Characterization of Three Varieties of Lathyrus Sativus L. by Fluorescent Karyotype and RAPD Analysis

Characterization of Three Varieties of Lathyrus sativus L. by Fluorescent Karyotype and RAPD Analysis

Laila Akter, Moontaha Mahbub, Md. Ahashan Habib and Sheikh Shamimul Alam*

Department of Botany, University of Dhaka, Dhaka 1000, Bangladesh

Received January 20, 2015; accepted July 20, 2015

Summary Three varieties of Lathyrus sativus L. released from Bangladesh Agricultural Research Institute (BARI), viz., BARI Khesari-1, BARI Khesari-2 and BARI Khesari-3, were studied cytoge- netically and at the molecular level by using RAPD for genomic characterization. The three varieties were found to posses 2n=14 chromosomes. The karyotype formulae of BARI Khesari-1 and BARI Khesari-2 are 8m + 6sm, while it is 10 m + 4sm in BARI Khesari-3. After orcein and CMA staining, one to two very small chromosome-like bodies were found in some cells of BARI Khesari-1, whereas no such body was observed after DAPI staining. Due to their unique features these can be considered as GC-rich heterochromatic “B-chromosomes.” In BARI Khesari-2, two pairs of satellites were found, while only one pair was present in BARI Khesari-3. The inverted position of satellited regions indicated the probable occurrence of homozygous inversion. An indication of paracentric inversion regarding DAPI-banding pattern was found in pair V of BARI Khesari-2 and thus could be considered as marker chromosomes for this variety. The three varieties have distinct CMA- and DA- PI-banding patterns. In BARI Khesari-2, a total of 12 C-bands were found on 10 chromosomes (out of 14 chromosomes). On the basis of band position and length, a deep correlation between C-banding and DAPI-banding pattern was found suggesting the heterochromatic nature of DAPI bands. Each variety showed different RAPD ﬁngerprinting with 59.09% polymorphisim. In addition, a number of variety-speciﬁc unique RAPD bands was observed. Therefore, each variety could be characterized on the basis of karyotype and RAPD analysis.

Key words Fluorescent banding, Karyotype, RAPD, Lathyrus sativus L.

The genus Lathyrus L. belongs to Fabaceae consisting of about 160 annual and perennial species (Allkin et al. 1986), some of which have agricultural importance. Lathyrus sativus L. is one of the most important food legumes in this genus. It is known in English as grasspea, blue sweet pea, chickling vetch or white vetch. The grasspea is an annual legume commonly grown for its grain and also used for fodder or green manure. The vegetative parts are utilized in the production of fodder or forage for animals. It includes several inbred cultivars widely cultivated across the world, viz., Bangladesh, China, In- dia, Burma, Nepal and Pakistan, as well as in Southern Europe, parts of Africa and South America (Duke 1981, Smartt 1990). There are few studies on the nutritional aspects of grasspea. According to Rahman et al. (1991), the values for Lathyrus sativus are as follows: energy 362.3 cal, protein 31.6%, fat 2.7%, nitrogen-free extract 51.8%, crude ﬁbre 1.1% and ash 2.2%. In addition, the crop has several ecological advantages including optimal growth in arid or semiarid environments, neutral to al- kaline soils, heavy clays and also high resistance to many pests compared to other forage legumes (Palmer et al. 1989, Kumari and Prasad 2005). Moreover, due to atmospheric nitrogen ﬁxing

