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Crop Science 40:683-687 (2000)
© 2000 Crop Science Society of America

CROP BREEDING, GENETICS & CYTOLOGY

Genetics and Cytology of Chromosome Inversions in Soybean Germplasm

R.G. Palmera, H. Sunb and L.M. Zhaob

a USDA ARS CICGR Unit and Departments of Agronomy and of Zoology/Genetics, Iowa State University, Ames, IA 50011-1010 USA
b Jilin Academy of Agricultural Sciences, Gongzhuling, Jilin Province 136100, People's Republic of China

rpalmer{at}iastate.edu


   ABSTRACT
TOP
 NOTES
 ABSTRACT
INTRODUCTION
 Materials and methods
 Results and discussion
 REFERENCES
 
One type of chromosome aberration, an inversion, results in the reverse orientation of genes on a chromosome. Inversions are very useful in genetic linkage tests and have been important in the evolution of certain species of animals and plants. In soybean, three accessions (PIs) with a paracentric chromosome inversion were identified. Our objective was to determine if the paracentric inversions identified in PI 597651 and PI 597652 [Glycine max (L.) Merr., cultivated species] and in PI 407l79 (G. soja Siebold & Zucc., wild annual species) were identical. The G. soja inversion was backcrossed into G. max cultivar Hark. The two G. max accessions from China were intercrossed, and based on pollen staining of F1 and F2 plants, were considered identical in chromosome structure. However, the G. soja accession had a chromosome structure different from the two G. max accessions. Meiotic studies confirmed the presence of the paracentric inversions. Crosses of PI 597651 with either cultivar Hark or Hark homozygous inversion gave F1 plants with two to three times as many meiotic cells with chromosome bridges as cells with laggards and fragments. However, crosses of PI 567652 with either cultivar Hark or Hark homozygous inversion gave F1 plants with about equal numbers of meiotic cells with bridges as cells with laggards and fragments. Therefore, cryptic structural differences between these two Chinese accessions might influence chromosome pairing, crossing over, and segregation. This might explain the different meiotic behaviors in crosses of the two Chinese accessions with Hark and Hark homozygous inversion.


   INTRODUCTION
TOP
 NOTES
 ABSTRACT
 INTRODUCTION
Materials and methods
 Results and discussion
 REFERENCES
 
CHROMOSOME ABERRATIONS may result from the loss, multiplication, or rearrangement of the genetic material. Chromosome inversions represent one type of rearrangement that occurs when chromosome segments become detached, inverted, and are reunited so that the gene segment contained therein is in reverse linear order in relationship to the rest of its chromosome (Rieger et al., 1991, p. 281–282). Muller (1940) proposed the following terminology: inversions including the centromere are pericentric and inversions limited to a single chromosome arm are paracentric. If the inverted region is large enough, the homologous chromosomes in an inversion heterozygote pair gene by gene and form a loop configuration that can be observed at pachytene. Crossovers within the loops of both pericentric and paracentric inversions result in chromatids with duplications and deficiencies. Plants heterozygous for either pericentric or paracentric inversions are characterized by pollen abortion as a result of crossing over within the inversion segment. The percentage of sterility is dependent on the amount of crossing over within the inversion segment (Morgan, 1950). Crossing over within paracentric loops alone or with a crossover in the interstitial segment leads to the appearance of chromatin bridges and fragments at anaphase I and II, and subsequently to aborted spores. Dicentric chromatids are responsible for bridges and acentric chromatids (fragments). By contrast, crossovers that occur within inversion loops of percentric inversion heterozygotes do not result in bridges and fragments. Therefore, the observation of bridges and fragments at meiotic anaphase is evidence for the presence of a paracentric inversion (McClintock, 1931).

