Genome Analysis of Finger Millet E. Coracana by Interspecific Hybridization Among Diploid Wild Species of Eleusine (Poaceae)

Genome Analysis of Finger Millet E. coracana by Interspecific Hybridization among Diploid Wild Species of Eleusine (Poaceae)

Rachayya M. Devarumath1, Subhash C. Hiremath2, Satyawada Rama Rao3*, Arun Kumar3 and Sangeeta Bewal3

1 Genetic Engineering and Molecular Biology Laboratory, Vasantadada Sugar Institute, Manjari (BK) Pune –12, India 2 Department of Botany, Karnataka University, Dharwad 580 003, India 3 Cytogenetics and Molecular Biology Laboratory, Department of Botany, J. N. V. University, Jodhpur 342 005, India

Received July 25, 2005; accepted October 24, 2005

Summary Four species of Eleusine viz. E. intermedia, E. floccifolia, E. indica and E. tristachya are wild diploid taxa (2n2x18) and are presumed to have contributed to the evolution of finger millet, E. coracana, the amphidiploid with AABB genome (2n2x36) and an important minor millet grown in Africa and South Asia. E. indica is known to have contributed the A genome to E. coracana, while the information regarding the other genome donor species is not forthcoming. The present investigations were undertaken to clearly understand the genome homology of 3 species viz. E. intermedia, E. floccifolia and E. tristachya vis-à-vis the A genome of E. indica and to analyze the interspecific relationship among these diploid species and their possible contribution to the evolution of E. coracana. Interspecific F1 hybrids of E. intermedia E. indica, E. intermedia E. floccifolia and E. tristachyaE. intermedia were produced and studied for morphological and cytological details. The experimental data revealed a high degree of homology at the interspecific level among all 4 species resulting in high frequency of bivalents and a very low frequency of univalents and multivalents. Inspite of good chromosome pairing and bivalent formation, a significant reduction in

pollen stainability (8–32%) has been recorded in the F1 hybrids. The data obtained on chromosome associations at diakinesis/ metaphase I of meiosis suggest that these diploid species form a close genetic assemblage within the genus Eleusine. Various factors affecting the pollen stainability and seed fertility are discussed in the light of available information on the subject.

Key words Cytogenetics, Eleusine species, Genome analysis, Interspeciﬁc hybrids, Morphology.

The genus Eleusine Gaertn. (tribe Eragrosteae, family Poaceae) comprises 9–12 species of predominantly African origin, including 3 polyploid taxa, viz. E. coracana, E. africana and E. kigeziensis. The species are grown in the wild except E. coracana, which is commonly known as ﬁnger millet and has been domesticated for cultivation in several parts of Africa and South Asia (Phillips 1972, Hiremath and Chennaveeraiah 1982). In India it is cultivated as a minor millet crop in dry areas of the Karnataka, Andhra Pradesh and Tamilnadu provinces in the South and Uttar Pradesh and Himachal Pradesh in the North. Finger millet is one of the important minor millets grown in the world providing highly nutritious grains which are high in protein, fats and minerals as compared to other popular crops like rice and sorghum (Barbeau and Hilu 1993). E. coracana is an allotetraploid species and is based on the basic chromosome number of 9 (2n4x36) with a probable genome constitution of AABB. The diploid species E. indica (2n2x18) is known to be the donor of the A genome to this species (Chennaveeraiah and Hiremath 1974a, Hiremath and Sali-