* Corresponding author, e-mail: [email protected] DOI: 10.1508/cytologia.80.457 458 L. Akter et al. Cytologia 80(4) capacity, the crop fits well into a long-term sustainable farming system (Campbell and Clayton 1997). In spite of these attributes, the excessive consumption causes irreversible paralysis of the lower limbs (neurolathyrism) due to the presence of a neurotoxin called β-N-oxalyl-L-αβ- diaminopropionic acid or ODAP. In Bangladesh, this pulse is called ‘Khesari.’ The first systematic collections of this species were made in 1979 in collaboration with ICRISAT. During 1980 and 1981 additional collections were made, financed by FAO. Unfortunately, the major part of these collections were either dam- aged or lost because of the lack of proper storage facilities (Sarwar et al. 1995). A number of local varieties of Lathyrus sativus with variable characteristics are available in Bangladesh. Bangladesh Agricultural Research Institute (BARI) has been collecting available germplasm around the country. These are mainly identified on the basis of plant morphology. BARI has long been trying to develop better varieties using the local germplasm. As a conse- quence, three BARI varieties were released. These are BARI Khesai-1, BARI Khesai-2 and BARI Khesai-3 differentiated only on the basis of flower color, ODAP percentage and some agronomic features. Characterization on the basis of morphology is not reliable since due to phenotypic plastic- ity, the variety may show slight morphological variation. For an improved breeding programme of Khesari, genomic knowledge is a pre-requisite. Moreover, the genetic purity test of these Khesari varieties is an urgent need because Khandaker et al. (2007) reported trisomy in a variety of lentil released from BARI. Therefore, an authentic characterization of these Khesari varieties is essen- tial. It is well known that karyotype signifies a very stable character and is specific for each specimen, giving preliminary genomic information about an organism (Schweizer 1976, Alam and Kondo 1995). It plays an important role in determining the taxonomic status of a taxon where the taxonomic parameters are insufficient. Previously, a few works have been done on the conventional karyotype analysis of Lathyrus sativus (Campbell et al. 1994, Karadag and Buyukburc 2000). These works were almost confined to 2n (2n=14) chromosome counts (Rees and Narayanan 1997, Ozcan et al. 2006). The conventional karyotype analysis is unable to express the differences among different varieties of a species since they possess similar 2n chromosome numbers and even other karyotype parameters (Khatun and Alam 2010, Khatun et al. 2011). Moreover, it is not possible to detect minute deletion, inversion, tandem duplication, etc. by conventional karyotype analysis. In such a case, some modern methods can be applied. Staining with DNA base specific fluoro- chromes (CMA and DAPI) is one of such methods. CMA binds with GC-rich repetitive sequences of the genome giving characteristic yellow color bands. On the other hand, DAPI binds to AT-rich repeats showing a characteristic blue color. Giemsa C-banding is another method for critical karyotype analysis. It usually helps to determine the location of constitutive heterochromatic regions on chromosomes. With the help of the above banding techniques it was possible to characterize karyotypes of different plant species (Schweizer 1976, Alam and Kondo 1995, Alam and Kondo 1996, Kondo and Hizume 1982, Jessy et al. 2005, Akter and Alam 2005, Islam and Alam 2011, Sultana et al. 2011). For molecular characterization, DNA fingerprinting by Random Amplified Polymorphic DNA (RAPD) is a method for characterizing germplasm authentically. This method has been used as a versatile tool for investigating various genomic aspects of organism (Williams et al. 1993, Alam et al. 2012, Jahan et al. 2012). It includes characterization of genetic variability, genome fingerprinting, genome mapping, gene localization, analysis of genome evolution, population genetics, taxonomy, etc. RAPD markers can provide a powerful approach to understand the organization and distribution of genetic resources in natural and managed populations. Therefore, an extensive research should be undertaken to obtain genomic information about 2015 Karyotype and RAPD of Three Lathyrus sativus L. Varieties 459 the three Lathyrus sativus varieties released by BARI. The aims of the present research work were: i. to determine the diploid chromosome number for each Lathyrus sativus variety, ii. to compare the orcein, CMA and DAPI karyotypes of the three varieties of Lathyrus sativus and iii. to determine the genetic polymorphism among the germplasm by RAPD.

Materials and methods

The following three released varieties of Lathyrus sativus were investigated in this study: BARI Khesari-1, BARI Khesari-2 and BARI Khesari-3. Seeds were collected from BARI and maintained in the Botanic Garden, Department of Botany, University of Dhaka, Bangladesh.

