INCIPIENT GENOME DIFFERENTIATION IN GOSSYPIUM. 11. COMPARISON OF 12 CHROMOSOMES IN G. HIRSUTUM,

HETEROZYGOUS TRANSLOCATIONS G. MUSTELINUM AND G. TOMENTOSUM USING

CLARE A. HASENKAMPF AND MARGARET Y. MENZEL

Department of Biological Science, Florida State University, Tallahassee, Florida 32306

Manuscript received October 29,1979 Revised copy received April 21,1980

ABSTRACT

Eight homozygous translocation lines (TT) of G. hirsutum marking 3 chro- mosomes of the A genome and 9 chromosomes of the D genome were crossed with G. hirsutum, G. mustelinum and G. tomentosum, all homozygous for the standard end arrangements (tt). Chiasma frequencies in the G. hirsutum Tt controls were compared with those in the G. hirsutum x G. mustelinum and the G. hirsutum x G. tomentosum Tt hybrids. Both nucleus-wide and region- specific chiasma frequencies were compared.-Some genome differentiation appears to have arisen between G. hirsutum and G. mustelinum. The G. hirsu- tum x G. mustelinum hybrids had a 1.8 to 1.9% reduction in the nucleus-wide chiasma frequency. Four of the eight TT lines showed a 3.4 to 10.5% reduc- tion in chiasmata in the hybrid translocation quadrivalents, suggesting that chromosomes 1, 21, 23 and 24 may have undergone localized genome differ- entiation. The two species may differ naturally in the end arrangement of two chromosomes, since a quadrivalent not due to experimentally introduced trans- locations was observed in 13% of the PMC‘s of two G. hirsutum x G. mute- linum hybrids.-Very little genome differentiation has occurred between G. hirsutum and G. tomentosum. In the G. hirsutum x G. tomentosum hybrids, the nucleus-wide estimates showed only a very small (0.1 to 0.2%), though statistically significant, lowering of the chiasma frequency, and there was no reduction in chiasma frequency in the more sensitive readings for specific translocation quadrivalents.

PECIES of the genus Gossypium L. are assigned to seven genome groups based upon the number of bivalents and chiasmata formed in interspecific hybrids

(PHILLIPS 1974). Chromosomes of intragroup hybrids synapse and form chias- mata more effectively than those of intergroup hybrids. However, even among species assigned to the same genome group, evidence may be found for a lower level of chromosome divergence, which has been termed “incipient differentia- tion” ( MENZEL, BROWN and NAQI 19 78).

T h e individual chromosomes of Gossypium genomes cannot at present be dis- tinguished morphologically in normal material, and linkage maps are available for only a small portion of any cotton genome (KOHEL 1972; 1978). However, chromosome translocations available in allotetraploid G. hirsutum (upland cot- Genetics 95: 971-983 August, 1980.

9 72 C. A. HASENKAMPF A N D M. Y. MENZEL

ton) may be used to mark individual chromosomes of counterpart genomes in interspecific hybrids. The rationale for this method of studying genome diver- gence was developed in some detail in the first paper of this series (MENZEL, BROWN and NAQI 1978).

The present paper compares the genomes of three allotetraploid species: G. hirsutum, 2(AD) G. tonentosum, .%(AD) 3; and G. mustelinum, 2(AD) 4. G. hirsutum behaves cytologically like a diploid in that multivalents are virtually never formed at meiosis, and monosomes and deficiencies are rarely transmitted through the pollen. G. tomentosum Nuttal ex Seeman and G. mustelinum Miers ex Watt are wild species (FRYXELL 1965; PICKERSGILL, BARETT and ANDRADE- LIMA 1975). The former is endemic to the Hawaiian islands, and the latter is found in the Rio Grande do Norte, Brazil. G. tomentosum is known to have the same chromosome end arrangement as G. hirsutum (GERSTEL and SARVELLA 1956). No previous cytological analysis of G. mustelinum has been reported, but it forms fertile hybrids with G. hirsutum and G. barbadense L. (STEPHENS and PHILLIPS 1972).

