Caryologia. International Journal of Cytology, Cytosystematics and Cytogenetics 73(3): 45-54, 2020 Firenze University Press www.fupress.com/caryologia ISSN 0008-7114 (print) | ISSN 2165-5391 (online) | DOI: 10.13128/caryologia-867 Caryologia International Journal of Cytology, Cytosystematics and Cytogenetics Citation: W.M. Amer, R.A. Hassan, A.S. Abdo (2020) Cytogenetic and molecu- lar studies of the Egyptian Capsella bursa-pastoris (Brassicaceae). Caryo- logia 73(3): 45-54. doi: 10.13128/caryolo- gia-867 Received: February 23, 2020 Accepted: April 19, 2020 Published: December 31, 2020 Copyright: © 2020 W.M. Amer, R.A. Hassan, A.S. Abdo. This is an open access, peer-reviewed article pub- lished by Firenze University Press (http://www.fupress.com/caryologia) and distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distri- bution, and reproduction in any medi- um, provided the original author and source are credited. Data Availability Statement: All rel- evant data are within the paper and its Supporting Information files. Competing Interests: The Author(s) declare(s) no conflict of interest. Cytogenetic and molecular studies of the Egyptian Capsella bursa-pastoris (Brassicaceae) Wafaa M. Amer, Rania A. Hassan*, Amany S. Abdo Botany and Microbiology Department, Science Faculty, Cairo University, Egypt * Corresponding authors. E-mail: raniaali@sci.cu.edu.eg; raniaaly2006@yahoo.com Abstract. Capsella bursa-pastoris  (Brassicaceae) is one of the most successful tetra- ploid species in the world. It showed high morphological diversity within Egyptian populations. Morphological investigations of herbarium specimens and fresh col- lected populations grouped them under three distinguished morphotypes (Lobed “L”; Simple “S” and Lobed-Simple “LS”) depending mainly on the basal leaves struc- ture. This high degree of phenotypic variation has received our critical attention. Until recently, the previous studies on C. bursa-pastoris  attributed its phenotypic variation to environmental factors. But in Egypt, these three morphotypes were traced in mixed populations along with the species geographical range, so the environmental factors have no influence on their distribution or phenotypic variation. Accordingly, our pri- mary concern in this study was to  determine the factors controlling this variation. The cytogenetic studies revealed that the three identified morphotypes are three dis- tinct genotypes with three different chromosome numbers: 2n=2x=16 (diploid) for “L”; 2n=3x=24 (triploid) for “S”; 2n=4x=32 (tetraploid) for “LS”. The triploid genotype “S” showed rare occurrence among the studied populations and is postulated to be a new record of a hybrid in Egypt. Karyotyping of the three genotypes showed significant differences in the genome and chromosomes relative lengths. Molecular study using cpSSR technique supported the cytogenetic results and differentiated the three studied genotypes. The retrieved results revealed that the phenotypic diversity within the Egyp- tian C. bursa-pastoris populations is genetically controlled. Keywords: Capsella bursa-pastoris, cytogenetic studies, genotypes, karyotyping, molecular study, phenoplasticity. INTRODUCTION Brassicaceae (Cruciferae) or mustard family is one of the largest Angio- sperm families, it comprises 3977 species and 341 genera and 52 tribes (Kief- er et al., 2014). One of the most important genera in the Brassicaceae is genus Capsella Medik. Molecular systematic studies confirmed that genus Capsella belongs to the tribe Camelineae (Neuffer et al., 2014). This genus is an excel- lent model for molecular evolutionary studies due to its phylogenetic rela- tions within the Brassicaceae. Studying its genetics, speciation and sympatric distribution is important for agricultural matters. Genus Capsella is represented worldwide by five species: the two self- compatible tetraploid C. bursa-pastoris (L.) Medik and C. thracica Velen 46 Wafaa M. Amer, Rania