Copyright 0 1988 by the Genetics Society of America

Identification of the “A” Genome of Finger Millet Using ChloroplastDNA

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 ( corocana subsp. coracana), an important cereal in East Africa and , 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 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 itis the only other annual diploid species inthe genus with a basic chromosome number of x = 9 like finger millet. Eight ofthe ten restriction endonucleases used had 16 toover 30 restriction sites per genome and were informative. E. coracana subsp. coracana and subsp. africana and E. indica were identical inall 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 ofthe problems associated IV -. with chromosome pairing and polyploidy.

INGER millet (Eleusinecoracana (L.) Gaertn. MATERIALS AND METHODS F subsp. coracana) is an important cereal inEast material:Three seed collectionswere used for each Africa and India(HILU and DEWET 1976a).Argu- of E. coracana subsp. coracana, (KH264, KH276, KH268), ments regarding theevolution of this cereal have long E. coracana subsp. africana (KH223, KH225, D&H 3695), prevailed. Finger millet is a tetraploid species with 2n E. indica (KH263, D&H3711, USDA217609), E. tristachya = 36 chromosomes. The crop is believed to be a direct (D&H3660, D&H3685, D&H33 17). The seed materials were either collected by the author (symbolized KH), or domesticatefrom the tetraploid E. caracana subsp. were obtained from the seed collections of J. M. J. DEWET africana (KENNEDY-O’BYRNE1957) Hilu and deWet and J. R. HARLAN(symbolized D&H) or the United States (HILU andDEWET 1976a, b; HILU, DEWETand SEIG- Department of Agriculture (symbolized USDA). Seeds were LER 1978; CHENNAVEERDIAH and HIREMATH 1974). sown in 28 X 53 cm flats with saw dust, and seedlings were However, the species that contributed the genomesof grown in a greenhouse. Three to four-week-old seedlings were placed overnight in the dark to destarch. Leaves were wild and domesticated finger millet are not known. then harvested and used either immediately or, incases Eleusine indica (L.) Gaertn. has previously been con- whenleaf material were not enough to conduct a DNA sidered as the progenitor of finger millet or as one extraction experiment, the leaves were washed,dried-frozen of the parental species (GREENWAY 1945;KENNEDY- in liquid nitrogen, and stored in a deep freezer at -70” O’BYRNE 1957;JAMESON 1970). Based on the lack of until being used for ct DNA extraction. Chloroplast DNA extraction and restriction endonucle- chromosomepairing in asynthetic hybrid between asedigestion: Freshleaves were weighed, washed thor- subsp. coracana and E. indica, CHENNAVEERDIAHand oughly with distilled water, and cut into about one inch HIREMATH(1 974) concluded thatE. indicacould have pieces. The ct DNAwas extracted from the fresh leaves not contributed any of the genomes of finger millet. following a modified version of the procedure of SALTZand Another possible genomic donor is Eleusine tristachya BECKMAN(1981). Frozen leaves were divided in about 10- since it is the only other annual diploid species with a 15-g portions, placed in amortar with liquid nitrogen, ground to a fine powder, and the different portions from basic chromosome number of x = 9 like finger millet. one species were pooled. The powdered leaf material was The objective of this study is to use restriction site then soaked in the extraction buffer until the frozen lumps homology of chloroplast(ct) DNA to evaluate the dissociated. Further extraction of ct DNA followedthe same genetic affinity of E. indica to wild and domesticated procedure used for the fresh material. Whenever necessary, finger millet. The ct DNA is a highly conserved mol- CsCl banding of the ct DNA was performed. Chloroplast DNA from each of the accessions represent- ecule and its potential use in phylogenetic studies has ing the four taxa were digested with the restriction endo- beendocumented in several studies (CURTISand nucleaseenzymes EcoRI, BamHI, AuaI, AvaII, HindIII, CLEGC1984; PALMER 1985; HILU 1987). XbaI, BglII, PstI, KpnI, and PvuII, following the suppliers

