Origin and some characters of the cotton monosomic lines of cotton
Simple sequence repeats (SSR) have been applied as useful markers for understanding cotton genetics. In the last decade, chromosome-deficient stocks of Gossypium hirsutum L. were used in the development of chromosome substitution lines for G. barbadense L., G. tomentosum Nuttall et Seemann, and G. mustelinum Watt chromosomes or chromosome segments. Several DNA markers have already been assigned to the individual chromosomes of G. hirsutum. We created new cotton monosomic lines in Uzbekistan after irradiation of seeds by thermal neutrons or pollen gamma‐irradiation to complement other global efforts in the development of cotton chromosome substitution lines. The primary objective of this chapter is to report the use of chromosome-specific SSR markers and a well-defined tester set of cotton translocation lines from the Cotton Cytogenetic Collection at Texas A&M AgriLife Research to confirm chromosome specificity of monosomic lines in Uzbekistan cytogenetic collection of cotton. Our results have assigned several different monosomic lines to the chromosomes 2, 4, 6, and telosome 11 At‐subgenome and chromosomes 18 and 20 or 22 Dt‐subgenome. These lines will be very useful in molecular mapping, the creation of substitution lines, and cotton breeding.
- cotton (Gossypium hirsutum L.)
- molecular markers
Monosomic plants can be used for the synthesis of the chromosome substitution (CS-B) lines . Such substitution lines are a useful means of interspecific introgression and for studying economically important genes. In upland cotton (
About 10 years ago, replacement and additional
Simple sequence repeats (SSR) of cotton have been applied widely as molecular markers for genetics, mapping, trait analysis, and germplasm diversity analysis and comparisons of specific individuals, lines, and populations. Several SSR markers associated with lint percentage have been located to chromosomes 12, 18, 23, and 26 using deletion analysis in aneuploid chromosome substitution lines. SSRs were used to analyze the genetic identities of specific hypoaneuploid interspecific F1 hybrids and disomic chromosome substitution intermediates genotypes, using the principles of deletion molecular analysis [10, 11]. Recent reports extended the systematic characterizations of additional hypoaneuploid chromosome substitution F1 hybrids and many chromosome substitution lines using chromosome-specific SSR-markers [6, 7].
A Cotton Cytogenetic Collection of Uzbekistan now includes 95 primary monosomics derived from the highly inbred
The primary objective of this chapter is to report the use of chromosome-specific SSR markers and a well-defined tester set of translocation lines of the USA collection to confirm chromosome specificity of monosomic lines in Uzbekistan collection.
2. Materials and methods
2.1. Plant material
Monosomic lines of the Uzbek Cytogenetic collection were developed in a common genetic background of the highly inbred line
A well-defined tester set of translocation lines of Cytogenetic Collection of the USA was kindly provided by Prof. D.M. Stelly through a USDA-Uzbekistan cotton germplasm exchange program. All plant materials were vegetatively maintained in the greenhouse of National University of Uzbekistan.
2.2. Cytological analyses
For studies of meiotic chromosome pairing at metaphase-I (MI) and sporad normality floral buds were collected in the morning, and after the removal of calyx and corolla, fixed 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 MI chromosome configurations in the microsporocytes, commonly known as “pollen mother cells” (PMCs) using the iron acetocarmine squash technique. Analyses of hybrid plant chromosomes were carried out on the basis of MI configurations. The development of F1 hybrid plant PMCs was assessed based on the cytological features observed at the tetrad stage. The meiotic index was calculated as the percentage of sporads that were normal tetrads. Pollen viability was estimated as percentage of mature pollen grains stainable in acetocarmine. All cytological observations were made using Biomed (Leica, Heerbrugg, Switzerland) and Axioskop A1 (Carl Zeiss, Germany) microscopes.
2.3. Identification of hybrid F1 monosomic translocation heterozygotes
Monosomes were chromosomally identified using translocation tests. For this purpose, the monosomic (2
2.4. DNA extraction and genotyping
Genomic DNAs were extracted from young leaf samples of cytogenetically identified F1 monosomic cotton hybrids using CTAB method . Extracted genomic DNAs were checked in 0.9% agarose electrophoresis, and DNA concentrations were diluted to a working concentration of 15 μl based on Hind III-digested λ-phage DNA 25 ng/μl. PCR amplification carried out in a 10 μl volume reaction mix containing 1.0 μl 10× PCR buffer, (with 25 mM Mg Cl2), 0.2 μl BSA, 0.08 μl dNTPs (25mM), 0.2 μl primer pairs, 0.1 μl Taq-polymerase, and 2 μl DNA template. PCR runs were conducted with an initial denaturation of DNA at 94°C for 2 min, followed by 35 cycles of 94°C (step 1) for 20 sec, 55°C (step 2) for 30 sec, and 72°C (step 3) for 50 sec. After 35 cycles, the extension temperature of 72°C was held for 7 min. The PCR products were visualized in 3.5% high-resolution agarose gel, stained with ethidium bromide and photodocumented using an Alpha Imager (Innotech Inc., USA) gel documentation system.