A. Hassan, Amany S. Abdo (2n=4x=32); the self-incompatible diploid C. grandiflora (Fauche & Chaub) Boiss; the two self-compatible diploid C. rubella Rent. and C. orientalis Klokov (Hurka et al., 2012; Neuffer et al., 2014). These species differ greatly in their geographical distribution, where, C. grandiflora is limited to northwestern Greece and Albania; C. rubella has a broad Mediterranean / central European distribu- tion; C. orientalis is found from Eastern Europe to Cen- tral Asia (Hurka et al., 2012); C. thracica is endemic to Bulgaria (Neuffer et al., 2014). Capsella bursa-pastoris (Shepherd’s Purse) is an annual to biennial species, extremely variable in size and leaf form, distinguished by terminal and axillary raceme inflorescences. Its silicula fruit is obcordate-obtriangular in shape (Amer et al., 2019). This species is the second most common flowering plant in the world (Zhou et al., 2001), grows as a common weed of agriculture in almost all countries of the world from tropical to subarctic hab- itats (Holm et al., 1979), and shows a high phenotypic plasticity (Korsmo, 1954; Holzner and Numata, 1982). Accordingly, the evolution of polyploidy and weediness in C. bursa pastoris is interesting to agricultural research (St Onge, 2010). Many taxonomic studies were carried out on this species depending on morphological characters and resulted into many species, subspecies, varieties, micro- species, biotypes, and segregates (Aksoy et al., 1998; Aksoy et al., 1999; Neuffer, 2011). A considerable amount of literature has attributed the phenotypic variation in C. bursa-pastoris to environ- mental or geographical factors like seasonality, tempera- ture, shade, rainfall, latitudinal and altitudinal gradients (Almquist, 1929; Neuffer, 1989; Neuffer and Bartelheim, 1989; Stace, 1989; Neuffer, 1990; Aksoy, 1996; Aksoy et al., 1999; Neuffer and Hoffrogge 2000). In Egypt, Capsella is a monospecific genus repre- sented by C. bursa-pastoris (Boulos, 1999). The field study and morphological investigations of this species – based on the leaves, inflorescence and fruit charac- ters – showed the presence of high degree of phenotypic variation and revealed the presence of three morpho- types namely: Lobed “L”, Simple “S” and Lobed-Simple “LS” (Amer et al., 2019). These three morphotypes were traced in mixed populations along with the species geo- graphical range, so the environmental factors have no influence on their distribution or phenotypic variation. Therefore, this study aims to determine the factors controlling the phenotypic variation of C. bursa-pastoris morphotypes through cytogenetic and molecular studies to clarify the impact of genetic diversity on their pheno- plasticity. MATERIALS AND METHODS Morphological study The morphological investigations of C. bursa-pasto- ris were carried on 36 old populations deposited as her- barium specimens in Cairo University Herbarium (CAI) and Assiut University Herbarium (ASTU). In addition to 66 fresh populations collected from Menoufia (Abu Sleem village), Faiyum (Sinnuris district, El Siliene) and El Saff regions during our field work conducted in 2016- 2018. The studied specimens (old & fresh) from differ- ent distribution localities are shown in Table 1. From 66 fresh populations, 25 specimens/ population were undergone morphological investigations using different morphological criteria of leaves, inflorescence and fruit. Acronyms of herbaria follow Thiers (2019). The morphological investigations distinguished three morphotypes of C. bursa-pastoris in all the stud- ied populations namely: (L) Lobed, (S) Simple and (LS) Lobed-Simple (Amer et al., 2019). Cytogenetic studies Sample preparation For cytogenetic studies, the specimens collected from Faiyum region (marked with * in Table 1) were selected to nullify the environmental factors. Seeds of 30 specimens representing the three morphotypes (10/ morphotype) were collected, soaked in distilled water for 2 