The Utilization of Translocation Lines and Microsatellite Markers for the Identification of Unknown Cotton Monosomic Lines The Utilization of Translocation Lines and Microsatellite Markers for the Identification of Unknown Cotton Monosomic Lines

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 A t ‐subgenome and chromosomes 18 and 20 or 22 D t ‐subgenome. These lines will be very useful in molecular mapping, the creation of substitution lines, and cotton breeding. for Mo11), (BNL3971, BNL3590, and GH‐198 for Mo16 and Mo19), (BNL1434, BNL1897, and BNL3971 for Mo93), and corresponding absence of the respective L‐458 allele. The results helped reveal the chromosomal identities of monosomes Mo11, Mo16, Mo19, and Mo93 based on known chromosomal locations of the respective SSR markers. Because the aforementioned SSR markers were assigned previously to chromosome 2 of the A t ‐subgenome of cotton [11, 19–


Introduction
Monosomic plants can be used for the synthesis of the chromosome substitution (CS-B) lines [1]. Such substitution lines are a useful means of interspecific introgression and for studying economically important genes. In upland cotton (Gossypium hirsutum L.), Kohel et al. [2] studied multiple traits of disomic substitution lines that contained chromosomes 6 or 17 of G. barbadense (L.).
About 10 years ago, replacement and additional G. barbadense chromosome substitution lines became available. These were developed similarly by hybridization of the same G. barbadense donor line to quasi-isogenic G. hirsutum hypoaneuploids, followed by iterative modified backcrossing and then inbreeding. Each resultant CS-B line was expected to be substituted for one chromosome or segment and to be largely though not necessarily completely devoid of unrelated G. barbadense segments [3,4]. The results revealed information on the association of specific chromosomes with genes for agronomic and fiber traits. More recently, substitution lines were created for G. tomentosum [5] and G. mustelinum [6], as well as for additional chromosomes of G. barbadense [7]. Saha et al. subsequently used the CS-B lines to identify chromosomal locations of important traits and beneficial genes and interactions [3,8,9].
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 F 1 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 F 1 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 G. hirsutum line "L-458" by irradiation of the seeds with thermal neutrons and pollen with gamma-rays [12]. The chromosome identities of these monosomic lines of the Uzbek Collection need to be determined to optimize the efficiency of collection maintenance and its use in breeding-related research, for example, development of 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 translocation lines of the USA collection to confirm chromosome specificity of monosomic lines in Uzbekistan collection.

Plant material
Monosomic lines of the Uzbek Cytogenetic collection were developed in a common genetic background of the highly inbred line G. hirsutum line L-458, which was created through multiple generations of self-pollination (F 20 ) of cultivar 108-F [13,14]. Because all of the 95 primary monosomics were isolated from a common genetic background, some differences observed among them can be attributed to the differences in their monosomic state [15]. Irradiation of seeds by thermal neutrons or pollen gamma-irradiation gave rise to most (76) of the monosomics. Other monosomic plants were detected among progenies of desynaptic plants (17) and translocation heterozygote plants (2). The primary monosomics were numbered according to their order of detection (Mo1-Mo95).
The G. barbadense line 3-79 is nonphotoperiod sensitive and is highly homozygous as it is originated as a doubled haploid [16]. It has been used extensively as parent in genetic studies, genomics [17], and as a donor parent for the substituted chromosome (CS) or chromosome segments from G. barbadense in our study. The overall method of the CS line development was discussed in previous reports [4,18].
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.

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 F 1 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.

Identification of hybrid F 1 monosomic translocation heterozygotes
Monosomes were chromosomally identified using translocation tests. For this purpose, the monosomic (2n = 51) lines from Uzbek Cytogenetic Collection were crossed as seed parent with translocation lines (2n = 52) of the tester set from USA Cotton Cytogenetic Collection provided by Texas A&M AgriLife Research. Floral buds of F 1 hybrid progeny were analyzed to identify individuals that were monosomic (2n = 51) and also heterozygous for the respective translocation, that is, monosomic translocation heterozygotes. To reveal "critical configurations" and detect common chromosomes among the chromosomes involved in interchanges with monosomes, the meiotic MI configurations were analyzed in heterozygotes of monosomic translocations heterozygous F 1 hybrids. Progeny modally forming 23II + IV + I were interpreted as indicating independence between chromosomes affected by monosomy and the translocation, whereas progeny modally forming 24II + III were interpreted as evidence of association between the monosome and one of the two translocation chromosomes. It was generally presumed that parent and progeny monosomic conditions were similar, that is, no monosomic "shift" occurred.

DNA extraction and genotyping
Genomic DNAs were extracted from young leaf samples of cytogenetically identified F 1 monosomic cotton hybrids using CTAB method [18]. 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.

