Karyotyping and Molecular Cytogenetic Studies Using ...

Karyotyping and Molecular Cytogenetic Studies

Using Repetitive Sequences in Lettuce

January 2020

WINNY DEWI WIDARMI

Graduate School of Horticulture

CHIBA UNIVERSITY

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CONTENTS

GENERAL INTRODUCTION ................................................................................................. 4

CHAPTER I ............................................................................................................................. 10

Characterization of Lettuce Chromosomes Based on Condensation Patterns and Physical

Mapping of 45S and 5S rDNAs Using Fluorescence in situ Hybridization ............................ 10

1.1 Introduction ..................................................................................................................... 12

1.2 Materials and Methods .................................................................................................... 16

1.3 Results ............................................................................................................................. 19

1.4 Discussion ....................................................................................................................... 22

1.5 Table and Figures ............................................................................................................ 26

CHAPTER 2 ............................................................................................................................ 30

Cytological variation of (AAG)7 repeat on lettuce chromosomes by fluorescence in situ

hybridization ............................................................................................................................ 30

2.1 Introduction ..................................................................................................................... 32

2.2 Materials and Methods .................................................................................................... 34

2.3 Results ............................................................................................................................. 36

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2.4 Discussion ....................................................................................................................... 37

2.5 Figures ............................................................................................................................. 40

CHAPTER 3 ............................................................................................................................ 43

Physical localization of rDNAs and microsatellite sequences on the chromosomes of Lactuca

saligna using fluorescence in situ hybridization ..................................................................... 43

3. 1 Introduction .................................................................................................................... 45

3.2 Materilas and Methods .................................................................................................... 47

3.3 Results ............................................................................................................................. 49

3.4 Discussion ....................................................................................................................... 51

3.5 Figures ............................................................................................................................. 54

GENERAL DISCUSSION ...................................................................................................... 58

REFERENCES ........................................................................................................................ 66

SUMMARY ............................................................................................................................ 77

ACKNOWLEDGMENTS ............................................................................................ 79

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GENERAL INTRODUCTION

Lettuce is an important vegetable crop species in the group of leafy vegetables

produced commercially in many countries worldwide. It is especially important as a

commercial crop in Asia, North and Central America, and Europe. China, U.S., Spain, Italy,

India and Japan are among the world’s largest producers (Lebeda et al. 2007; Mou 2008). An

annual plant in the daisy family, Compositae (Asteraceae), tribe Lactuceae of subfamily

Cichorioideae (Křístková et al. 2008). Due to its economic importance, most current studies

of the genus Lactuca have focused on lettuce cultivars and the species that can be easily

crossed to domesticated lettuce (Koopman et al. 1998; Zohary 1991), including studies of

phylogeny and trait breeding. However, the broader phylogenetic relationships of

domesticated lettuce and wild lettuce species remain unclear, including the taxonomic

boundary of the genus Lactuca L. itself.

Cultivated lettuce (Lactuca sativa L.) is a diploid (2n= 18) species and there are three

wild species in the subsection Lactuca, L. serriola, L. saligna, and L. virosa; all are 2n = 18

and self-fertilizing and important genitors for lettuce breeding (Michelmore et al. 1994;

Koopman et al. 1998). The estimated genome size of L. sativa was 2.7 Gb, with repetitive

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sequences accounting for 74.2% of the genome (Michelmore et al. 1994; Koopman et al.

1998; Reyes-Chin-Wo, S. et al. 2017).

L. saligna is considered one of the most important species in the lettuce genepool

(Lebeda et al. 2007). It has come to play a considerable part in lettuce resistance breeding to

Bremia lactucae (downy mildew pathogen) (Lebeda et al. 2002, 2007; Beharav et al. 2006).

The wild relatives of agricultural crops conserved in genebank collections constitute a broad

genetic base that can provide essential materials for plant breeding (Lebeda et al. 2007).

In the last dozen years, relationships among the cultivated lettuce and its allied species

have been studied using molecular techniques (Kesseli et al. 1991; Hill et al. 1996;

Witsenboer et al. 1997; Koopman et al. 1998). These analyses clarify that the closest species

of the cultivated lettuce is L. serriola.

Cytological studies, karyotype analysis (Lindqvist 1960; Haque and Godward 1985;

Koopman and De Jong, 1996; Matoba et al. 2007) and chromosome banding (Koopman et al.

1993), have been demonstrated the relationships among lettuce and its closely related species.

However, they observed metaphase chromosomes which are in highly condensed condition.

Components of metaphase chromosomes, i.e., number, length and arm ratio, had been

commonly used to identify the karyotype of species, except for maize and rice pachytene

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chromosomes. Metaphase chromosomes of horticultural plants in particular is relatively small

compared to those of other plant species, it had been difficult to discriminate individual

chromosomes.

Fukui (1986) and Fukui and Mukai (1988) used the first-generation chromosome

image analyzing system (CHIAS) and observed specific condensation patterns (CPs) in small

plant chromosomes at the somatic prometaphase. So far, chromosomes have been

characterized by CP in saltbush (Fukui and Mukai, 1988), rice (Fukui and Iijima, 1991), and

soybean (Yanagisawa et al. 1991). CP considered to be a useful procedure to characterized

morphological traits and identify the chromosomes in many plant species.

Fluorescence in situ hybridization (FISH) techniques provide the phylogenetic

information in closely related taxa, and FISH with ribosomal RNA genes and telomeric

sequences has been widely utilized for karyotyping and studying chromosomal organization

and evolution in many plant species, e.g., spinach (Ito et al. 2000); rose (Akasaka et al.

2003); lettuce (Matoba et al. 2007); and cucumber (Zhang et al. 2012). Combinations of

FISH and condensation pattern has not been carried out in the lettuce chromosomes.

FISH with repetitive DNA probes results in chromosome-specific hybridization

pattern that allows identification of all chromosomes within a species, making this technique

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a powerful tool for the karyotyping and studying chromosomal organization and evolution in

many plant species (Jiang and Gill 2006; Mukai et al. 1993; Pedersen and Langridge 1997).

Karyotype analysis and chromosomal distribution of 5S and 18S rDNAs were detected by

FISH method (Matoba et al. 2007). The karyotype analysis revealed the dissimilarity

between L. virosa and L. sativa, L. serriola, L. saligna.

Microsatellites or simple sequence repeats (SSRs) are tandem repeats of DNA motifs

composed of short repeated units about 1-6 nucleotides, that can be found anywhere in the

genome, both in protein-coding and noncoding regions (Toth et al. 2000). Because of their

high mutability, microsatellites are thought to play a significant role in genome evolution by

creating and maintaining quantitative genetic variation (Tautz et al. 1986; Kashi et al. 1997).

Microsatellite markers can be developed as polymerase chain reaction-based markers

and used as probes in the fluorescence in situ hybridization (FISH) which is a crucial tool for

plant molecular cytogenetic researches (Jiang and Gill 2006). Microsatellites have been

widely used for chromosome identification and study of genome organization and evolution

in different plant species e.g. Triticeae (Cuadrado et al. 2008), Allium (Monkheang et al.

2016) and Panax ginseng (Waminal et al. 2018). As far as we know, no previous FISH

experiments using microsatellites sequences as probes have been reported in L. saligna.

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The distribution of microsatellite AAG on the chromosomes of wheat, barley, and

related species in Triticeae are well reported, the sequence (AAG)5 was used for in situ

hybridization to identify different chromatin classes in wheat (Cuadrado et al. 2000, 2008).