hours, and germinated at room temperature. Root tips of about 1 cm length were treated with colchicine (C22H25NO6, 0.025 %) for 2 hours at room temperature, and washed thoroughly with distilled water. Fixation was done using ethyl alcohol: glacial acetic acid (3:1, v/v). Samples were washed thoroughly with water and hydro- lyzed using 1 N HCl at 64° C for 5 minutes. The slides were prepared by squashing the root tips using 45% ace- tic acid and stained with aceto orcein solution. Chromosome count and karyotyping Chromosome count was performed on mitotic meta- phase cells. For each morphotype, ten clearly observable metaphase cells from ten individuals were selected and photographed using standard and high resolution auto- mated karyotyping software processing (Leica CW4000). Metaphase chromosomes of each morphotype were placed in pairs, arranged and numbered in order of size, with keeping in view the centromere position to consti- 47Cytogenetic and molecular studies of the Egyptian Capsella bursa-pastoris (Brassicaceae) tute a karyotype. The length of the short arm (p) and the long arm (q) was measured for each chromosome, and the total length (TL=p+q) was calculated. The relative length (RL) of the chromosomes (TL / sum TL × 100) and the mean relative length (MRL) of each chromosome pair were calculated. The centromeric index (CI) was estimated by (P / TL x 100), the mean centromeric index (MCI) was calculated to represent the centromeric index value of a particular chromosome pair, then the chromo- somes were classified according to Levan et al. (1964). Molecular study From t he sa me 30 specimens col lected f rom Faiyum region (Table 1) for cy togenetic studies, 14 specimens representing the three morphotypes were chosen for molecular study (4 Lobed, 4 Simple and 6 Lobed-Simple). DNA extraction A total genomic DNA was extracted from 1 g young leaves using CTAB (cetyl-trimethyl ammonium bro- mide) extraction buffer procedure described by Doyle and Doyle (1990) and modified by Allen et al. (2006). PCR reactions and data analysis For each 25 μl PCR reaction, add 12.5 μl Dream Taq Green PCR Master Mix (2X), 1 μl Forward primer (5 -́ GCC TAC CGC ATC GAA ATA GA-3´), 1 μl Reverse Table 1. Collected specimens of Capsella bursa-pastoris (L.) Medik. in Egypt with their geographical distribution (arranged from North to South). Collection & Herbarium Date Longitude Latitude Locality Amer 8312 (CAI) 18.1.1987 30°26’33” 31°25’15” Beheira Province, Rosetta Fahmy 963 (CAI) 2.5.1988 27°14’55” 31°21’23” Mersa Matruh, El Sallum road Fayed & El Naggar s.n. (ASTU) 15.3.1984 30°00’44” 31°17’00” Alexandria, El Montazha Amer 16225 (CAI) 6.3.1988 30°32’58” 31°12’17” Beheira Province, Mahmudiya Abdel Fattah & Abdel Aziz s.n. (CAI). 19.3.1974 31°23’00” 31°01’53” El Mansoura Gun Romée 443 (CAI) 12.3.1968 30°55’33” 30°47’13” Tanta Amer 1515 (CAI) 18.3.1982 31°48’59” 30°43’19” Sharkiya, Faqus G. Täckholm s.n. (CAI) 7.1.1927 31°12’46” 30°41’40” Barrage (Zifta) El Naggar s.n. (ASTU) 30.1.1985 31°11’11” 30°27’19” Banha, Kafor Mousa El Bakry 2708 (CAI) 29.4.1981 31°33’43” 30°24’57” Bilbeis Chrtek, Kosinova & Slavikova s.n. (CAI) 4.4.1977 31°17’00” 30°12’01” Bahtim El Batanony s.n. (CAI) 7.2.1957 31°14’14” 30°07’26” El Menoufia Amer et al. s.n. (CAI) 27.1.2017 31°12’54” 30°06’45” El Menoufia, Abu Sleem village El Hadidy s.n. (CAI) 17.1.1952 31°11’58” 30°04’52” Imbaba El Hadidy s.n. (CAI) 12.1.1956 31°12’27” 30°01’39” Giza, Faculty of Science farm Taher El sayed s.n. (CAI) 19.11.1926 31°11’45” 30°01’12” Giza, in clover fields Chrtek & Kosinova s.n. (CAI) 13.4.1971 31°12’29” 30°01’05” Giza, Faculty of Agriculture farm Chrtek & Kosinova s.n. (CAI) 1.4.1971 31°13’12” 30°00’35” Giza, El Harraniya village Chrtek, Kosinova & Imam s.n. (CAI) 27.4.1967 31°15’48” 29°35’21” El Saff, fields along the road Amer et al. s.n. (CAI) 23.4.2018 31°15’17” 29°34’57” El Saff Abd El Ghani 5820 (CAI) 13.3.1983 30°51’27” 29°24’48” Faiyum, Sinnuris