Copyright 0 1988 by the Genetics Society of America

Identification of the “A” Genome of Finger Millet Using Chloroplast DNA

Khidir W. Hilu

Department of Biology, Virginia Polytechnic Institute and State University, Blacksburg, Virginia 24061 Manuscript received May 26, 1987

Accepted October 2, 1987

ABSTRACT Finger millet (Eleusine corocana subsp. coracana), an important cereal in East Africa and India, is a

tetraploid species with unknown genomic components. A recent cytogenetic study confirmed the direct origin of this millet from the tetraploid E. coracana subsp. africana but questioned Eleusine indica as a genomic donor. Chloroplast (ct) DNA sequence analysis using restriction fragment pattern was used to examine the phylogenetic relationships between E. coracana subsp. coracana (domesticated finger millet), E. coracana subspecies africana (wild finger millet), and E. indica. Eleusine tristachya was included since it is the only other annual diploid species in the genus with a basic chromosome number of x = 9 like finger millet. Eight of the ten restriction endonucleases used had 16 to over 30 restriction sites per genome and were informative. E. coracana subsp. coracana and subsp. africana and E. indica were identical in all the restriction sites surveyed, while the ct genome of E. tristachya differed consistently by at least one mutational event for each restriction enzyme surveyed. This random survey of the ct genomes of these species points out E. indica as one of the genome donors (maternal genome donor) of domesticated finger millet contrary to a previous cytogenetic study. The data also substantiate E. coracana subsp. africana as the progenitor of domesticated finger millet. The disparity between the cytogenetic and the molecular approaches is discussed in light of the problems associated

I V - . with chromosome pairing and polyploidy.

F INGER millet (Eleusine coracana (L.) Gaertn. subsp. coracana) is an important cereal in East

Africa and India (HILU and DEWET 1976a). Argu- ments regarding the evolution of this cereal have long prevailed. Finger millet is a tetraploid species with 2 n = 36 chromosomes. The crop is believed to be a direct domesticate from the tetraploid E. caracana subsp. africana (KENNEDY-O’BYRNE 1957) Hilu and deWet (HILU and DEWET 1976a, b; HILU, DEWET and SEIG- LER 1978; CHENNAVEERDIAH and HIREMATH 1974). However, the species that contributed the genomes of wild and domesticated finger millet are not known. Eleusine indica (L.) Gaertn. has previously been con- sidered as the progenitor of finger millet or as one of the parental species (GREENWAY 1945; KENNEDY- O’BYRNE 1957; JAMESON 1970). Based on the lack of chromosome pairing in a synthetic hybrid between subsp. coracana and E. indica, CHENNAVEERDIAH and HIREMATH (1 974) concluded that E. indica could have not contributed any of the genomes of finger millet. Another possible genomic donor is Eleusine tristachya since it is the only other annual diploid species with a basic chromosome number of x = 9 like finger millet.

The objective of this study is to use restriction site homology of chloroplast (ct) DNA to evaluate the genetic affinity of E. indica to wild and domesticated finger millet. The ct DNA is a highly conserved mol- ecule and its potential use in phylogenetic studies has been documented in several studies (CURTIS and CLEGC 1984; PALMER 1985; HILU 1987).

Genetics 11% 163-167 (January, 1988)

MATERIALS AND METHODS

Plant material: Three seed collections were used for each of E. coracana subsp. coracana, (KH264, KH276, KH268), E. coracana subsp. africana (KH223, KH225, D&H 3695), E. indica (KH263, D&H3711, USDA217609), E. tristachya (D&H3660, D&H3685, D&H33 17). The seed materials were either collected by the author (symbolized KH), or were obtained from the seed collections of J. M. J. DEWET and J. R. HARLAN (symbolized D&H) or the United States Department of Agriculture (symbolized USDA). Seeds were sown in 28 X 53 cm flats with saw dust, and seedlings were grown in a greenhouse. Three to four-week-old seedlings were placed overnight in the dark to destarch. Leaves were then harvested and used either immediately or, in cases when leaf material were not enough to conduct a DNA extraction experiment, the leaves were washed, dried-frozen in liquid nitrogen, and stored in a deep freezer at -70” until being used for ct DNA extraction.

