CYTOLOGIA
Online ISSN : 1348-7019
Print ISSN : 0011-4545
Regular Article
Impact of Structural Heterozygosity on Pollen Fertility and Chiasma Frequency in Leontopodium jacotianum from the Cold Deserts of Ladakh (Jammu and Kashmir), India
Younas Rasheed Tantray , Vijay Kumar Singhal, Nissar Ahmad Khan, Raghbir Chand Gupta
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Keywords: Male meiosis, Structural heterozygosity, Cold desert, Jammu and Kashmir, Tetraploid, Leontopodium jacotianum
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2019 Volume 84 Issue 3 Pages 251-254

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Abstract

Meiotic studies have been carried out on three plants of Leontopodium jacotianum from Cold deserts of Ladakh. The chromosome number of n=24 counted in all the plants adds a new tetraploid cytotype for the species. The plants collected from Zoji-La showed a normal meiotic course leading to high pollen fertility. However, the plants collected from Sankoo region of cold deserts of Ladakh, Jammu, and Kashmir showed abnormal meiosis caused by structural heterozygosity for reciprocal translocations. The structural heterozygosity was observed for the first time in the species. Chiasma frequency was also calculated by using one-way analysis of variance (ANOVA) in the plants to see the effect of reciprocal translocations. Besides reciprocal translocation, the plant also showed various meiotic abnormalities in the form of early disjunction of bivalents, chromatin bridges, and laggards. Structural heterozygosity and other meiotic abnormalities in the plant seem to be responsible for a reduction in pollen fertility.

Leontopodium jacotianum Beauv. (family Asteraceae) is a perennial stoloniferous herb forming mats, grayish tomentose, stolons slender, elongate, usually branched terminated by a loose rosette of leaves. Leaves are lanceolate or oblong-lanceolate. Capitula are usually densely crowded. In India, the species is found in Himalayas between 3000–5000 m in Jammu and Kashmir, Uttar Pradesh and Sikkim and grows on alpine slopes and meadows. The flowering period is long, and starts in May and continues up to September.

During cytomorphological surveys in the wild plants of Western Himalayas, we have analyzed the individuals of L. jacotianium collected from cold deserts of Ladakh division of Jammu and Kashmir State. All the three plants depicted the same chromosome number, 2n=48 and exist at 4x level. However, one plant showed the presence of structural heterozygosity for reciprocal translocations. The aims of the present study were to analyze in detail the meiotic course and to correlate the occurrence of pollen sterility to structural heterozygosity. The aim was also to see the effect of structural heterozygosity on chiasma frequency.

Materials and methods

Materials for cytological analysis and pollen fertility have been collected from Zoji-La, at altitude 3000 m (PUN 619780 and Sankoo, at altitude 3400 m (PUN 62504, 62505) in the Ladakh division of Jammu and Kashmir. The voucher specimens of the studied plants were deposited in the Herbarium, Department of Botany, Punjabi University Patiala (PUN).

For meiotic studies, young floral heads were fixed in a freshly prepared Carnoy’s fixative (ethanol, chloroform, acetic acid in a volume ratio of 6 : 3 : 1) for 24 h. Materials were subsequently transferred to 70% ethanol and stored in a refrigerator at 4°C. Slides of meiocytes were prepared by squashing the developing anthers in 1% acetocarmine. A total of 150 pollen mother cells (PMCs) were observed for meiotic chromosome counts and meiotic abnormalities at prophase I, metaphase I (M I), anaphase I (A I), telophase I (T I) and sporads. Pollen viability was estimated by squashing the anthers in a glycerol–acetocarmine mixture (1 : 1). Well-filled pollen grains with stained nuclei were taken as fertile while shriveled and unstained pollen grains were counted as sterile. Photomicrographs were taken from the freshly prepared slides of PMCs with well-spread chromosome, sporads and pollen grains with a digital imaging system of Leica Qwin. For statistical analysis, ANOVA is applied by using GraphPad prism 8.0 software (http://www.graphpad.com/).

