The use of aneuploid lines significantly increases the effectiveness of molecular-genetic analysis and the development of superior quality breeding lines via substitutions by alien chromosomes. To date, however, a complete set of aneuploid series for each cotton chromosome is not available. Here, we present the development of a monosomic stock collection of cotton (
The cultivated
Cultivated allotetraploid cotton,
In Uzbekistan, independent investigations on creation of cytogenetic lines in
Monosomic lines were developed in a common genetic background of the highly inbred line L-458
Two types of irradiation, thermal neutron irradiation of seeds and pollen
All M1 plants as well as some M2 plant families with multiple seeds were grown under field conditions. All the progeny of abnormal plants with a few seeds from M2 and M3 generations, seeds of desynaptic plants, monosomic translocation heterozygotes, and selfed or outcrossed monosomic plants were germinated on moist filter paper in petridishes at 28°C. Plants were then transplanted into plastic pots with soil. All seedlings were transplanted to greenhouse land soil, and when the first true leaves emerged, transplants were immediately irrigated.
For studies of chromosome pairing at metaphase-I (MI) of meiosis, flower buds were fixed in the morning, after the removal of calyx and corolla, in a solution of 96% alcohol and acetic acid (7 : 3). Buds were kept at room temperature for 3 days then immersed in fresh fixative and stored at 4°C. For cytological preparations, buds were rinsed in tap water before being examined for meiotic associations in the pollen mother cells (PMCs) using the iron acetocarmine squash technique [
Transmission of the monosomes in M4–M6 progenies was studied in selfed or outcrossed populations of the monosomic plants. We studied transmission of monosomes in 33 selfed and 12 outcrossed progenies that are detailed in Table
Between 1987 and 2007, we developed a total of 92
The origin of the cotton primary monosomics from cytogenetic collection developed in Uzbekistan (radiation).
Dose of irradiation (Gy) | Number of primary monosomics* | Monosomic lines** | ||
M1 | M2 | M3 | ||
Irradiation of seeds by thermal neutrons | ||||
15 | 3 | 1 | 0 | |
25 | 0 | 1 | 0 | |
27 | 0 | 1 | 2 | Mo1 |
35 | 1 | 2 | 0 | Mo56, Mo62 |
Total | 4 | 4 | 2 | 3 |
Irradiation of pollen by gamma rays | ||||
10 | 5 | 4 | 2 | Mo10, Mo39, Mo40, Mo50, Mo81, Mo82 |
15 | 4 | 9 | 1 | Mo3, Mo31, Mo53 |
20 | 11 | 8 | 3 | Mo4, Mo7, Mo11, Mo22, Mo27, Mo28, Mo34, Mo35, Mo36, Mo66, Mo75, Mo89 |
25 | 14 | 3 | 0 | Mo9, Mo13, Mo15, Mo16, Mo17, Mo19, Mo38, Mo46, Mo48, Mo76, Mo77 |
Total | 34 | 24 | 6 | 32 |
*A total number of primary monosomics in Table
Chromosome pairing at meiotic metaphase-I observed in PMCs and pollen fertility in the cotton desynaptic parental plants (DPPs) and their monosomics (Mo) progenies.
Material | Mo | Chromosome number | Chromosome associations | Pollen fertility | ||||
Total number of cells | Number of univalents |
Frequency of chromosome | Total number | Fertility, (%) | ||||
univalents | bivalents | |||||||
1609/66-22 | Mo55 | 51 | 22 | 1–3 | 1.45 ± 0.18 | 24.77 ± 0.09 | 466 | 93.99 ± 1.10 |
1609/66-4 | Mo69 | 51 | 33 | 1 | 1.00 ± 0.00 | 25.00 ± 0.00 | 499 | 98.27 ± 0.58 |
1063/63-133 | Mo70 | 51 | 25 | 1–3 | 1.08 ± 0.08 | 24.96 ± 0.04 | 639 | 95.15 ± 0.85 |
1063/63-134 | Mo71 | 51 | 24 | 1 | 1.00 ± 0.00 | 25.00 ± 0.00 | 352 | 96.02 ± 1.04 |
1063/63-135 | Mo72 | 51 | 21 | 1 | 1.00 ± 0.00 | 25.00 ± 0.00 | 632 | 96.87 ± 0.69 |
1063/63-136 | Mo73 | 51 | 30 | 1–3 | 1.47 ± 0.15 | 24.77 ± 0.08 | 612 | 98.53 ± 0.49 |
1570/149-318 | Mo78 | 51 | 18 | 1 | 1.00 ± 0.00 | 25.00 ± 0.00 | ||
1570/149-137 | Mo85 | 51 | 16 | 1 | 1.00 ± 0.00 | 25.00 ± 0.00 | 984 | 97.97 ± 0.45 |
179/212 | Mo87 | 51 | 20 | 1 | 1.00 ± 0.00 | 25.00 ± 0.00 | 217–693 | 40.09 ± 3.33–92.93 ± 0.97 |
356/85 | Mo58 | 51 | 33 | 1–3 | 1.12 ± 0.08 | 24.94 ± 0.04 | 685 | 81.02 ± 1.50 |
356/86 | Mo59 | 51 | 24 | 1 | 1.00 ± 0.00 | 25.00 ± 0.00 | 222–1292 | 2.25 ± 1.00–70.12 ± 1.27 |
356/87 | Mo60 | 51 | 22 | 1 | 1.00 ± 0.00 | 25.00 ± 0.00 | 307 | 38.44 ± 2.78 |
356/88-14 | Mo79 | 51 | 22 | 1 | 1.00 ± 0.00 | 25.00 ± 0.00 | 666 | 92.94 ± 0.99 |
