Karyotype Variations in Pisum Sativum Ect. Abyssinicum

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Caryologia International Journal of Cytology, Cytosystematics

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Abyssinicum C. Conicella & A. Errico To cite this article: C.

Errico (1990) Karyotype Variations in Pisum Sativum Ect.

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1 Caryologia International Journal of Cytology, Cytosystematics and Cytogenetics ISSN: (Print) (Online) Journal homepage: Karyotype Variations in Pisum Sativum Ect. Abyssinicum C. Conicella & A. Errico To cite this article: C. Conicella & A. Errico (1990) Karyotype Variations in Pisum Sativum Ect. Abyssinicum, Caryologia, 43:1, 87-97, DOI: / To link to this article: Published online: 30 Jan Submit your article to this journal Article views: 258 View related articles Citing articles: 7 View citing articles Full Terms & Conditions of access and use can be found at

2 CARYOLOGIA Vol. 43, n. 1: 87-97, 1990 KARYOTYPE VARIATIONS IN PISUM SATIVUM ECT. ABYSSINICUM C. CONICELLA and A. ERRICO * C.S. peril Miglioramento Genetico degli Ortaggi C.N.R., Portici; * lstituto di Agronomia Generale e Coltivazioni Erbacee, Universita di Napoli, Portici, Italy. SUMMARY - P. sativum ect. abyssinicum was studied by karyotype and meiotic analyses. Two lines were investigated and resulted to be different from the standard P. sativum karyotype and from each other. Line 2 showed a reciprocal interchange involving chromosomes III and IV and both lines showed pericentric inversions in chromosome VI that involved different segments. INTRODUCTION Pisum abyssinicum, previously classified as a species, was regarded as an ecotype of P. arvense by LAMPRECHT (1966); afterwards, it was considered as synonym of P. sativum in respect to which abyssinicum differentiated into a morphologically distinct form under cultivation and selection in a different geographical area (SMARTT 1984). In this paper, it is suggested to regard abyssinicum as an ecotype of P. sativum in accordance with the classification proposed by LAMPRECHT and to consider as current species name P. sativum instead of P. arvense. Genetical and cytological analyses performed on crosses between P. abyssinicum and P. sativum evidenced the presence of chromosome rearrangements in P. abyssinicum: VoN RosEN (1944) showed both a reciprocal translocation and an inversion in one of the translocated chromosomes; SACCARDO (1971) found only one interchange; LAMPRECHT (1964) through genetical analyses ascertained two independent translocations involving the chromosomes I/VII and IV/VI; in a previous paper (CoNICELLA et al. 1985) one reciprocal interchange involving the chromosomes III/IV in an abyssinicum line was identified. In this paper, the karyotypes of two lines of the ect. abyssinicum are investigated in comparison with a standard line of P. sativum. Contribution no. 44 from the Vegetable Breeding Center, C.N.R., Portici, Italy.

88 CONICELLA and ERRICO MATERIAL AND METHODS Seeds of the Lamprecht's standard line 110 of Pisum sativum and of two accessions of the ecotype abyssinicum coming from Blixt's collection (Widar

3 88 CONICELLA and ERRICO MATERIAL AND METHODS Seeds of the Lamprecht's standard line 110 of Pisum sativum and of two accessions of the ecotype abyssinicum coming from Blixt's collection (Widar Weibull, Sweden) named line 1 and line 2 in this work were germinated on filter paper in Petri dishes in a growth chamber at 24 C. The main and secondary root tips were collected when about 1 em long. Root tips were pretreated in aqueous solution of 0.05% 5-7- dibromo-8-hydroxyquinoline for 16 hours at 3 C and then fixed in 1:3 acetic acidethanol for at least 2 hours. Staining of the mitotic chromosomes was obtained by Schiff's reagent after hydrolysis with HC15N for 55 min. at room temperature. For each karyotype, at least 6-7 good metaphase plates were selected and photographed at a magnification of 400 X. Total chromosome length and the lengths of short and long arms and of satellites, when visible, were measured in millimeters on the photographs at a final magnification of 2200 X. For each chromosome pair, the short/long arm ratio and the relative length as percentage of total diploid complement length were calculated. Crosses were performed between the two lines of ect. abyssinicum and six lines belonging to a translocation tester set kindly given by Dr. Lamm (Sweden). Crosses between the two abyssinicum lines and the standard karyotype line 110 were also done. On F1 hybrids meiotic analysis in metaphase I was carried out on mm long flower buds fixed for 48 hours with a solution of 1:3 proprionic acid-ethanol, to which a small amount of ferric chloride had been added as a mordant. Acetocarmine staining was performed on PMC for meiotic analysis and on mature pollen grains for fertility. RESULTS The most frequent diakinesis chromosome configurations and the pollen fertility percentages off 1 hybrids between the two lines of ect. abyssinicum and the lines with known translocations and the standard line L.llO are reported in Table 1. As to the line 1 of ect. abyssinicum one ring-of-4 chromosomes resulted to be the most frequent configuration in the crosses with the translocation testers; no association was observed in the cross between line 1 and the control (Fig. 1a). About 50% pollen fertility was found as average in the F1 plants heterozygous for one interchange; it is noteworthy the value of 67% pollen fertility in the cross with the test line. In the line 2 of ect. abyssinicum two associations of four chromosomes (Fig. 1b) and chain-of-6 chromosomes (Fig. 1c) were evidenced in the crosses with the translocated lines; one association of four chromosomes was found in the cross with the control. The karyotypes of Lamprecht's line 110 Pisum sativum and of the two lines of the abyssinicum ecotype were studied (Fig. 2). Both the lines of abyssinicum have a diploid chromosome number of 2n = 14 as L.llO (Fig. 3). Table 2 reports the mean values with standard deviation of the relative