Chloroplast DNA extraction and restriction endonucle- ase digestion: Fresh leaves were weighed, washed thor- oughly with distilled water, and cut into about one inch pieces. The ct DNA was extracted from the fresh leaves following a modified version of the procedure of SALTZ and BECKMAN (1981). Frozen leaves were divided in about 10- 15-g portions, placed in a mortar with liquid nitrogen, ground to a fine powder, and the different portions from one species were pooled. The powdered leaf material was then soaked in the extraction buffer until the frozen lumps dissociated. Further extraction of ct DNA followed the same procedure used for the fresh material. Whenever necessary, CsCl banding of the ct DNA was performed.

Chloroplast DNA from each of the accessions represent- ing the four taxa were digested with the restriction endo- nuclease enzymes EcoRI, BamHI, AuaI, AvaII, HindIII, XbaI, BglII, PstI, KpnI, and PvuII, following the suppliers

164 K. W. Hilu

1 2 3 4 5 1 2 3 4 5 1 2 3 4 5 1 2 3 4 5

A B C D FIGURE I.-Chloroplast DNA of Elnrsine species digested with four representative restriction endonucleases. Each set of five lanes

represent A-Hind111 fragment size marker ( I ) , and chloroplast DNA restriction fragments of E. coracana subsp. coracana (2). E. coracana subsp. africana (3), E. indica (4). and E. fritfachya (5) . The DNA was digested with the restriction endonucleases BamHI (A), AuaI (B), BgfII (C), and PsfI (D). The fragments were resolved on 1 % agarose. Fragment size resulting from digestion of the chloroplast DNA of the four Eleusine taxa with these four restriction endonucleases and the other six enzymes are listed in Table 1.

instructions. About 0.5-pg aliquot samples of the DNA fragment digests were run on 1 % agarose gels, stained with ethedium bromide, and photographed under ultraviolet light. The fragment sizes were estimated by comparison to the X-Hind111 standards.

RESULTS

Among the ten restriction endonuclease enzymes used, PvuII and KpnI were not informative in the sense that they did not reveal mutational events spe- cific to any of the four taxa including E. tristachya. This is not surprising since PvuII and KpnI normally produce a few cuts and had only 10-1 1 restriction sites in these ct DNA genomes of Eleusine. The re- maining eight restriction endonucleases were inform- ative; generating over 20 restriction DNA fragments for the most part (Figure 1 and Table 1). If we assume that the restriction sites of these endonucleases are disjunct, then the ten endonucleases have randomly surveyed a total of 231 six-base restriction sites, amounting to 1386 nucleotide base pairs of the about 125 kbp (as estimated from restriction fragment sizes) of the genomes. This represents over 1% random survey of each ct genome.

The restriction endonuclease sequence data consis- tently show that E. indica is identical to both subspecies of E. coracana. The E. coracana-E. indica group dif- fered from E. tristachya in the presence of at least one

mutational event (Figure 1, Table 1). The exact na- ture of the mutational events (i.e., whether a deletion or an extra site) in the ct genomes of E. tristachya and the other species is sometimes hard to assess without DNA hybridization and mapping of the genomes. However, in certain cases reasonably accurate infer- ences can be made from comparing the endonuclease restriction patterns. For instance in the case of BamHI, E. tristachya fragment pattern lacks the 18.0 DNA fragment found in the ct DNA of E. coracana-E. indica group but possesses two extra DNA fragments, a 15.8 kbp and a 1.9 kbp fragments (Figure 1 and Table 1). The most likely explanation here is that an extra site is present in the 18.0 fragment resulting in the 1.9 and a co-migrating 15.8 fragments. The issue of the type of the mutational differences observed between E. tristachya and the other species of Eleusine will be the subject of a forthcoming publication.