Cytogenetic studies of interspecific hybrids between the cultivated soybean G. max and the wild annual soybean G. soja revealed the presence of one or more paracentric inversions (Ahmad et al., 1977a, 1977b, 1979, 1983). These interspecific hybrids exhibited marked differences in chromosome behavior and fertility depending on parents and temperature. In general, summer-grown plants had a higher frequency of meiotic irregularities than did winter-grown plants (Ahmad et al., 1977b). Plants grown in controlled environment cabinets confirmed the effect of high temperature causing more irregularities in chromosome behavior (Ahmad et al., 1983). Furthermore, differences among cross-combinations involving two G. max parents with two G. soja parents were genotype specific (Ahmad et al., 1983). Hybrids within the perennial wild soybean species have shown the presence of paracentric inversions (Singh and Hymowitz, 1985; Hymowitz et al., 1991).

In a search for chromosome aberrations among cross-combinations of G. max germplasm, Delannay et al. (1982) and Palmer (1985) reported that seven of 626 combinations gave F1 hybrids with 10 to 30% pollen sterility. A similar study with G. soja germplasm showed that 20 suspected inversions from 142 cross-combinations gave 10 to 40% pollen sterility (Delannay et al., 1982; Palmer, 1985). The G. soja accessions from Japan and South Korea had a higher frequency of suspected inversions than did accessions from China, Taiwan, and Russia. One G. soja accession from South Korea, PI 407179, has been confirmed to differ from G. max by a paracentric inversion (Harper, 1992). This PI was backcrossed to the cultivar Hark (Weber, 1967) to create a near-isogenic line homozygous for the paracentric inversion (Palmer, 1981, unpublished data).

Sun et al. (1991) have identified paracentric inversions in two Chinese G. max landraces, Wei Da Yu and Sun Wu Xiao Bai Mei. These landraces have been entered into the USDA soybean germplasm collection as PI 597651 and PI 597652, respectively.

Our objective was to determine if the paracentric inversions identified in G. soja PI 407179 and in G. max PI 597651 and PI 597652 were identical.


   Materials and methods
TOP
 NOTES
 ABSTRACT
 INTRODUCTION
 Materials and methods
Results and discussion
 REFERENCES
 
The two Chinese G. max landraces, Wei Da Yu and Sun Wu Xiao Bai Mei, were identified previously as differing from other G. max lines by a chromosome inversion (Sun et al., 1991). The two Chinese homozygous inversion genotypes were used directly in cross-pollinations. PI 407179 (G. soja) was identified as differing from G. max by a chromosome inversion (Delannay et al., 1982; Palmer, 1985). The inversion from PI 407179 was backcrossed into cultivar Hark (BC6) to develop a near-isogenic line homozygous for the inversion. Selection for inversion heterozygosity was practiced between each backcross by identifying plants expressing partial pollen sterility. The three homozygous inversion lines (PI 597651, PI 597652, and Hark homozygous inversion from PI 407179) were each crossed to Hark and were intercrossed with each other to create three homozygous inversion x homozygous inversion combinations.

The F2 plants from the three homozygous inversion lines x Hark were grown at the Bruner Farm near Ames, IA, which has Clarion and Nicollet loam soils (fine-loamy, mixed, superactive, mesic Typic Hapludoll and fine-loamy, mixed, superactive, mesic Aquic Hapludoll). These plants were used for pollen analyses. The F1 plants used for meiotic analyses were grown in the glasshouse at Ames, at 24 ± 2°C (dark) and 29 ± 2°C (light). Day length was 14 h of light and 10 h of darkness. The photosynthetic irradiance range was 270 to 500 µmol photon m-2 s-1. At Gongzhuling, China, the F1 plants used for meiotic analyses were grown in 5-L pots in fine-silty, mixed, superactive mesic Typic Hapludoll soil outside during the summer under natural photoperiod. Temperatures were 17 ± 2°C (dark) and 22 ± °2C (light).

Fertile and heterozygous chromosome inversion plants were identified by squashing anthers in an aqueous solution of I2KI (Jensen, 1962, p. 203). Anthers from fertile plants gave densely stained reddish-brown pollen grains, whereas anthers from heterozygous inversion plants gave both well-stained pollen grains and aborted (smaller and lightly stained) pollen grains. Five hundred pollen grains were counted from each plant examined.