Chromosome-specific SSR primer pairs were collected according to recently published genetic mapping papers [11, 19–27]. For each chromosome, we chose four loci that were polymorphic between L-458 (
3.1. Cytogenetic characteristics of the aneuploid hybrid F1 plants
We crossed 46 monosomic and two monotelodisomic lines of
Hybrid F1 plants had intermediate phenotypes, including strong effects on plant shape and size; clustering (flower positions and density); number, sizes and shapes of leaves, bracts, and stipules; sizes and number bract teeth; coloration; number and sizes of external nectarines; and boll shape. Additional phenotypic effects by monosomy were super-imposed on these general phenotypic effects of interspecific hybridity.
Meiotic metaphase-I analysis of 56 monosomic hybrid F1 plants revealed that 46 plants exhibited modal chromosome pairing of 25 bivalents and one univalent. Seven monosomic hybrid F1 plants were characterized with the presence of additional univalents and their average univalent frequencies per cell among them ranged from 1.06 ± 0.06 in Mo67xPima 3-79 to 1.22 ± 0.15 in Mo11xPima 3-79. Three univalents were observed in some PMCs of the seven monosomic hybrid F1 plants from Mo7, Mo11, Mo27, Mo67, Mo72, Mo75 and Mo89. Three monosomic hybrid F1 plants from families Mo17xPima 3-79 formed three univalents in many PMCs, and they accordingly exhibited relatively high average univalent frequencies per cell, which ranged from 1.24 ± 0.13 to 1.45 ± 0.15. The similarity of relative univalent sizes across the affected cells (24 II + 3 I) suggested that the extra pair of univalents in all of these cells arose from the same pair of chromosomes. A similar effect was noted in the parental monosomic plant in selfed Mo17 progenies, and could indicate that Mo17 is affected by more than simple monosomy. The three monosomic hybrid F1 plants (Mo17xPima 3-79) also seemed to differ in their morphology, which suggests the possibility that one or more of them arose following a univalent shifts due to irregular chromosome disjunction in meiosis of the maternal Mo17 parent.
Meiotic products were characterized by examining sporads in most of the monosomic hybrid families, looking for example for the frequency of normal tetrad conformation. Meiotic index, originally proposed by Love  for evaluation of meiosis in wheat, reports the normal tetrad percentage and is an indicator of meiotic stability. Most of monosomic hybrid F1 plants had a higher meiotic index (more than 90%) than that of the control hybrid F1 plants (L-458xPima 3-79—89.25 ± 0.58%), which indicated that their univalent chromosome underwent regular disjunction. However, the meiotic index values of four of the monosomic hybrid F1 plants (Mo48xPima 3-79, Mo59xPima 3-79, Mo62xPima 3-79, Mo92xPima 3-79) were characterized ranging from 89.78 ± 0.50% to 85.96 ± 1.29%. Two monosomic hybrid F1 plants showed an increase of percentage of tetrads with micronuclei to 4.41 ± 0.45% (Mo59xPima 3-79) and 4.53 ± 0.37% (Mo92xPima 3-79) in comparison with control hybrid F1 plants (L-458xPima 3-79—1.02 ± 0.06%), which demonstrated disturbances in monosome disjunction and formation of imbalanced gametes.
Pollen viability after acetocarmine staining was studied in monosomic hybrid families. Pollen viability was high in most monosomic hybrid F1 plants. However, nine monosomic hybrid F1 plants had reduced pollen viability and showed early haplo-deficient microspore abortion prior to mature pollen stage. Two monosomic hybrid F1 plants had small reductions in pollen viability (up to 20%), four monosomic hybrid F1 plants had more reductions in pollen viability (up to 30%), and three monosomic hybrid F1 plants had reductions in semisterile pollen (up to 50%). Thus, some of monosomic hybrid F1 plants had decreased meiotic index and decreased pollen viability.