Cytogenetical study Healthy roots were collected and pretreated with 8-hydroxyquinoline (0.002 M) for 1.40 h at 20–25°C followed by 15 min of fixation in 45% acetic acid at 4°C. These were then hydrolyzed in a mixture of 1 N HCl and 45% acetic acid (2 : 1) at 60°C for 15 s. The root tips were stained and squashed in 1% aceto-orcein. For CMA- and DAPI-banding, Alam and Kondo’s (1995) method was used with slight modification. After hydrolysing and dissecting, the materials were squashed with 45% acetic acid. The cover glasses were removed quickly from dry ice and allowed to air dry for at least 24 h before study. The air-dried slides were first pre-incubated in McIlvaine’s buffer (pH 7.0) for 30 min followed by Distamycin A (0.1 mg/mL) treatment for 10 min. The slides were rinsed mildly in McIl- vaine’s buffer supplemented with MgSO4 (5 mM) for 15 min. One drop of CMA (0.1 mg/mL) was added to the materials for 15 min in a humid chamber and then rinsed with McIlvaine’s buffer with

MgSO4 for 10 min. The slides were mounted in 50% glycerol and kept at 4°C overnight before observation. These were observed under a Nikon (Eclipse 50i) fluorescent microscope with a blue violet (BV) filter cassette. For DAPI-staining, after 24 h of air drying, the slides were first pre- incubated in Mcllvaine’s buffer (pH 7.0) for 27 min and treated in Actinomycin D (0.25 mg/mL) for 10 min in a humid chamber. The slides were immersed in a DAPI solution (0.01 mg/mL) for 20 min and mounted with 50% glycerol. These were observed under a Nikon (Eclipse 50i) fluorescent microscope with a ultraviolet (UV) filter cassette. The Giemsa staining method proposed by Alam and Kondo (1995) was followed with minor modification. The 24-h air-dried slides were incubated in a 5% aqueous solution of Ba(OH)2·8H2O for 5 min at 60°C. The slides were rinsed with distilled water for 10 min. After rinsing the slides were kept in 2×SSC for 1 h at 60°C. The slides were again rinsed with distilled water for 10 min followed by air drying for 1 h and stained with 6% Giemsa (R66, Gurr) at pH 6.8 for 4 h. The slides were rinsed with distilled water for 10 min, air dried for 30 min and finally mounted with phos- phate buffer.

DNA isolation Leaves were harvested and total genomic DNA was extracted by using the modiﬁed CTAB method (Doyle and Doyle 1987). The DNA concentration was quantiﬁed through a spectrophotom- eter (Analylikjena, Specord 50, Germany). The A 260/280 readings for DNA samples were 1.6–1.8.

PCR ampliﬁcation and primer survey The PCR reaction mixture contained 2 µL of template DNA (25 ng), 18.8 µL of de-ionized distilled water, 2.5 µL of Taq buffer A 10×(Tris with 15 mM MgCl2), 1.0 µL of primer (10 µM), 0.5 µL of dNTPs (2.5 mM) and 0.2 µL of Taq DNA polymerase (5 U/µL) for a total of 25 µL. PCR ampli- 460 L. Akter et al. Cytologia 80(4)

ﬁcation was done in an oil-free thermal cycler (Biometra UNOII, Germany) for 46 cycles after an initial denature at 94°C for 5 min, denature at 94°C for 1 min, annealing at 34–36°C for 30 s, extension at 72°C for 3 min and ﬁnal extension at 72°C for 5 min. Four random primers such as OPA-1 (5′-CAG GCC CTT C-3′), OPA-2 (5′-TGC CGA GCT G-3′), OPA-10 (5′-GTG ATC GCA G-3′) and primer-23 (5′-GTC AGG GCA A-3′) were used in the present study for screening.