4 TABLE 1 - Most frequent diakinesis chromosome configurations and pollen fertility (P.F.) in the crosses between two lines of «Pisum sativum» ect. «abyssinicum» and six lines with known translocations. TABLE 2 - Mean and standard deviation of the relative lengths (r.l.) and of the short/long arm ratio (a.r.) of the chromosomes of the haploid set of «Pisum sativum» (L.llO) and of two lines of ect. «abyssinicum» (1.1, 1.2). Pisum sativum Pisum sativum Pisum sativum Chromosome L. 110 ect. abyssinicum L. 1 ect. abyssinicum L. 2 pair. n. r.l. % a.r. r.l. % a.r. r.l. % a.r. I 13.24± ± ± ± ± ±0.06 II 11.77± ± ± ± ± ±0.05 III 14.24± ± ± ± ± ±0.02 IV 14.25± ± ± ± ± ±0.02 v ± ± ± ± ± ±0.05 VI 14.04± ± ± ± ± ±0.08 VII ± ± ± ± ± ±0.03 I 2! "; 00 '[) Standard P.F. T(1-5)a P.F. T(3-5)b P.F. T(5-7)a P.F. T(4-6)a P.F. T(2-6)a P.F. T(3-7)a P.F. karyotype % % % % % % P. sativum ect. abyssinicum L. 1 P. sativum ect. abyssinicum L. 2 7II 63 5II+ liv 46 5II+ liv 47 5II+ liv 52 5II+ liv 47 5II+ liv 44 5II+ liv 48 3II+2IV 39 4II+ 1VI 44 3II+2IV 38 4II+1VI 44 3II+2IV 39 43

5 Fig Metaphases I. a) 6II + 21 in the cross between the line 1 of abyssinicum and the line 110 of Pisum sativum (1400 X) b) 31I + 21V in the cross between the line 2 of abyssinicum and the line T(2-6)a (500 x) c) 4II + 1Vl in the cross between the line 2 of abyssinicum and the line T(4-6)a (1700 X).

KARYOTYPE IN PISUM SATIVUM ECT. ABYSSINICUM 91 Fig. 2. - Karyotypes of Pisum sativum and of two lines of ect. abyssinicum (2000 x ).

6 KARYOTYPE IN PISUM SATIVUM ECT. ABYSSINICUM 91 Fig Karyotypes of Pisum sativum and of two lines of ect. abyssinicum (2000 x ). lengths and arm ratios of the seven chromosome pairs numbered according to BLixT (1959) in the line 110 in the two lines of abyssinicum. The karyotype of the line 1 of ect. abyssinicum differs from the standard for the presence of one more metacentric pair - pair VI - that is submetacentric in the line 110. The comparison between the line 2 of ect. abyssinicum and the line 110 shows differences in the relative length values of the chromosome pair III (14.98% versus 14.24%) and pair IV (13.71% versus 14.25%) and in the arm ratio of the pair VI (0.26 versus 0.55). Student's t analysis (Table 3) shows that the differences between the karyotypes of Pisum sativum and ect. abyssinicum 1.1 are significant for TABLE 3 - Student's t values for the differences in the chromosome relative length (r.l.) and arm ratio (a. r.) between «Pisum sativum» and «P. sativum» ect. «abyssinicum» L.l. Chromosome d.f. r.l. d.f. a.r. I II III IV *** ** v VI *** VII P<O.Ol=**- P<O.OOl=***

Fig. 3. - Mitotic metaphases of Pisum sativum 1.