DISCUSSION

The chloroplast DNA restriction sequence data show that E. coracana subsp. caracana, E. coracana subsp. africana, and E. indica share a common ct genome. The ct DNA of these species have complete sequence homology with respect to the nucleotides recognized by the ten restriction endonucleases. Frag- ment convergence in migration that arise from differ-

Finger Millet Genome Identification 165

TABLE I

Restriction fragments of E. cwacana subsp. coracana and subsp +ana, E. indica and E. ttistachyca produced by ten restriction endonucleases

BamHI XbaI Bgl1 I PstI Hind111 AuaI EcoRI AuaII PUUII KpnI

E.c: Et.’ E x . E.t. E.c. E.t. E.c. E.t. EL. E.t. E.c. E.t. E.c. E.t. E.c. E.t. E.c. E.t. E.c. E.t.

18.0 - 23.1 23.1 23.1 23.1 24.8 24.8 8.9 8.9 24.9 24.9 (14.5) (14.5) 7.8 7.8 24.8 24.8 21.5 21.5 - 15.8 19.7 19.7 9.1 9.1 23.1 23.1 8.6 8.6 11.6 11.6 7.9 7.9 5.1 5.1 21.5 21.5 19.9 19.9

15.8 15.8 18.6 18.6 7.7 - 16.4 16.4 8.3 8.3 11.0 11.0 4.8 4.8 (3.6) (3.6) (15.6) (15.6) 18.8 18.8 11.4 11.4 (9.4) (9.4) - 7.6 12.0 12.0 7.8 7.8 8.1 8.1 4.0 4.0 3.4 3.4 13.1 13.1 17.7 17.7 8.8 8.8 6.8 6.8 6.6 6.6 11.2 - 7.7 7.7 6.2 6.2 3.9 3.9 3.2 3.2 9.4 9.4 13.1 13.1 7.5 7.5 6.0 - (5.6) (5.6) - 10.7 7.3 7.3 5.9 - 3.8 3.8 (3.1) (3.1) 7.6 7.6 8.2 8.2 6.1 6.1 - 5.9 5.5 5.5 8.9 8.9 7.1 7.1 - 5.7 3.5 3.5 3.04 3.04 4.9 4.9 6.9 6.9 5.4 5.4 5.3 5.3 4.75 4.75 (7.5) (7.5) 6.7 6.7 5.6 5.6 3.0 - (2.7) (2.7) 4.3 4.3 5.9 5.9

(5.1)d (5.1) 4.4 4.4 4.5 4.5 5.4 5.4 6.2 6.2 4.8 4.8 2.9 (2.9) 2.6 - 1.1 1.1 2.2 2.2 5.0 5.0 4.0 4.0 4.0 4.0 5.3 5.3 5.9 5.9 (4.4) (4.4) 2.8 2.8 - 2.57 2.1 2.1 4.4 4.4 3.8 3.8 3.9 3.9 4.7 - 5.5 5.5 4.0 4.0 2.5 2.5 2.3 - 0.8 0.8 3.5 3.5 3.3 3.3 3.85 3.85 - 4.66 4.9 4.9 3.8 3.8 2.4 2.4 2.26 2.26 3.2 3.2 3.1 3.1 (3.6) (3.6) 3.2 3.2 3.6 3.6 3.6 - 2.3 2.3 2.1 -

