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Crop Science 40:1543-1551 (2000)
© 2000 Crop Science Society of America

CROP BREEDING, GENETICS & CYTOLOGY

Primary Trisomics in Soybean

Origin, Identification, Breeding Behavior, and Use in Linkage Mapping

S.J. Xu, R.J. Singh, K.P. Kollipara and T. Hymowitz

Dep. of Crop Sciences, Univ. of Illinois, 1102 South Goodwin Avenue, Urbana, IL 61801 USA

soyui{at}uiuc.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 Materials and methods
 Results
 Discussion
 REFERENCES
 
A primary trisomic is an excellent cytogenetic tool for locating Mendelian genes on a particular chromosome and for associating a conventional genetic linkage group with a specific chromosome. The objectives of this study were to produce and identify 20 primary trisomics of soybean [Glycine max (L.) Merr.] and use them for associating linkage groups with specific chromosomes. Primary trisomics isolated from the progenies of asynaptic and desynaptic mutants, male sterile lines, neutron-irradiated plants, and tissue culture-induced sterile mutants were backcrossed (BC4) with soybean cv. Clark 63. They were identified at pachytene on the basis of the diagnostic landmarks such as association of the extra chromosome with its normal homologues in a trivalent configuration, distribution of euchromatin and heterochromatin, total chromosome length, arm ratios, and by examining chromosome pairing at metaphase I in F1 plants with 2n = 42 chromosomes isolated from crosses among primary trisomics. The F1 plants from similar primary trisomics exclusively showed 21 II and those from dissimilar primary trisomics exhibited microsporocytes with 20 II + 2 I, 1 III + 19 II + 1 I, and 2 III + 18 II configurations. Thus, 20 primary trisomics were tentatively identified and were designated as triplo 1 through triplo 20. Female transmission of the extra chromosome in 20 primary trisomics ranged from 27.3% (triplo 20) to 58.5% (triplo 9). On the basis of the modification ratio (17:1) of a gene located on the extra chromosome in primary trisomic analysis, three marker genes Eul (seed urease), Lx1 (lipoxygenase-1), and P2 (puberulent) were located on chromosomes 5, 13, and 20, respectively. The ultimate aim of this project is to develop by means of primary trisomics a universal cytogenetic map of soybean by associating existing classical and several molecular maps with the specific chromosomes.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
Materials and methods
 Results
 Discussion
 REFERENCES
 
PRIMARY TRISOMIC ORGANISMS contain a normal chromosome complement plus an extra complete standard chromosome. Cytogenetic maps have been developed in several diploid crops by means of primary trisomics. A large number of classical and molecular genes were associated with specific chromosomes in these crops by either modification of genetic ratios or dosage effects from extra chromosomes in primary trisomics (Singh, 1993).

Because of its small chromosomes, soybean cytogenetics has lagged far behind that of other major crops. Soybean contains 40 almost identical somatic chromosomes. Special cytological skills are needed to obtain well-spread mitotic and meiotic preparations. Approximately 250 morphological and isozyme markers are available and to date, 20 classical genetic linkage groups consisting of 68 genetic markers have been published (Palmer and Shoemaker, 1998). The soybean molecular maps now include more than 1000 RFLP (restriction fragment length polymorphism), RAPD (random amplified polymorphic DNA), SSR (simple sequence repeat) and AFLP (amplified fragment length polymorphism) markers (Cregan et al., 1999). However, except for classical linkage group 8 (Sadanaga and Grindeland, 1984), none of the other genetic linkage groups nor any molecular maps have been associated with specific chromosomes. Thus, soybean lacks a unified cytogenetic and molecular map.

Palmer (1976) made an early attempt to develop primary trisomics of soybean. A number of aneuploid lines were isolated from different meiotic mutants and male sterile lines (Palmer, 1974; Palmer and Heer, 1976; Palmer and Skorupska, 1994; Sadanaga and Grindeland, 1979). However, only five primary trisomics were characterized and arbitrarily designated as Tri A, Tri B, Tri C, Tri D, and Tri S (Gwyn and Palmer, 1989; Gwyn et al., 1985; Palmer, 1976; Sadanaga and Grindeland, 1984; Skorupska et al., 1989). These primary trisomics were morphologically indistinguishable. The five known primary trisomics and additional lines with 2n = 41 chromosomes were not identified karyotypically.