The oligonucleotide sequence (AAG)7 was used for genome and chromosome identification

via in situ hybridization in cultivated barley and related species of the Triticeae (Poaceae)

(Pedersen et al. 1996). The sequence (AAG)9 was used for the complementary identification

of all A-genome chromosomes in diploid and polyploid wheat (Badaeva et al. 2015, 2016).

The sequence (AAG)6 was firstly applied to the species of Zanthoxylum to perform a more

accurate karyotype analysis of Z. armatum (Luo et al. 2018). Probing AAG repeats using in

situ hybridization is a useful diagnostic tool in cytogenetics and for understanding genome

organization.

The aims of the present studies are (1) to identify and discriminate of lettuce

chromosomes in prometaphase cells through karyotype analysis based on condensation

patterns using CHIAS IV, also localize the sites for 45S and 5S rDNAs by FISH method, (2)

to perform FISH using (AAG)7 as the probe in order to clarify the possibility of using the

microsatellite repetitive sequences for discrimination and/or identification of the

chromosomes in Lactuca species, and (3) to perform FISH using nine SSRs and 45S and 5S

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rDNA sequences as the probes, on the mitotic, and meiotic diakinesis and pachytene

chromosomes of L. saligna in order to identify the distribution of the repetitive sequences.

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CHAPTER I

Characterization of Lettuce Chromosomes Based on Condensation Patterns and

Physical Mapping of 45S and 5S rDNAs Using Fluorescence in situ Hybridization

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Summary

Lactuca sativa L. is one of the most important reference species for genome research

in Compositae. In the present study, fluorescence in situ hybridization (FISH) and

chromosome image analysis system IV (CHIAS IV) were applied to obtain detailed

information of the characteristics of lettuce chromosomes. One lettuce cultivar (L. sativa

L.) and three wild species L. serriola, L. saligna and L. virosa were used to analyze

chromosome lengths, arm ratios and degrees of chromosomal condensation at prometaphase.

Locations of 45S and 5S rRNA genes were determined by FISH technique. Our results

showed that nine pairs of homologous chromosomes could be discriminated for all four

species by their morphological characteristics, condensation patterns (CPs) and localization

of rRNA loci. Statistical analysis showed that the total length of L. saligna chromosomes

was significantly the shortest among the species tested. The present study is the first for

physical mapping of condensed regions and rDNAs using CPs and FISH of prometaphase

chromosomes in lettuce. The information provided here would be valuable for practical

breeding and genetics of lettuce.

Key words: CHIAS IV, Condensation pattern, FISH, Lettuce, rDNA.

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1.1 Introduction

Cultivated lettuce, Lactuca sativa L. (2n = 18), is one of the most important

vegetables in the group of leafy plants produced commercially in many countries worldwide.

World production of lettuce in 2016 was more than 26 million tons, and those were mainly

produced in Asia, North and Central America and Europe (FAO 2017,

http://faostat.fao.org/site/567/DesktopDefault.aspx?PageID=567#ancor). L. sativa is an

annual plant in the subtribe Lactucinae, tribe Lactuceae (Cichorieae), subfamily

Cichorioideae of the family Compositae (Asteraceae) (Křístková et al. 2008). However, there

are uncertainties about the phylogenetic relationships within the genus Lactuca, mainly due

to the complex and variable morphological characters of the species (Zhen et al. 2017).

Lebeda et al. (2004, 2009) divided the genus into seven sections and two geographical groups

based on taxonomic and biogeographical criteria, i.e., Lactuca (subsection Lactuca and

Cyanicae), Phaenixopus, Mulgedium, Lactucopsis, Tuberosae, Micranthae and Sororiae, and

African and North American groups.

There are three wild species in the subsection Lactuca, namely, L. serriola, L.

saligna and L. virosa; all being 2n = 18, self-pollinated plants and important genetic

resources for lettuce breeding (Michelmore et al. 1994, Koopman et al. 1998). It is estimated

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that L. serriola, was at first domesticated in Egypt more than 4000 years ago, and was the

ancestral species of the present cultivated lettuce (Harlan 1986).

So far, many cytological studies, karyotype analyses, chromosome banding and

FISH techniques were carried out to demonstrate the relationships among the cultivated

lettuce and its closely related species. Lindqvist (1960) was the first who studied the most

comprehensive chromosome morphology of Lactuca species. He observed distinct

differences in metaphase chromosome morphology between L. virosa and the other three

species, L. sativa, L. serriola and L. saligna and found that the karyotypes of L. sativa and L.

serriola were identical, and the measurements of the chromosomes revealed certain

dissimilarities between these two species and L. saligna. Haque and Godward (1985)

described the somatic metaphase chromosome pairs on the basis of their lengths, centromere

positions and presence or absence of satellites of three cultivars of L. sativa, L. serriola and a

cultivar of Cichorium intybus. He found slight differences in chromosome lengths, the L.

sativa cultivars and L. serriola being distinguishable by their mean lengths. Koopman et al.

(1993) obtained chromosome banding patterns with C- and N- bandings, and AgNO3 staining

in somatic metaphase of four Lactuca species and found out that L. sativa and L. serriola had

almost identical chromosome morphology, and L. saligna differed only slightly from them,

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but L. virosa was quite distinct from the other species. Koopman and de Jong (1996) applied

a numerical analysis to study the evolutionary relationships among L. sativa, L. serriola, L.

saligna and L. virosa using karyotypes and relative DNA content. They found that L. sativa

and L. serriola were very closely related, and L. saligna differed significantly from L.

sativa/serriola only in its relative DNA amount. L. virosa differed from L. saligna and L.

sativa/serriola for all parameters. They also found the largest difference between L. saligna

and L. virosa, and considered that L. saligna and L. virosa are advanced species that evolved

in different directions.

Matoba et al. (2007) examined karyotypes and chromosomal distribution of 5S and

18S rDNAs in L. sativa, L. serriola, L. saligna and L. virosa by FISH method. The karyotype

analysis revealed dissimilarity between L. virosa and the other three species, and it was

difficult to distinguish L. sativa and L. serriola based solely on karyotype analysis. FISH

result showed one 5S rDNA and two 18S rDNA loci in all four species. They revealed that

both 5S and 18S rDNA loci of L. sativa, L. serriola and L. saligna were identical in their

numbers and locations. However, the locations of rDNAs of L. virosa were different from

those of the other species.

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Since metaphase chromosomes of horticultural plants are relatively small compared

to those of other plant, e.g., wheat and barley, it has been rather difficult to discriminate their

individual chromosomes. In lettuce, all cytological studies, karyotype analyses, and

chromosome banding have been conducted mainly using metaphase chromosomes, which are

in highly condensed condition (Matoba et al. 2007). Fukui (1986) and Fukui and Mukai

(1988) developed the CHIAS through measuring chromosome condensation patterns (CPs) in

small plant chromosomes at prometaphase. So far, chromosomes of many species have been

characterized with CHIAS, e.g., in saltbush (Fukui and Mukai 1988), rice (Fukui and Iijima

1991), soybean (Yanagisawa et al. 1991), tea plant (Furukawa et al. 2017), etc. CP is

considered to be a useful and reliable parameter to characterize morphological traits and

identify the chromosomes in many plant species, having small chromosomes. On the other

hand, FISH technique provides phylogenetical information in closely related taxa, and FISH

technique using ribosomal RNA genes and telomere sequences as the probes have been

widely applied for karyotyping and studying chromosomal organization and evolution in

many plant species.

Combination of CP and FISH analyses have provided novel aspects of chromosome

images via physical localization of repetitive sequences on the elongated prometaphase

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chromosomes. The aim of the present study is to identify and discriminate lettuce

chromosomes in prometaphase cells through karyotype analysis based on the CPs using

CHIAS IV and determining the locations of 45S and 5S rDNAs by FISH method.