* Corresponding author, e-mail: [email protected] 428 R. M. Devarumath et al. Cytologia 70(4) math 1992) while the identity of diploid species contributing the B genome is still elusive (Hire- math and Salimath 1992). On the other hand a few more diploid species namely E. floccifolia, E. intermedia, E. tristachiya, all based on x9 (2n2x18) are reported to have contributed in the evolution of E. coracana (Bisht and Mukai 2001, 2002). While E. intermedia, E. tristachiya are presumed to have contributed A genome, Bisht and Mukai (2001) have recently carried out molecular cytogenetic studies in E. floccifolia and concluded that this is the most possible B genome donor of E. coracana. However, these observations are contrary to earlier reports based on crossability studies (Chennaveeraiah and Hiremath 1974a, Salimath et al. 1995) which indicate more affinity of E. floccifolia with species like E. tristachya possessing the A genome. Similarly, the exact contribution of E. intermedia, a mainly African diploid species with the highest 2C DNA content (Hiremath and Salimath 1991) among all the diploid taxa, in the evolution is unknown, although it is reported to be a bridge species between annual (E. indica) and perennial (E. floccifolia) species of Eleusine (Bisht and Mukai 2001). Thus, there is a great deal of ambiguity regarding interspecific relationship among diploid taxa of Eleusine, which still remains as a major problem. To resolve such ambiguity an interspecific hybridization program involving all the 4 diploid species mentioned above, has been undertaken with the objective of determining the genomic status of these taxa. The present investi- gation constitutes morphological and cytological analysis of F1 hybrids from 3 different crosses involving E. indica, E. intermedia, E. flociifolia and E. tristachya. These crosses were successful in only 3 combinations E. intermediaE. indica, E. intermediafloccifolia and E. tristachya E. intermedia. The genomic homology and differentiation pattern were assessed based upon the crossability, degree of meiotic chromosome pairing and fertility factors in the species and their F1 hybrids.

Materials and methods Seeds of various species of Eleusine were obtained either from the International Live Stock Centre of Africa, Ethiopia, or from the United States Department of Agriculture, USA. Crossing experiments were conducted in the open field with potted plants, and reciprocal crosses were invari- ably made in all the combinations. Hybridizations were carried out according to the technique of Richardson (1958) with suitable modifications. Morphological characters were used to identify the hybrid seedlings; confirmation was carried out later by cytological analysis. Morphological data for various attributes were collected both for parents and hybrids from mature plants. The morphological comparison of quantitative characters of parents and their F1 hybrid is based on Anderson’s met- roglyph analysis of Eleusine as proposed by Chennaveeraiah and Hiremath (1974b). For meiotic observations, spikes of parents and F1 hybrids were fixed in Carnoy’s fluid (6: 3 : 1 ethanol-chloro- form-acetic acid) and the pollen mother cells (PMCs) were stained with 2% acetocarmine. For per cent pollen stainability, the pollen grains were stained in 1 : 1 glycerine:actocarmine mixture and 3 slides were scored on the average for stainable pollens.

Results

Crossability and morphology of parents and F1 hybrids Interspecific hybrids were successfully produced, when E. intermedia was used as a female parent in 2 crosses involving E. indica and E. floccifolia, where it was involved as male parent in the cross with E. tristachya. The reciprocal crosses in all the 3 combinations did not yield F1 hybrids. In E. intermediaE. indica, from 125 crosses performed 15 seeds were obtained, out of which 2 seeds germinated and the seedlings grown till maturity. In case of E. intermediaE. floccifolia out of 180 crosses made, only 18 seeds were obtained, out of which 8 germinated and grew to maturity. 2005 Genome Analysis of Finger Millet E. coracana 429