Gel electrophoresis The ampliﬁed products were separated electrophoretically on 1% agarose gel. The gel was prepared using 1.0 g of agarose powder containing 8 µL of ethidium bromide (10 mg/mL) and 100 mL of 1×TAE buffer. Agarose gel electrophoresis was conducted in 1×TAE buffer at 50 V and 100 mA for 1.5 h. DNA ladder (1 kb) was electrophoresed alongside the RAPD reactions as a marker. DNA bands were observed on a UV-transilluminator and photographed by a gel documentation system.

Scoring and data analysis The PCR products were analyzed after gel electrophoresis. The photographs were critically discussed on the basis of presence (1) or absence (0), size of bands and overall polymorphism of bands.

Results and discussion

Karyotype analysis The three varieties were found to possess 2n=14 chromosomes (Figs. 1–3 and 11–13). A similar 2n chromosome number was reported earlier by different authors (Rees and Narayanan 1997, Ozcan et al. 2006). Therefore, the 2n chromosome number of the three varieties released by BARI correlated with the earlier reports. Darlington and Wylie (1995) reported 2n=28 and 2n=42 in the somatic cells of Lathyrus pratensis and L. palustris ssp. palustris, respectively. This result indicated tetraploidy and hexaploidy of the two Lathyrus spp., respectively. However, no tetraploid or hexaploid was found in the BARI-released Khesari varieties. The karyotype formulae of BARI Khesari-1 and BARI Khesari-2 are 8m+6sm, while it is 10 m + 4sm in BARI Khesari-3 (Table 1). Similar karyotype formulae were reported by Badr et al. (2009) for different germplasms of Lathyrus sativus collected from Canada, Afghanistan and Hun- gary. No gradual decrease in individual chromosome length was found in these three BARI varieties. A sharp decrease in chromosomal length was not reported by earlier workers either (Rees and Narayanan 1997, Ozcan et al. 2006). This indicated that the species conserved its gross karyotype beyond the geographical boundary.

B-chromosomes? In BARI Khesari-1, a very small chromosome-like body was found in some cells. The number of this kind of chromosome-like body was not fixed and varied from one to two per cell. This body was very small at about 0.73 µm in length and thus could not be arranged with the normal complements in the respective karyotype (Figs. 1a, b and 11, arrows). The above features suggest that this chromosome-like body is a “B-chromosome”. Badr et al. (2009) confidently reported B- chromosome in Lathyrus sativus. Generally, B-chromosomes are heterochromatic in nature. As a result there is no impact of its presence or absence on the morphology of the plant. BARI Khesari-1 does not show any morphological differences due to the presence of such small chromosomes. Af- ter CMA-staining, a well-fluoresced small chromosome-like body was found in a few cells (Figs. 4 and 14, arrow). However no such body was observed in any cell after DAPI staining (Fig. 7). Therefore, the B-chromosomes of Lathyrus sativus are GC-rich and heterochromatic in nature. 2015 Karyotype and RAPD of Three Lathyrus sativus L. Varieties 461

Figs. 1–23. Orcein-, CMA- and DAPI-stained mitotic metaphase and RAPD analysis of three varieties of Lathyrus sativus L. 1 (a, b). Orcein-stained mitotic metaphase chromosomes of BARI Khe- sari-1 (arrow showing probable B-chromosome), 2. Orcein-stained mitotic metaphase chromosomes of BARI Khesari-2, 3. Orcein-stained mitotic metaphase chromosomes of BARI Khesari-3 (arrow indicating satellite), 4. CMA-stained mitotic metaphase of BARI Khesari-1 (arrow showing probable B-chromosome), 5. CMA-stained mitotic metaphase of BARI Khe- sari-2, 6. CMA-stained mitotic metaphase of BARI Khesari-3 (arrow indicating satellite), 7. DAPI-stained mitotic metaphase chromosomes of BARI Khesari-1, 8. DAPI-stained mitotic metaphase chromosomes of BARI Khesari-2, 9. DAPI-stained mitotic metaphase chromosomes of BARI Khesari-3 (arrow indicating satellite), 10. Giemsa-stained mitotic metaphase of BARI Khesari-2, 11. Orcein karyotype of BARI Khesari-1 (arrow showing probable B-chromosome), 12. Orcein karyotype of BARI Khesari-2 (arrow indicating satellite), 13. Orcein karyotype of BARI Khesari-3 (arrow indicating satellite), 14. CMA karyotype of BARI Khesari-1 (arrow showing probable B-chromosome), 15. CMA karyotype of BARI Khesari-2, 16. CMA karyotype of BARI Khesari-3 (arrow indicating satellite), 17. DAPI karyotype of BARI Khesari-1, 18. DAPI karyotype of BARI Khesari-2 (arrow showing marker chromosome), 19. DAPI karyotype of BARI Khesari-3 (arrow indicating satellite), (Bar=5 µm), 20. RAPD analysis with primer OPA-1, 21. RAPD analysis with primer OPA-2, 22. RAPD analysis with primer OPA-10, 23. RAPD analysis with primer-23. 462 L. Akter et al. Cytologia 80(4)