Genetics 11% 163-167 (January, 1988) 164 K. W.Hilu 12345123451234512345

A B C D FIGUREI.-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 mutational event (Figure 1, Table 1). The exact na- fragment digestswere run on 1 % agarose gels, stained with ture of the mutational events (i.e., whether a deletion ethediumbromide, and photographedunder ultraviolet light. The fragment sizes were estimated by comparison to or an extrasite) in the ct genomes of E. tristachya and the X-Hind111 standards. the other species is sometimes hard to assess without DNA hybridization and mapping of the genomes. RESULTS However, in certain cases reasonably accurate infer- ences can be made from comparing the endonuclease Among the ten restriction endonuclease enzymes restriction patterns.For instance in the case of BamHI, used, PvuII and KpnI were not informative in the E. tristachya fragmentpattern lacks the 18.0 DNA sense that they did not reveal mutational events spe- fragment foundin the ct DNA of E. coracana-E. indica cific to any of the four taxa including E. tristachya. group butpossesses two extra DNA fragments, a 15.8 This is not surprising since PvuII and KpnI normally kbp and a 1.9 kbp fragments (Figure 1 and Table 1). produce a few cuts and had only 10-1 1 restriction The most likely explanation here is that an extrasite sites in these ct DNA genomes of Eleusine. The re- is present in the 18.0 fragment resulting in the 1.9 maining eight restriction endonucleases were inform- and a co-migrating 15.8 fragments. The issue of the ative; generating over 20 restriction DNA fragments type of the mutational differences observed between for themost part (Figure 1 and Table1). If we assume E. tristachya and the other species of Eleusine will be that the restriction sites of these endonucleases are the subject of a forthcoming publication. disjunct, then the ten endonucleases have randomly surveyed a total of 231 six-base restriction sites, DISCUSSION amounting to 1386 nucleotide base pairs of the about 125 kbp (as estimated from restriction fragmentsizes) The chloroplast DNA restrictionsequence data of the genomes. Thisrepresents over 1% random show that E. coracana subsp. caracana, E. coracana survey of each ct genome. subsp. africana, and E. indica sharea common ct The restriction endonuclease sequence dataconsis- genome. The ct DNA of these species have complete tently show that E. indica is identical to both subspecies sequence homology with respect to the nucleotides of E. coracana. The E. coracana-E. indica group dif- recognized by the tenrestriction endonucleases. Frag- fered from E. tristachya in the presence of at least one ment convergencein migration that arise from differ- F inger Millet Genome Identification Genome Millet Finger 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.’ Ex. 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.c.E.t. E.t. 18.0 - 23.123.1 23.1 23.124.8 24.8 8.9 8.9 24.9 24.9 (14.5) (14.5) 7.87.8 24.824.8 21.5 21.5 - 15.8 19.723.1 9.119.79.1 8.6 8.6 11.6 11.6 7.9 5.15.1 7.9 21.5 21.519.9 19.9 15.8 15.8 18.618.6 7.7 - 16.416.4 8.3 8.3 11.0 11.0 4.8 4.8 (3.6) (3.6) (15.6) (15.6) 18.818.8 11.4 11.4 (9.4) (9.4) - 7.612.012.0 7.8 7.8 8.1 8.1 4.0 4.0 3.4 3.4 13.113.1 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 9.49.4 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 6.94.94.9 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 5.94.3 5.9 (5.1)d (5.1)4.4 4.5 5.45.4 4.5 6.2 6.2 4.8 4.8 2.9 (2.9) 2.6 - 2.21.1 1.1 2.2 5.04.0 4.05.0 4.0 4.0 5.35.9 5.3 2.85.9 2.8 (4.4) (4.4) -2.1 2.12.57 3.8 3.8 3.9 3.9 4.74.4 3.9 3.94.4 3.8 3.8 - 2.35.5 2.5 2.5 5.5 4.0 4.0 - 0.8 0.8 3.5 3.5 3.3 3.3 3.85 3.852.262.26 2.4 2.4 3.8- 3.8 4.9 4.9 4.66 3.2 3.2(3.6)(3.6) 3.1 3.1 3.63.23.6 3.6 3.2 -2.1 2.3 2.3 - (3.1) (3.1)3.42.9 2.9 - 2.7 2.7 (3.3) 3.5(3.3) - 2.12.1 - 2.04 2.8 2.8 1.9 1.9 1.9 2.8 2.8 - 3.3 2.8 2.82.0 2.0 2.0 - 2.0 3.4 2.4 2.4 1.9 -2.5 2.5 (2.7) (2.7) 3.373.37 1.91.8 1.8 1.9 2.1 1.8 1.82.1 1.8 2.1 2.3 2.3 2.331.8 1.8 2.33 3.1 3.1 (1.75) (1.75) 2.0 2.0 1.3 1.32.2 2.2 - 2.3 2.7 2.7 (1.62)1.5 1.5(1.62) - 2.11.9 1.1 1.1 2.1 1.58 2.5 2.52.28 2.28 1.581.4 1.4 1.0 1.0 1.9 1.9 1.9 1.9 1.9 1.71.9 1.91.7 1.0 1.0 2.2 2.2 1.55 1.551.3 1.3 1.6 1.6 1.7 1.7 (1.2) (1.2) 1.4 1.41.5 2.1 1.5 2.1 1.41.5 1.4 1.51.1 1.1 1.3 (1.27)1.3 (1.27)1.6 1.6 1.3 1.3 1.3 1.5 (1.3) 1.4(1.3) 1.4 1.0 1.21.5 1.2 1.0 0.9 0.9 1.31.0 1.3 1.0 1.4 1.4 1.1 1.1 0.9 0.9 0.8 0.8 1.12 1.12 1.12 0.8 0.8 0.9 1.3 1.31.9 1.9 0.80.9 0.8 0.61.08 0.6 1.08 1.1 0.8 0.8 0.76 1.10.8 0.8 0.76 0.7 0.90.7 0.7 0.9 0.7 0.7 0.8 0.8 0.6 0.6 0.60.5 0.8- 0.8 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 ing data suggest that subspecies africana is the direct this taxonomic level and their impact on interpreta- progenitor of finger millet. Based on morphological tion of the data is minimal when a large number of and flavonoidchemistry studies, HILU and DEWET nucleotide restriction sites are surveyed. Therefore, (1976b) and HILU, DEWET and SEIGLER(1978) this study provides support for the domestication of reached the same conclusion. These conclusions are finger millet from E. coracana subsp. africana. How- further supporte‘d by this ct DNA study. However, in ever, contrary to a previous cytogenetic study (CHEN- CHENNAVEERDIAHand HIREMATHcytogenetic study, NAVEERDIAH and HIREMATH1974), the ct DNA se- the hybrids obtained from crossing subspecies cora- quence data suggest that E. indica is one of the ge- cana and E. indica were highly sterile and their 27 nomic donors of the tetraploids wild and domesticated chromosomes behaved as univalents in 97% of the finger millet. microspore mother cells. Based on the lack of chro- CHENNAVEERDIAHand HIREMATH(1 974) per- mosome pairing, CHENNAVEERDIAHand HIREMATH formed crossesbetween domesticated finger millet concluded that E. indica could have not contributed (subsp. coracana) and both subspecies africana and E. any of the genomes of finger millet. indica. The hybrids obtained from crossing subspecies The cytogenetic data and theconclusion concerning coracana and subspecies africana had good seed fertil- the phylogenetic relationship between E. coracana and ity and their 36 chromosomes formed 18 bivalents in E. indica, however,have little sup.port from plant 87% of the microspore mother cells. Chennaveerdiah morphology. E. indica and wild finger millet (subsp. and Hiremath concluded that the chromosome pair- africana) are very similar morphologically (HILUand 166 K. W. Hilu