Immature floral buds for meiotic analyses were collected from fertile and heterozygous inversion glasshouse-grown plants (Ames, IA) during the first 6 h of the light cycle. The buds were fixed immediately in either 3:1 absolute ethanol/chloroform saturated with ferrous acetate or 6:3:2 absolute ethanol/chloroform/propionic acid saturated with ferrous acetate. The buds were fixed for a minimum of 24 h, washed twice with 70% ethanol, and stored at 4°C. For microscopic examination, the buds were rinsed with distilled water, placed in 45% propionic acid, and stained with propionocarmine. The microsporocytes were scored for frequencies of bridges and fragments. Pollen grains and anther squashes were viewed on a Zeiss Standard WL Research microscope (Carl Zeiss, Thornwood, NY) using an MC63 Photomicrographic Camera Attachment (Brinkmann Instruments, Des Plaines, IL) and Kodak Techpan film (Eastman Kodak Co., Rochester, NY).1

At Gongzhuling, China, all five cross-combinations were evaluated cytologically. Immature floral buds for meiotic analyses were collected and fixed similarly as in Ames, IA. Pollen grains and anther squashes were viewed on a Leitz orthoplan microscope (Leica, Deerfield, IL) with a 35-mm camera attachment and Kodak Techpan film.


   Results and discussion
TOP
 NOTES
 ABSTRACT
 INTRODUCTION
 Materials and methods
 Results and discussion
REFERENCES
 
Pollen
The intercrosses of the two Chinese landraces gave completely fertile pollen for all the F1 and F2 plants (Table 1) . These two landraces have the identical chromosome inversion, consistent with the observations of Sun et al. (1991).


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  		Table 1 Pollen fertility of F1 plants and F2 plants from cross-pollinations of homozygous chromosome inversion soybean plants

 
The three homozygous inversion lines were crossed to cultivar Hark (N N) and pollen was classified from F1 and F2 plants (Fig. 1A and 1B) . The cross of Hark (N N) with Hark homozygous inversion (In In) gave F1 plants with about 25% pollen sterility (Table 1). The two Chinese landraces crossed with Hark (N N) had about 18% F1 pollen sterility (Table 1).



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  		Fig. 1 Pollen grains and meiotic cells of F1 soybean plants from cross-combinations of homozygous chromosome inversion plants. (A) Fertile pollen grains from Hark (N N). (B) Fertile and sterile pollen grains from PI 597652 x Hark (N N). (C) Late anaphase I from Hark (N N) x Hark (In In) (homozygous inversion) with two fragments. (D) Anaphase I from Hark (N N) x Hark (In In) with bridge. (E) Dicentric bridge in anaphase I from PI 597651 x Hark (N N) (F) Anaphase I from PI 597651 x Hark (In In) with a bridge and one fragment. Bars for Fig. 1A and 1B = 9 µm, and for Fig. 1C, 1D, 1E, and 1F = 9.2 µm

 
The F2 Hark cross-combination (N N x In In) gave 46 plants with very low percentage of pollen sterility and 46 plants with a level of pollen sterility similar to the F1 plants (Table 1). A few F2 plants had slightly lower or slightly higher pollen sterility. The two Chinese landraces crossed with Hark (N N) gave F2 plants that were distributed into three pollen sterility categories (Table 1).

The two Chinese landraces crossed with Hark (In In) gave F2 plants that were distributed into four sterility categories (Table 1). These pollen sterility categories would be expected to represent no inversion, the Chinese landrace inversion, the Hark (G. soja) inversion, and the two different inversions. The percentage of pollen sterility is dependent on the amount of crossing over within the inverted region. A pollen sterility class of 31 to 40% would include F2 plants with variation in crossover frequencies (Dowrick 1957). Suppression of crossing over by asynapsis and nonhomologous pairing would lower the number of crossovers, thereby reducing the percentage of pollen abortion (McClintock, 1931; Maguire, 1966). A pollen sterility class of 41 to 50% would represent those plants with both paracentric inversions.