1.2 Materials and Methods

Plant materials and chromosome sample preparation

The seeds of Lactuca sativa cv. 'Patriot', L. serriola, L. saligna and L. virosa

investigated were supplied from Nagano Vegetable and Ornamental Crops Experimental

Station, Japan. Root tips of 1 cm long were collected from the seedlings or young plants

grown for a few weeks after seedling stage in 500-mL pots. The root tips were fixed in the

solution of 3 ethanol : 1 acetic acid (v/v) at 4°C for at least one day before chromosome

preparation. Fixed root tips were washed three times with distilled water, and were stored in

70% ethanol at 4°C until use. Chromosome preparations were made through enzymatic

maceration, where root tips were digested with an enzymatic mixture [4% cellulase

Onozuka RS (Yakult), 2% pectolyase Y-23 (Kyowa), and 1% pectinase (Sigma)] at 37°C for

45-90 min. Squashing was made in 45% acetic acid on the glass slides and the slides were

stored at -80°C more than 12 h, and the coverslips were removed with a razor blade.

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Chromosomes were stained with 1% (v/v) Giemsa (Merck) in 1/15 M sodium phosphate

buffer (pH 6.8) for 2 min and air dried.

DNA probe preparation

The 45S rDNA (pTa71, Gerlach and Bedbrook 1979) and 5S rDNA (Fukui et al.

1994) were used as the probes for FISH analyses. The 5S rDNA was amplified from the

genomic DNA of L. sativa by polymerase chain reaction (PCR) using coding regions of 5S

rDNA as the primers (5’-GATCCCATCAGAACTCCGAAG-3’ and

5’-CGGTGATTTAGTGCTGGTAT-3’) (Fukui et al. 1994). PCR products with about 300 bp

were inserted into the EcoRV site of pZErO-2 Vector (Invitrogen) according to the

manufacturer’s instruction. The plasmid inserted with 5S rDNA unit was used as probe. The

45S and 5S rDNAs were labeled with DIG-Nick Translation Mix (Roche), and Biotin-Nick

Translation Mix (Roche) according to manufacturer’s instruction, respectively.

FISH analysis

Chromosome slides were re-fixed in 1% paraformaldehyde in PBS for 10 min at room

temperature. Procedures for FISH were followed as described by Kikuchi et al. (2008). The

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10 µL of hybridization cocktail containing 50% formamide, 10% dextran sulfate, 2×SSC, and

100-500 ng labeling probe was applied to the slide, covered with a coverslip, and then

denatured for 3 min at 80°C on a heat block. After incubation at 37°C for one day, the slide

was washed and briefly dried. Detection of the biotin- and DIG-labeled probes was

performed with biotinylated streptavidin-FITC (Vector Laboratories) and

anti-digoxigenin-rhodamine (Roche), respectively. The slides were counter-stained with 5

µg/mL, 4,6-diamidino-2-phenylindole (DAPI) in a VectaShield antifade mounting medium

(Vector Laboratories). All FISH images were captured with an Olympus BX53 fluorescence

microscope equipped with a CCD camera (CoolSNAP MYO; Photometrics). The FISH

images were processed by MetaMorph, Meta imaging series version 7.8 (Molecular Devices,

San Jose) and edited with Photoshop CC 2017 (Adobe).

Image analysis

CHIAS IV (http://www2.kobe-u.ac.jp/~ohmido/index03.htm) was used to analyze the

images of the lettuce prometaphase chromosomes. Giemsa-stained chromosome images were

converted to 8-bit grey scale and the fluorescence signal images were captured as

red-green-blue (RGB) images with 8-bit grey levels for the construction of the ideogram.

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Homologous chromosomes were detected by observing morphological characteristics and

CPs, and designated as Chromosomes 1 to 9 according to their lengths, excluding satellite.

Data for arm lengths and degrees of condensation of nine pairs of chromosomes were

collected in six chromosome spreads. The arm ratio was calculated as the length of the long

arm divided by the length of the short arm (Levan et al. 1964). The comparison of the mean

lettuce chromosome lengths among species was done by Tukey’s t-test. For statistical

analysis, StatPlus ver. 6 (AnalystSoft Inc., Walnut) was used.

1.3 Results

Morphology of lettuce prometaphase chromosomes

Squashed prometaphase chromosomes stained with Giemsa of the four species are

shown in Fig. 1-1. In all species, chromosome pairs could be discriminated with their

morphologies and condensation patterns; heavily condensed chromosome regions were

recognizable as densely stained regions and were distinguished from weakly as loosely

condensed regions.

Table 1 shows numerical data of the prometaphase chromosomes. The lengths of the

chromosomes varied from 5.34 to 13.24 µm in four species. The four longest chromosomes

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were submetacentric, and the others were metacentric and submetacentric. The total

chromosome lengths of L. sativa and L. serriola were almost the same (82.99 µm and 82.94

µm, respectively), L. saligna had the shortest total lengths (70.04 µm), and L. virosa had the

longest total lengths (85.54 µm).

Physical mapping of 45S and 5S rDNAs

Physical locations of the 45S and 5S rDNAs on the chromosomes are shown in Fig.

1-2. Two pairs of 45S rDNA signals were detected at the secondary constrictions and

satellites on the short arms of the chromosomes of all species used. A pair of 5S rDNA

signals were detected at the interstitial regions of the short arms of chromosomes of all

species. A pair of 5S signals were localized carrying one of the two pairs on 45S rDNA

signal.

Characterization of lettuce chromosomes

Quantitative ideograms of the prometaphase chromosomes of the four lettuce species

generated by CHIAS IV based on the characteristics of CPs, chromosome lengths, and FISH

signals are shown in Fig. 1-3. It was clarified that the distal regions of the short arms and

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long arms of all lettuce chromosomes studied were relatively heavily condensed than the

proximal and interstitial regions.

Characteristics of the chromosomes of each species were as follows; i) L. sativa:

Chromosomes 1 and 5 showed heavy condensation in the long arms. Chromosome lengths of

this species ranged from 6.20 to 12.65 µm (Table 1). Chromosome 1 to 6 were

submetacentric and Chromosomes 7 to 9 were metacentric. Two pairs of 45S rDNA signals

were detected at the end of the short arms of chromosomes 3 and 5. One pair of 5S rDNA

signals was detected in the interstitial regions of the short arms of Chromosome 5. ii) L.

saligna: the chromosome lengths of this species were the shortest among the species used,

ranging from 5.34 to 11.09 µm (Table 1-1). Chromosomes 1 to 5 were submetacentric and

Chromosomes 6 to 9 were metacentric. Four large 45S rDNA signals were observed at the

end of the short arms of Chromosomes 3 and 5. Two 5S rDNA signals were detected in the

interstitial regions of the short arms of Chromosome 5. Chromosome 4 and 5 had condensed

region on the short arm, while Chromosome 9 had condensed region on the long arm. All

chromosomes had condensed regions in the centromeric regions except Chromosome 6. iii)

L. serriola: the interstitial regions were slightly condensed, except Chromosome 6, which

were highly condensed. The chromosome lengths ranged from 6.01 to 12.61 µm (Table 1-1).