district, El Siliene Amer et al. s.n. (CAI) 27.1.2017 30°51’27” 29°24’48” *Faiyum, Sinnuris district, El Siliene Abd El Ghani 5234 (CAI) 8.3.1983 30°48’56” 29°21’20” Faiyum district, Beni Saleh Abd El Ghani 5320 (CAI) 8.3.1983 30°27’11” 29°19’16” Faiyum district, in clover fields Fayed et al. s.n. (ASTU) 2.5.2010 34°18’25” 27°56’48” Southern Sinai, Farsh Elias Zareh & Fayeds.n. (ASTU) 5.12.1990 31°12’05” 27°10’20” Assiut, Sohag East road Zareh s.n. (ASTU) 5.12.1990 31°20’18” 27°02’44” Assiut, El- Matmar Zareh & Fayed s.n. (ASTU) 30.1.1991 31°22’02” 26°57’05” Assiut, Sedfa Zareh s.n. (ASTU) 28.2.1962 32°00’10” 26°14’08” El-Balliana, Sohag *Specimens subjected to cytogenetic and molecular studies. 48 Wafaa M. Amer, Rania A. Hassan, Amany S. Abdo primer (5 -́ CAA GAA AGT CGG CCA GAA TC-3´), 2 μl Template DNA, and complete to 25 μl by water (nuclease-free). The PCR was performed using the recommended thermal cycling conditions: one cycle of initial denatura- tion at 95°C for 5 minutes, 35 cycles of denaturation at 95°C for 45 seconds followed by annealing at 57°C for 45 seconds then extension at 72°C for 60 seconds, and one cycle of final extension at 72°C for 10 minutes. The reaction products were separated by electropho- resis on 1.6 % agarose gel in 1x TBE buffer and run in the same buffer at 100 V for 1 hour, and visualized by staining with 0.5 µg/ml of ethidium bromide and photo- graphed under UV light. The cpSSR locus ATCP31017 was sequenced and DNA was amplified using the previously mentioned primers (Castro et al., 2014). The amplified fragments were sequenced in ABI377 DNA sequencer (ABI, USA). Then BLAST programs were used for searching DNA databases for sequence similarities. Mega software was used to carry out multiple sequence alignment and cal- culate genetic distances among studied taxa. Neighbour- joining dendrogram was constructed showing the genet- ic relationships among 14 specimens of the three studied genotypes. RESULTS Morphological diversity The morphological investigations and taxonomic revision of 36 old herbarium specimens and 66 recently collected populations of C. bursa-pastoris based on 25 morphological characters including plant height, basal and cauline leaves features, as well as inflorescence and fruit characters (Amer et al., 2019). The most differen- tial characters were that of the basal leaves. The results of that study (Amer et al., 2019) revealed the presence of three morphotypes in Egypt namely: Lobed “L” with all basal leaves are lobed, Simple “S” in which all basal leaves are simple, and Lobed-Simple “LS” in which basal leaves are mixed lobed with simple (Figure 1). The three identified morphotypes were co-distrib- uted and traced in the field as mixed populations along with the species geographical range. Where, the “LS” morphotype was the most common type and showed the highest phenotypic variation, while the “S” morpho- type showed rare occurrence in all studied localities. The environmental factors such as shading, temperature, soil type and rainfall showed no influence on the distribu- tion of these morphotypes (Amer et al., 2019). Figure 1. Morphological diversity within the Egyptian C. bursa pastoris genotypes; L: Lobed, S: Simple, LS: Lobed-Simple. 49Cytogenetic and molecular studies of the Egyptian Capsella bursa-pastoris (Brassicaceae) Cytogenetic analysis Chromosome number Chromosome count for each morphotype of C. bur- sa-pastoris was done in the mitotic metaphase (Figure 2). The three studied morphotypes recorded three different chromosome numbers. Accordingly, they are treated as three distinct genotypes. The Lobed “L” genotype has diploid chromosome set of 2n=2x=16. The Simple “S” genotype has triploid chromosome set of 2n=3x=24 with minor aneuploidy in chromosome no. 6 and 8 (2n=3x- 2=22). This triploid genotype is recorded for the first time in Egypt. While the Lobed-Simple “LS” genotype recorded the presence of tetraploid chromosome set of 