Cytogenetic characteristics of the aneuploid hybrid F 1 plants
We crossed 46 monosomic and two monotelodisomic lines of G. hirsutum from Uzbek cytogenetic collection of cotton and a G. barbadense doubled haploid line Pima 3-79. Among resulting progeny, the aneuploid hybrid F 1 plants were detected in 37 interspecific F 1 progeny families based on hybrid phenotypes and meiotic metaphase-I configuration analyses. In five of the aneuploid hybrid F 1 families, three hybrid monosomic plants were detected in each progeny, in 11 aneuploid hybrid F 1 families two hybrid monosomic were isolated, and in the remaining 21 hybrid families, one aneuploid hybrid F 1 plant was detected. These results showed differences in the ease monosomic detection in various hybrid backgrounds and/or differences in maternal transmission rates for various monosomes. Deficiencies for one chromosome arm occurred in the progenies of two monotelodisomic hybrid F 1 plants.
Hybrid F 1 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 F 1 plants revealed that 46 plants exhibited modal chromosome pairing of 25 bivalents and one univalent. Seven monosomic hybrid F 1 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 F 1 plants from Mo7, Mo11, Mo27, Mo67, Mo72, Mo75 and Mo89. Three monosomic hybrid F 1 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 F 1 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.
Pollen viability after acetocarmine staining was studied in monosomic hybrid families. Pollen viability was high in most monosomic hybrid F 1 plants. However, nine monosomic hybrid F 1 plants had reduced pollen viability and showed early haplo-deficient microspore abortion prior to mature pollen stage. Two monosomic hybrid F 1 plants had small reductions in pollen viability (up to 20%), four monosomic hybrid F 1 plants had more reductions in pollen viability (up to 30%), and three monosomic hybrid F 1 plants had reductions in semisterile pollen (up to 50%). Thus, some of monosomic hybrid F 1 plants had decreased meiotic index and decreased pollen viability.

Molecular marker analysis
For molecular analysis of the monosomic hybrid F 1 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 G. hirsutum L., we aimed to utilize chromosome-specific SSR markers to identify and reconfirm the chromosome specificity identities of monosomic lines of our collection, based on SSR content monosomic substitution F 1 hybrids created by crossing with the doubled haploid G. barbadense L. line 3-79. Detection and genotyping of SSR markers were straightforward and in a manner described in previous reports that utilized a PCR amplification of chromosome-specific markers in the genomic DNAs of hybrid plants. To localize SSR loci to chromosomes, we screened monosomic hybrid F 1 plants for the L-458 allele using labeled and/or unlabeled primers. For SSR loci located at sites other than the chromatin deficient segment, the L-458 marker would be present and F 1 hybrids would exhibit heterozygous phenotype. In comparison, if an SSR locus was on the segment deficient from the hypoaneuploid hybrid F 1 plant, the electropherogram would lack the L-458 allele and exhibit hemizygous pattern for the donor allele from G. barbadense Pima 3-79.  Because all of these SSR markers previously were assigned to chromosome 4 A t -subgenome of cotton [11,[19][20][21][22][23][24][25][26][27], the results indicated that the unknown monosomes Mo7, Mo31, Mo38, Mo59, Mo66, Mo69, Mo70, Mo71, Mo72, Mo73, Mo75, Mo76, Mo79, Mo81, Mo89, and Mo90 all involved monosomy for chromosome 4 of the cotton A t -subgenome (Figure 2).
Using the similar approach, monosomic line Mo48 was assigned to the chromosome 18 of the D t -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 D t -subgenome using chromosome-specific SSR marker-JESPR235 genotyping experiment with monosomic F 1 substitution hybrids (Mo17x Pima 3-79) [19,26], because of this SSR marker was assigned to these two chromosomes.
For instance, monosomic lines of the chromosome 2 A t -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.

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 F 1 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) [29]. 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 F 1 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 F 1 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 A t -subgenome of cotton using SSR markers (BNL3971, BNL3590, and GH-198), we can now confidently tell that the monosome Mo19 from chromosome 2 A t -subgenome ( Table 1).
The cytological analyses of the two hybrid combinations involving two tester lines (Mo67xTT3R-5R,  showed that monosome Mo67 is not chromosome 3, 5, 9, or 25 because the modal MI pairing configuration of its monosomic F 1 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 F 1 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 A t -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 F 1 hybrid of Mo75xTT4R-15L MI pairing was 24 II + 1 III. Molecular marker data suggested that the monosome Mo75 is chromosome 4 A t -subgenome (Table 1; Figure 7).  Thus, out of one monotelodisomic and 24 monosomic lines identified using chromosomespecific SSR markers, three monosomes were confirmed by means of the translocation test. From 24 identified monosomic lines, four are chromosome A t -2, 16 are chromosome A t -4, two are chromosome A t -6, and one each is chromosome D t -18, chromosome D t -20 or 22 and telosome A t -11.

Discussion
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 A t -subgenome and two chromosomes were designated chromosomes 18 and 20 or 22 D t -subgenome. One telosome was designated as a telosome 11 A t -subgenome.
Twenty other monosomes were identified as duplicates of three of aforementioned monosomes (chromosomes 2, 4, and 6). Chromosome 4 of the A t -subgenome was recovered more frequently (16 times), than chromosome 2 of the A t -subgenome (4 times) and chromosome 6 of the A tsubgenome (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 D t -subgenome and one telosome as a telosome 11 A t -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][31][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. [16] 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.

Conclusion
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.