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Chromosomes 1 to 7 were submetacentric and Chromosomes 8 and 9 were metacentric. The

positions of the rDNA signals in the karyotype were similar to those of L. sativa and L.

saligna, in which four 45S rDNA signals were observed at the end of the short arms of

Chromosomes 3 and 5. A pair of 5S rDNA signals were detected in the interstitial regions of

the short arms of Chromosome 5. iv) L. virosa: chromosome length of this species was the

longest among the species used, ranging from 6.21 to 13.24 µm (Table 1). Chromosomes 1 to

4, and 6, 8 and 9 were submetacentric, and Chromosomes 5 and 7 were metacentric.

Chromosomes 2 and 7 had highly condensed regions along the whole chromosomes.

Locations of the 45S and 5S rDNAs were different from the other species, namely, two pairs

of 45S and one pair of 5S rDNAs located at the end of the short arm of Chromosome 5 and

interstitial region of Chromosome 7, respectively.

1.4 Discussion

In the present study, homologous chromosome pairs in four Lactuca species were

identified by observing prometaphase chromosomes stained with Giemsa solution.

Condensed chromosome regions at prometaphase were recognized as densely stained regions

distinguish from other regions. Our results of karyotype were coincident with the previous

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reports (Lindqvist 1960, Haque and Godward 1985, Koopman et al. 1993, Koopman and de

Jong 1996, and Matoba et al. 2007); the karyotypes of L. sativa and L. serriola were almost

similar to each other and they seemed to be closely related. Although it was reported that the

karyotypes of them were difficult to distinguish based solely on the karyotype analysis of

metaphase chromosomes (Matoba et al. 2007), the present results suggest that the use of

prometaphase chromosomes are helpful to discriminate the chromosomes based on the CPs in

L. sativa and L. serriola.

It is considered that the total chromosome length of a plant is reflected with the

genome size of it. The genome sizes of L. sativa, L. saligna, L. serriola and L. virosa were

reported to be 2.7 Gb, 2.2 Gb, 2.7 Gb and 3.3 Gb, respectively (Koopman 2000,

Reyes-Chin-Wo et al. 2017). The L. virosa genome is larger than those of the other lettuce

species. Here, we can find a clear correlation between the genome sizes and the total

chromosome lengths. The difference of the genome sizes might be due to the difference in

the amounts of condensed region on the interstitial and distal regions of short and long arms

of the chromosomes. The majority of L. virosa chromosomes exhibited highly condensed

(Figs. 1-1 and 1-3) and the longest chromosome length (Table 1) in comparison with the

other three species. It is suggested that genome sizes of the plants are strongly correlated with

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chromosome length mainly due to the amount of repetitive DNA sequences in Lilium (Du et

al. 2017) and Nigella damascena (Kuznetsova et al. 2017). Therefore, the present results

suggest that the abundance of repetitive DNA in the genomes may contribute to the genome

sizes in Lactuca species, and it is reflected as the CPs of the chromosomes.

Karyotyping using CHIAS IV and FISH mapping facilitated the construction of

quantitative and physical ideograms of plant chromosomes. Through combining these two

techniques, comprehensive lettuce chromosome maps would be constructed using DNA

sequences.

The loci of the 45S and 5S rDNA of L. sativa, L. serriola and L. saligna were

identical in their numbers and locations, except the locations of rDNAs of L. virosa was

different from those of the other species. This result was similar to the previous study by

Matoba et al. (2007) who used 18S and 5S rDNAs as the probes on metaphase chromosomes.

As the conclusion, the present study is the first for physical mapping of condensed

regions and rDNAs using CP and FISH of prometaphase chromosomes in lettuce. The

development of a quantitative chromosome map as shown in the present report is

fundamental for physical mapping of the genes and quite useful not only for basic genome

research and chromosome analysis but also practical breeding and genetics of lettuce. In

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future studies, other probes prepared from different kinds of repetitive sequences, single copy

genes and DNA markers would be localized for the construction of precise physical

chromosome maps of lettuce.

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1.5 Table and Figures

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Fig. 1-1. Giemsa-stained prometaphase chromosomes of L. sativa (A), L. saligna (B), L.

serriola (C) and L. virosa (D). Numerals indicate the chromosome numbers according to

their length (see text for explanation). Bars = 10 µm

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Fig. 1-2. FISH prometaphase chromosome spreads (A, C, E, G) and karyotypes (B, D, F,

H) of the lettuce showing 45S (red) and 5S (green) rDNAs signals in. L. sativa (A), L. saligna

(B), L. serriola (C) and L. virosa (D). Chromosomes are arranged according to their length

in karyotypes. Bars = 10 µm

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Fig 1-3. Ideogram of prometaphase chromosomes of L. sativa (A), L. saligna (B), L. serriola

(C) and L. virosa (D), constructed by CHIAS IV. Numerals denote the chromosome

numbers according to their chromosome lengths (see text for details). Black, gray, and white

areas represent chromosome regions with heavy, intermediate and slight condensation,

respectively. The gaps in the middle of the chromosomes indicate the centromere regions.

Red and green circles demonstrate 45S and 5S rDNA loci, respectively.

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CHAPTER 2

Cytological variation of (AAG)7 repeat on lettuce chromosomes by

fluorescence in situ hybridization

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Summary

Cytological studies using fluorescence in situ hybridization (FISH) technique

provides phylogenetical information in closely related taxa and have been widely applied for

karyotyping and studying chromosomal organization and evolution in plant species. In the

present study, FISH using a microsatellite sequence of (AAG)7 as the probe was performed in

order to discriminate the chromosomes in four Lactuca species, i.e., L. sativa, L. serriola, L.

saligna, and L. virosa. Different distribution patterns of (AAG)n signals were shown on the

chromosomes in the four Lactuca species studied, suggesting that these signals could be used

as cytogenetic landmarks for chromosome identification in Lactuca species.

Key words FISH; microsatellite; (AAG)7 repeat; lettuce

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2.1 Introduction

Cultivated lettuce (Lactuca sativa L.) is an economically important member of the

genus Lactuca, which includes closely related wild species, L. serriola, L. saligna and L.

virosa. All of them have 2n = 2x =18 chromosomes, and the estimated genome size of L.

sativa is 2.7 Gb, with repetitive sequences accounting for 74.2% of the genome (Koopman et

al. 1998; Michelmore et al. 1994; Reyes-Chin-Wo et al. 2017). So far, karyotype analyses

were carried out to demonstrate the relationships among the cultivated lettuce and its closely

related species (Haque and Godward 1985; Koopman and De Jong 1996; Lindqvist 1960;

Matoba et al. 2007). Koopman et al. (1993) applied chromosome banding techniques to

discriminate the chromosomes of Lactuca species. Widarmi et al. (2019) and Matoba et al.

(2007) applied FISH technique using 45S and 5SrDNAs to identify the chromosomes of

Lactuca species. From these studies, it was clarified that chromosomal characteristics of L.

sativa and L. serriola were almost similar to each other, while cytological features of L.

saligna are slightly different from L. sativa/L. serriola, and L. virosa is significantly different

from the other species.

Fluorescence in situ hybridization (FISH) using repetitive DNA sequences as the

probes results in chromosome-specific hybridization patterns that allows identification of all

33

chromosomes within a species, making this technique a powerful tool for the karyotyping and

studying chromosomal organization and evolution in many plant species ((Mukai et al. 1993;

Pedersen and Langridge 1997; Jiang and Gill 2006).

The distribution of microsatellite (AAG)n on the chromosomes of wheat, barley,

and related species in Triticeae were well studied. The sequence (AAG)5 was used as the

probe for in situ hybridization to identify different chromatin classes in wheat (Cuadrado et al.

2000; 2008). The oligonucleotide sequence (AAG)7 was used for genome and chromosome

identification via in situ hybridization in cultivated barley and related species of the Triticeae

(Poaceae) (Pedersen et al. 1996) The sequence (AAG)9 was used for the complementary

identification of all A-genome chromosomes in diploid and polyploid wheat (Badaeva et al.