2n=4x=32 (Figure 2). Karyotype analysis The karyotyping data of these three genotypes are provided in Figure 3 and Table 2. The retrieved results showed that the chromosomes are small in size, the total genomic length ranges from 43.48 µm in the Lobed “L” genotype to 67.76 µm in Lobed-Simple “LS” genotype, while the Simple “S” genotype has an intermediate value of 61.46 µm. Furthermore, the chromosomes are highly vari- able referring to their mean relative length (MRL), as shown in Figure 4. The chromosome pair 1 is the long- est in the three genotypes, its length ranges from 3.21 µm in Lobed-Simple “LS” genotype to 4.48 µm in Sim- ple “S” one, and its mean relative length (MRL) ranges from 4.74% in Lobed-Simple “LS” genotype to 7.99% in Lobed “L” one. The length of the shortest chromosome pair 8 ranges from 1.54 µm in Simple “S” to 1.74 µm in Lobed “L, and its mean relative length (MRL) ranges from 2.35% in Simple “S” genotype to 3.99% in Lobed “L” with an intermediate value of 2.52% in Lobed-Simple “LS” genotype. The eight chromosome pairs were grouped based on the centromere position into four types: acrocentric, metacentric, submetacentric and subtelocentric (Table 2). The chromosome pairs from 1 to 4 are metacentric in all the studied genotypes. The chromosome pair no. 5 is metacentric in genotypes “L” and “S”, while it is acrocen- tric in “LS” genotype. The chromosome pair 6 is acro- centric in “L” genotype, while it is submetacentric in “S” and “LS” genotypes. The chromosome pair 7 appeared acrocentric in “L” genotype, submetacentric in “S” gen- otype, and metacentric in “LS” genotype. The chromo- some pair 8 is metacentric in “L” and “S” genotypes, while subtelocentric in “LS” genotype. Molecular analysis Gel electrophoresis of the PCR amplification prod- ucts of 14 studied specimens (4 Lobed, 4 Simple and 6 Lobed-Simple) produced 14 bands of good quality. The statistical results of cpSSR locus ATCP31017 sequences developed a Neighbour-joining dendrogram (Figure 5) that separated the genotypes “L”, “S”, and “LS” into three genetic clusters. The first cluster includ- ed four specimens (L1-4) that represented the Lobed “L” genotype (2n=16). In this cluster, specimens L1 and L3 showed high genetic similarity to each other. The sec- ond cluster included also four specimens (S5-8) that represented the Simple “S” genotype (2n=24). In this cluster, specimens S5 and S7, and specimens S6 and S8 showed high genetic similarity to each other. The third cluster included six specimens (LS9-14) that represented the Lobed-Simple “LS” genotypes (2n=32). In this clus- ter, specimen LS9 showed the lowest genetic similarity Figure 2. Photomicrographs of well spread mitotic metaphase in the three genotypes of C. bursa pastoris: L: Lobed diploid with 2n=2x=16; S: Simple triploid with 2n=3x=24; LS: Lobed-Simple tetraploid with 2n=4x=32. 50 Wafaa M. Amer, Rania A. Hassan, Amany S. Abdo with other specimens. While specimens LS10 and LS12, also LS13 and LS14 showed high genetic similarity to each other. The DNA sequences of the studied 14 specimens of C. bursa-pastoris were registered on the National Cent- er for Biotechnology Information (NCBI) under the following accession numbers MN602606, MN602607, M N6 026 08, M N6 026 09, M N6 02610, M N6 02611, M N6 02612 , M N6 02613, M N614131, M N614132 , MN614133, MN614134, MN614135 and MN614136. Table 2. Karyotyping data for the studied C. bursa pastoris genotytpes, L: lobed, S: simple, LS:lobed-simple, A: Acrocentric, M: Metacentric, SM: Submetacentric, ST: Subtelocentric. L S LS Long arm (q) µm 1 1.74±0.05 2.74±0.63 1.63±0.05 2 1.58±0.00 1.97±0.12 1.29±0.16 3 1.58±0.10 1.37±0.10 1.10±0.26 4 1.37±0.10 1.23±0.04 1.05±0.10 5 1.37±0.00 1.19±0.09 1.63±.38 6 2.32±0.00 1.51±0.25 1.34±0.24 7 2.21±0.00 1.23±0.23 0.95±0.15 8 1.08±0.50 0.84±0.49 1.35±0.22 Short arm (p) µm 1 1.74±0.05 2.10±0.32 1.58±0.11 2 1.58±0.00 1.86±0.16 1.21±0.16 3 1.53±0.05 1.33±0.11 0.95±0.11 4 1.32±0.05 