The following questions will be addressed: (1) Has any chromosome diver- gence occurred between G. hirsutum and the two wild species? (2) If divergence has occurred, has it occurred similarly in all chromosome regions (generalized genome differentiation) or to different extents in the 3 A and 9 D genome chro- mosomes studied here (localized genome differentiation) ? Preliminary ac- counts of this work have been published (HASENKAMPF and MENZEL 1978; HASENKAMPF et a1 1979).

MATERIALS AND METHODS

The following translocations were used (see MENZEL and BROWN 1978a): T(H1R; H16R) line 4672, T(H2R; H14R) line 2B-1, T(H4L; H19R) line 10-5Ka, T(H14L; H23) line 2777, T(H15R; H16L) line 8-5Ga, T(H19R; H24R) line 2786, T(H2OR; H21L) line 7-3F and T(H20L; H22R) line DP-4. For brevity, these will hereafter be referred to as T1;16, T2; 14, etc. These translocation lines were isolated and the chromosomes involved were identified by BROWN and co-workers at the Texas Agricultural Experiment Station (BROWN 1980). They all originated from cultivated upland cotton, but are not on a uniform genetic background.

Each experiment consisted of a cross of each homozygous TT line with G. tomentosum, G. mustelinum, and G. hirsutum (line TM-1 with standard tt end arrangements). The 2 ( A D ) Tt plants served as controls; the chiasma frequencies found in these plants were compared with those obtained for hybrids between the TT lines and the two wild species. In all crosses, the T T lines were used as the ovule parent. The TM-1 pollen was taken from several plants of this highly inbred line. Pollen from G. tomentosum was obtained from a single plant grown from seeds of the Texas Agricultural Experiment Station stock G253, which has been inbred for several generations. Pollen of G. mustelinum was taken from a single plant from a strain collected at Caico, Brazil, by I. L. Gridi-Papp and inbred for only one or two generations. The hybrids were planted in the field at the Mission Road Research Facility in May 1977. The hybrid nature of the plants was verified by morphological examination.

Buds were collected in September and October, 1977, and fixed in a fresh 3:l mixture of 95% ethanol and glacial acetic acid. Slides were prepared by the aceto-carmine squash technique. Those with pollen mother cells (PMC’s) at metaphase I (MI) were systematically scored until 100 cells had been analyzed from each pedigree or until the material was exhausted,

ALLOTETRAPLOID COTTON GENOMES 973 The following information was recorded for each of the 2,947 PMC‘s analyzed: (1) the num-

ber of one- and two-chiasma bivalents (11) among the 24 pairs of chromosomes not involved in the translocation (used to generate the mean number of chiasmate bivalent arms for each type of progeny); (2) the number of chiasmata present per association of translocated chromosomes (used to generate the mean number of chiasmata per quadrivalent); and (3) the specific regions of the quadrivalent (IV) in which chiasmata occurred. Using T20;22 as an example, a typical PMC for the control and two interspecific hybrids are given in Figure 1. The modal heterozy- gous translocation configuration within G. hirsuium is shown in Figure 2. The number and location of chiasmata are deduced from the type of multivalent formed (Figure 3). For six of the lines, certain regions were indistinguishable from each other. In such cases, the two regions were considered as one in which zero, one or two chiasmata could be scored. For example, region U and c of T14;23 could not be distinguished, and chiasma tallies were recorded as zero, one or two for region U + c. The method of data collection and collation was elaborated by MENZEL., BROWN and NAQI (1978) and HASENKAMPP (1979).

FIGURE l.-Photomicr3eraphs of typical MI PMC‘s. Figures 1 x 4 are translocation hetero- zygotes, using T20;22 as an example. Figure 1A is 2(AD),Tt with 24 two chiasma (PX) II’s, 1 IV with chiasmata at U, c, b and d. Figure 1B is (AD),(AD),Tt with 24 2X 11’s 1 IV with chiasmata at U, c, b and d. Figure 1C is (AD),(AD),Tt with 22 2X 11’s and 2 1X 11’s (3 chro- mosomes marginally in focus), I IV with chiasmata at U, c, b and d Figure 1D is (AD),(AL)),tt with 22 2X 11’s. 2 1X II’s and 1 IV.

