeding and cytogenetics.Adv. Agron. 20,49-89. Burton, G. W., and Werner, B. K. (1991). Genetic markers to locate and transfer heterotic chromosome blocks for increased pearl millet yields. Crop Sci. 31,576579. Burton, G. W., and Wilson, J. P. (1995).Identification and transfer of heterotic chromosome blocks for forage yield in short-day exotic pearl millet landraces. Crop Sci. 35, 1184-1 187. Burton, G. W., Hanna, W. W., and Powell, J. B. (1980). Hybrid vigor in forage yields of crosses between pearl millet inbreds and their mutants. Crop Sci. 20,744-747. Busso, C. S., Liu, C. S., Hash, C. T., Witcombe, J. R., Devos, K. M., deWet, J. M. J., and Gale, M. D. (1995). Analysis of recombination rate in female and male gametogenesis in pearl millet (Pennisetum glaucum) using RFLP markers. Theoc Appl. Genet. 90,242-246. Carman, J. G.. and Wang, R. R-C. (1992). Apomixis in Triticeae. In “Proc. of Apomixis Workshop,” pp. 26-29. National Technical Service, Springfield, VA. Chase, A. (1921).The Linnaean concept of pearl millet. Am. J . Bor. 8 , 4 1 4 9 . Cherney, J. H., Axtell, J. D., Hassen, M. M., and Anliker, K. S. (1988).Forage quality characterization of a chemically induced brown-midrib mutant in pearl millet. Crop Sci. 28,783-787. Chittenden, L. M., Shertz, K. F., Lin, Y-R., Wing, R. A., and Paterson, A. H. (1994). RFLP mapping of a cross between Sorghum bicolor and S.propinquum, suitable for high-density mapping, suggests ancestral duplication of Sorghum chromosomes. Theor:Appl. Genet. 87,925-933. Clegg, M. T., Rawson, J. R. Y., and Thomas, K. (1984). Chloroplast DNA variation in pearl millet and related species. Genetics 106,449461, Dave, H. R. (1987). Pearl millet hybrids. Proc. Intl. Pearl Millet Workshop, pp. 121-126. Desdi, M. C. (1959). A naked flower mutant in pearl millet. Sci. Culture 25,207-208. De Vincente, M. C., and Tanksley, S. D. (1991). Genome-wide reduction in recombination of backcross progeny derived from male versus female gametes in an interspecific cross of tomato. Theoc Appl. Genet, 83, 173-178. Dujardin, M., and Hanna, W. W. (1984).Microsporogenesis, reproductive behavior, and fertility in five Penniserum species. Theoc Appl. Genet. 67,197-201. Gepts, P., and Clegg, M. T. (1989). Genetic diversity in pearl millet (Pennisetumglaucum [L.] R. Br.) at the DNA sequence level. J. Hered. SO, 203-208. Gerlach, W. L., and Bedbrook, J. R. (1979).Cloning and characterization of ribosomal RNAgenes from wheat and barley. Nucleic Acids Res. 7, 1869-1885. Goyal, R. D. (1962).A “spreading” mutant in Bajra (Pennisetum typhoides S Kc H). Sci. Culture 28, 437438. Gupta, S. C. (1995). Inheritance and allelic study of brown midrib trait in pearl millet. J. Hered. 86, 301-303.