7 Fig Mitotic metaphases of Pisum sativum (a), Pisum sativum ect. abyssinicum 1. 1 (b), Pisum sativum ect. abyssinicum 1. 2 (c) (2000 X).

8 KARYOTYPE IN PISUM SATIVUM ECT. ABYSSINICUM 93 TABLE 4 - Student's t values for the differences in the chromosome relative length (r.l.) and arm ratio (a.r.) between «Pisum sativum» and «P. sativum» ect. «abyssinicum» L.2. Chromosome d.. r.l. d.. a.r. I II III * ** IV * ** v VI *** VII P<0.05=*- P<0.01=**- P<0.001=*** the relative length and the arm ratio of the chromosome pair IV and for the arm ratio of chromosome VI. Significant differences were found between the line 110 and the line 2 of ect. abyssinicum in the relative lengths and in the arm ratios of the pairs III and IV and in the arm ratio of the pair VI (Table 4). The idiograms of the two abyssinicum lines in comparison with the line 110 are shown in Figs The shortest and longest possible length of the inverted segments on the chromosome pair VI in the lines 1 and 2 are shown in Fig. 6; the lower and upper breaking points have been established hypothetically on the basis of centromere position and short arm length I I- 6 I- B I- 0 II Ill IV v VI VII CIRMJSaoE PAIR Fig Diagrammatic representation of the karyotypes of P. sativum (A) and of abyssinicum line 1 (B).

9 94 CONICELLA and ERRICO I- B 12 I ; c II IIJ IV v VI VII ORJ'olmE PAIR Fig Diagrammatic representation of the karyotypes of P. sativum (A) and of abyssinicum line 2 (C). a:: fl "' e,. II! Gl fl 3.., c s 'C % 0 s 3.. " Fig Dammatic representation ofthe chromosome VI of P. sativum (centromere is at zero). The smallest G and the longest E7n possible inverted segments are represented in abyssinicum line 1 (on the left) and line 2 (on the right).

10 KARYOTYPE IN PISUM SATIVUM ECT. ABYSSINICUM 95 DISCUSSION The results of our karyotypic analysis in Pisum sativum agree with those reported by BuxT (1958): based on the arm ratio the karyotype consists of two pairs of metacentric chromosomes (m) - pairs I and II - and five pairs of submetacentric chromosomes (sm), two of which with satellites on their longer arms - pairs IV and VII. The comparison of the karyotype of the ect. abyssinicum line 1 with the standard line 110 has evidenced a variation in the arm ratio of chromosome pair VI without altering chromosome length and a decrease in the length and arm ratio of pair IV. An asymmetrical pericentric inversion probably changed the submetacentric pair VI into a metacentric one. The formation of at least one single crossing over within the inversion produces gametes with deficiency and duplication and it causes the fall in pollen fertility in the F 1 with the control line. As to chromosome IV, it is difficult to decide whether the cause of the alteration in morphology of this chromosome pair is a small structural change like a deletion. VoN RosEN (1944) hypothesized that the phenomenon «varying linkage» evidenced in P. abyssinicum was associated to some small chromosome rearrangements, e.g. small inversions, deletions, deficiencies which are presumedly present in this ecotype. The karyotype analysis of the ect. abyssinicum line 2 has shown variations in the morphology of the pairs III, IV and VI. The relative lengths of pairs III and IV have respectively increased and decreased, nearly in the same proportion, in comparison with the standard line. The short arms of the two pairs are involved in these variations because the arm ratio of pair III has increased and that of the pair IV has been reduced. The analysis of the chromosome configurations at diakinesis in F 1 hybrids between line 2 and the translocation testers has shown the presence of one interchange involving the chromosome pairs III and IV. It seems, therefore, that the variations in the pairs III and IV depend on an interchange, that involves a longer segment of the short arm of pair IV, and a shorter segment of the short arm of pair Ill. The karyotype analysis allowed both the confirmation of the translocated pairs and the identification of the arms involved in the interchange. It allowed to identify an inversion on the chromosome pair VI also. The decreased arm ratio arose by an asymmetrical pericentric inversion, that «shifted» the centromere in more terminal position in respect to the control. Both lines of the ect. abyssinicum have the chromosome VI involved in a pericentric inversion, that involves different segments. The pericentric inversion is the determining factor of the chromosome polymorphism of pair VI, that could be characterized by heterochromatic

96 CONICELLA and ERRICO regions susceptible to breakage, but the lack of good Giemsa banding technique in Pisum does not allow verify this hypothesis (LAMM 1981; WoLFF 1985). The ect.