3.2. Molecular marker analysis
For molecular analysis of the monosomic hybrid F1 plants, the principles of deletion molecular analysis were used [10, 11]. Considering that many DNA markers have already been assigned to the individual chromosomes of
Our results revealed that four monosomic hybrid F1 plants (Mo11xPima 3-79, Mo16xPima 3-79, Mo19xPima 3-79, and Mo93xPima 3-79) deficient for unknown chromosome(s) showed the presence of only
SSR-based deficiency analysis of the six monosomic hybrid F1 plants (Mo70xPima 3-79, Mo71xPima 3-79, Mo76xPima 3-79, Mo81xPima 3-79, Mo89xPima 3-79, and Mo90xPima 3-79), each deficient for an unknown chromosome showed the presence of only
Further, our results revealed that two monosomic hybrid F1 plants (Mo13xPima 3-79 and Mo67xPima 3-79), each of which deficient for unknown chromosome, showed differential presence of polymorphic
Using the similar approach, monosomic line Mo48 was assigned to the chromosome 18 of the Dt-subgenome of cotton based on polymorphism of BNL3280 marker from chromosome 18. Monosomic line Mo17 was assigned to the chromosome 20 or 22 of the Dt-subgenome using chromosome-specific SSR marker—JESPR235 genotyping experiment with monosomic F1 substitution hybrids (Mo17x Pima 3-79) [19, 26], because of this SSR marker was assigned to these two chromosomes.
A monotelodisomic line recovered among progeny of Mo21 and was identified using the chromosome-specific SSR markers—BNL3442, Gh246, CIR212 that were polymorphic monosomic F1 substitution hybrids (Telo21x Pima 3-79). This result suggested that monotelodisomic line Telo21 is chromosome arm 11 of the At-subgenome of cotton because these SSR markers are from the arm of chromosome arm 11 of cotton [11, 19–27].
Thus, we identified chromosomes specificities for 24 monosomic and one monotelodisomic line using F1 substitution hybrids and chromosome-specific SSR markers. Results demonstrated the differential rates of monosome occurrence and/or recovery among the cotton chromosome complement. In our Uzbekistan-based experiments, chromosome 2, 4, 6, 11, 18, and 20 or 22 were recovered as monosomic/monotelodisomic individuals. The identities of 11 additional F1 substitution hybrids have yet to be identified, namely Mo1, Mo4, Mo9, Mo28, Mo39, Mo50, Mo62, Mo82, Mo84, Mo91, and Mo92. Individual cotton chromosome deficiency had a specific influence in plant morphology and some characters such as bolls (Figure 4).
For instance, monosomic lines of the chromosome 2 At-subgenome including Mo11, Mo16, Mo19, and Mo93, all have a similar phenotypic syndrome that includes features such as dwarf plant architecture; small, narrow, and dense leaf; short sympodia and small and round bolls.
Monosomic lines of the chromosome 4 At-subgenome including Mo7, Mo31, Mo38, Mo59, Mo66, Mo69, Mo70, Mo71, Mo72, Mo73, Mo75, Mo76, Mo79, Mo81, Mo89, and Mo90, all have such characters as bushy plant; leaves usually with wavy margins; long peduncle and bolls; and long bolls. Monosomic lines of the chromosome 6 At-subgenome including Mo13 and Mo67—both have bushy plant; short sympodia; small and rounded bolls; and late flowering (Figure 5).
Monosomic plants deficient for a copy of chromosome 11 of the At-subgenome (Mo21) have dwarf plant architecture; short sympodia; small leaves; and small and defectiveed bolls with curved apical tips. Monosomic plants deficient for a copy of chromosome 18 of the Dt-subgenome (Mo48) have dwarf plant architecture, short sympodia, small leaves and bolls that are smaller and rounder. Monosomic plants deficient for a copy of chromosome 20 or 22 of the Dt-subgenome (Mo17) have dwarf plant architecture; small leaves; short sympodia; small, defective, unsymmetrical bracteoles; late flowering and set fewer seed.