Table 1. Comparative orcein-, CMA- and DAPI-karyotype analysis of three varieties of Lathyrus sativus L.

No. of Centromeric No. of % of GC-rich No. of % of AT-rich Variety 2n satellite formulae CMA-bands repeats DAPI-bands repeats

BARI Khesari-1 14 ̶ 8m + 6sm 06 8.74 10 06.17 BARI Khesari-2 14 4 8m + 6sm 04 6.40 13 10.43 BARI Khesari-3 14 2 10 m + 4sm ̶ ̶ 07 04.63

m=metacentric chromosomes, sm=sub-metacentric chromosomes

Satellites In BARI Khesari-2, after orcein-staining, one satellite was found on the short arm of both members in pair II and V (Fig. 12, arrows). However, only a pair of satellites was found on the short arms (pair V) in BARI Khesari-3 after both orcein and CMA staining (Figs. 13 and 16, arrows). Satellites in Lathyrus sativus L. were reported on the long arms by Seijo and Fernandez (2003). The position of the satellites found in this study was different from the previous work. The change of the satellited regions indicated the occurrence of homozygous inversion. The above-mentioned satellites were dully visible after CMA staining although not ﬂuoresced. Generally, satellites are NORs and thus are rich in GC-repeats (Schierwater et al. 1994). The satellite portions were not ﬂuoresced, indicating the absence of GC-rich repeats.

Fluorescent banding Six and four CMA bands were found in BARI Khesari-1 and BARI Khesari-2, respectively (Figs. 4, 5 and 14, 15, Table 1). No CMA band was found in BARI Khesari-3 (Figs. 6, 16, Table 1). CMA bands represent GC-rich repeats (Schierwater et al. 1994). The absence of CMA bands revealed that BARI Khesari-3 does not have much GC-rich repeats compared to the other two varieties (Figs. 6, 16). The number of DAPI-positive bands found in BARI Khesari-1, BARI Khesari-2 and BARI Khesari-3 was 10, 13 and 7, respectively (Figs. 7–9, 17–19, Table 1). Except for two bands in BARI Khesari-2, all the DAPI-positive bands were located at the terminal regions of the respective chromosomes (Figs. 8, 18). DAPI-positive bands revealed the presence of AT-rich repeats (Schierwater et al. 1994). The three varieties possessed more AT-rich repeats in comparison with GC-rich repeats (Table 1).

Marker chromosomes Heteromorphicity regarding DAPI-banding pattern was found in pair V of BARI Khesari-2. In this pair, one member has two terminal bands while the other member has two such bands, one at the terminal and another below the centromere of the long arm (Fig. 18, arrow). This banding pattern indicated a paracentric inversion. This kind of banding pattern was absent not only in the other pairs of this variety but also in the other two varieties. Therefore, these two chromosomes could be considered as marker chromosomes for BARI Khesari-2. Moreover, a centromeric CMA- positive band was found in a member of pair VII in BARI Khesari-2 (Fig. 15). After counter staining with DAPI, a DAPI-positive band was found exactly at the same location (Fig. 18). This feature revealed the tandem existence of AT- and GC-rich repeats (Alam et al. 2012). Thus this chromosome could be used as another marker chromosome of BARI Khesari-2.