Pachytene analysis is an extremely useful tool for identifing individual chromosomes in plant species having small chromosomes. By pachytene analysis, primary trisomics were successfully characterized in maize (Zea mays L.), rice (Oryza sativa L.), and tomato (Lycopersicon esculentum Mill.) (Singh, 1993). In soybean, Singh and Hymowitz (1988) identified individual soybean chromosomes by pachytene analysis and constructed the first cytological map based on chromosome length and euchromatin and heterochromatin distribution. Thirteen of the possible 20 primary trisomics were identified by pachytene analysis (Singh and Hymowitz, 1991; Ahmad et al., 1992; Ahmad and Hymowitz, 1994). To identify all 20 possible primary trisomics, we examined numerous unidentified aneuploid lines with 2n = 41 chromosomes at pachytene. Thirteen primary trisomics published previously were also reexamined. The objectives of this paper are to report the origin, identification, and breeding behavior of 20 primary trisomics of soybean, and present three examples of their use in locating genes on chromosomes are presented.


   Materials and methods
TOP
 ABSTRACT
 INTRODUCTION
 Materials and methods
Results
 Discussion
 REFERENCES
 
The sources of the primary trisomics were the progenies of the 37 aneuploid lines with 2n = 41 (33 lines), 2n = 42 , and 2n = 43 chromosomes (Table 1) . These lines were initially isolated from several meiotic mutants (asynaptic and desynaptic) and male sterile lines. The pedigrees of 33 lines are known and four lines are of unknown origin. Most aneuploid lines were provided by Dr. R.G. Palmer, USDA, ARS, Iowa State University, Ames, IA. Lines T190-39, T190-47-1, and T190-47-3 were generated in the Soybean Cytogenetics Laboratory, University of Illinois.


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  		Table 1 List of aneuploid lines as source of primary trisomics in soybean

 
The cytological methods for identifying primary trisomics were essentially the same as reported previously (Singh and Hymowitz, 1991; Ahmad et al., 1992; Ahmad and Hymowitz, 1994). Two to four plants with 2n = 41 and one with 2n = 40 from each of the aneuploid lines were identified and grown to maturity in the greenhouse. General morphology of the trisomic plants and their disomic sib plant was compared during vegetative and reproductive stages. Primary trisomics were identified on the basis of trivalent configurations at pachytene. They were designated in descending order as triplo 1 (carrying the longest chromosome) to triplo 20 (carrying the shortest chromosome). The triplo number assignment, and parental sources in the designated 20 primary trisomics are listed in Table 2 .


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  		Table 2 The 20 primary trisomics (2n = 41) identified by pachytene analysis in soybean

 
Some primary trisomics were crossed with one another by the hybridization procedure described by Singh et al. (1998). Young flower buds were emasculated and pollinated immediately with pollen from male plants. All the F1 plants with 2n = 42 were identified cytologically and were grown in the greenhouse. Chromosome pairing was examined at metaphase I. Photomicrographs were taken on high contrast film Kodak Technical Pan 2415 (Eastman Kodak Company, Rochester, NY) by means of same procedure described by Xu et al. (2000b).

Photomicrographs had final magnification 1000x and were printed on Kodak Polycontrast III RC Glossy Paper at 3 to 4x.

The 20 primary trisomics were of diverse origins and needed to be transferred into a uniform genetic background. Soybean cv. Clark 63 was used as a recurrent parent because it is adapted to central Illinois (maturity group IV) and produces abundant pollen grains in a greenhouse. During the backcrosses, primary trisomic plants were generally used as female parents in consecutive crosses with Clark 63 until each of the trisomics reached to BC4 generation. In a few cases when only one trisomic plant was recovered, the trisomic plant was reciprocally crossed with Clark 63 to ensure enough trisomic plants in next generation.

In genetic marker analysis, primary trisomics were crossed with a triple null genotype [ti (Kunitz trypsin inhibitor absent), eu1 (urease absent), and sp1 (ß-amylase activity absent) (Garcia-Orbegozo and Hymowitz, 1989)] and two marker stocks PI 408251 [lx1 (lipoxygenase-1 absent) (Hildebrand and Hymowitz, 1981)] and T31 [p2 (puberulent) (Palmer and Kilen, 1987)]. All primary trisomics used in crosses were analyzed for presence or absence of urease by the procedure of Kloth et al. (1987) and for lipoxygenase-1 by the procedure of Hildebrand and Hymowitz (1981). The primary trisomics were all homozygous for Eu1 (seed urease present), Lx1 (Lipoxygenase-1 present) and P2 (pubescent).