DEWET1976a), and thetwo taxa have been confused TIS and CLECC 1984; PALMER1985). Its utility in until KENNEDY-O’BYRNE (1 957) recognized E. afri- tracing phylogenies particularly at the polyploid level cana as adifferent species based on its tetraploid has been well documented in amphiploid species be- nature anda few morphological characters. Neverthe- longing to genera such as Triticum and Aegilops (Bow- less, the only fairly consistent qualitative morphologi- MAN, BONNARDand DYER1983; TSUNEWAKIand OCI- cal differences between the two species are seed or- HARA 1983), Brassica (ERICKSON,STRAUS and BEVERS- namentation and ligule type (PHILLIPS1972; HILU DORF 1983; PALMERet al. 1983),and Coffea (BER- and DEWET 1976a); the latter character occasionally THOU, MATHIEUand VEDEL1983). The endonuclease breaks down. restriction studies and the maternal mode of inherit- The disparity between the traditional cytogenetic ance of the ct DNA were so effective that in the case and thechloroplast molecular studies raises an impor- of the allotetraploid species Aegilops triuncialis, its two tant point regarding interpretation of phylogenetic morphological races were confirmed to be the result affinities particularly at the polyploid level. In their of reciprocal crosses between the same parents (OCI- cytogenetic study, CHENNAVEERDIAHand HIREMATH HARA and TSUNEWAKI1982). Therefore, the ct ge- (1 974) did not mention the numberof crosses made. nome is a more convenient, less time consuming, and However, throughout their text they spoke in a sin- a reliable tool for inferring phylogenetic relationships gularterm for each cross combination, giving the in polyploid species compared with traditional cyto- impression that there was one cross per species com- genetic studies. bination. They also stated that cautionshould be The chloroplast and its genome are predominantly exercised because their conclusion is based onthe maternally inherited in flowering (KIRK and pairing behaviors in 45 microspore mother cells only. TILNEY-BASSETT1978; SEARS1980, 1983; WHATLEY DEWEY(1 982) asserts that conclusions concerning 1982). This could imply that E. indica is the maternal phylogenetic affinities may be faulty if based on scanty parent of wild and domesticated finger millet. If E. information such as pairing in a single hybrid, and coracana is truly an allopolyploid, the other genome that only extensive cytogenetical studies are meaning- donor will still need to be identified. ful in this regard. More important, lack of chromo- This project was supported by the United States Agency for some pairing does not necessarily always indicate lack International Development grant DPE-5542-G-SS-7032-0and the of genomic similarities (DEWET and HARLAN1972). International Board for Plant Genetic Resources grant 85/49. The Meiosis is a highly coordinated set of processes that author is grateful to J. M. J. DEWETand J. R. HARLAN andthe U.S. are under the control of a large number of simply Department of Agriculture for providing some of the seed material. inheritedgenes (BAKERet al. 1976; JACKSON and CASEY1980; KAUL and MURTHY 1985). Quite often LITERATURECITED mutations in those regulating genes result in various BAKER,B. S., A. T. C. CARPENTER,M. S. E~POSITO,R. E. 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