C-banding A total of 12 C-bands were found on 10 chromosomes (out of 14 chromosomes) in BARI Khe- sari-2 (Fig. 10, Table 2). Ten chromosomes had terminal C-bands. A centromeric band and a terminal band were found in a chromosome. In another chromosome, a band was present just below the centromere (Fig. 10). The banding pattern indicated that the heterochromatins are mostly dis- 2015 Karyotype and RAPD of Three Lathyrus sativus L. Varieties 463

Table 2. Giemsa C-banding analysis of BARI Khesari-2.

No. of C-bands C-banded region Variety Terminal Centromeric Below centromere Total length (µm) %

BARI Khesari-2 10 1 1 2.76 10.42

Table 3. Compilation of RAPD analysis in three varieties of Lathyrus sativus L.

No. of No. and size (bp) Size range Polymorphisms Primer code Total band polymorphic of germplasms speciﬁc (bp) (%) band unique band

OPA-1 9 650–1800 9 1400 bp in BARI Khesari-2 100 OPA-2 13 430–2500 7 2500, 500 bp in BARI Khesari-2 2100, 53.85 1600, 1000 bp in BARI Khesari-3 OPA-10 6 400–2000 6 400 bp in BARI Khesari-2 1000 bp in 100 BARI Khesari-3 Primer-23 16 150–2400 4 2400 bp in BARI Khesari-1 800 bp in 25 BARI Khesari-3

Grand total: 44 150–2500 26 10 59.09 tributed at the terminal regions of this variety. Heterochromatins have a tendency to remain in the equilocal region (Sumner 1990). Therefore, in this variety the heterochromatins have a tendency to accumulate at the terminal regions. A deep correlation between C-banding and DAPI-banding patterns was found in BARI Khe- sari-2. Out of 13 DAPI bands, 11 were present at the terminal regions. The other two bands were at the centromeric regions (Fig. 18). Most of the C-bands were at the terminal region of the respective chromosomes. The total length of the C-banded region was 2.76 µm, which covered about 10.42% of the total chromatin length. This is almost similar to those of the DAPI-banded region (Table 2). Therefore, the C- and DAPI-banding patterns of BARI Khesari-2 revealed that DAPI-banded regions were probably heterochromatic in nature. In other words, C-banded regions were AT-rich.

RAPD fingerprinting In this study, a total of 16 RAPD primers were screened on template DNA of three BARI varieties. Only 4 out of 16 were selected because they produced multiband fingerprinting which was easily scorable and reproducible. The primer sequence, band size and banding pattern of the three varieties are shown in Table 3. The four primers generated 44 distinct bands, of which 26 were considered as polymorphic. Band size ranging from 150–2500 bp of PCR amplification products scored for all primers. Light and bright bands were produced in the RAPD reactions. Light bands were produced from low homology between the primer and the pairing site on the DNA strand (Thormann et al. 1994). In this study, 59.09% polymorphism was recorded. Therefore, the three varieties used in this study were quite diverse from each other. The diversification would be useful for an improved breeding programme of Lathyrus sativus L. in Bangladesh. In addition to polymorphism, 10 unique RAPD sequences were identified using four different primer combinations. The term unique sequence means that the sequence found in a variety with a certain primer was absent in other varieties (Figs. 20–23, Table 3). The unique bands were stable and specific for the respective varieties and thus could be used as a tool for characterization. From the foregoing discussion it is clear that the three BARI varieties of Lathyrus sativus L. could be characterized authentically by combined cytogenetical and molecular analyses. 464 L. Akter et al. Cytologia 80(4)

Acknowledgements

This research was partly supported by a grant from the Ministry of Science and Technology, People’s Republic of Bangladesh.

References

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