The morphological marker w1 (white flower) was previously located on the extra chromosome of Tri S by the translocation test (Sadanaga and Grindeland, 1984) and v2 (variegated leaf) on the extra chromosome of Tri A (Honeycutt et al., 1990). Tri A and Tri S were later identified as triplo 5 and triplo 13 based on pachytene analysis (Singh and Hymowitz, 1991). To confirm the chromosomal locations of v2 and w1, triplo 5 and triplo 13 were crossed with lines T312 (v2v2) and T161 (w1w1), respectively.

Primary trisomics were used as female parents in a majority of the crosses. However, most primary trisomics were used as male parents in crosses with T31 because T31 was a poor pollinator. The chromosome numbers of F1 seeds were counted and two plants with 2n = 41 and one plant with 2n = 40 from each of the crosses were grown to maturity in the greenhouse. The F2 seeds from selfed F1 trisomic plants were scored for segregation of isozyme marker genes Eu1/eu1 and Lx1/lx1. The F2 seeds from crosses with T31 and T312 were germinated in sand trays. The segregation of pubescent versus puberulent and normal leaves versus variegated leaves were scored at seedling stage about 3 wk post emergence. The F2 seeds from crosses of triplo 13 with T161 were germinated, the hypocotyl color (purple versus green) of seedlings was scored, and the seedlings were transplanted into 15-cm clay pots (2 to 3 plants/pot). The flower colors (purple versus white) were recorded after flowering.

Chi-square analysis was performed by the Statistical Analysis System (SAS Institute, 1992) to test if the genetic segregation ratios of marker genes fit the disomic (D) ratio (3:1) or a trisomic (T) ratio (17:1). The trisomic segregation ratio of 17:1 is based on the 50% transmission rate of an extra chromosome from the primary trisomic F1s with duplex (AAa) genotype (Singh, 1993).


   Results
TOP
 ABSTRACT
 INTRODUCTION
 Materials and methods
 Results
Discussion
 REFERENCES
 
Twenty possible primary trisomics were isolated from 37 aneuploid lines . The aneuploid lines originated from the progenies of asynaptic and desynaptic mutants, neutron-irradiated plants, tissue culture-induced sterile mutants, and monogenic male sterile lines (Table 1). Since some aneuploid lines were heterozygous for the male sterile trait and segregated for fertility and sterility, primary trisomics were selected only from the plants with normal meiotic pairing and high pollen and seed fertility.

Because original primary trisomics were of different genetic backgrounds, it was difficult to evaluate morphological alterations. In BC3 and BC4 generations with Clark 63, several primary trisomics exhibited some unique diagnostic features. Triplo 1 showed the largest pods (Fig. 1a) and seeds (Fig. 1b) among the 20 primary trisomics and they were about one-third larger than those of the disomic sib . Triplo 1 possessed more vigorous vegetative growth and larger branches and dark-green leaves than disomics. Triplo 13 showed shorter nodes, and smaller pods and seeds compared with its disomic sib. Grey-colored saddle seeds in triplo 17 differentiated it from its disomic sib and other primary trisomics. Seventeen primary trisomics did not express obvious differences in morphology from their disomic sibs.



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  		Fig. 1 Pods and seeds of triplo 1 and its disomic in soybean. a. Pods (left) of disomic (top) and triplo 1 (bottom). b. Seeds of disomic (top) triplo 1 (bottom)

 
Somatic metaphase chromosomes of soybean are almost indistinguishable except for a pair of nucleolus organizer chromosomes (Fig. 2a) . Triplo 13 for the nucleolus organizer chromosome contained three satellite chromosomes (Fig. 2b). Selected trisomic plants were analyzed cytologically at pachytene. The common trivalent configuration observed at pachytene was that the two chromosomes completely paired end-to-end while the third chromosome paired with its homologues mainly at the centromeric region and was unpaired for most of its length. Other trivalent configurations were nonhomologous pairing of the extra chromosome or three by three associations along a large portion of three homologues. The 20 primary trisomics were tentatively identified and designated on the basis of pachytene analysis (Table 2). Representative trivalent configurations of triplo 1 to triplo 20 are shown in Fig. 3 .