356/88-15 | Mo80 | 51 | 20 | 1 | 1.00 ± 0.00 | 25.00 ± 0.00 | 447 | 99.78 ± 0.22 |
356/88-5 | Mo84 | 51 | 39 | 1–3 | 1.41 ± 0.13 | 24.79 ± 0.07 | 322 | 98.14 ± 0.75 |
356/88-2 | Mo91 | 51 | 19 | 1–3 | 1.11 ± 0.11 | 24.95 ± 0.05 | 65–726 | 53.85 ± 6.18–89.81 ± 1.12 |
DPP: desynaptic parental plants showed in bold-faced letters.
Examples of monosomic plants along with original parental line: (a) control plant L-458; (b) Mo71; (c) Mo80; (d) Mo16. Meiotic configuration of these plants was shown in Figure
Similar analyses of cotton plants from seed irradiation with thermal neutrons at doses of 15, 25, 27, and 35 Gy revealed fewer primary monosomics. There were only 10 plants from three generations studied after irradiation; moreover, four of them were also interchanged heterozygotes. In the M1 generation, there were only 4 chromosome-deficient plants, 3 of them from the 15 Gy dose and one from the 35 Gy dose. Similarly, 4 monosomic plants were isolated in M2 generation in all 4 doses and two monosomic plants were identified from M3.
Previous results demonstrated that pollen
In addition to traditional radiation-induced cotton monosomics, we used the desynaptic effects which have been found to be a useful source of aneuploidy in other crops. Although desynaptic plants in different crops are usually sterile or show extremely low fertility, the desynaptic cotton plant 1063/63-13 had semisterile pollen due to different numbers of unpaired univalents (from 2 to 28) in PMCs. This desynapsis level may be regarded as intermediate. Another five desynaptic plants were characterized with weak desynapsis level and formed from 2 to 12 univalents. As a result, 16 primary monosomics were isolated from the progenies of 6 desynaptic plants and one unexamined plant from the desynaptic plant progeny (Table
All the initial desynaptic plants differed by their number of unpaired chromosomes (from 2 to 28 univalents). Disruptions in unpaired chromosome disjunction led to a random univalent distribution between the cell division poles, forming numerous tetrads with micronuclei (to
It should be noted that the use of an asynaptic genotype in tobacco [
Meiotic metaphase-I analysis of 92 cotton primary monosomics revealed modal chromosome pairing with 25 bivalents and one univalent in 38 plants. Forty-nine monosomic plants were characterized with the presence of additional univalents. Thus, in 32 monosomics, the formation of three univalents in some PMCs was observed due to lack of pairing of single pair of chromosomes. Three monosomics formed five univalents in some PMCs suggesting the absence of pairing in two chromosome pairs. Another five monosomics were characterized with the presence of unpaired chromosomes in 20–30% PMCs. In 8 chromosome-deficient plants, a strong desynaptic effect was detected as they formed from 3 to 11 univalents in 40–60% PMCs studied. In the monosomic plant Mo52, detected in M2 generation after pollen
Results reported on wheat [
In seven of our monosomes (Mo6, Mo7, Mo19, Mo30, Mo56, Mo61, and Mo62), besides univalents and bivalents, rare trivalents (from
Preliminary evaluation of subgenome assignment showed that translocations in two monosomic plants (Mo9; Mo22) might have been of the At-genome because of their large quadrivalents. Three other monosomics had small quadrivalents (Mo1, Mo54, and Mo63) that apparently originated from the Dt-genome. The remaining chromosome-deficient plants had quadrivalents of medium size, and their subgenome localization should be determined by genome analysis using the D-genome diploid plants. Ten monosomics with simultaneous translocation heterozygosity were characterized with low quadrivalent frequency, and only two of them (Mo23 and Mo24) had high quadrivalent frequencies (to
Analyses of the sizes of monosomes revealed medium univalent size in 43 monosomics (Figure
Meiotic configurations in primary monosomics in cotton
Analysis of tetrads was carried out for 87 primary monosomics of our collection. Most of the monosomics (73 or 83.91%) had a higher meiotic index (more than 90%) than that of the control plants (
Analyses of tetrads and pollen fertility in cotton primary monosomics (Mo) with reduced meiotic index.