974 C. A. HASENKAMPF AND M. Y. MENZEL

FIGURE 2.-Photomicrographs of each translocation line’s modal configuration in translocation heterozygotes. Modal configurations for a given translocation line were the same whether G. hirsutum, G. tomentosum or G. mustelinum was the pollen parent. The numbers 1 through 13 are used to designate chromosomes of the A genome, 14 through 26 to designate those of the D genome. Figure 2A-T1;16 chiasmata present at a, c, e, f and b or d Figure 2B-T2;14 chiasmata at a, c, d and e. Figure 2C-T4;19 chiasmata at U, c, b and d . Figure 2D-T14; 23 chiasmata at a, c, e, f and b or d. Figure 2E-T15;16 chiasmata atg, c, b, d, and e or f. Figure 2F-T19;24 chiasmata a t U, C, b and d. Figure 2GT20;Pl chiasmata at a, c, b, d and e or f. Figure 2H-T20;22 chiasmata at a, c, b and d .

The use of translocations permits a comparison of chiasma frequencies for particular chro- mosomes in addition to comparisons for the entire nucleus. Also, in the translocation configura- tion, a maximum of two chiasmata can be detected in the arm involved in the translocation; whereas, in the other bivalents of the complement, only the presence or absence of chiasmata, equivalent to a maximum of one chiasma per arm, can be detected.

Besides the crosses already mentioned, several other types were attempted: G. hirsutum ‘I’M-1 x G. tomentosum, G. mustelinum x G. tomentosum and G. hirsutum TM-I x G. mute- h u m . Hybrid seeds of G. hirsutum TM-1 x G. tomentosum were not obtained. Although vig- orous G. mustelinum x G. tomentosum hybrids were obtained, they have not bloomed in the 26 months since their planting. Cells from the G. hirsutum ‘I’M-1 x G. mustelinum hybrids were analyzed for the presence of multivalents and the number of chiasmate bivalent arms.

All photomicrographs were made with a Zeiss Photomicroscope 111 using High Contrast Copy film. The film was developed in D19 using the standard range of times and temperatures.

RESULTS

The mean number of chiasmate I1 arms (Table 1) was the statistic used in assessing the overall similarity between G. hirsutum and the two wild species. Two-way analyses of variance compared the mean number of chiasmate I1 arms of the interspecific hybrids with that of the controls (Table 2). Thus, the species being crossed with the TT line was considered as one of the “treatments.”

Hybrids combining ( A D ) I and (AD), had a significantly lower average number of chiasmate I1 a r m s than the controls (P < 0.00001, Table 2). Although significant, the differences were small. On the average, G. mustelinum hybrids

ALLOTETRAPLOID COTTON GENOMES 975

C c a a

b

a c a C

d

a c a C

E

G

F

FIGURE 3.-Figures 3A-F are characteristic MI configurations in translocation heterozygotes when chiasmata occur in the regions indicated. Figure 3G is a schematic of the presumed pachy- tene quadrivalent with regions a to f demarcated. The letters a and c designate the two unbroken arms, b and d the translocated segments of the broken arms distal to the breakpoints, and e and f the interstitial regions of the broken arms. When A-D translocations are used, a, b and e are used to designate the regions of the D chromosomes and c, d and f the regions of the A chromosomes (MENZEL and BROWN 1978a). When D-D translocations are used, a, b, and e are used to designate the regions of the lower numbered chromosome.

had a 1.8 to 1.9% reduction in chiasma frequency (the 95% confidence interval estimate being 0.846 to 0.912 fewer chiasmata).

In the hybrids combining ( A D ) and ( A D ) 3, a statistically significant, but very small, reduction in chiasmata was also found when compared with the con- trol ( P < 0.05, Table 2). An average reduction in chiasma frequency of 0.1 to

976 C. A. HASENKAMPF A N D M. Y. MENZEL

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978 C. A. HASENKAMPF A N D M. Y. MENZEL

TABLE 2

Analysis of variance of mean no. chiasmate 11 arms in 2 (AD) Tt controls and (AD) I (AD) Tt or (AD) ~ (AD) Tt interspecific hybrids

Source of variation

Within cell Species TT Line Species by TT Line Plant within species by TT Line

Control us. ( A D ) , ( A D ) , Tt

Mean F Sign. of D.F. square value F(P)

21 0.061 - - 1 11.393 209.241 P<O.OOOOl 7 0.415 7.620 P<0.0003 7 0.359 6.598 P<0.0007

22 0.055 0.887 P=0.610

Mean F Sign. of

30 0.029 - - 1 0.078 5.586 P<0.05 7 0.063 4.474 P<0.005 7 0.022 1.546 P>O.10

D.F. square value F(P)

24 0.014 0.486 P=O.963

0.2% was observed (the 95% confidence interval estimate being 0.054 to 0.082 fewer chiasmata).