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  		Fig. 2 Photomicrographs of mitotic metaphase chromosomes in soybean. a. A cell with 2n = 40 chromosomes in a disomic plant (arrows indicate two satellite chromosomes). b. A cell with 2n = 41 chromosomes in a primary trisomic (triplo 13) for nucleolus organizer chromosomes (arrows indicate three satellite chromosomes). Bar = 10µm

 


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  		Fig. 3 Photomicrographs of trivalent configurations at pachytene in primary trisomics of soybean (arrowheads and arrows indicate the centromeres and extra chromosomes, respectively). a. Triplo 1. b. Triplo 2. c. Triplo 3. d. Triplo 4. e. Triplo 5. f. Triplo 6. g. Triplo 7. h. Triplo 8. i. Triplo 9. j. Triplo 10. k. Triplo 11. l. Triplo 12. m. Triplo 13. n. Triplo 14. o. Triplo 15. p. Triplo 16. q. Triplo 17. r. Triplo 18. s. Triplo 19. t. Triplo 20. Bar = 10µm

 
Among the designated 20 primary trisomics, six new primary trisomics, including triplo 7 (Fig. 3g), triplo 9 (Fig. 3i), triplo 11 (Fig. 3k), triplo 17 (Fig. 2q), triplo 18 (Fig. 3r), and triplo 20 (Fig. 3t), were found from the aneuploid lines A84C1-5-8-5, MD76, T170, A84C1-5-11-18, KS:TH745, and A88A-21, respectively (Table 2). For the 13 primary trisomics reported previously (Singh and Hymowitz, 1991; Ahmad et al., 1992; Ahmad and Hymowitz, 1994), five were confirmed: triplo 1 (Fig. 3a), triplo 3 (Fig. 3c), triplo 4 (Fig. 3d), triplo 5 (Fig. 3e), and triplo 13 (Fig. 3m). However, on the basis of new cytological evidence, an appropriate rearrangement and substitution of other primary trisomics were made (Table 2). The primary trisomics originated from four aneuploid lines SRF-70: 11, SRF-70:TH216, A88B-13, and SRF-70:120 were changed from triplo 8, triplo 10, triplo 16, and triplo 19 to triplo 6 (Fig. 3f), triplo 12 (Fig. 3e), triplo 10 (Fig. 3j), and triplo 15 (Fig. 3o), respectively. Aneuploid line MD70, which was previously identified as the duplicate of triplo 10, was changed to triplo 14 (Fig. 3n). Previously designated triplo 2 (SRF-70:28) and triplo 6 (KS:TH772) were identified as duplicates of triplo 5 and triplo 3, respectively. The new triplo 2 (Fig. 3b), triplo 8 (Fig. 3h), triplo 16 (Fig. 3p), and triplo 19 (Fig. 3s) were identified from aneuploid lines KS:TH 747, A84C1-5-10-15, T190-47-1, and T177, respectively.

The primary trisomics had the meiotic pairing configurations of either 1 III + 19 II (Fig. 4a) or 20 II + 1 I (Fig. 4b) at metaphase I in a majority of microsporocytes. To verify the pachytene analysis results, 23 F1 hybrids with 2n = 42 chromosomes were produced from a large number of crosses among primary trisomics. The meiotic chromosome pairing revealed that the frequency of 21 II in hybrids SRF-70:28 x triplo 5 (Tri A) (Fig. 5a) , triplo 3 (KS:TH775) x KS:TH772, and triplo 17 (A84C1-5-11-18) x A84C1-5-4-8-1 ranged from 73 to 92% (Table 3) , suggesting that both lines in the hybrids carry the same extra chromosome. Likewise, only a small number of the microsporocytes showed meiotic pairing of 20 II + 2 I (8 to 20%). Therefore, these three hybrids were revealed as tetrasomics. In contrast, the other 20 hybrid combinations showed meiotic pairing of 20 II + 2 I (38 to 76%, Fig. 5b), 1 III + 19 II + 1 I (18 to 50%, Fig. 5c), and 2 III + 18 II (0 to 16%, Fig. 5d) (Table 3). This observation confirmed that the extra chromosomes of the two primary trisomics were dissimilar.



