Chromosome Count, Male Meiotic Behavior and Pollen Analysis of Eight Populations of Trachyspermum ammi (L.) Sprague (Apiaceae) from Iran

Abstract

Trachyspermum ammi (L.) Sprague (Ajwan) is one of the most important industrial and medicinal plants which belongs to the family Apiaceae. A meiotic study of eight populations from Iran was performed. All populations were diploid n=9 (2n=2x=18) forming mainly bivalents with some amount of quadrivalent and hexavalent possibly due to the occurrence of heterozygote translocations. The ANOVA test of meiotic characters showed a significant difference (p＜0.05) in chiasma frequency as well as chromosome associations in Ajwan populations. Chromosome migration or cytomixis and syncyte formation and tripolar structure in telophase II were also reported for the first time in natural populations in Iran and led to diploid gamete formation or 2n pollen grains in almost all populations were studied. Cluster analysis of meiotic data showed the distinctness of the populations in their meiotic behavior.

Trachyspermum ammi (L.) Sprague (Ajwain) is one of the most important industrial and medicinal plants which belongs to the family Apiaceae. It is a diploid and annual grassy plant with open pollination reproductive system (Malhotra and Vijay 2004). It is originated in the Middle East and distributed in Egypt, Iraq, Iran, Afghanistan, Pakistan, Eastern India, and North Africa (Zahin et al. 2010, Dalkani et al. 2012, Dwivedi and Kumar 2017, 2018). Ajwain seeds are used in traditional medicine as an analgesic, stimulant, carminative, anti-nausea, anti-asthma, antihypertensive, antispasmodic and topically to treat rheumatic pain, and also various other pharmacological activities (Ranjan et al. 2011, Zarshenas et al. 2013). Ajwain is an important plant in the production of essential oils such as thymol and carvacrol. The important compounds in Ajwain seeds are thymol, γ-terpinene, p-cymene, and α-pinene (Zarshenas et al. 2014).

In general, despite the presentence of a lot of knowledge about the medicinal properties of Ajwain, there have been very limited cytogenetic data on the plant and no report from Iran. As far as our information Ajwain is a diploid species and has 18 chromosomes (2n=2x=18) that are small and almost identical in size (Shubhada et al. 1984). Therefore, cytogenetic studies are difficult in this species (Chattopadhyay and Sharma 1990). Recently, in research induced syncyte via cytomixis using gamma rays has been reported in Ajwain (Dwivedi and Kumar 2018). Newly the presence of maximum of two B-chromosomes is reported in PMCs of diploid species which were retained in induced autotetraploid plants after the treatment of 0.2% colchicine at the seedling stage. Meiotic behavior and chromosome associations were also analyzed in diploid and autotetraploid carrier plants (Dwivedi and Kumar 2019). Therefore, the present study provides chromosome count, meiotic behavior, and pollen analysis of eight populations of Ajwain for the first time in Iran.

Materials and methods

In the present research meiotic studies were performed on eight native populations of Ajowan from Iran (Table 1). Seeds of each population were collected from different regions of Iran and were cultivated in the research greenhouse. Young flower buds of each population were harvested at 8–10 AM and fixed in glacial acetic acid: ethanol (1 : 3) for 24–48 h. Then the fixed flower buds were washed three times with distilled water and preserved in 70% ethanol at 4°C in a refrigerator (Nouroozi et al. 2010). For cytological preparations, 2% aceto-orcein was used with the squash technique. To study chiasma frequencies and distributions, approximately 100 PMCs were analyzed at diakinesis. Five hundred PMCs were analyzed at the metaphase and anaphase stages to study chromosome segregations. To determine pollen fertility minimum of 1,000 pollen grains were checked for each population using 2% aceto-orcein : 50% glycerin (1 : 1) for 30 min. Complete, round, and fully stained pollen grains were supposed as fertile while shrunken, incomplete pollen grains with no stain were considered infertile (Dwivedi and Kumar 2019). To reveal a significant distance among different populations, analysis of variance (ANOVA) followed by the least significant distances (LSD) were performed on meiotic characters. To group populations according to similarities in meiotic behavior, UPGMA and WARD clustering methods, as well as ordination based on principal component analysis (PCA), were used (Romesburg 1984). Statistical analyses used SPSS ver. 20

Table 1. Ajwain populations studied and their localities in Iran.