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Hanna, W. W. (1987). Utilization of wild relatives of pearl millet. Proc. Inrl. Pearl Millet Workshop, pp. 33-42. Hanna, W. W. (1989).Characteristics and stability of a new cytoplasmic nuclear male-sterile source in pearl millet. Crop Sci. 29, 1457-1459. Hanna, W. W. (1990). Transfer of germplasm from the secondary to the primary gene pool in Pennisetum. Theor:Appl. Genet. 80,20&204. Hanna, W. W. (1993).Registration of pearl millet parental lines Tift 8677 and A,/B I Tift 90D,El. Crop Sci. 33, 1119. Hanna, W. W. (1995).Use of apomixis in cultivar development. Adv. Agron. 54,333-350. Hanna, W. W. (1997). Registration of Tift 8593 pearl millet genetic stock. Crop Sci. 37, 1412. Hanna, W. W., and Bashaw, E. C. (1987).Apomixis: Its identification and use in plant breeding. Crop Sci. 27, 1136-1 139. Hanna, W. W., and Burton, G. W. (1992).Genetics of red and purple plant color in pearl millet. J. Hered. 83,386-388. Hanna, W. W., and Dujardin, M. (199 I). Role of apomixis in building and maintaining genome comKimber, ed.), pp. binations. In “Proc. 2nd Intl. symp. on Chromosome Engineering in Plants” (G. 112-1 17. University of Missouri, Columbia, MO. Hanna, W. W., and Monson, W. G. (1980).Yield, quality, and breeding behavior of pearl millet X Napier grass interspecific hybrids. Agron. J. 72,358-360. Hanna, W. W., Wells, H. D., Burton, G. W., Hill, G. M., and Monson, W. G. (1988). Registration of Tifleaf 2 pearl millet. Crop Sci. 28, 1023. Hanna, W. W., Dujardin, M.. and Monson, W. G. (1989).Using diverse species to improve quality and yield in the Pennisetum genus. Proc. Intl. Grassl. Congr: 16,403404. Hanna, W. W., Dujardin, M., Ozias-Akins, P., and Arthur, L. (1992). Transfer of apomixis in Pennisefum. In “Proc. of Apomixis Workshop,” pp. 30-33. National Technical Service, Springfield, VA. Hanna, W., Dujardin. M., Ozias-Akins, P., Lubbers, E., and Arthur, L. (1993).Reproduction, cytology, and fertility of pearl millet X Penniserurn squamularum BC, plants. J. Hered. 84,213-216. Hanna, W. W., Hill, G. M., Gates, R. N., Wilson, J. P., and Burton, G. W. (1997). Registration of ‘Tifleaf 3’ pearl millet. Crop Sci. 37, 1388. Harlan, J. R. (1971).Agricultural origins: Centers and noncenters. Science 174,468474. Helentjaris, T., Slocum, M., Wright, S. Shaefer, A,. and Neinhuis, J. (1986). Construction of genetic linkage maps in maize and tomato using restriction fragment length polymorphisms. Theor: Appl. Genet. 72,76 1-769. Hitchcock, A. S., and Chase, A. (1951). “Manual of the Grasses of the United States,” 2nd ed. U.S. Dept. Agric. Misc. Publ. 200, Washington, DC. Hulbert. S. H., Richter, T. E., Axtell, J. D., and Bennetzen, J. L. (1990).Genetic mapping and characterization of sorghum and related crops by means of maize DNA probes. Proc. Nut Acad. Sci. USA 87,425 14255. Hussey, M. H., Bashaw, E. C., Hignight, K. W., Wipff, J., and Hatch, S. L. (1993).Fertilization of unreduced female gametes: A technique for genetic enhancement within the Cenchrus-Pennisetum agamic complex. Proc. Intl. Grussl. Cong. 17,404-405. International Crops Research Institute for the Semi-Arid Tropics (ICRISAT). (1996). Improving the unimprovable: Succeeding with pearl millet. ICRISATReport, May. Jauhar, P. P. (1968). Inter- and intra-genomal chromosome pairing in an interspecific hybrid and its bearing on basic chromosome number in Pennisefum. Genefica39,360-370. Jauhar, P.P. (1970a). Haploid meiosis and its bearing on phylogeny of pearl millet, Pennisefum typhoides Stapf et Hubb. Geneticcr 41,532-540. Jauhar. P. P. (1970b).Chromosome behaviour and fertility of the raw and evolved synthetic tetraploids of pearl millet, Pennisetum typhoides Stapf et Hubb. Geneticu 41,407424.

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