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  		Fig. 4 Meiotic pairing configurations at metaphase I in primary trisomics. a. A cell with one trivalent (arrow) and 19 bivalents in triplo 20. b. A cell with 20 bivalents and one univalent in triplo 20. Bar = 10 µm

 


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  		Fig. 5 Meiotic pairing configurations at metaphase I in 42-chromosome F1 hybrids from crosses between primary trisomics. a. A cell with 21 bivalents in F1 hybrid of SRF-70:28 x triplo 5. b. A cell with 20 bivalents and two univalents (arrows) in F1 hybrid of triplo 2 x triplo 5. c. A cell with one V-shaped trivalent (indicated by a large arrow) 19 bivalents and one univalent trivalent (indicated by a small arrow) in F1 hybrid of triplo 2 x triplo 5. d. A cell with one chain trivalent (indicated by arrow) and one V-shaped trivalent (indicated by arrow) and 18 bivalents in F1 hybrid of triplo 2 x triplo 5. Bar = 10 µm

 

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  		Table 3 Chromosome pairing at metaphase I of meiosis in 2n = 42 hybrids from crosses among primary trisomics in soybean

 
Average female transmission rates of the 20 primary trisomics was 42% with a range of 27 (triplo 20) to 59% (triplo 9) (Table 4) . In addition, one monosomic plant was found in BC4 of triplo 6 x Clark 63 (Xu et al., 2000a). The female transmission rates were calculated from backcross progenies (F1 to BC4) of the primary trisomics x Clark 63 and from F1 hybrids with various marker stocks. Therefore, the populations were highly heterozygous and the heterozygosity might favor the higher female transmission rates (Singh, 1993).


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  		Table 4 Female transmission rate (%) of 20 primary trisomics (2n = 41) in soybean

 
The segregation ratios (Table 5) of the marker genes in F2 progenies from triplo 5 x eu1, triplo 13 x lx1, and triplo 20 x p2 agree with a trisomic ratio of 17:1, which is expected on 50% transmission of the extra chromosome. This indicates that loci Eu1, Lx1, and P2 are located on chromosomes 5, 13, and 20, respectively. The segregation data for other F2 populations fit the disomic ratio of 3:1, except that the ratios of the F2 progenies from triplo 3 x lx1, triplo 6 x lx1, and triplo 1 x p2 significantly deviated from 3:1 (Table 5). The segregation ratio in F2's from triplo 3 x lx1 and triplo 1 x p2 fit a ratio of 1:1 with {chi}2 values 0.444 and 0.404 , respectively, and triplo 6 x lx1 give an approximate ratio of 6:1 . Heterozygosity of the loci, expressivity of the recessive traits, population size, or other unknown factors may cause the significant deviation of segregation from either disomic or trisomic ratios.


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  		Table 5 Genetic segregation ratios for marker genes in F2 of primary trisomics of soybean

 
Among 220 F2 plants from the cross of triplo 5 x T312 (v2v2), 212 plants had normal leaves and eight had variegated leaves. Forty-one of 45 F2 plants from cross of triplo 13 x T161 (w1w1) had purple flowers and four plants had white flowers. The segregation data agree with a trisomic ratio of 17:1 for v2 and w1 , confirming that the v2 and w1 loci are located on chromosomes 5 and 13, respectively.


   Discussion
TOP
 ABSTRACT
 INTRODUCTION
 Materials and methods
 Results
 Discussion
REFERENCES
 
Primary trisomics may originate from various sources in plants. Generally, when autotriploids can be produced, they are the best source for primary trisomics. The complete or incomplete set of primary trisomics has been isolated in many diploids species from the progenies of autotriploids (Singh, 1993). The autotriploids are usually produced by crossing tetraploids with diploids and also occasionally occur in natural populations. However, crosses between tetraploid and diploid plants have not been successful in producing autotriploids in soybean (Hu, 1968; Porter and Weiss, 1948; Sadanaga and Grindeland, 1981; Zhang and Palmer, 1990a). We obtained two autotriploid plants after a large number of crosses between tetraploid and diploid plants by using improved embryo rescue techniques. Both plants died at the seedling stage because they were too weak to survive after transplanting. Chen and Palmer (1985) obtained spontaneous autotriploids from progenies of male sterile (ms1) lines. However, the spontaneous autotriploids have not been used to produce primary trisomics in soybean (Palmer and Skorupska, 1994).

Thus far, the source of primary trisomics in soybean has been the aneuploid lines derived from asynaptic and desynaptic mutants (Palmer, 1974; Palmer and Heer, 1976), male sterile (ms) lines (Chen and Palmer, 1985; Sadanaga and Grindeland, 1981; Zhang and Palmer, 1990b), neutron-irradiated plants (Sadanaga and Grindeland, 1979), and tissue culture-induced sterile mutants (Graybosch et al., 1987; Palmer and Skorupska, 1994). The aneuploids are generally found among open-pollinated seeds from sterile meiotic mutants and male sterile (ms) plants. They can also be produced directly by crossing the meiotic mutants and genetic male sterile lines with normal plants. We also have generated several aneuploid lines by crossing `Funman' sterile plants with Clark 63.