Mo | Dose of irradiation, (Gy) | Microsporocytes | Pollen fertility | |||
Total number of microsporocytes | Meiotic index* | Tetrads with micronuclei, % | Total number of pollen grains | Fertility, % | ||
Mo4 | 20 | 1777 | 68.32 ± 1.10 | 6.87 ± 0.60 | 475 | 93.47 ± 1.13 |
Mo6 | 20 | 1654 | 87.85 ± 0.80 | 2.48 ± 0.38 | 207 | 74.40 ± 3.03 |
Mo8 | 15 | 695 | 80.29 ± 1.51 | 2.01 ± 0.53 | 593 | 61.72 ± 2.00 |
Mo16 | 25 | 2110 | 76.07 ± 0.93 | 21.56 ± 0.90 | 185 | 5.43 ± 1.67 |
Mo21 | 15 | 2129 | 89.24 ± 0.67 | 5.87 ± 0.51 | 387 | 18.58 ± 1.98 |
Mo23 | 20 | 765 | 88.76 ± 1.14 | 8.37 ± 1.00 | 132 | 81.82 ± 3.36 |
Mo28 | 20 | 858 | 87.18 ± 1.14 | 2.10 ± 0.49 | 450 | 70.67 ± 2.15 |
Mo34 | 20 | 1981 | 88.54 ± 0.72 | 2.68 ± 0.36 | 358 | 46.23 ± 2.64 |
Mo37 | 20 | 1317 | 81.85 ± 1.06 | 0.91 ± 0.26 | 185 | 82.16 ± 2.81 |
Mo52 | 15 | 1326 | 89.00 ± 0.86 | 2.56 ± 0.43 | 135 | 72.60 ± 3.84 |
Mo57 | 15 | 2684 | 87.15 ± 0.65 | 3.95 ± 0.38 | 325 | 0 |
Mo74 | 15 | 2295 | 89.32 ± 0.64 | 4.44 ± 0.43 | 340 | 0 |
Mo88 | 27 | 972–1785 | 47.94 ± 1.60/95.52 ± 0.49 | 0.62 ± 0.25/0.22 ± 0.11 | 983 | 91.96 ± 0.87 |
Mo90 | 20 | 203–2068 | 12.32 ± 2.31–98.69 ± 0.25 | 6.90 ± 1.78–0 | 2326 | 93.98 ± 0.49 |
L-458 | 0 | 2190 | 95.11 ± 0.46 | 1.42 ± 0.25 | 3200 | 96.44 ± 0.33 |
L-458 is original parental (control) genotype; meiotic index is a percentage of normal tetrads in all sporad.
Transmission of the monosomes in the progenies of cotton monosomics (Mo) under greenhouse conditions.