11 96 CONICELLA and ERRICO regions susceptible to breakage, but the lack of good Giemsa banding technique in Pisum does not allow verify this hypothesis (LAMM 1981; WoLFF 1985). The ect. abyssinicum appears to be cytologically heterogenous with two distinct karyotypes differing for an interchange and an inversion; likewise in pumilio karyotypical differences were found in the accessions from southern and northern Israel (BEN-ZE'Ev and ZoHARY 1973). The results of this work show that abyssinicum is cytologically characterized by an inversion on the pair VI and a III/IV interchange; reciprocal translocations are very common in the genus Pisum, where also the wild forms pumilio and elatius were found to be characterized by a IV/VI interchange (BEN-ZE'Ev and ZoHARY 1973). An inversion was already evidenced by VoN RosEN (1944) in abyssinicum but inversions are a less frequent rearrangement in Pisum, where they were reported in nuclear (SuTTON 1937) and in chloroplast pumilio genome (PALMER et al. 1985). The pericentric inversions can play an important role in karyotype evolution, as already evidenced in some species of Drosophila (DoBZHANSKY 1970), where the inversions tend to accumulate in a single chromosome or chromosome arm. This study and the literature data do not allow conclusions about the evolutionary significance of the pericentric inversions in the ect. abyssinicum. SMARTT has classified abyssinicum as synonym of P. sativum in his last taxonomic work (1984). It is known that semi-sterility barriers are present in the crosses between sativum and abyssinicum which are due to the chromosome rearrangements that we have ascertained; therefore, according to our results, abyssinicum, together withpumilio and elatius, is to be considered as an ecotype or a botanical variety of P. sativum. Acknowledgments. - review of the manuscript. We are particularly grateful to Prof. Francesco D'AMATO for critical REFERENCES BEN-ZE'Ev N. and ZoHARY S., Species relationships in the genus Pisum L. Israel]. Bot., 22: BuxT S., Cytolo of Pisum. II. The normal karyotype. Agri Hort. Genet., 16: , Cytolo of Pisum. III. Investigation of five interchange lines and coordination of linkage groups with chromosomes. Agri Hort. Genet., 17: CoNICELLA C. and ERRICo A., Identification of the chromosomes involved in translocations of P. abyssinicum and P. fulvum. Eucarpia Meeting on Pea Breeding, Sorrento (Italy), June 10-15, pp DoBZHANSKY TH., Genetics and the evolutionary process, New York, Columbia University Press. LAMM R. and MiRA valle R.J., A translocation tester set in Pisum. Hereditas, 45:

12 KARYOTYPE IN PISUM SA11VUM ECT. ABYSSINICUM 97 LAMM R., Contributions to the chromosomal nomenclature of Pisum. Pisum Newsletter, 4: 22. -, The relationship among some interchange lines in Pisum. Pisum Newsletter, 6: 29. -, Giemsa C-banding and silver-staining for cytological studies in Pisum. Hereditas, 94: LAMPRECHT H., Partielle Sterilitat und Chromosomenstruktur bei Pisum. Agri Hort. Genet., 22: , Die Entstehung der Arten und hoheren Kategorien. Springer-Verlag, Wien. PALMER].D., JoRGENSEN R.A. and THOMPSON W.F., Chloroplast DNA variation and evolution in Pisum: patterns of change and phylogenetic analysis. Genetics, 109: 195. RosEN G. VoN, Artkreuzung in der Gattung Pisum, insbesondere Zwischen P. sativum L. und P. abyssinicum Braun. Hereditas, 30: SACCARDO F., Crosses among Pisum species. Pisum Newsletter, 3: 38. SMARTT B.]., Evolution of grain legumes. I. Mediterranean pulses. Expl. Agric., 20: SuTTON E., Cytological studies of Pisum. I. Structural hybridity in P. humile. Ann. Bot., N.S., 1: WoLFF G., The usefulness of the C-banding method in identifying Pisum translocation lines. Nucleus, 28: 3-7. Received 17 September 1988; revision accepted 21 June 1989

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