(3.1) (3.1) 2.9 2.9 3.4 - 2.7 2.7 (3.3) (3.3) 3.5 - 2.1 2.1 - 2.04 2.8 2.8 1.9 1.9 - 3.3 2.8 2.8 - 3.4 2.0 2.0 2.0 2.0 2.4 2.4 1.9 - 2.5 2.5 (2.7) (2.7) 3.37 3.37 1.9 1.9 1.8 1.8 2.1 2.1 1.8 1.8 2.3 2.3 2.33 2.33 3.1 3.1 1.8 1.8 (1.75) (1.75) 2.0 2.0 1.3 1.3 2.2 2.2 - 2.3 2.7 2.7 (1.62) (1.62) 1.5 1.5 - 1.9 1.1 1.1 2.1 2.1 2.28 2.28 2.5 2.5 1.58 1.58 1.4 1.4 1.7 1.7 1.0 1.0 1.9 1.9 1.9 1.9 2.2 2.2 1.55 1.55 1.3 1.3 1.6 1.6 1.7 1.7 1.5 1.5 2.1 2.1 1.4 1.4 (1.2) (1.2) 1.4 1.4 1.5 1.5 (1.27) (1.27) 1.6 1.6 1.3 1.3 1.1 1.1 1.3 1.3 1.4 1.4 (1.3) (1.3) 1.5 1.5 1.2 1.2 1.0 1.0 0.9 0.9 1.3 1.3 1.1 1.1 1.4 1.4 1.0 1.0 0.9 0.9 0.8 0.8 1.12 1.12 1.9 1.9 1.3 1.3 0.9 0.9 0.8 0.8 0.6 0.6 1.08 1.08 0.8 0.8 1.1 1.1 0.8 0.8 0.76 0.76

0.7 0.7 0.9 0.9 0.7 0.7 0.5 - 0.8 0.8 0.6 0.6

0.7 0.7 0.6 0.6

0.55 0.55 ~~~~~ ~ ~~~

(I E.c. = restriction DNA fragments of E. coracana subsp. coracana and subsp. africana, and E. indica. * E.t. = restriction DNA fragments of E. tristachyu.

“Missing restriction DNA fragments. ( ) Two comigrating (doublets) restriction DNA fragments defined by higher staining intensity.

ent mutational events is possible, but very unlikely at this taxonomic level and their impact on interpreta- tion of the data is minimal when a large number of nucleotide restriction sites are surveyed. Therefore, this study provides support for the domestication of finger millet from E. coracana subsp. africana. How- ever, contrary to a previous cytogenetic study (CHEN- NAVEERDIAH and HIREMATH 1974), the ct DNA se- quence data suggest that E. indica is one of the ge- nomic donors of the tetraploids wild and domesticated finger millet.

CHENNAVEERDIAH and HIREMATH (1 974) per- formed crosses between domesticated finger millet (subsp. coracana) and both subspecies africana and E. indica. The hybrids obtained from crossing subspecies coracana and subspecies africana had good seed fertil- ity and their 36 chromosomes formed 18 bivalents in 87% of the microspore mother cells. Chennaveerdiah and Hiremath concluded that the chromosome pair-

ing data suggest that subspecies africana is the direct progenitor of finger millet. Based on morphological and flavonoid chemistry studies, HILU and DEWET (1976b) and HILU, DEWET and SEIGLER (1978) reached the same conclusion. These conclusions are further supporte‘d by this ct DNA study. However, in CHENNAVEERDIAH and HIREMATH cytogenetic study, the hybrids obtained from crossing subspecies cora- cana and E. indica were highly sterile and their 27 chromosomes behaved as univalents in 97% of the microspore mother cells. Based on the lack of chro- mosome pairing, CHENNAVEERDIAH and HIREMATH concluded that E. indica could have not contributed any of the genomes of finger millet.

The cytogenetic data and the conclusion concerning the phylogenetic relationship between E. coracana and E. indica, however, have little sup.port from plant morphology. E. indica and wild finger millet (subsp. africana) are very similar morphologically (HILU and

166 K. W. Hilu

DEWET 1976a), and the two taxa have been confused until KENNEDY-O’BYRNE (1 957) recognized E. afri- cana as a different species based on its tetraploid nature and a few morphological characters. Neverthe- less, the only fairly consistent qualitative morphologi- cal differences between the two species are seed or- namentation and ligule type (PHILLIPS 1972; HILU and DEWET 1976a); the latter character occasionally breaks down.