Mo | Total no. of plants | No. of studied plants | Disomics (26II) | Monotelodisomics (25II+1t) | Monosomics (25II+1I) | Transmission (%) | No. of progenies |
---|---|---|---|---|---|---|---|
Transmission of monosomes studied in outcrossed progenies | |||||||
(Mo1)* | 6 | 6 | 5 | 0 | 1 | 16.67 | 1 |
(Mo2) | 6 | 6 | 5 | 0 | 1 | 16.67 | 1 |
Mo3 | 29 | 24 | 23 | 0 | 1 | 4.17 | 3 |
Mo4 | 45 | 29 | 27 | 0 | 2 | 6.9 | 2 |
(Mo10) | 7 | 7 | 6 | 0 | 1 | 14.29 | 2 |
Mo11 | 52 | 34 | 24 | 0 | 10 | 29.41 | 4 |
Mo13 | 27 | 21 | 18 | 0 | 3 | 14.29 | 3 |
Mo15 | 44 | 42 | 41 | 0 | 1 | 2.38 | 5 |
(Mo28) | 5 | 5 | 4 | 0 | 1 | 20.00 | 1 |
Mo56 | 26 | 26 | 24 | 0 | 2 | 7.69 | 4 |
Mo63 | 19 | 13 | 11 | 0 | 2 | 15.38 | 2 |
(Mo74) | 12 | 7 | 6 | 0 | 1 | 14.29 | 1 |
Transmission of monosomes studied in selfed progenies | |||||||
Mo7 | 29 | 19 | 14 | 0 | 5 | 26.32 | 2 |
Mo9 | 48 | 34 | 32 | 0 | 2 | 5.88 | 3 |
Mo16 | 22 | 18 | 10 | 0 | 8 | 44.44 | 3 |
Mo17 | 33 | 31 | 24 | 1 | 6 | 19.35 | 9 |
Mo19 | 38 | 31 | 24 | 1 | 6 | 19.35 | 4 |
(Mo27) | 9 | 9 | 7 | 0 | 2 | 22.22 | 2 |
Mo31 | 25 | 25 | 16 | 0 | 9 | 36.00 | 5 |
Mo35 | 24 | 23 | 21 | 0 | 2 | 8.70 | 3 |
Mo38 | 17 | 17 | 14 | 0 | 3 | 17.65 | 2 |
Mo40 | 33 | 33 | 32 | 0 | 1 | 3.03 | 2 |
Mo42 | 30 | 25 | 21 | 0 | 4 | 16.00 | 2 |
(Mo48) | 11 | 11 | 9 | 0 | 2 | 18.19 | 3 |
Mo50 | 37 | 26 | 20 | 0 | 6 | 23.07 | 3 |
Mo60 | 16 | 14 | 10 | 0 | 4 | 28.57 | 3 |
Mo61 | 41 | 18 | 16 | 1 | 1 | 5.56 | 3 |
Mo62 | 61 | 24 | 17 | 0 | 7 | 29.17 | 4 |
Mo66 | 31 | 31 | 20 | 0 | 11 | 35.48 | 3 |
Mo67 | 40 | 35 | 32 | 0 | 3 | 9.38 | 4 |
Mo69 | 18 | 18 | 13 | 0 | 5 | 27.78 | 2 |
Mo70 | 18 | 18 | 15 | 0 | 3 | 16.67 | 3 |
Mo71 | 20 | 20 | 13 | 0 | 7 | 35.00 | 2 |
Mo72 | 23 | 23 | 16 | 0 | 7 | 30.43 | 2 |
Mo73 | 28 | 24 | 18 | 0 | 6 | 25.00 | 2 |
Mo75 | 35 | 15 | 11 | 0 | 4 | 26.67 | 3 |
Mo76 | 31 | 18 | 14 | 0 | 4 | 22.22 | 4 |
(Mo77) | 22 | 10 | 6 | 0 | 4 | 40.00 | 1 |
Mo79 | 31 | 22 | 16 | 0 | 6 | 27.27 | 3 |
Mo80 | 48 | 20 | 17 | 0 | 3 | 15.00 | 3 |
Mo81 | 21 | 12 | 9 | 0 | 3 | 25.00 | 2 |
(Mo82) | 17 | 11 | 7 | 0 | 4 | 36.36 | 2 |
Mo84 | 31 | 18 | 10 | 0 | 8 | 44.44 | 2 |
Mo85 | 48 | 26 | 25 | 0 | 1 | 3.85 | 2 |
Mo89 | 47 | 21 | 17 | 0 | 4 | 19.05 | 3 |
Meiotic index decrease in 6 monosomics (Mo16, Mo28, Mo52, Mo74, Mo88, and Mo90) could be explained with the presence of additional univalents at meiotic metaphase-I. In contrast, meiotic index decrease in 4 monosomics (Mo8, Mo21, Mo23, and Mo57) was connected with simultaneous translocation heterozygosity that led to chromosome disjunction disturbances and the production of tetrads with micronuclei. However, meiotic index decrease in 3 monosomics with the modal chromosome pairing (Mo4, Mo34, and Mo37) and increase of number of tetrads with micronuclei in Mo4 (to
Monosomic plants with a reduced meiotic index also differed from each other by pollen fertility. Complete pollen sterility was recorded in two monosomics (Mo57 and Mo74); however, partial female fertility provided seed set from outcrossing. Such strong variability of pollen fertility in the monosomics with reduced meiotic index was explained with both cytogenetic status influence and specific radiation action especially in 8 monosomics isolated from M1 generation after irradiation.
Pollen fertility after acetocarmine staining was studied in 90 primary cotton monosomics isolated mainly from different types of irradiation. Although the acetocarmine-based pollen fertility considered relatively insensitive method, it is widely used (i.e., see [
Percentage distribution of pollen fertility for 79 cotton monosomic plants. Note: the remaining 11 monosomics were not included in the histogram due to their varied pollen fertility level in different flowers.