Results

All the three plants of L. jacotianum from the cold deserts of Ladakh revealed the same gametic chromosome number of n=24 which was ascertained from the presence of 24 bivalents at diakinesis and M I (Fig. 1A, B) and 24 : 24 segregation of chromosomes at A I (Fig. 1C). In one plant from Sankoo (PUN 61978), the PMCs showed the presence of multiple associations of four-six chromosomes. Analysis of chromosomal associations on the basis of well-spread chromosomes in 100 PMCs (Table 1) revealed that 72 PMCs showed regular 24 bivalents while the remaining 28 PMCs depicted the presence of quadrivalents (ring or chain, Fig. 1D, E) or hexavalents (chain, Fig. 1F, G). Further analysis revealed that 5.17% of the chromosomes were involved in reciprocal translocations while the rest constitute the perfect 24 bivalents. The PMCs in the plant also depicted some meiotic abnormalities in the form of an early disjunction of some bivalents (Fig. 1H), laggards (Fig. 1I) and chromatin bridges (Fig. 1J). Consequent to these meiotic abnormalities, the studied individuals showed a 15% reduction in pollen fertility (Fig. 1k). Chiasma frequency was also calculated in the accessions without structural heterozygosity showing perfect 24 bivalent formation and in the accession with structural heterozygosity to see the effect of reciprocal translocations on chiasma frequency. The chiasma frequencies per PMC and per bivalent were found to be higher in the plant showing structural heterozygosity (43.92±2.42, 1.83) than those without any reciprocal translocation (39.46±9.31, 1.64) (Table 1). However, the chiasma frequency in the PMCs depicting perfect 24 bivalents in both the accessions does not show any significant differences.

Fig. 1. Male meiosis in L. jacotianum. (A) A PMC with 24 bivalents at diakinesis. (B) A PMC with 24 bivalents at M I. (C) A PMC depicting equal 24 : 24 chromosomal segregation at A I. (D) A PMC showing 22II + 1IV (ring type) at M I (arrowed). (E) A PMC showing 20II +1IV (chain type) (arrowhead) +1IV (ring type) at M I (arrowed). (F) A PMC showing 21II +1VI (chain type) at diakinesis (arrowed). (G) A PMC showing 21II + 1VI (chain type) at M I (arrowed). (H) A PMC showing early disjunction of a bivalent (arrowed). (I) A PMC showing laggards (arrowed). (J) A PMC showing chromatin bridge (arrowed). (K) Fertile and sterile pollen grains. Scale bar=10 µm.
Table 1. Analysis of chromosomal associations and chiasma frequency in L. jacotianum.
AccessionsNumber of PMCs analyzedChromosomal associationsChiasma per PMC* Chiasma per bivalent
BivalentsQuadrivalentsHexavalents
RingRodRingChainChain
With structural heterozygosity (PUN 61978)PMC with 24 bivalents721052/1728———24–481.61
676/172838.61±9.58
(60.88%)
(39.12%)
PMCs with bivalents and multivalents28486/67262/67228161241–481.83
(72.32%)(9.23%)(8.33%)(4.76%)(5.36%)43.92±2.42
Number and % of chromosomes involved in chromosomal associations1003076/4800112/480064/480072/4800——
1476/4800(2.33%)(1.34%)(1.5%)
(64.08%)(30.75%)
Normal
PUN 62504PMCs with 24 bivalents802480/3840———24–481.64
1360/384039.46±9.31
PUN 62505PMCs with 24 bivalents50(64.42%)(35.41%)—24–481.62
1660/2400——39.06±11.0
740/24001
(69.17%)(30.83%)

*One way ANOVA value: p-value=0.0031; R2=0.8358; p＜0.05: i.e., chiasma frequency is significantly different between different accessions

Discussion

The presently recorded chromosome number of n=24 is new chromosome number for the species from India and adds a new tetraploid cytotype to the previous diploid cytotype (2n=24) from Pakistan (Khatoon and Ali 1988). Khatoon and Ali (1988) have also reported the dysploid chromosome number of 2n=28 in the species. It thus appears that the species is quite active in evolution depicting both intraspecific euploidy (2x, 4x) and dysploidy (2n=24, 28).