The TT line used as the ovule parent in each cross was considered to be the other “treatment” in the two-way analyses. In both the G. mustelinum and G. tomentosum hybrids, the ‘IT line used as the ovule parent does appear to have a n effect on the overall chiasma frequency ( P < 0.0003 and 0.005, respectively, Table 2). The variation from plant to plant within a particular pedigree was not significant ( P = 0.610 and P = 0.963), or at least was no greater than the variation that occurred among different samples from the same plant.

The mean number of chiasmata per multivalent was used in assessing the homology of particular pairs of chromosomes. For each lT line used, two one-way analyses were performed; one compared the ( A D ) l ( A D ) , Tt hybrid to the control, the other compared the ( A D ) ( A D ) Tt hybrid to the same control.

Significant differences were found between the controls and the ( A D ) , ( A D ) , Tt hybrids in four of the eight translocation lines studied (Table 3). Translocation line T1;16 showed a significant difference at the 0.0056 probability level, and the 95% confidence interval estimate of the difference indicates a 4.2 to 4.9% re- duction in the mean number of chiasmate regions per IV in the interspecific hy- brid. Similarly, the other three ‘IT lines had the following P values and percent reduction: (1) T14;23, P = 0.0005, % reduction = 8.6 to 10.5; (2) T19;24, P = 0.0007, % reduction = 3.4 to 3.7; and (3) T20;21, P = 0.0360, % reduction = 5.4 to 7.0. In the comparisons involving G. tomentosum hybrids, none of the differences between controls and hybrids were significant at the 0.05 probability level.

For each PMC analyzed, the regions of the quadrivalent in which a chiasma occurred were also recorded (Table 4). For each region, a x2 analysis was done comparing the frequency of chiasmata in the controls and interspecific hybrids. The tabulated data from which the means and frequencies of Tables 1 and 4 were generated can be found in HASENKAMPF 1979 (APPENDIX). Only three regions were fopnd to have significant reductions. In the comparisons of the ( A D ) ( A D ) , Tt hybrids, a significant difference was found only in T20;22 for the interstitial segments of chromosomes 20 and 22 ( P = 0.0003), In the comparisons of the

ALLOTETRAPLOID COTTON GENOMES 9 79

TABLE 3

Results of comparisons of 2(AD) I Tt controls to (AD) (AD), Tt and 20 (AD) I (AD) Tt interspecific hybrids using one-way analysis of variance of mean number chiasmde regions

per ZV and x 2 analysis of frequency of chiasmata per region studied

2(AD), Tt us. ( A D ) , ( A D ) , Tt (A)

One-way analysis of mean no. Xmate regions per IV

x*-analysis of freq. Xma per region __--

Between group Within ~ u p Region with P P Regions ’IT line D.F. Meansq. D.F. Mean sq. Value Value Compared sign. cliff.

T1;16 T2;14 T4;19 T14;23 T15;16 T19;24 T20;21 T20;22

0.1626 0.0011 0.1972 0.9643 0.2653 0.0612 0.2389 0.1853

7 4 4

10 5 4 5 4

0.0105 0.0092 0.0619 0.0374 0.1291 0.0007 0.0295 0.0447

15,5232 0.1234 3.1868

25.7656 2.0540

89.7135 8.101 7 4.1456

0.0056 0.7431 0.1488 0.0005 0.2112 0.0007 0.0360 0.1 114

2(AD), Tt us. ( A D ) , ( A D ) , Tt

none none none none none none none

e+f

One-way analysis of mean no. Xmate regions per IV

xa-analysis of freq. Xma per region - - - - ~

Between group Within group -- F P Regions Region with TTline D.F. Mean sq. D.F. Meansq. Value Value -Pared sign. diff.