Taken together, more than half of parental monosomics had partial or complete pollen sterility. After irradiation in M1 generation, pollen grain overabortion might be the result of radiation physiologic effect. However, transmission of the character to the next generations in daughter monosomics of higher generations (M4–M6) was obviously connected with gene(s) responsible for pollen development located in the monosome chromosomes (Mo22, Mo39, and Mo46). In due course, the abortive pollen detection in 6 monosomic lines suggested a polygenic control of pollen development in cotton
We used the individual seed weight method to facilitate monosome cytotype detection. According to Douglas’ opinion [
The reproduction of the monosomics was studied in the selfed and outcrossed progenies under field and greenhouse conditions. Comparative analysis of the cotton monosomics reproduced in the field revealed stronger morphological differences in comparison with disomic sibs as well as monosomics reproduced in the greenhouse. As a result, monosomics were reproduced in 18 progenies under field conditions. However, because of limited vegetation period, we did not manage to analyze most of the progenies and determine exact transmission frequency in the field. Eleven of them were reproduced later under greenhouse condition whereas 7 monosomics (Mo22, Mo34, Mo36, Mo39, Mo45, Mo46, and Mo53) have not been reproduced at this time. Progenies of 77 different monosomics were studied in the greenhouse. Table
Similarly, the rate of the transmission of the haplo-deficient gamete varied from low (4%) in a monosome 9 to high (49%) in a monosome 4 from the USA Cytogenetic Collection and 3 monosomics (for chromosomes 3, 9, and 16) usually transmitted with the lowest frequencies [
Deficiencies for one chromosome arm occurred in the progenies of seven monosomics. Thus, in three monosomic progenies (Mo17, Mo19, and Mo61) that differed with respect to monosome transmission rates (Table
High heteromorphic bivalent frequency was observed in all of the six monotelodisomics (to
Meiotic metaphase-I in monotelodisomic plant from progeny Mo21, showing 25 normal bivalents and monotelodisomic bivalent (including one normal chromosome plus one telosome). The arrow indicates the monotelodisomic bivalent. Note that the background of figures was cleaned using Adobe Photoshop CS 2 version 9.0.
Transmission in the progenies of 12 translocation heterozygous monosomics revealed daughter monosomic plants in only 6 progenies. Moreover, in two of them (Mo9 and Mo22), the daughter monosomics had no quadrivalents, and one progeny (Mo61) had heterozygous translocation, whereas two monosomics from the Mo63 and Mo54 progenies were translocation homozygotes. There were quadrivalent associations in F1BC1 hybrids from the crosses with standard line L-458. This suggests a positive role of the chromosome interchanges in these monosomics because of a selective advantage of the gametes containing single-chromosome deficiencies and the interchange between two other chromosomes in hetero- and homozygous conditions, respectively.
According to a transmission study in 12 other monosomic families, disomic plants with desynaptic effect were detected due to desynapsis in parental plants and spontaneous desynaptic gene mutations in parental plants. Thus, analysis of monosomic progenies under field and greenhouse conditions provided daughter monosomic reproduction in 52 different families. However, the detection of other aberrations in 6 of them requires reanalysis of their progenies to isolate monosomics without any other karyotype disturbances.
The following selective behavior features were revealed in the cotton monosomics studied: karyotypic heterogeneity of progenies due to production of gametes with
We developed a new set of cotton monosomic stocks through radioactive irradiation of single genotype of L-458 cotton line. The results demonstrated detection of new unique desynaptic cotton plants in which progeny produced monosomics with high frequency. We observed the very rare occurrence of univalent misdivisions because of monosome stability in the unique genetic background. Our results demonstrate that light seed weight is not a universal marker for monosomy in cotton, and we detected possible univalent shifts in three monosomic progenies. Our observations with the development of reproductive organs of some monosomic plants suggested chromosome localization of genetic factors that control male gametophyte viability in the deficient chromosomes. Chromosomal identification of these new monosomic cotton stocks, using modern genetics methods (e.g., [
This work was partially supported by research Grants nos. 38/96, 28/98, 26/2000, and F.4.1.15 from the Committee for Science and Technology of the Republic of Uzbekistan. The authors thank Dr. Ian Dundas, University of Adelaide, Australia, and Dr. Masoud Sheidai, Shahid Beheshti University, Tehran, Iran for their critical reading of the paper and suggestions. They also thank Dr. Eric J. Devor, Iowa State University, USA, and Dr. Johnie N. Jenkins, USDA-ARS, Mississippi, USA, for their critical review and edition of the paper.