In a third combination, E. intermedia was involved as male parent with E. tristachya. From135 hand pollinations performed, 16 seeds were collected, out of which only 2 germinated and survived till maturity. The estimation of the crossability percentage ranged between 10 to 12% in the above 3 crosses. E. intermedia, E. floccifolia, E. indica and E. tristachya are distinct species which can be iden- tified through their unique morphological characteristics. The F1 hybrids obtained in 3 crosses showed characteristics which were intermediate in nature or similar to one of the parents (Figs. 1–7, Table 1). In the E. intermedia E. indica cross, the F1 hybrid resembled the female parent in the majority of the characters studied except for the color of style and anthers. However, the number of spikes/inflorescence showed intermediate value between the 2 parents. An interesting aspect of the cross E. intermedia E. floccifolia is that the F1 hybrid had more resemblance with the male parent in many of the characters except for the number of spikes/inflorescence, in which it showed an intermediary value between the parents. The spikelet length in F1 hybrid showed significant increase as compared to the better parent. The only trait, in which the hybrid was similar to the female parent, was the width of the spike. In the E. tristachya E. intermedia cross, the F1 hybrid resembled the female parent only in spike width, while it showed a tendency towards the male parent in many of the other traits. The F1 hybrid was conspicuous in having an intermediate value for the number of spikes/inflorescence and an enhanced spikelet length of 7.0 mm, even when compared with the better parent.

Cytology of parents and their F1 hybrids Meiotic data of parents and their F1 hybrids have been summarized in Table 2 and illustrated in Figs. 8–13. All species involved in the interspecific hybridization are diploids with 2n18. In E. intermedia and E. floccifolia although the majority of the PMCs showed normal bivalent formation, a few cells had a mixture of univalents and bivalents. But the number of univalents never exceeded 2 per cell. In E. indica and E. tristachya all the cells analyzed at diakinesis/metaphase-I revealed normal bivalent formation (9II). The average chromosome association for E. intermedia, E. floccifolia, E. indica and E. tristachya was 8.82 II0.20 I, 8.80 II0.20 I, 9 II and 9 II, respectively. The disjunction of bivalents/chromosomes at anaphase I and II by and large had been normal. PMCs analysed in the E. intermediaE. indica cross have shown a mixture of univalents, bivalents, trivalents and quadrivalents. PMCs in this hybrid on the average had 0.01 IV0.03 III8.77 II0.28I. The number of univalents and bivalents ranged from 4–7 and 2–9, respectively, whereas the number of trivalents and quadrivalents never exceeded 1 per PMC. Lagging univalents at anaphase I and occurrence of micronuclei at telophase II were characteristic of this hybrid. In E. intermediaE. floccifolia the PMCs have shown a mixture of univalents, bivalents and quadrivalents, but no incidence of trivalents. The average meiotic configuration was 0.11 IV 8.70 II0.29 I. The number of univalents , bivalents and quadrivalents ranged from 0–2, 7–9 and 0–1, respectively. Occasional laggards were recorded at anaphase I. The F1 hybrid of E. tristachya E. intermedia was characteristic in exhibiting an almost diploid-like behavior in chromosome associations at metaphase I except for a single stray quadriva- lent observed in one cell. The average number of chromosome association was 0.01IV 8.54 II0.44 I. Anaphase I and II were more or less normal with regular disjunction of bivalents/chromosomes.

Pollen stainability The diploid parental species have shown pollen stainability of 85% in E. intermedia, 90% in E.

ﬂoccifolia and 99% in both E. indica and E. tristachya. However, the F1 hybrids produced involving these species showed drastic reduction in pollen stainability which ranged between a meager 8% to 430 R. M. Devarumath et al. Cytologia 70(4)