Complete and incomplete primary trisomic series have been morphologically and cytologically identified in a number of diploid plant species (Singh, 1993). Morphological alterations of primary trisomics from their normal diploid sibs caused by the extra chromosomes may provide the most convenient differentiation of different primary trisomics in a diploid species. Therefore, the primary trisomic lines or plants are usually grouped into different morphological types prior to cytological identification. The seven primary trisomics in barley (Hordeum vulgare L.) were established on basis of classification of seven morphological types: Bush, Slender, Pale, Robust, Pseudo-normal, Purple, and Semierect (Tsuchiya, 1967).

It is well known that primary trisomics in tomato (Rick et al., 1964) and in rice (Khush et al., 1984) were precisely designated on basis of pachytene analysis. However, distinguishing morphological features facilitated pachytene analysis in both cases. The identification of the primary trisomics in rice was conducted on the trisomic lines, which had been categorized into 12 morphological groups. Eleven of the 12 rice primary trisomics can be identified morphologically from one another and from diploid plants (Khush et al., 1984).

Since original aneuploid lines used in this study have diverse genetic backgrounds and belong to different maturity groups, it was difficult to evaluate the morphological alterations caused by extra chromosomes among original primary trisomics. After 20 primary trisomics were transferred into a genetic background of Clark 63, several primary trisomics expressed distinctive changes in morphology, such as large pods in triplo 1, short internodes of triplo 13, and blue saddle of triplo 17. These morphological changes provide reliable confirmation of these primary trisomics. We observed that some slightly modified traits in trisomic condition became more pronounced in the tetrasomic condition. This may reflect the polyploid nature of soybean. Tetrasomic plants from triplo 13 had very small pods and seeds and tetrasomics from triplo 17 had compressed pods and grey-colored seeds. Gwyn and Palmer (1989) indicated that two extra chromosomes (tetrasomics, double trisomics) could induce a characteristic syndrome of morphological changes that is statistically detectable. Therefore, one of the approaches to distinguish primary trisomics morphologically is to isolate tetrasomics from 20 primary trisomics.

Soybean is not a model species for cytological studies (Singh and Hymowitz, 1991) because the 40 small chromosomes are indistinguishable from one another except for one pair of satellited chromosomes. Ladizinsky et al. (1979) analyzed soybean somatic chromosomes by using a Giemsa C-banding technique, and the chromosomes failed to produce informative banding patterns for identification of individual chromosomes. Pachytene analysis is an excellent tool for identification of primary trisomics in many diploid crops (Singh, 1993). The effectiveness of pachytene analysis varies in different species. The identification of extra chromosomes at pachytene was less complicated in maize, rice, and tomato; relatively convenient in potato (Solanum tuberosum L.); but difficult in sorghum (Sorghum vulgare Pers.) because nonhomologous pairing of chromosomes interfered with the process of the identification (Singh, 1993). Thus far, pachytene analysis has been the only feasible method for characterization of primary trisomics in soybean.

We tentatively identified 20 primary trisomics of soybean on the basis of the association of the extra chromosome with its normal homologues in a trivalent configuration, chromosome length, arm ratios, and distribution of euchromatin and heterochromatin at pachytene. In soybean primary trisomics, some trivalents showed nonhomologous pairing where the two arms of the extra chromosome paired. But, this type of nonhomologous pairing is not a major obstacle because we analyzed the trivalents that represent the typical features of a specific chromosome.

However, a high number of chromosomes probably make the pachytene analysis in soybean more difficult than other species such as maize, potato, rice, sorghum, and tomato. It is known that the trivalents are best studied when they are completely separated from the rest of the chromosomes (Rick et al., 1964). But the separation of the trivalents at pachytene was extremely difficult in soybean because of the high degree of chromosome entangling. Thus, the number of trivalents applicable to chromosome identification was small compared with other plant species. Moreover, the complete separation of the trivalents from the rest of the chromosomes requires the extreme flattening and scattering of the chromosome preparation and this usually causes chromosome stretching (Rick et al., 1964). Chromosome stretching was very common in the soybean, which often caused some uncertainty in chromosome identification. Because of these inherent difficulties and disadvantages of soybean cytology, the reliability of the designated 20 primary trisomics based on pachytene analysis should be tested and confirmed by other approaches.