1.19±0.04 1.00±0.11 5 1.21±0.05 1.16±0.10 0.22±0.10 6 0.26±0.15 0.70±0.39 0.45±0.44 7 0.21±0.00 0.74±0.35 0.84±0.06 8 0.66±0.34 0.70±0.39 0.36±0.15 Total length (p+q) µm 1 3.48±0.10 4.84±0.95 3.21±0.16 2 3.16±0.00 3.83±0.28 2.5±0.32 3 3.11±0.15 2.70±0.20 2.05±0.37 4 2.69±0.15 2.42±0.08 2.05±0.21 5 2.58±0.05 2.35±0.19 1.84±0.48 6 2.58±0.15 2.21±0.64 1.79±0.58 7 2.42±0.00 1.97±0.55 1.79±0.21 8 1.74±0.54 1.54±0.79 1.71±0.37 Mean relative length (MRL) 1 7.99 7.38 4.74 2 7.27 5.83 3.69 3 7.14 4.12 3.03 4 6.18 3.69 3.03 5 5.94 3.59 2.72 6 5.94 3.37 2.64 7 5.57 2.99 2.64 8 3.99 2.35 2.52 Mean centromeric index (MCI) 1 50 44.41 49.13 2 50 48.55 48.41 3 49.23 49.18 45.67 4 49.02 49.17 48.82 5 46.88 49.25 12.3 6 9.94 30.69 25.01 7 8.68 36.63 47.16 8 40.11 47.53 21.93 Type 1 M M M 2 M M M 3 M M M 4 M M M 5 M M A 6 A SM SM 7 A SM M 8 M M ST Fig. 3. Karyotypes of C. bursa-pastoris genotytpes. L: Lobed dip- loid (2n=2x=16); S: Simple triploid (2n=3x=24); LS: Lobed-Simple tetraploid (2n=4x=32). Fig. 4. Mean relative length (MRL) of each chromosome pair in the three studied genotypes of C. bursa-pastoris. 51Cytogenetic and molecular studies of the Egyptian Capsella bursa-pastoris (Brassicaceae) DISCUSSION The taxonomic revision of Capsella bursa-pasto- ris in Egypt revealed the presence of a high degree of phenoplasticity in all the studied populations (old and fresh) and resulted in three distinctive morphotypes (“L” Lobed, “S” Simple and “LS” Lobed-Simple) based mainly on the basal leaves structure (Amer et al., 2019). These morphotypes were traced as mixed populations in all the studied localities (Table 1), so the environ- mental factors have no influence on their distribution or phenoplasticity (Amer et al., 2019). However, posi- tive correlation between the degree of genetic hetero- geneity and environmental variability was reported by Aksoy et al. (1998), Neuffer et al. (1999), Neuffer and Hurka (1999) and Castro et al. (2014). In addition to Han et al. (2015) who reported that C. bursa-pastoris populations from different regions are polymorphologi- cally different. The study of genetic diversity of C. bursa-pastoris in Egypt has received little attention so far. In this study, we tried to elucidate the role of genetic diversity within the Egyptian morphotypes in their phenoplasticity. The chromosome counting of the identified mor- photypes recorded three different chromosome numbers (2n=16 for Lobed “L”, 3n=24 for Simple “S”, 4n=32 for Lobed-Simple “LS”), so they are treated here as geno- types. Shull (1909) was the first to distinguish between four biotypes with different leaf types (simplex, rhom- boidea, tenuis, and heteris) and cleared the correla- tion between leaf shape and chromosome number. He recorded the tetraploid number for simplex and rhom- boidea biotypes, while heteris and tenuis were diploid. Nonetheless, the Egyptian morphotypes are not equiva- lent to Shull’s biotypes (Amer et al., 2019). Worldw ide, t he tet raploid C . bursa-pa stor i s (2n=4n=32) is one of the most successful plants that have high polymorphic level (Han et al., 2015). Similarly in Egypt, the tetraploid “LS” genotype is the most com- mon and diverse type with a wide range of leaf forms on the same plant (Amer et al., 2019). This is supported by Shull (1929), Löve and Löve (1956), Davis (1965), Raj (1965), Hsu (1968), Svensson (1983) and Hurka (1984). The diploid chromosome number (2n=2x=16) recorded in Lobed “L” genotype, was early recorded in Europe by Bosbach and Hurka (1981), in Greece by Svensson (1983), and in Kashmir by Jeelani et al. (2013). Although C. bursa-pastoris is a self-compatible spe- cies, different percentages of outcrossing were recorded by many authors: Aksoy et al. (1998) recorded 1-2%, Hurka et al. (1989) recorded 3-12%, and Hurka and Neuffer (1997) recorded up to 20%. In reviewing the literature, no data were found on the triploid “S” genotype (2n=3x=24) which is recorded for the first time in