Figs. 1–13. Morphology and cytology (meiosis) of diploid species of Eleusine and their F1 hybrids. 1–7), 1) Morhphology of spikes. E. intermedia. 2) E. floccifolia. 3) E. indica. 4) E. tristachya. 5) E. intermediaE. indica. 6) E. intermediaE. floccifolia. 7) E. tristachyaE. intermedia. 8–13) Cytology of F1 hybrids. 8) E. intermedia E. indica. Diakinesis with 9 II. 9) Diakinesis with 1 III5 II5 I. 10) E. intermediaE. floccifolia. Diakinesis with 9 II. 11) Diakinesis with 1 IV7 II. 12) E. tristachyaE. intermedia. Diakinesis with 9 II. 13) Diakinesis with 8 II2 I. 2005 Genome Analysis of Finger Millet E. coracana 431 pollen E. intermedia E. intermedia 1 F ybrids h 1 ybrids h 1 E. floccifolia E. tristachya parents and their F E. folccifolia E. tristachya 1 F Chromosome Associations Eleusine E. indica E. intermedia Morphological characters of diplod E. indica E. intermedia 1 Univalents Bivalents Trivalents Quadvalents %of F Chromosome associations at diakinesis/metaphase I in PMCs of parents and their F Table 1. No. Mean Range No. Mean Range No. Mean Range No. Mean Range stability E. intermedia Table 2. 69 19 0.27 4–7 604 8.75 2–9 1 0.01 0–1 1 0.01 0–1 8 90 – – – 810 9.0 – – – – – – – 89 80 35 0.43 2–6 683 8.53 6–9 – – – 1 0.01 0–1 15 104 31 0.29 0–2 904 8.69 7–9 – – – 10 0.09 0–1 32 150105 30145 21 0.20 – 0.20 2–4 0–2 – 1323 924 8.82 – 8.80 8–9 1305 8–9 – 9.0 – – – – – – – – – – – – – – – – 85 – 90 – 99 analysed E. intermedia No. of cells E. indica E. floccifolia E. intermedia Characters Species/hybrids HabitHeight TallLeaf surfaceLeaf marginNo. of spikes/inflorescence widthSpike TallSpiklet length ManyColor of styleColor of anthers Perennial HairyGlumes color Short No hair More Perennial Narrow Medium Hairy Color less No hair Yellow Tall Annual Few Medium Medium Green Purple Scabrous No hair Purple Perennial Narrow Tall Many Short Non-waxy Purple Green No hair Perennial Purple fairs wooly With Narrow Waxy Medium hairs wooly With Few Color less Green Perennial Tall No hair Yellow Medium Color less Long Annual Waxy Green No hair More Short Yellow Medium Color less Perennial Glabrous Short No hair Color less Grey Few Broad Yellow Tall Perennial Hairy Colorless Short Yellow Broad More Grey Color less Tall Hairy Long Yellow Narrow Many Green Yellow Medium Green Green E. intermedia E. intermedia E. floccifolia E. indica E. tristachya E. intermedia E. tristachya 432 R. M. Devarumath et al. Cytologia 70(4)

32% in these crosses (Table 2). The F1 hybrids in all the cross combinations were found to be com- pletely seed sterile.

Discussion Origin, evolution and domestication of finger millet has been subject of considerable interest, but for long remained an enigma for both cytologists and plant breeders. The allotetraploid nature of E. coracana (AABB, 2n4x36) and the probable donor species of A and B genomes have been debated extensively by various workers (Mehra 1963, Chennaveeraiah and Hiremath 1974a, Hilu and de Wet 1976, Hiremath and Salimath 1992, Bisht and Mukai 2001, 2002). The pantropical weedy species E. indica was initially considered as direct progenitor, but it could not be accepted due to the difference in ploidy level of both the species. Nevertheless, E. indica is now confirmed as one of the A genome donor species based on cytogenetical analysis (Hiremath and Salimath 1992) and chloroplast DNA restriction analysis of diploid and polyploid species (Hilu 1988). However, the identification of the B genome donor species could not be accomplished due to lack of information on genome candidature in the diplod taxa of allied genera. The other allotetraploid species, E. africana has been thoroughly investigated and confirmed to be the actual direct progenotor species of finger millet (Chennaveeraiah and Hiremath 1974a, Bisht and Mukai 2001, 2002). Indeed it is now believed that finger millet had originated as result of selection and further domestication of a large grain mutant from E. africana populations (Chennaveeraiah and Hiremath 1974a). There are certain other diploid species like E. intermedia, E. floccifolia and E. tristachya, which exhibit close interrelationships with each other and presumed to be the possible donor species of A and B genome to E. coracana. The present investigations on interspecific hybridization involving these species has revealed the existence of homology among the genomes of these species. Earlier Salimath et al. (1995) have reported high degree of homology between E. indica, E. floccifolia and E. tristachya. Chennaveeraiah and Hiremath (1973) found homology between E. tristachya and E. floccifolia. They were able to produce successful F1 hybrids in all the combinations. However, E. intermedia, a mainly African diploid species with highest 2C DNA content among all the diploid taxa, has not been investigated for its genomic relationship with other species. Thus the present investigations are an attempt to record the genome homology of this important species vis-à-vis other diploid species.