2015, 2016). Also the sequence (GAA)6 was used to perform a more accurate karyotype

analysis of Zanthoxylum armatum (Luo et al. 2018). Thus, probing AAG repeats for in situ

hybridization is seems to be a useful diagnostic tool in cytogenetics in many plant species.

In the present investigation, we aim to perform FISH using (AAG)7 as the probe in

order to clarify the possibility of using the microsatellite repetitive sequences for

discrimination and/or identification of the chromosomes in Lactuca species.

34



Seeds of L. sativa ‘Shinano Hope’, L. saligna, L. serriola, and L. virosa were kindly

provided by Nagano Vegetable and Ornamental Crops Experimental Station, Japan. They

were germinated and planted at the Graduate School of Horticulture, Chiba University,

Matsudo, Japan. Root tips of 1-2 cm long were collected from the seedlings and young plants

after a few weeks growth in 500-mL pots. The root tips were fixed in 3 ethanol : 1 acetic acid

(v/v) at 4°C for least 1 day before preparation. Fixed root tips were washed three times with

distilled water, and the samples were stored in 70% ethanol at 4°C. Chromosomes

preparations were made through enzymatic maceration, i.e., root tips were digested with 10

µl enzymatic mixture (4% Cellulase Onozuka RS, Yakult; 2% Pectolyase Y-23, Kyowa; and

1% Pectinase from Aspergillus, Sigma) at 37°C for 45-90 minutes. Squashing was made in

45% acetic acid and the slides were stored at -80°C for more than 12 h. The glass covers

were removed by forceps for the following FISH experiment.


35

The (AAG)7 probe was labelled with digoxigenin-11-dUTP (Roche) by terminal

deoxynucleotidyl transferase (TdT) (Thermo Fisher Scientific) following the manufacturer’s

instructions.

FISH analysis

Chromosome slides were re-fixed in 1% paraformaldehyde/1× PBS for 10 min at

room temperature. Procedures for hybridization and detection were followed as those

described by Kikuchi et al. (2008). The slides were counter-stained with

4,6-diamidino-2-phenylindole (DAPI) in a VectaShield antifade solution (Vector

Laboratories). All FISH images were captured with an Olympus BX53 fluorescence

microscope with a CCD camera (CoolSNAP MYO; Photometrics). The FISH images were

processed by Metamorph, Metavue imaging series version 7.8 and edited with Adobe

Photoshop CC 2017. CHIAS IV (http://www2.kobe-u.ac.jp/~ohmido/index03.htm) was used

for the construction of the ideogram.

36

2.3 Results

Images of mitotic prometaphase of four Lactuca species visualized after FISH

using (AAG)7 sequence as the probe are shown in Figure. 2-1. Different distribution

patterns of (AAG)n signals were shown in all four Lactuca species studied. In L. sativa and L.

serriola, FISH with (AAG)7 sequences revealed dispersed distribution patterns with one pair

of bright signals, respectively. While in L. saligna and L. virosa, distinct signals with

different intensities were observed in two pairs of chromosomes of L. saligna and five pairs

of chromosomes of L. virosa.

Figure 2-2 shows FISH karyotypes of four Lactuca species, which were arranged

according to the order of the lengths of the chromosomes based on the FISH images shown in

Figure 2-1. Quantitative ideograms of the prometaphase chromosomes of the four lettuce

species generated by CHIAS IV based on the characteristics of condensation patterns,

chromosome lengths, and FISH signals are shown in Fig. 2-3. Dispersed signal distribution

was observed in prometaphase chromosomes of L. sativa, with one pair of strong

pericentromeric signals on Chromosome 1. L. serriola also showed similar dispersed

distribution as those of L. sativa chromosomes, with one pair of small telomeric signals on

Chromosome 2. By contrast, distinct signals were observed on L. saligna and L. virosa

37

chromosomes. In L. saligna, Chromosomes 3 and 8 showed clear signals at the interstitial

regions of the long arm and subtelomeric region of the short arm, respectively. While, in L.

virosa, Chromosome 1 had four major signals, two on the proximal region of the short and

long arms, and two on the interstitial region of the long arm. Chromosome 3 showed two

pairs of signals, one on the interstitial region and one on the subtelomeric region of the long

arm. Chromosomes 2 and 5 showed one pair of clear signals on the long arms of the

chromosomes and Chromosome 4 had one pair of signals on the proximal region of the

chromosome. With their distribution patterns, Chromosomes 1 to 5 could be clearly

discriminated by using (AAG)7 probe.

2.4 Discussion

Schmidt and Heslop-harrison (1996) demonstrated that microsatellites, representing

a substantial fraction of the genome, showed chromosome-specific amplification in plants.

The repeat AAG presented different in situ hybridization patterns that provided cytogenetic

landmarks for chromosome identification in barley, Hordeum vulgare ssp. vulgare (Carmona

et al. 2013). Also, Sonah et al. (2011) described that AAG repeat units are major contributors

to the genomes of dicots.

38

As shown in the previous reports (Lindqvist 1960; Koopman and De Jong 1996),

the karyotype analyses indicated the closest relationship between L. sativa and L. serriola.

Our results showed that karyotype of L. sativa was similar to that of L. serriola, since the

dispersed signal distribution patterns of (AAG)7 repeats were similarly observed in both

species studied. However, L. sativa and L. serriola could be distinguished easily by the

presence of the AAG pericentromeric and telomeric signals on the Chromosomes 1 and 2,

respectively. On the other hand, L. saligna and L. virosa showed their specific karyotype

features with AAG signals compare to the other two species (Figure 2). Lindqvist (1960),

Haque and Godward (1985), Koopman et al. (1993), and Koopman and De Jong (1996)

described that cytological features of L. saligna and L. virosa are different from those of L.

sativa/L. serriola. Based on the sequence comparisons of chloroplast DNAs in Lactuca

species, Wei et al. (2017) revealed that L. sativa/L. serriola group was different from that of

L. saligna/L. virosa. This fact indicates that the distribution of microsatellite sequence on the

chromosomes reflects the phylogenetic differentiation of the species in the genus Lactuca.

Cytological analyses on the distribution of microsatellite sequences would provide insight

into the phylogenetic differentiation of the species in the genus Lactuca. In conclusion, the

39

AAG repeat presented different in situ hybridization patterns that provided cytogenetic

landmarks for chromosome identification in Lactuca species.

40

2.5 Figures

Figure 2-1. FISH of (AAG)7 (red) probe in four Lactuca species. (a-c) L. sativa cv. ‘Shinano

Hope’ showing dispersed signals distribution in all chromosomes and two bright signal sites

of (AAG)7, (d-f) L. serriola showing dispersed signals distribution in all chromosomes with

one pair of weak signals, (g-i) L. saligna showing bright signals in two pairs of

chromosomes, (j-l) L. virosa showing strong signals. Scale bar=10 µm

41

Figure 2-2. FISH karyotypes of four Lactuca species with (AAG)n signals. Homologous

pairs of chromosomes were arranged and numbered according to their lengths. Horizontal

lines show the positions of the centromeres. Scale Bar=10 µm

42

Figure 2-3. FISH ideogram of prometaphase chromosomes of L. sativa (a), L. serriola (b), L.

saligna (c) and L. virosa (d) with (AAG)n signals (red) constructed by CHIAS IV.