Population	Voucher No.	Location	Geographical region of Iran	Longitude	Latitude	Altitude (m)
Arak	14492	Arak	Center	49°40′E	34°00′N	1,735
Ardabil	10569	Ardabil	Northwest	48°18′E	38°15′N	1,332
Esfahan	38913	Isfahan	Center	51°43′E	32°39′N	1,615
Birjand	38473	Birjand, South Khorasan	East	59°13′E	32°53′N	1,461
Rafsanjan	23023	Kerman, Rafsanjan	South	56°05′E	30°30′N	1,545
Sarbisheh	37477	Sarbisheh,South Khorasan	East	59°40′E	32°30′N	1,827
Shiraz	12313	Shiraz	South	55°00′E	29°30′N	1,508
Karaj	906	Karaj	North	51°00′E	35°48′N	1,312

Results and discussion

All populations studied here were diploid and the chromosome number 2n=2x=18 which confirms the previous reports (Shah 1953, Sharma and Ghosh 1954, Thengane and Dnyansagar1984, Dwivedi and Kumar 2019) (Table 2, Fig. 1). In all the populations studied synozetic knobe were observed instead of leptotene and pachytene stages in prophase I (Fig. 1A). According to our results, the highest value of intercalary chiasma as well as ring bivalent (1.54 and 5.46, respectively), and the lowest value of terminal and total chiasma, as well as rod bivalents (10.22, 11.76 and 2.72), were observed in Ardabil population. Shiraz population had the highest value of terminal and total chiasma as well as rod bivalent (13.7, 14.7, and 5.74, respectively). Multivalents such as quadrivalent and hexavalent were usually formed in most of the populations studied, ranging from one to many but univalent formations only were observed in the Ardabil population (Table 2). The highest value of quadrivalent formation (0.42) was observed in the Rafsanjan population while the lowest value (0.22) was perceived in the Karaj population. The highest value of hexavalent formation (0.04) was observed in the Ardabil population while no hexavalent was observed in two populations of Birjand and Shiraz (Table 2). The ANOVA test (Table 3) showed a significant difference (p＜0.05) in chiasma frequency as well as chromosome associations in populations. Significant differences observed in chiasma frequency and distribution among populations studied may illustrate their genetic differences partially (Quicke 1993) and possible significant variation in the genes controlling chromosome pairing in the species. Such changes have been reported in populations of different species and are recognized as a means for producing new forms of recombination impressing the variability within natural populations in an adaptive route (Rees and Jones 1977). Multivalent formation such as quadrivalent and hexavalent in metaphase I in diploid species may represent the occurrence of heterozygote translocations between two or three pairs of non-homologous chromosomes. Such structural variations may increase the number of genetic changes in gametes by forming new genetic associations and linkage, which may be used for compatibility to diverse environmental conditions. Ardabil population presents the highest hexavalent and intercalary chiasma formation and the significant correlation between multivalent formation and intercalary chiasmata asserts such a suggestion, increasing the number of intercalary chiasmata may increase the genetic recombination by involving the genes present in the middle parts of chromosomes in recombination and lead to increasing genetic diversity of gametes in next generations. The occurrence of chromosome stickiness, disorganized chromosomes was observed in most populations studied (Table 4, Fig. 1). The sticky chromosomes were observed in both stages of metaphases I and II. The number of chromosomes that were involved in stickiness ranged from two to many forming an entire clumping of chromosomes. The highest percentage of metaphase I stickiness was observed in Rafsanjan (8.85) and Arak (8.13) populations respectively (Fig. 1N). Also, the highest value of metaphase II stickiness occurred in Ardabil (3.97) followed by the Birjand population (3.54). The highest value of clumping in metaphase I was observed in Arak (6.91) followed by Karaj populations (6.22). The highest value of clumping and disorganized chromosome occurrence in metaphase II were observed in the Ardabil population (7.95 and 2.25, respectively). The highest percentage of disorganized chromosomes in metaphase I was observed in the Arak population (6.91). The highest value of bridge formation in anaphase I occurred