Egypt. Its rare presence within C. bursa-pastoris populations comparing with the other two genotypes (Amer et al., 2019) may support its hybrid origin. The lack of chromosome no. 6 and 8 (aneuploidy, 2n=3x-2=22) in some individuals of this genotype sup- ports our postulation. Bretagnolle and Thompson (1995) cleared that the triploid offspring are typically sterile due to problems in chromosomal pairing and segrega- tion during meiosis, which may cause aneuploid gametes and result in sterility. Karyotype analysis showed that the chromosomes are small in size, this result agrees with Schmidt and Bancroft (2011) who reported that mitotic chromosomes of crucifer species are generally very small in size. The karyotyping of the three genotypes (Fig. 3 & Table 2) showed distinctive variations within genotypes in the Fig. 5. Diagonal matrix of the genetic distances and Neighbour-joining dendogram showing the genetic relationship among 14 specimens of the three studied genotypes (L, S and LS). 52 Wafaa M. Amer, Rania A. Hassan, Amany S. Abdo genome and chromosomes mean relative lengths (MRL) in addition to the centromere position. Our results are supported by Guerra (2008) who claimed that the kar- yological data in taxonomy contribute to evaluate the genetic relationships among species or populations and lead to better understanding of the way they diverged from each other. As reported by Amer et al. (2019), the “LS” popula- tions were large in size, up to 80 cm length with inflo- rescence up to 75 cm, while “L” and “S” populations were small in size (up to 50 cm long with inflorescence up to 40 cm). These results indicate that the growth rate of tetraploid populations is greater than that of other genotypes, and agree with Neuffer’s finding (1989) that the rate of growth was greater for the tetraploid groups. On the other hand, the small size of the diploid “L” and the triploid “S” genotypes can be explained by deletion of redundant genes which can result in downsizing of the genome, as reporteded early by Devos  et al. (2002), Blanc and Wolfe (2004), Vitte and Bennetzen (2006). The molecular results achieved by sequencing the cpSSR locus ATCP31017 of 14 specimens of C. bursa-pas- toris genotypes support the cytogenetic results, where the neighbour-joining dendrogram (shown in Figure 5) sepa- rated the studied genotypes “L”, “S”, and “LS” into three distinctive genetic clusters. The first cluster included four specimens for the diploid “L” genotype. In this cluster, specimens L1 and L3 showed high genetic similarity with each other, this result may be reflected from high mor- phological similarity (both specimens had plant length up to 50 cm and inflorescence up to 40 cm and similar cauline leaves). The second cluster included four speci- mens for the triploid “S” genotype. In this cluster, speci- mens S5 and S7 showed high genetic similarity reflected from morphological similarities with each other (plant length up to 50 cm, its inflorescence up to 40 cm). More- over, specimens S6 and S8 showed high genetic similar- ity as both samples had very small plant size (stem length was up to 20 cm, its inflorescence was up to 15 cm). The third cluster included six specimens for the tetraploid “LS” genotype. In this cluster, specimen LS9 had the low- est genetic similarity with the other specimens. This may be due to its very large size (stem length was up to 80 cm, its inflorescence was up to 75 cm) and presence of both simple and lobed cauline leaves on the same plant. On the other hand, specimens LS10 and LS12 were geneti- cally similar (both specimens had large plant size and simple cauline leaves). Also, specimens LS13 and LS14 were genetically similar, as both specimens had small plant size and simple cauline leaves. The Lobed-Simple “LS” genotype with the highest phenotypic plasticity (as shown in Fig. 1), also showed high genetic diversity (Fig. 5). 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