Numerals denote the chromosome numbers according to their chromosome lengths. Black,

gray, and white areas represent chromosome regions with heavy, intermediate and slight

condensation, respectively. The gaps in the middle of the chromosomes indicate the

centromere regions. Homologous pairs of chromosomes were arranged and numbered

according to their lengths. Horizontal lines show the positions of the centromeres.

43

CHAPTER 3

Physical localization of rDNAs and microsatellite sequences on the chromosomes of

Lactuca saligna using fluorescence in situ hybridization

44

Summary Lactuca saligna L. is one of the wild species in the genus Lactuca and is

considered to be the most important genetic resources in the lettuce genepool. In the present

study, fluorescence in situ hybridization (FISH) was applied to localize 45S and 5S rDNA

sequences and nine microsatellites on mitotic, and meiotic diakinesis and pachytene

chromosomes of L. saligna. FISH using 45S and 5S rDNAs as the probes provided high

resolution FISH images. Insertion of 5S rDNA into the region of 45S rDNA was also found

on Chromosome 5. This is the first report of high-resolution FISH with 45S and 5S rDNAs

on pachytene chromosomes of L. saligna. Of the nine microsatellite probes tested, (AAC)7,

(ACG)7, (CAC)7, and (CG)10 yielded no signals. The other five microsatellite probes, (AG)10,

(AC)10, (GCC)7, (ACT)7, and (AAG)7 successfully produced FISH signals. The signals of

(AG)n, (AC)n, and (GCC)n were found to be distributed across the prometaphase

chromosomes. In contrast, the signals of (ACT)n and (AAG)n showed chromosome specific

patterns, indicating these signals could be used as chromosomal markers of L. saligna. Our

FISH images using rDNAs and microsatellites as the probes provided valuable information

for discrimination of chromosomes in L. saligna, and it would be beneficial for future

cytogenetic studies on Lactuca genomes.

Key words L. saligna, FISH, microsatellites, rDNAs

45

3. 1 Introduction

Willow-leaf lettuce (Lactuca saligna L.) is a diploid wild species with chromosome

number n = 9, with the features of annual life cycle, characteristic glabrous stems, spike-like

inflorescences, sessile capitules, and almost linear steam leaves (Zohary 1991). L. saligna is

considered to be one of the most important species in the lettuce genepool (Lebeda et al.

2007). It has come to play a considerable role in lettuce resistance breeding to Bremia

lactucae (downy mildew pathogen) (Lebeda et al. 2002, 2007; Beharav et al. 2006).

Fluorescence in situ hybridization (FISH) technique using ribosomal RNA genes

and telomere sequences as the probes have been widely applied for karyotyping and studying

chromosomal organization and evolution in many plant species. Karyotype analyses and

chromosomal distribution of 5S and 18S rDNAs in L. sativa, L. serriola, L. saligna and L.

virosa by FISH method were examined by Matoba et al. (2007) and Widarmi et al. (2019).

These analyses revealed the dissimilarity among the karyotypes of L. virosa, L. sativa, L.

serriola and L. saligna. The FISH result showed that although the loci of the both rDNAs of

L. sativa, L. serriola and L. saligna were identical, those of L. virosa were different from

other species.

46

Microsatellites or simple sequence repeats (SSRs) are tandem repeats of DNA motifs

composed of short repeated units of 1-6 nucleotides, that can be found anywhere in the

genome, both in protein-coding and noncoding regions (Toth et al. 2000). Because of their

high mutability, microsatellites are thought to play a significant role in genome evolution by

creating and maintaining quantitative genetic variation (Tautz et al. 1986; Kashi et al. 1997).

Microsatellite markers were developed as polymerase chain reaction-based markers

and used as probes in fluorescence in situ hybridization (FISH) technique which is a crucial

tool for plant molecular cytogenetic researches (Jiang and Gill 2006). Microsatellites have

been widely used for chromosome identification and studies of genome organization and

evolution in different plant species e.g. Triticeae (Cuadrado et al. 2008), Allium (Monkheang

et al. 2016) and Panax ginseng (Waminal et al. 2018). As far as we know, no previous FISH

experiments using microsatellite sequences as the probes have been reported in Lactuca

species.

In the present study, we aimed to perform FISH using nine SSRs and 45S and 5S

rDNA sequences as the probes, on the mitotic, and meiotic diakinesis and pachytene

chromosomes of L. saligna in order to identify the distribution of the repetitive sequences.

47



Seeds of L. saligna were provided by Nagano Vegetable and Ornamental Crops

Experimental Station, Nagano, Japan. They were germinated in petri dishes in room

temperature and planted in pots in a greenhouse. Mitotic cells were obtained from root tips of

1-2 cm long collected from the seedlings and young plants grown in 500-mL pots for a few

weeks, whereas meiotic cells were obtained from immature flower buds of 0.8-3.0 mm in

size. The root tips and flower buds were fixed in 3 ethanol : 1 acetic acid (v/v) at 4°C for 5

days before chromosome preparation. Fixed root tips and flower buds were washed three

times with distilled water, and stored in 70% ethanol at 4°C until use. Chromosome

preparations were made through enzymatic maceration; root tips were digested with 10 µL

enzymatic mixture (4% Cellulase Onozuka RS, Yakult; 2% Pectolyase Y-23, Kyowa; and 1%

Pectinase from Aspergillus, Sigma) at 37°C for 45-90 min. Squashing was made in 60%

acetic acid and the slides were stored at -80°C for over 12 h until observation


48

Wheat 45S rDNA (pTa71) and 5S rDNA were used as the probes for FISH analyses

(Gerlach and Bedbrook 1979; Fukui et al. 1994; Widarmi et al. (in press)). 5S rDNA was

amplified from Lactuca sativa L. genomic DNA by polymerase chain reaction (PCR) using

coding regions of 5S rDNA as the primers (Fukui et al. 1994; Widarmi et al. (in press)). PCR

products with expected size of 301 bp were cloned into pZErO-2 Vector (Invitrogen)

according to manufacturer’s instruction. Both 45S and 5S rDNA probes were labeled with

Biotin-Nick Translation Mix (Roche), whereas 45S rDNA was labeled with DIG-Nick

Translation Mix (Roche) according to the manufacturer’s instructions. The (AAC)7, (ACG)7,

(ACT)7, and (CAC)7 probes were end-labelled with biotin-16-dUTP (Roche) and the (AG)10,

(CG)10, (AC)10, (AAG)7, and (GCC)7 probes with digoxigenin-11-dUTP (Roche) by terminal

deoxynucleotidyl transferase (TdT) (Thermo Fisher Scientific) following the manufacturer’s

instructions.

FISH analysis

Chromosome slides were re-fixed in 1% paraformaldehyde/1× PBS for 10 min at

room temperature. Procedures for hybridization and detection were followed as described by

Kikuchi et al. (2008). Biotin and digoxigenin labeled probes were detected using a

49

biotinylated streptavidin-FITC (vector laboratories) and anti-digoxigenin rhodamine (Roche),

respectively. The slides were counter-stained with 4,6-diamidino-2-phenylindole (DAPI) in a

VectaShield antifade solution (Vector Laboratories). All FISH images were captured with an

Olympus BX53 fluorescence microscope with a CCD camera (CoolSNAP MYO;

Photometrics). The FISH images were processed by Metamorph, Metavue imaging series

version 7.8 and edited with Adobe Photoshop CC 2017. In the karyotype analyses,

chromosomes were arranged and numbered according to their lengths.