T1;16 1 0.0029 10 0.0150 0.1962 0.6672 U , c, b+d,e, f none T2;14 1 0.0002 8 0.0067 0.0341 0.8582 a , c , d , e , f none

T14;23 1 0.0001 9 0.0338 0.0022 0.9636 a+c,b+d,e+f none T15;16 1 0.0803 5 0.0343 2.3413 0.1865 a+c,b+d,e+f none T19;24 1 0.0015 5 0.0005 3.2173 0,1328 a+c,b+d,e+f none T20;21 I 0.0017 8 0.0149 0.1146 0.7436 a+c, b+d,e+f none T20;22 I 0.0064 2 0.0004 16.0000 0.0572 aft, b+d,e+f none

T4;19 1 0.0011 7 0.0633 0.0169 0.9003 U , c ,b ,d , e d, e

( A D ) 1 ( A D ) 3 Tt hybrids, a significant difference was found in the broken arm distal to the breakpoint of chromosome 4 (region d ) and in the interstitial region of chromosome 19 (region e ) ( P = 0.044 and 0.0194, respectively). Region e showed an increase in chiasmata, while region d showed a decrease.

In analyzing the ( A D ) , ( A D ) hybrids, a second small quadrivalent was oc- casionally observed, suggesting the possibility of a naturally occurring difference in end arrangement between G. hirsutum and G. mustelinum. This second quad- rivalent was found in hybrids with all of the translocation lines used in this study. Also, 95 PMC’s were analyzed from two hybrids of a cross of G. hirsutum (line TM-1 standard end arrangements) with G. mustelinum. A quadrivalent was ob- served in approximately 13% of these PMC’s. An example of this quadrivalent is shown in Figure 3D.

980 C. A. H A S E N K A M P F A N D M. Y. M E N Z E L

ALLOTETRAPLOID COTTON GENOMES 98 1

DISCUSSION

A measurable level of genome differentiation is apparent in G. hirsutum X G . mustelinum hybrids. From a nucleus-wide estimate of the mean number of chiasmate I1 arms, a minimum estimate of a 1.8 to 1.9% reduction was obtained. This was confirmed by the even larger differences (3.4 to 10.5%) found by the more sensitive estimates of numbers of chiasmate regions per IV. Another evi- dence of chromosome divergence is the appearance of a quadrivalent in 13% of the PMC’s analyzed of the G. hirsutum (standard end arrangement) X G. mus- telinum hybrids. This suggests that there may be a difference in the chromosome end arrangements of the two species. A second small quadrivalent was occasion- ally seen in interspecific hybrids that combined G. mustelinum with each of the eight TT lines used in this study. Therefore, none of the 12 chromosomes marked by these eight translocations are involved in the naturally occurring translocation. The small size of the IV suggests that it involves at least one of the unmarked D genome chromosomes (17 ,18 ,25 ,26 ) . Since all hybrids were from a single plant of G. mustelinum, it is not known whether this structural arrangement is char- acteristic of the species. Further study of this end arrangement is in progress { H A ~ E N K A M P F , unpublished).

The G. hirsutum X G. tomeMOsum hybrids also differed statistically from the controls in chiasma frequencies. However, the reduction in chiasma frequency was so low (0.1 to 0.2%) that the differences may be of little biological signifi- cance. The more sensitive estimates of the numbers of chiasmate regions per IV showed no reductions.

The very small chiasma reduction observed in the G. hirsutum x G. tomento- sum hybrid suggests that, from the standpoint of recombination, any of the studied chromosomes of G. tomentosum would be an accessible source of genes for ge- netic breeding programs involving G. hirsutum. Even though G. hirsutum x G. mustelinum hybrids exhibit a reduction in chiasma frequency varying from 1.8 to 10.5%, depending on method used and chromosomes tested, these differences are still relatively small. Thus, with the possible exception of the interstitital region of chromosomes 20 and 22, all regions of G. mustelinum tested show a high degree of homology with G. hirsutum. Since genetic exchange still occurs with a fairly high frequency, breeding programs might profitably utilize G. mustelinum as a source of genetic variation.