3.3. Identification and numeration of the unknown monosomes
We have begun a new effort to identify cotton monosomic lines of the Uzbek cytogenetic cotton collection using a well-defined tester set of translocation lines of the cytogenetic collection of the USA, kindly provided by Prof. D.M. Stelly, Texas A&M University, USA, through USDA–Uzbekistan cotton germplasm exchange program. We performed numerous sexual crosses between monosomic lines of our collection and tester-translocation lines, but some monosomic lines were distinguished by low crossing characteristics and few seed set. Monosomes were identified by analyzing meiotic metaphase I configurations of monosomic translocation heterozygous F1 hybrids. When monosome of such a hybrid involves neither of the translocated chromosomes, each metaphase I cell typically includes a quadrivalent and a monosome (23 II + 1 IV + 1 I). In contrast, when the monosome involves one of the translocated chromosomes, a trivalent typically occurs (24 II + 1 III) .
Cytological analyses of the two hybrid combinations involving two tester set lines (Mo19xTT6L-7L, Mo19xTT10R-11R) showed that monosome Mo19 is not chromosome 6, 7, 10, or 11 because the modal MI pairing configuration in the respective monosomic F1 hybrids included 23 II + 1 IV + 1 I. In contrast, the tests with translocation lines TT2L-6R and TT2R-8Rb showed that the monosome Mo19 is chromosome 2, because the modal MI pairing configurations in the monosomic F1 hybrids of Mo19xTT2L-6R and Mo19xTT2R-8Rb M I were 24 II + 1 III (Figure 6).
Since the monosomic line Mo19 had common chromosome 2 in the two translocation lines and it was assigned to chromosome 2 At-subgenome of cotton using SSR markers (BNL3971, BNL3590, and GH-198), we can now confidently tell that the monosome Mo19 from chromosome 2 At-subgenome (Table 1).
The cytological analyses of the two hybrid combinations involving two tester lines (Mo67xTT3R-5R, Mo67xTT9R-25) showed that monosome Mo67 is not chromosome 3, 5, 9, or 25 because the modal MI pairing configuration of its monosomic F1 hybrids was 23 II + 1 IV + 1 I. The test with translocation line TT6L-7L showed, however, that the monosome Mo67 could be from chromosome 6 or 7, because in the monosomic F1 hybrid of Mo67xTT6L-7L MI the modal MI pairing configuration was 24 II + 1 III. Molecular marker data suggested that the monosome Mo67 must be chromosome 6 of the At-subgenome (Table 1).
The cytological test of the hybrid combination involving one tester line (Mo75xTT4R-15L) showed that monosome Mo75 could be from chromosome 4 or 15, because in the monosomic F1 hybrid of Mo75xTT4R-15L MI pairing was 24 II + 1 III. Molecular marker data suggested that the monosome Mo75 is chromosome 4 At-subgenome (Table 1; Figure 7).
|Mo11||Pollen irradiation||1991||Medium||A 2||BNL3590, GH-198|
|Mo16||Pollen irradiation||1991||Medium||A 2||BNL3971, BNL3590, GH198|
|Mo19||Pollen irradiation||1991||Large||A 2||BNL3971, BNL3590, GH198|
|Mo93||Pollen irradiation||2007||Medium||A 2||BNL1434, BNL1897, BNL3971|
|Mo7||Pollen irradiation||1990||Medium||A 4||BNL2572, CIR122, CIR048|
|Mo31||Pollen irradiation||1993||Medium||A 4||BNL2572, CIR122, CIR048|
|Mo38||Pollen irradiation||1993||Large||A 4||BNL2572, CIR122, CIR048|
|Mo59||Desynapsis||1996||Medium||A 4||BNL2572, CIR122, CIR048|
|Mo66||Pollen irradiation||1995||Medium||A 4||BNL2572, CIR122, CIR048|
|Mo69||Desynapsis||1997||Medium||A 4||BNL2572, CIR122, CIR048|
|Mo70||Desynapsis||1997||Medium||A 4||BNL2572, CIR122, GH-107|
|Mo71||Desynapsis||1997||Medium||A 4||BNL2572, CIR122, GH-107|
|Mo72||Desynapsis||1997||Medium||A 4||BNL2572, CIR122, CIR048|
|Mo73||Desynapsis||1997||Medium||A 4||BNL2572, CIR122, CIR048|
|Mo75||Pollen irradiation||1999||Medium||A 4||BNL2572, CIR122, CIR048|
|Mo76||Pollen irradiation||2001||Medium||A 4||BNL2572, CIR122, GH-107|
|Mo79||Desynapsis||2002||Small||A 4||BNL2572, CIR122, CIR048|
|Mo81||Pollen irradiation||2003||Medium||A 4||BNL2572, CIR122, GH-107|
|Mo89||Desynapsis||2003||Medium||A 4||BNL2572, CIR122, GH-107|
|Mo90||Pollen irradiation||2003||Small||A 4||BNL2572, CIR122, GH-107|
|Mo13||Pollen irradiation||1991||Large||A 6||BNL1064, BNL2884, BNL3650, CIR203, Gh032,Gh039, Gh082, TMB1538|
|Mo67||Translocation||1996||Large||A 6||BNL1064, BNL2884, BNL3650, CIR203, Gh032,Gh039, Gh082, TMB1538|
|Telo Mo21||Pollen irradiation||1991||Large||A 11||BNL3442, GH246, CIR212|
|Mo48||Pollen irradiation||1994||Small||D 18||BNL3280|
|Mo17||Pollen irradiation||1991||Medium||D 20 or 22||JESPR235|
Thus, out of one monotelodisomic and 24 monosomic lines identified using chromosome-specific SSR markers, three monosomes were confirmed by means of the translocation test. From 24 identified monosomic lines, four are chromosome At-2, 16 are chromosome At-4, two are chromosome At-6, and one each is chromosome Dt-18, chromosome Dt- 20 or 22 and telosome At-11.