3.3 Results

Physical locations of 45S and 5S RNA genes on the chromosomes of L. saligna are

shown in Fig. 3-1. Two large pairs of 45S rDNA signals were detected in the interphase cells

at the nucleolus organizer regions (NOR) and were on the short arms of the prometaphase

chromosomes. A pair of 5S rDNA signals were detected at the interstitial regions of the short

arms of the chromosomes. It was also found another 5S rDNA site co-localizing with one site

of 45S rDNA loci.

Fig. 3-2 shows FISH karyotypes at mitotic prometaphase and meiotic diakinesis

stages. Two pairs of 45S rDNA signals could be localized at the ends of the short arms of

50

Chromosome 3, and Chromosome 5, respectively. Two 5S rDNA signals were localized in

the interstitial regions of the short arms of Chromosome 5. Moreover, Chromosomes 5 had

another 5S rDNA site co-localizing with 45S rDNA loci.

FISH experiment using 45S and 5S rDNAs on the pachytene chromosomes

provided a much higher resolution of the signals located on the corresponding mitotic and

meiotic bivalent chromosomes (Fig. 3-3). Two clear 45S rDNA signals and one 5S rDNA

signal were detected in the pachytene chromosomes. Moreover, another 5S rDNA site was

found clearly to be co-localized with 45S rDNA. (Fig. 3-3d).

After FISH using nine microsatellite sequences as the probes, (AAC)7, (ACG)7,

(ACT)7, (CAC)7, (AG)10, (CG)10, (AC)10, (AAG)7, and (GCC)7 , only five, (AG)10, (AC)10,

(GCC)7, (ACT)7, and (AAG)7 were successful in producing FISH signals. The signals of

(AG)n, (AC)n, and (GCC)n were found to be distributed across the prometaphase

chromosomes of L. saligna (Fig. 3-4a-c). In contrast, FISH signals of (ACT)n and (AAG)n

showed intensively limited localization on the chromosomes. Two pairs of signals of (ACT)n

were localized at the centromeric and interstitial regions of Chromosomes 2, and one pair of

the signals at the centromeric regions of Chromosome 5 (Fig. 4d). One pair of signals of

(AAG)n were found at the interstitial regions of the long arms of Chromosome 3, and three

51

pairs of the signals at the interstitial regions of the short arms of Chromosomes 6, 8, and 9

(Fig. 3-4e).

3.4 Discussion

In the present study, two pairs of 45S rDNA signals were detected at the nucleolus

organizer regions (NOR) on the short arms of the Chromosomes 3 and 5, and a pair of 5S

rDNA signals were detected at the interstitial regions of the short arms of Chromosome 5.

Our results confirmed the results of Matoba et al. (2007). However, it was clarified that

meiotic diakinesis and pachytene chromosomes of L. saligna provide much higher resolution

of FISH analysis, namely, new 5S rDNAs signal were detected (Figs. 3-1, 3-2 and 3-3). This

is the first report of high-resolution FISH with 45S and 5S rDNA on pachytene chromosome

of L. saligna.

In the present study, insertion of 5S rDNA into the region of 45S rDNA was found on

Chromosome 5 (Fig2. 3-1, 3-2, and 3-3). This phenomenon is considered accidentally

occurred by transposon-like DNA-mediated or retrotransposon-like RNA-mediated

transposition in some lower eukaryotes (Drouin and de Sa 1995; Sone et al. 1999). By using

molecular cytogenetics, Southern blot hybridization, PCR-based techniques and DNA

52

sequencing, 5S genes can be embedded within the 45S multigene family in angiosperms

(Asteraceae; Garcia et al. 2009, 2010). These authors suggested that the 5S gene integration

within the 45S multigene family probably occurred once in Asteraceae, but might have

repeatedly occurred elsewhere during plant evolution. Also, it was reported that rDNA loci

can rapidly change places on chromosomes (Dubcovsky and Dvorak 1995). Our present

finding of the insertion of 5S rDNA into 45S rDNA seems to have taken place during the

divergence in evolution.

Schmidt and Heslop-Harrison (1996) demonstrated that microsatellites, representing a

substantial fraction of the genome, showed chromosome-specific amplification in plants. In

the present work, only five, (AG)10, (AC)10, (GCC)7, (ACT)7, and (AAG)7 were successful in

producing FISH signals. Cuadrado and Jouve (2007) reported that AG and AC repeat sites

were remarkably similar and uniformly dispersed along the euchromatic portion of metaphase

chromosomes of human, barley and Drosophila melanogaster. Also, Dou et al. (2016) found

that the distribution of GCC repeat sites were dispersed in metaphase chromosome of

Hordeum brevisubulatum. It was found that the (AG)10, (AC)10, and (GCC)7, repeats gave

dispersed distribution in almost all chromosomes of L. saligna. Therefore, it might be

53

possible that these three repetitive sequences are universally distributed among the eukaryote

chromosomes including L. saligna.

On the other hand, it was found that (ACT)n, and (AAG)n could be used as

chromosomal markers of L. saligna, since the homologous chromosome could be

distinguished based on the karyotype of prometaphase chromosomes and the distribution

patterns of the SSRs hybridized with these two probes. Dou et al. (2016) also showed that the

SSRs, (ACT)n and (AAG)n, are ideal markers for chromosome identification because of their

abundance and their large number of polymorphic sites across individual chromosomes of H.

bogdanii

As the conclusion, locations of 45S and 5S rDNAs and five SSRs, (AG)n, (AC)n,

(GCC)n, (ACT)n, and (AAG)n were physically mapped on the chromosomes of L. saligna.

The FISH results presented here using rDNA and SSRs as the probes provide beneficial

information that might be used for future cytogenetic studies and could contribute physical

assembly of the L. saligna genome.

54

3.5 Figures

Figure 3-1. Physical mapping of 45S and 5S rDNAs in Lactuca saligna L. (a) A mitotic

interphase cell, (b) chromosomes at mitotic prometaphase, and (c) meiotic diakinesis,

showing nine bivalents and two pairs of 45S rDNA (red) and one pair of 5S (green) rDNA

signals. Yellow arrows show co-localization of 45S and 5S rDNAs. Scale Bar=10 µ

55

Figure 3-2. FISH karyotype of L. saligna using 45S and 5S rDNA as the probes (upper:

mitotic prometaphase chromosomes, lower: meiotic metaphase chromosomes, arranged

according to their sizes and morphology from No. 1-9), showing two pairs of 45S rDNA loci

(red) and one pair of 5S rDNA loci (green). The images of the mitotic and diakinesis

chromosomes were separated out from Fig. 1.The yellow arrows indicate co-localization of

45S and 5SrDNAs. Scale bar=10 µm

56

Figure 3-3. High resolution physical mapping of 45S and 5S rDNAs on pachytene

chromosomes of L. saligna. (a) DAPI , (b) 45S rDNA, (c) 5S rDNA, (d) Merged pictures

showing three 45S rDNA loci (red) and two 5S (green) rDNA loci. Yellow arrow indicates

co-localization of 45S and 5srDNAs. Scale bar=10 µm

57

Figure 3-4. FISH patterns using microsatellite probes (a) (AG)10 (red), (b) (AC)10 (green), (c)

(GCC)7(red), (d) (ACT)7 (green), and (e) (AAG)7 (red), on prometaphase chromosomes of L.

saligna. Arrows indicate fluorescent sites of (ACT)n (d) and (AAG)n (e). Scale bar=10 µm

58

GENERAL DISCUSSION

Cultivated lettuce (Lactuca sativa L.) belongs to the genus Lactuca and is an

important vegetable worldwide, which has been a problematic genus in terms of its

taxonomic circumscription and phylogenetic affinities for more than a century. Over the past

decades, there have been many controversies about the phylogeny of Lactuca species, due to

the morphological characters of Lactuca species are complex and diverse, adding up the

difficulty of studying phylogenetic relationships within Lactuca.