in Arak (9.67) followed by the Isfahan population (8.01) respectively. The highest percentage of tripolar formation was observed in the Rafsanjan population (Fig. 1M). Cytomixis was occurred in different stages of meiosis in almost all populations studied. The maximum amount of chromosome migration was observed in prophase I in syncytic knot and diakinesis substages. The highest percentage of cytomixis (4.86) and syncyte (12.21) was observed in the Rafsanjan population, while the lowest value of the same occurred in the Ardabil population (0.87) and (4.34), respectively (Table 4). The meiotic abnormalities mentioned above may result in aneuploid or unreduced (2n) pollen grains (Brownfield and Kohler 2011). Pollen grains fertility may range from 88.5% in Arak to 98.12% in Birjand populations. Except for Sarbisheh population, in the rest of the populations unreduced or diploid pollen formation was observed. The mean size of normal or haploid pollen grain was 19.49 µm, while unreduced or diploid pollen grain mean size was 24.91 µm, in populations studied. Meiotic abnormalities such as chromosome stickiness and laggard chromosomes are reported for the first time in Ajwain. Correct chiasma formation and spindle orientation are responsible for maintaining the bivalent leading to the normal chromosome segregation (Bione et al. 2000). Therefore, any defect in the formation of chiasmata can cause the formation of laggard chromosomes. Environmental and genetic factors, as well as the genomic-environmental interaction, have been considered as the reason for chromosome stickiness in different plant species (Pessim et al. 2015). The chromatin migration occurred in different stages of meiosis in populations studied. The migration of chromatin among the adjacent meiocytes occurs through cytoplasmic connections originating from the pre-existing system of plasmodesmata formed within the tissues of the anther. The plasmodesmata become completely plugged off by the deposition of callose, but in some cases they persist during meiosis and increase in size, forming conspicuous inter-meiocyte connections or cytomictic channels that permit the transfer of chromosomes. However, malfunctioning of certain genes during cell division failed to plug these pores which persist till the later stages to form intercellular channels through which transfer of nuclear material and other cell contents may take place (Dwivedi and Kumar 2018). Cytomixis is considered to be of less evolutionary importance but it may lead to the production of aneuploid plants with certain morphological characteristics or produce unreduced gametes (Falistocco et al. 1995). Unreduced gamete formation is of evolutionary importance leading to the production of plants with higher ploidy levels. Cytomixis, by producing aneuploid pollen grains, may also be responsible for the reduction in pollen fertility of the Ajwain populations studied. The occurrence of giant pollen grains has been used as an indication of the production of 2n pollen (Bretagnolle and Thompson 1995). Unreduced gametes are known to produce individuals with higher ploidy levels through a process known as sexual polyploidization (Barcaccia et al. 2021), which has been considered as the major route to the formation of naturally occurring polyploids. The occurrence of multipolar cells and chromosome migration might be considered as the possible mechanisms of unreduced meiocytes and pollen grain formation in Ajwain. Different cluster analyses of populations based on meiotic characters were produced. Two major clusters (Fig. 2). The first major cluster consists of two sub-clusters. Population Sarbisheh, Karaj, Esfahan and Birjand form the first sub-cluster and Arak, Rafsanjan and Shiraz form the second sub-cluster. Ardabil population form the second major cluster. Principle component analysis of meiotic data in the studied populations confirms the cluster analyses (Fig. 3). The factor analysis of meiotic data determined that the first two components comprise about 85.31% of the total variance. Terminal and total chiasmata and ring bivalents in the first factor and quadrivalent and hexavalent formations in the second factor showed the highest positive correlations (r=＞85%) and therefore may be considered as the most variable meiotic characters in Ajwain populations studied.