The phenomenon of structural heterozygosity for reciprocal translocations is regarded as a source of structural polymorphism of chromosomes and, served as excellent cytogenetic tools and are being utilized in breeding different crops by geneticists and plant breeders (Müntzing and Prakhen 1941, Hrishi et al. 1969, Mahama et al. 1999, Auger and Birchler 2002). The reciprocal translocation is the most frequent type of chromosome mutations in crop plants (Sadanaga and Newhouse 1982, Singh 2003). These structural changes also help in adaptation and speciation of plant species (Stebbins 1971, King 1993, Rieseberg 2001, Rieseberg and Willis 2007). It is assumed that structural changes of chromosomes in such species constitute an important part of the adaptive genetic system (Gill and Singhal 1998) and have played an important role in evolution. The structural heterozygosity in plants has a great potential for the creation and conservation of specific gene combinations which are usually characterized by the reduced reproductive capacity and presence of multivalents (Sharma and Gohil 2011).

Associations of more than two chromosomes in a diploid taxon or four chromosomes in a tetraploid might indicate that at least partial homology of chromosomes extends to some non-homologous pairs. It is possible either due to the hybrid nature of taxon or heterozygosity for translocations (Kumar et al. 2015). Burnham (1956) opined that such structural rearrangements of chromosomes are the main source of intraspecific variation. Presently, multiple associations of chromosomes due to reciprocal translocations have been reported for the first time in L. jacotianum. The reciprocal translocation is the phenomenon of chromosomal rearrangements between non-homologous chromosomes, resulting in structural heterozygotes. Such structural heterozygotes occasionally produce duplications or deficiencies resulting in non-viable and sterile pollen grains as reported in a number of flowering plants viz. Citrus jambhiri (Singhal and Gill 1981), Artemisia parviflora (Gupta et al. 2010), A. absinthium (Malik et al. 2010), Euphorbia pilosa (Saggoo and Farooq 2011) Astragalus chlorostachys (Rana et al. 2012), Saxifraga diversifolia (Kumar and Singhal 2013), Achillea millefolium (Singhal et al. 2014), Tanacetum artemisioides (Singhal et al. 2016), Anthoxanthum odaratum (Singhal and Kumari 2017), Arthraxon hispidus (Kumari and Singhal 2018), Fagophyrum aculatum and Deutzia staminea (Gupta et al. 2018a, b).

Chiasma frequency is useful for estimation of genetic recombination in a population especially for those organisms whose genetic analysis is difficult or impossible to perform (Colombo 1992). Chiasma formation is a crucial and delicate stage during meiosis (Sjödin 1970). Increase in chiasma frequency in the PMCs of accession of L. jacotianum depicting multiple chromosomal associations seems to be the consequence of structural heterozygosity as has also been reported in Achillea millefolium, (Singhal et al. 2014), Anemone rivularis (Kumar et al. 2015) and Anthoxanthum odoratum (Singhal and Kumari 2017).

Acknowledgment

Authors wish to thank the University Grant Commission, New Delhi for providing financial assistance under the DRS, SAP I, II & III, and ASSIST Programme and also awarding Juniour Research Fellowship to Younas Rasheed Tantray (Award letter no. 2121430298 12/8/2015). Authors are also thankful to Head, Department of Botany, Punjabi University, Patiala for providing necessary laboratory, herbarium, and library facilities. Thanks are also due to In-charge IPLS-DBT project (BT/PR 4548/INF/22/146/2012) for laboratory facilities.

References
 
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