Primary trisomics of soybean could be confirmed by meiotic pairing analysis of the 2n = 42 chromosome hybrids produced from the intercrosses among primary trisomics. Theoretically, the meiotic pairing configurations in the 42-chromosome plants differ depending on their tetrasomic (carrying two identical extra chromosomes) or double trisomic (carrying two different extra chromosomes) nature (Ahmad and Hymowitz, 1994). Tetrasomics are expected to have meiotic pairing patterns of 21 II and 1 IV + 19 II, whereas double primary trisomics have 20 II + 2 I, 1 III + 19 II + 1 I, and 2 III + 18 II. Using this approach, Gwyn and Palmer (1989) proved that Tri A, Tri B, Tri C, and Tri D are different, but Tri D and Tri E carry the same extra chromosome. Ahmad et al. (1992) and Ahmad and Hymowitz (1994) analyzed 12 hybrids from the crosses involving some of the 13 reported primary trisomics. In the present study, 20 F1 hybrids were produced. However, these hybrids represent only a small portion of all possible cross combinations among the 20 primary trisomics. Therefore, further confirmations on the 20 designated primary trisomics are needed by making additional new crosses among the primary trisomics.

This study revealed that the 20 primary trisomics averaged 42% female transmission, with a range of 27 (triplo 20) to 59% (triplo 9). The results were similar to the female transmission rates of Tri A (34%), Tri B (45%), and Tri C (39%) reported by Palmer (1976). Triplo 1 (Tri C) and triplo 5 (Tri A) in our study had 47 and 37% female transmission, respectively. The slight increases of the rates in these two primary trisomics probably resulted from the heterozygosity of the trisomic populations. We noticed that BC1 and BC2 generations showed an increased frequency of trisomic individuals. Heterozygosity is generally favorable for transmission of extra chromosomes. Apparently, primary trisomics of soybean had a higher female transmission than those in diploid species (Singh, 1993).

We have not studied the male transmission rates of the 20 primary trisomics in detail. It seems that all the primary trisomics can transmit their extra chromosomes through pollen because tetrasomics were isolated from selfed progenies of all the original primary trisomics (Xu et al., 1998) and double primary trisomics could be isolated from the F1 hybrids between the trisomics. In the present study, F1 plants with 2n = 41 chromosomes were isolated from the crosses of T31 () with primary trisomics (). Palmer (1976) reported that pollen transmission was 27% for Tri A, 22% for Tri B, 43% for Tri C, and intercrosses among three trisomics produced 13% 2n = 42 progeny. However, primary trisomics hardly transmit their extra chromosomes through pollen in a majority of diploid species (Singh, 1993). High female and male transmission rates of soybean primary trisomics are most likely related to polyploid nature of the species.

Primary trisomics are good materials for locating a gene on a particular chromosome, verifying the independence of linkage groups, and associating the genetic linkage groups with the individual chromosomes (Singh, 1993). Honeycutt et al. (1990) located the variegated leaf mutant gene v2 on the extra chromosome of Tri A (triplo 5). Hedges and Palmer (1991) located an isozyme marker, dia 1 (diaphorase), on the extra chromosome of Tri D by using starch gel electrophoresis. Tri D was identified as triplo 4 by pachytene analysis (Singh and Hymowitz, 1991). Sadanaga and Grindeland (1984) located the w1 (white flower) locus on the satellite chromosome using translocation test. In the present study, we confirmed that the marker genes v2 and w1 are located on chromosome 5 and 13, respectively. We further located two seed protein genes eu1 and lx1 and a morphological marker gene p2 on chromosome 5, 13, and 20, respectively. Thus far, as determined by primary trisomics, only four of 20 soybean chromosomes have been associated with genetic markers.

We are continuing genetic tests by using biochemical, morphological, and molecular markers. The 20 primary trisomics will be finally confirmed when different markers are placed on all 20 chromosomes by using the primary trisomics. This study will eventually associate conventional genetic linkage groups and several molecular maps with the cytological maps as has been done for maize, rice, and tomato.


   ACKNOWLEDGMENTS
 
Research was supported by the Illinois Agricultural Experiment Station and the Illinois Soybean Program Operating Board grants No. 93-19-131-3 HY and 97-23-182-3 HY.

Received for publication December 6, 1999.


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