Table 2. Meiotic characteristics of populations.

Population	2n	RB	RoB	QUD	HEX	UNI	IX	TEX	TOX
Arak	18	3.48	4.62	0.4	0.02	0	1.32	12.98	14.3
Ardabil	18	5.46	2.72	0.34	0.04	0.02	1.54	10.22	11.76
Esfahan	18	4.48	3.94	0.24	0.02	0	1.14	11.8	12.94
Birjand	18	5.16	3.24	0.3	0	0	1.08	11.1	12.18
Rafsanjan	18	3.4	4.7	0.42	0.02	0	0.7	13.66	14.36
Sarbisheh	18	4.48	3.9	0.26	0.02	0	1.4	11.52	12.92
Shiraz	18	2.62	5.74	0.32	0	0	1	13.7	14.7
Karaj	18	4.96	3.54	0.22	0.02	0	1.28	11.22	12.5

RB: Ring bivalent, RoB: Rod bivalent, QUD: Quadrivalent, Hex: Hexavallent, UNI: Univallent, IX: Intercalary chiasma, TEX: Terminal chiasma, TOX: Total chiasma.

　

Fig. 1. Representative meiotic cells in populations studied. A-Synozetic knot in Ardabil population. B-Diakinesis cell in Ardabil population showing ring and rod bivalents. C-Diakinesis cell in Rafsanjan population showing ring and rod bivalents. D-Diakinesis cell in Rafsanjan population showing seven bivalents and 1quadrivalent. E-Diakinesis cell in Ardabil population showing seven bivalents and one quadrivalent. F-Diakinesis cell in Birjand population showing seven bivalents and 1quadrivalent. G-Diakinesis cell in Shiraz population showing seven bivalents and one quadrivalent. H-Meiocyte with 18 bivalents showing syncyte phenomenon in Rafsanjan population. I-Meiocyte with eighteen bivalents showing syncyte phenomenon in Shiraz population. J-Meiocyte with eighteen bivalents showing syncyte phenomenon in Arak population. K-Metaphase I showing disorganized chromosome. L-Anaphase I cell in the Isfahn population showing laggard chromosome. M-Tripolar cell in Sharbisheh population. N-Chromosome stickiness in the Karaj population. O-Normal tetrad formation in Birjand population. P and Q-Unreduced pollen grains in Sabzevar and Shiraz populations. R and S-Fertile and Non-fertile pollen grains. Scale bar=10 µm.

Table 3. ANOVA test of meiotic characteristics in populations.

Character		Sum of squares	df	Mean square	F	Sig.
Ring bivalent	Between Groups	13.748	7	1.964	26.613	0.000
	Within Groups	0.590	8	0.074
	Total	14.339	15
Rod bivalent	Between Groups	12.604	7	1.801	30.570	0.000
	Within Groups	0.471	8	0.059
	Total	13.075	15
Univalent	Between Groups	0.074	7	0.010	0.522	0.796
	Within Groups	0.161	8	0.020
	Total	0.234	15
Quadrivalent	Between Groups	0.002	7	0.000	0.657	0.703
	Within Groups	0.004	8	0.000
	Total	0.006	15
Hexavalent	Between Groups	0.001	7	0.000	1.000	0.493
	Within Groups	0.001	8	0.000
	Total	0.002	15
Intercalary chiasmata	Between Groups	0.964	7	0.138	1.231	0.385
	Within Groups	0.895	8	0.112
	Total	1.859	15
Terminal chiasmata	Between Groups	22.916	7	3.274	11.297	0.001
	Within Groups	2.318	8	0.290
	Total	25.235	15
Total chiasmata	Between Groups	17.110	7	2.444	18.846	0.000
	Within Groups	1.038	8	0.130
	Total	18.148	15

Table 4. Meiotic behavior of populations.

Number	Population	n	Stickiness (%)		Clumping (%)		Disorganized chromosome (%)		Anaphase bridge (%)	Laggard chromosome (%)		Tripolar (%)	Cytomixis (%)	Syncyte (%)	Normal pollen size (µ)	2n pollen size (µ)	Pollen fertility (%)	2n pollen (%)
			MI	MII	MI	MII	MI	MII	AI	AI	AII
1	Arak	9	8.13	2.85	6.91	0	6.93	1.91	9.67	0	4.16	4.94	4.28	10.71	18.8	24.41	88.5	0.7
2	Ardabil	9	5.29	3.97	3.72	2.27	2.11	7.95	0.75	3.78	0	3.71	0.87	4.34	20.51	24.35	96.5	1
3	Esfahan	9	6.09	3.12	3.55	1.56	2.03	0	8.01	1.41	0	9.6	2.10	8.41	20.31	25.94	96.31	1.5
4	Birjand	9	4.79	3.54	0.68	0	4.71	0	0	1.34	0	4.92	2.21	7.21	18.93	24.52	98.12	0.8
5	Rafsanjan	9	8.85	0	7.96	0	3.54	0	4.42	2.65	0	10.73	4.86	12.21	19.72	23.74	97.4	0.8
6	Sarbisheh	9	5.68	3.12	3.41	2.02	2.27	0	0	1.96	0	4.02	3.52	7.33	16.95	NO	91.6	NO
7	Shiraz	9	5.21	0	4.31	0	5.11	2.12	1.21	1.36	0	5.21	3.33	10.66	20.12	26.19	96.3	2
8	Karaj	9	7.88	3.01	6.22	2.12	4.14	2.31	1.49	0.99	5.05	6.5	4.66	10.12	20.6	25.22	89.4	0.5

　

Fig. 2. UPGMA dendrogram of populations based on meiotic data.

Fig. 3. PCA plot of populations based on meiotic data.

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