The minimum genetic length of an inversion may be estimated from pollen abortion frequencies (Morgan, 1950). As a correction, the percentage of pollen abortion occurring in homozygous plants should be deducted from the percentage of pollen abortion in heterozygous inversion plants. The control F1 plants grown in Ames, IA had 1% aborted pollen. Therefore, based on pollen data, the minimum genetic length of this inversion is 25.2% - 1.0% = 24.2%. This calculation considers that single crossovers and the average of two-strand, three-strand, and four-strand doubles will produce tetrads with two normal and two aborted microspores (Burnham, 1962). The two Chinese landraces also had about 1% aborted pollen in control plants. Thus the minimum genetic length for this inversion should be reduced by about 1% and becomes 16.5% for PI 597651 and 17.7% for PI 597652.

Cytology
The cross-combination Hark (N N) x Hark (In In) was grown in a glasshouse at Ames, IA, and in the field at Gongzhuling, China. In the Iowa environment, bridges plus fragments were observed in 19.4% of the meiocytes (Fig. 1C and 1D, Table 2) . This contrasts to only 2.8% meiotic aberrations for the same cross-combination grown in the China environment (Table 2). Differences in growth conditions may be the explanation for the apparent discrepancy in the number of meiotic aberrations between the same genotype grown in two different environments.


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  		Table 2 Meiotic irregularities of F1 plants from cross-pollinations of homozygous inversion soybean plants

 
More than 100 meiocytes were examined cytologically for the Hark (N N) x Hark (In In) cross-combination. Different scientists made the observations, but that variable should not account for the sevenfold variation in values. Temperature is known to influence crossing over in plants (Dowrick 1957). Percentage of pollen sterility is dependent on the number of crossovers that occur within inversion loops (Morgan 1950). Ahmad et al. (1977b, 1983) demonstrated conclusively that high temperatures increased meiotic aberrations in soybean inversion heterozygotes. Our data from the Hark (N N) x Hark (In In) plants, grown in two contrasting environments, probably are the result of the effect of temperature differences on meiosis. The higher temperatures in Iowa seemed to increase the frequency of bridges plus laggards and fragments. Our data are consistent with the observations of Ahmad et al. (1977b).

In the cross PI 597651 x Hark (N N), almost twice as many cells with bridges were recorded as cells with laggards and fragments (Fig. 1E, Table 2). However, in the cross PI 597652 x Hark (N N), about equal numbers of cells with bridges were recorded as cells with laggards and fragments (Table 2).

Almost three times as many cells with bridges were recorded as cells with laggards and fragments in the cross PI 597651 x Hark (In In) (Fig. 1F, Table 2). About equal numbers of cells with bridges were recorded as cells with laggards and fragments in the cross PI 597652 x Hark (In In) (Table 2).

The meiotic aberrations observed in the two cross-combinations with PI 567651 resulted in more bridges than laggards and fragments (Table 2). Different configurations of dicentric bridges and fragments at anaphase I and II are expected and depend on the types of crossovers within the inversion loop (Singh, 1993). Crossover configurations that gave rise to dicentric chromatids were consistently higher in cross-combinations with PI 567651 than with PI 567652. In addition, cryptic structural differences in chromosomes (Stephens, 1950) between the two Chinese G. max landraces may have influenced chromosome pairing in the crosses with Hark (N N) and its near-isogenic homozygous inversion line (In In).


   NOTES
TOP
 NOTES
ABSTRACT
 INTRODUCTION
 Materials and methods
 Results and discussion
 REFERENCES
 
The first two authors contributed equally to this work. This is a joint contribution, Journal Paper J-18412, of the Iowa Agriculture and Home Economics Experiment Station, Ames, IA (project 3352), supported by Hatch Act and State of Iowa, and from the United States Department of Agriculture, Agricultural Research Service, Corn Insects and Crop Genetics Research Unit.

1 The mention of a trademark or proprietary product does not constitute a guarantee or warranty of the product by the USDA or Iowa State University and does not imply its approval to the exclusion of other products that may be suitable. Back

Received for publication July 28, 1999.


   REFERENCES
TOP
 NOTES
 ABSTRACT
 INTRODUCTION
 Materials and methods
 Results and discussion
 REFERENCES
 




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