The disparity between the traditional cytogenetic and the chloroplast molecular studies raises an impor- tant point regarding interpretation of phylogenetic affinities particularly at the polyploid level. In their cytogenetic study, CHENNAVEERDIAH and HIREMATH (1 974) did not mention the number of crosses made. However, throughout their text they spoke in a sin- gular term for each cross combination, giving the impression that there was one cross per species com- bination. They also stated that caution should be exercised because their conclusion is based on the pairing behaviors in 45 microspore mother cells only. DEWEY (1 982) asserts that conclusions concerning phylogenetic affinities may be faulty if based on scanty information such as pairing in a single hybrid, and that only extensive cytogenetical studies are meaning- ful in this regard. More important, lack of chromo- some pairing does not necessarily always indicate lack of genomic similarities (DEWET and HARLAN 1972). Meiosis is a highly coordinated set of processes that are under the control of a large number of simply inherited genes (BAKER et al. 1976; JACKSON and CASEY 1980; KAUL and MURTHY 1985). Quite often mutations in those regulating genes result in various kinds of meiotic abnormalities including lack of ho- mologous chromosome pairing and complete sterility. Asynapis, desynapsis, and diploidization gene muta- tions have been reported in several plant species (BEA- DLE 1983; LI, PAO and LI 1945; RILEY and CHAPMAN 1958; GAUTHIER and MCGINNIS 1968; KIMBER 1961; RAMACE and HERNANDEZ-SORIANO 1972; JAUHAR 1975). In fact, an induced asynaptic mutation has already been reported in finger millet (SEETHARAM et al. 1973, 1975). These types of mutations can sup- press or promote chromosome pairing, making phy- logenetic interpretations based on chromosome pair- ing rather difficult at the polyploid level. In addition to gene mutations that influence chromosome pairing, structural rearrangements in the genome could also lead to misleading chromosome pairing such as the case of wheats (JOHNSON and DHALIWAL 1978).

The above constraints imposed on using cytogenetic studies in interpreting phylogenetic affinities are cir- cumvented by using the DNA sequence homology approach. The chloroplast genome has the advantage of being a slowly evolving genome with very low incidence of parallel and convergent evolution (CUR-

TIS and CLECC 1984; PALMER 1985). Its utility in tracing phylogenies particularly at the polyploid level has been well documented in amphiploid species be- longing to genera such as Triticum and Aegilops (Bow- MAN, BONNARD and DYER 1983; TSUNEWAKI and OCI- HARA 1983), Brassica (ERICKSON, STRAUS and BEVERS- DORF 1983; PALMER et al. 1983), and Coffea (BER- THOU, MATHIEU and VEDEL 1983). The endonuclease restriction studies and the maternal mode of inherit- ance of the ct DNA were so effective that in the case of the allotetraploid species Aegilops triuncialis, its two morphological races were confirmed to be the result of reciprocal crosses between the same parents (OCI- HARA and TSUNEWAKI 1982). Therefore, the ct ge- nome is a more convenient, less time consuming, and a reliable tool for inferring phylogenetic relationships in polyploid species compared with traditional cyto- genetic studies.

The chloroplast and its genome are predominantly maternally inherited in flowering plants (KIRK and TILNEY-BASSETT 1978; SEARS 1980, 1983; WHATLEY 1982). This could imply that E. indica is the maternal parent of wild and domesticated finger millet. If E. coracana is truly an allopolyploid, the other genome donor will still need to be identified.

This project was supported by the United States Agency for International Development grant DPE-5542-G-SS-7032-0 and the International Board for Plant Genetic Resources grant 85/49. The author is grateful to J. M. J. DEWET and J. R. HARLAN and the U.S. Department of Agriculture for providing some of the seed material.

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Communicating editor: M. T. CLEGG