The utilization of the microsatellite markers and translocation lines for the identification of unknown cotton monosomic lines in our study provided the unified identification and numeration of the chromosomes and chromosome arm for 24 aneuploid lines of the Uzbek cytogenetic collection of cotton. On the basis of molecular-genetic and cytogenetic tests involving translocations, three of the six monosomes were designated chromosomes 2, 4, and 6 of the At-subgenome and two chromosomes were designated chromosomes 18 and 20 or 22 Dt-subgenome. One telosome was designated as a telosome 11 At-subgenome.
Twenty other monosomes were identified as duplicates of three of aforementioned monosomes (chromosomes 2, 4, and 6). Chromosome 4 of the At-subgenome was recovered more frequently (16 times), than chromosome 2 of the At-subgenome (4 times) and chromosome 6 of the At-subgenome (2 times) during the pollen irradiation in different doses and in progeny of the desynaptic plants (Table 1). The other chromosomes were recovered only once (chromosomes 18 and 20 or 22 Dt-subgenome and one telosome as a telosome 11 At-subgenome). These data demonstrated differences between the studied chromosomes on the response to irradiation treatments, and/or differences in their ability to survive as embryos and plants, and/or their ability to be maternally transmitted through nullisomic maternal gametes and gametophytes. It is possible that the centromeric region of the cotton chromosome 4 is more susceptible to treatments, and/or this chromosome is under less severe selection against monosomy and thus deficiencies are recovered and transmitted more frequently in the natural and irradiated populations. Identification of other monosomic lines using similar strategy is in progress in our laboratories.
According to our previous results, two monosomics lines (Mo11 and Mo19) from our collection may be involved the same monosome and were homologous because they had chromosomal associations with the same translocation line Tr16 of Uzbek cytogenetic collection [12, 15].
There are many challenges in genetic and molecular-genetic analyses of the complex genomes of cotton [5, 30–32], and the development of novel aneuploid lines for tetraploid cotton will be important to solve mapping of molecular markers in the cotton genome. Our results, which are largely based on radiation-induced aneuploidy, paralleled the findings of Endrizzi et al.  showed that chromosomes 4 and 6 are the two chromosomes that occur most frequently as spontaneous monosomes in natural populations of cotton. A challenge in attaining complete genome coverage, that is, monosomes for all chromosomes may be that some chromosomes may contain genes that are unique and essential to the zygote, plant, functional megaspore or megagametophyte. For these chromosomes, it may not possible to recover or sexually utilize monosomes; for others, the difficulty is likely to range from relatively easy to challenging.
The results presented in this report suggest that microsatellite markers are facile and useful tools for cytogenetic analysis of the cotton chromosome deficient plants. The utilization of the microsatellite markers for the identification of unknown cotton monosomic lines provided an opportunity to foster the identification and numeration of the chromosomes and chromosome arm for 24 aneuploid lines of the Uzbekistan cytogenetic collection of cotton. These markers can also be readily used for the development of new cotton chromosome substitution lines and germplasm introgression.
This work was partially supported by research Grants F-5-31 and KA-8009 from the Committee for Science and Technology of the Republic of Uzbekistan. We thank the administration of Center of Genomics and Bioinformatics, Academy of Science of Uzbekistan for the continued support of collaborative research and being an instrumental for the molecular experiments conducted in this study.