From the analysis of morphological data it is amply clear that the F1 hybrid, instead of exhibiting intermediary characters as expected, have inherited most of the characters from E. intermedia in the E. intermediaE. indica and E. tristachyaE. intermedia crosses irrespective of whether it is involved as male or female parent. This indicates the possibility of E. intermedia possessing most of the dominant genes/ alleles for various genetic marker characteristics and it can successfully in- herit these to F1 hybrids. Further the number of spikes/inﬂorescence is the only character which ex- hibited consistently intermediate nature in all 3 crosses which indicates that this important yield related trait is polygenic in nature. Our cytological analysis in four diploid Eleusine species revealed the occurrence of univalents in 2 species namely E. intermedia and E. ﬂoccifolia. This may be the a direct consequence of preco- cious separation of rod bivalents as reported in a number of plants (Sybenga1972, Kumar and Rao 2002, Kumar et al. 2002). The other 2 species E. indica and E. tristachya, showing normal bivalent formation in all analyzed cells can be correlated to the existence of complete homology between pairing partners at zygotene and are devoid of structural heterozygosity. Contrary to expectations, a very high frequency of bivalents at diakinesis/metaphase I in the

PMCs of all the 3 F1 hybrids is quite intriguing and suggest the possibility of identical genetic make up leading to normal pairing behaviour of chromosomes at zygotene. Although multivalents were recorded in the form of trivalents and quadrivalents in few cells, but their mean values remained ex- 2005 Genome Analysis of Finger Millet E. coracana 433 tremely low and their number never exceeded one per cell wherever encountered. Therefore, it is quite obvious to conclude that E. intermedia, E. floccifolia and E. tristachya and E. indica are close- ly related to each other in terms of genome homology. E. indica is confirmed as donor species of E. coracana contributing A genome. Therefore we suggest that all the 3 species should be considered as possible A genome donor for E. coracana. Bisht and Mukai (2001) suggested E. floccifolia as the most probable B genome donor based upon their observations and results obtained through in situ hybridization techniques. However, if this was the case in cross involving E. intermediaE. floccifolia, the present investigations should have yielded a highly reduced bivalent frequency and more univalents per PMC. But our observations are quite different and on the average mostly bivalents were encountered in the PMCs (8.70II) while the univalent frequency was very low (0.29I). It is therefore suggested that the genomic status of E. floccifolia should be thoroughly reinvestigated in the light of the present findings. Inspite of good chromosome pairing and bivalent formation, a significant reduction in pollen stainability (8–32%) of the F1 hybrids has been recorded. The reduced fertility in the F1 hybrids of all the above crosses may be correlated to cryptic structural hybridity affected by reciprocal translo- cations, a phenomenon which has been reported in many species, e.g. E. tristachya, E. floccifolia, E. indica (Chennaveeraiah and Hiremath 1973, Salimath et al. 1995) and in Avena damascena and A. canariensis as reported as by Lagget (1984). Thus the data obtained in our present investigations on morphology and chromosome associations at diakinesis/metaphase I of meiosis suggest that these diploid species form a close genetic assemblage with in the genus Eleusine. Cryptic structural hybridity might have adversely affected the fertility of F1 hybrids as already reported earlier by various workers.

Acknowledgement We (RMD & SRR) are thankful to Prof. S. N. Raina of CMC laboratory, Department of Botany, Delhi University, Delhi for his constant encouragement.

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