In a study of flavonoid distributions in Gossypium, PARKS et al. (1975) ob- served that the allotetraploids were very similar to each other, with one major ex- ception: Gossypiun barbadense and G. tomentosum lack the flavonal rutinosides that are present as major constituents in G. hirsutum and G. mustelinum. G. bar- badense is most similar chromatographically to the D genome species G. klotzschi- anum, while G. hirsutum is most similar to G. raimondii. These observations led PARKS and co-workers to speculate that G. klotzschianum contributed the D genome to G. barbadense and G. tomentosum. The present cytological data do not support this interpretation, since the chromosomes of G. tomentosum are more similar to those of G. hirsutum than are those of G. mustelinum.

982 C. A. H A S E N K A M P F A N D M. Y. M E N Z E L

It may be noted that the genome of G. hirsutum appears more similar to that of the Hawaiian wild cotton G. tomentosum than to that of G. mustelinum. The latter is sympatric with G. hirsutum over at least a part of its range in Northeast- ern Brazil. It may be that sympatry has fostered the initiation of genome differ- entiation between these two closely related allotetraploids.

With regard to whether the chromosome divergence found is generalized or localized, the data suggest some degree of localization. In the ( A D ) l ( A D ) , hy- brids, four of the eight translocation lines had a significantly lower mean number of chiasmata per quadrivalent. The eight translocations can be divided into four pairs, each pair having one chromosome in common. That is, chromosomes 14, 1 4 1 9 and 20 were each involved in two different translocations: (1) T1;16 and T15;16, (2) T2;14 and T14;23, ( 3 ) T4;19 and T19;24, and (4) T20;21 and T20;22. In each case, only one of the two showed a reduction in the mean num- ber of chiasmata per quadrivalent in the (AD)1 ( A D ) , hybrids. This suggests that in each pair of cases one of the unshared chromosomes had undergone a measure- able degree of genome differentiation. For example, since T1;16 showed a signifi- cant difference and T15;16 did not, unshared chromosome 1 is a likely candidate for genome differentiation. By this reasoning, chromosomes 1,21,23 and 24 may have undergone some differentiation between G. hirsutun and G. mustelinum. This possibility could be tested further by using translocations that allow identi- fication of specific regions of these four chromosomes.

When the chiasma frequencies for each specific region of the marked chromo- somes were compared, no significant differences were detected in any of the four translocations that had a lower overall chiasma frequency. Failure to detect differences for any of the specific regions of a particular translocation, despite an overall reduction in the number of chiasmata per quadrivalent, may be due to a uniform distribution of the reduction to all regions or to the generally less power- ful nature of the x2 test to reveal small differences, or both.

Another instance of apparent localized differentiation in the ( A D ) ( A D ) hybrids involved the interstitial regions e + f of T20;22, which showed a highly significant and rather large (40%) reduction in chiasma frequency. Among the ( A D ) (AD) hybrids, those with T4; 19 showed localized differences. The inter- stitial region of chromosome 19 (region e) showed an increase in chiasma fre- quency, while the broken arm of chromosome 4 distal to the breakpoint (region d ) showed a decrease. The significance of the observed increase in region e and decrease in region d is unknown; it may be due to random error, since the sample from the control was small ( n = 36).

In conclusion, incipient genome differentiation appears to exist between G. hirsutum and G. mustelinum. Some localized regions of chromosome differentia- tion may be present. However, further studies of other chromosomes and other translocations marking the same chromosomes are needed to assess whether the apparently localized differentiation is a function of specific chromosomes or of the particular translocations studied.

ALLOTETRAPLOID COTTON GENOMES 983

Supported in part by Grant No. DEB77-24476 from the National Science Foundation. The data are from a thesis submitted to Florida State University in partial fulfillment of the require- ments for the M.S. degree. We thank M. S. BROWN and others a t Texas Agricultural Experiment Station for supplying the cotton stocks, KAREN GRAFFIUS, LINDA DERBY, BENVENUTO ANDREAN and GLADYS CHAPMAN for technical assistance, and BETTY BROWN, DR. D. MEETER and DR. P. LACAYO of the F. S. U. Statistical Consulting Center for help with statistical analysis.

LITERATURE CITED

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Corresponding editor: R. L. PHILLIPS

MENZEL, M. Y., M. S. BROWN and S. NAQI, 1978