FISH-based karyotyping is a powerful cytogenetics tool to study chromosome

organization, behavior, and chromosome evolution that provide phylogenetical information in

closely related taxa and have been widely applied for karyotyping and studying chromosomal

organization and evolution in many plant species. The pattern of hybridization signals greatly

facilitates chromosome identification and accurate karyotype analysis (Kato et al. 2004,

Paesold et al. 2012, Wolny et al. 2013). Furthermore, comparative studies of plant repetitive

sequences are useful for the investigation of evolutionary relationships among plant species

(Menzel et al. 2008). Various combinations of repetitive sequences, both those occurring in

tandem and forming large clusters, such as ribosomal genes, telomeric repeats, and satellite

59

DNAs, including centromere-specific repeats, and those having a dispersed distribution

typical for transposable elements have been cytogenetically mapped by FISH

(Heslop-Harrison 2000, Schwarzacher 2003).

In this thesis I presented karyotyping and molecular chromosome studies using rDNA

and microsatellite sequences in lettuce. FISH and CHromosome Image Analyzing System IV

(CHIAS IV) were applied to obtain detailed information of the characteristics of lettuce

chromosomes, which were described in the Chapter 1. One lettuce cultivar (L. sativa L.) and

three wild species L. serriola, L. saligna and L. virosa were used to analyze chromosome

lengths, arm ratios and degrees of chromosomal condensation at prometaphase. Since

metaphase chromosomes of horticultural plants are relatively small compared to those of

other plants, e.g., wheat and barley, it has been rather difficult to discriminate their individual

chromosomes. In lettuce, all cytological studies, karyotype analyses, and chromosome

banding have been conducted mainly using metaphase chromosomes, which are in highly

condensed condition (Matoba et al. 2007). Prometaphase chromosomes, which are two times

longer and loosely condensed, provide a significantly better resolution for fluorescence in situ

hybridization than metaphase chromosomes. Our results showed that nine pairs of

60

homologous chromosomes could be discriminated for all four species using their

morphological characteristics, condensation patterns (CPs) and localization of rRNA loci.

In chapter 2, it is shown the distribution of (AAG)7 of Lactuca species was performed

to clarify the possibility of using the microsatellite repetitive sequences for discrimination

and/or identification of the chromosomes in Lactuca species.

Different distribution patterns of (AAG)n signals were shown in all four Lactuca

species studied. In L. sativa and L. serriola, FISH with (AAG)7 sequences revealed dispersed

distribution patterns with one pair of bright signals, respectively. While in L. saligna and L.

virosa, distinct signals with different intensities were observed in two pairs of chromosomes

of L. saligna and five pairs of chromosomes of L. virosa. Schmidt and Heslop-harrison

(1996) demonstrated that microsatellites, representing a substantial fraction of the genome,

showed chromosome-specific amplification in plants. The repeat AAG presented different in

situ hybridization patterns that provided cytogenetic landmarks for chromosome

identification in barley, Hordeum vulgare ssp. vulgare (Carmona et al. 2013). Also, Sonah et

al. (2011) described that AAG repeat units are major contributors to the genomes of dicots.

As shown in the previous reports (Lindqvist 1960; Koopman and De Jong 1996), the

present karyotype analyses indicated the closest relationship between L. sativa and L. serriola.

61

Our results showed that karyotype of L. sativa was similar to that of L. serriola, since the

dispersed signal distribution patterns of (AAG)7 repeats were similarly observed in both

species studied. On the other hand, L. saligna and L. virosa showed their specific karyotype

features with AAG signals compare to the other two species. Lindqvist (1960), Haque and

Godward (1985), Koopman et al. (1993), and Koopman and De Jong (1996) described that

cytological features of L. saligna and L. virosa are different from those of L. sativa/L.

serriola. Based on the sequence comparisons of chloroplast DNAs in Lactuca species, Wei et

al. (2017) revealed that L. sativa/L. serriola group was different from that of L. saligna/L.

virosa. This fact indicates that the distribution of microsatellite sequence on the

chromosomes reflects the phylogenetic differentiation of the species in the genus Lactuca.

The distribution of the repetitive sequences on mitotic interphase, prometaphase,

and meiotic diakinesis and pachytene chromosomes of L. saligna were determined in Chapter

3. In the present study, two large pairs of 45S rDNA signals were detected in the interphase

cells at the nucleolus organizer regions (NOR) and were on the short arms of the

prometaphase chromosomes. A pair of 5S rDNA signals were detected at the interstitial

regions of the short arms of the chromosomes. It was also found another 5S rDNA site

co-localizing with one site of 45S rDNA loci. Our results confirmed the results of Matoba et

62

al. (2007). However, it was clarified that meiotic diakinesis and pachytene chromosomes of

L. saligna provide much higher resolution of FISH analysis, namely, new 5S rDNAs signal

were detected (Figs. 3-1, 3-2 and 3-3). This is the first report of high-resolution FISH with

45S and 5S rDNA on pachytene chromosome of L. saligna.

In the present study, insertion of 5S rDNA into the region of 45S rDNA was found on

Chromosome 5. This phenomenon is considered accidentally occurred by transposon-like

DNA-mediated or retrotransposon-like RNA-mediated transposition in some lower

eukaryotes (Drouin and de Sa 1995; Sone et al. 1999). By using molecular cytogenetics,

Southern blot hybridization, PCR-based techniques and DNA sequencing, 5S genes can be

embedded within the 45S multigene family in angiosperms (Asteraceae; Garcia et al. 2009,

2010). These authors suggested that the 5S gene integration within the 45S multigene family

probably occurred once in Asteraceae, but might have repeatedly occurred elsewhere during

plant evolution. Also, it was reported that rDNA loci can rapidly change places on

chromosomes (Dubcovsky and Dvorak 1995). Our present finding of the insertion of 5S

rDNA into 45S rDNA seems to have taken place during the divergence in evolution.

As demonstrated by Schmidt and Heslop-Harrison (1996) that microsatellites,

representing a substantial fraction of the genome, showed chromosome-specific amplification

63

in plants. In the present work, only five, (AG)10, (AC)10, (GCC)7, (ACT)7, and (AAG)7 were

successful in producing FISH signals. Cuadrado and Jouve (2007) reported that AG and AC

repeat sites were remarkably similar and uniformly dispersed along the euchromatic portion

of metaphase chromosomes of human, barley and Drosophila melanogaster. Also, Dou et al.

(2016) found that the distribution of GCC repeat sites were dispersed in metaphase

chromosome of Hordeum brevisubulatum. It was found that the (AG)10, (AC)10, and (GCC)7,

repeats gave dispersed distribution in almost all chromosomes of L. saligna. Therefore, it

might be possible that these three repetitive sequences are universally distributed among the

eukaryote chromosomes including L. saligna.

On the other hand, we found that (ACT)n, and (AAG)n could be used as chromosomal

markers of L. saligna, since the homologous chromosome could be distinguished based on

the karyotype of prometaphase chromosomes and the distribution patterns of the SSRs

hybridized with these two probes. Dou et al. (2016) also showed that the SSRs, (ACT)n and

(AAG)n, are ideal markers for chromosome identification because of their abundance and

their large number of polymorphic sites across individual chromosomes of H. bogdanii

Chromosome painting on mitotic and meiotic chromosomes with various tandem and

dispersed repeats in lettuce revealed that some of the repeats can be used in the identification

64

of individual chromosomes. The 45S rDNA hybridized to chromosomes 3 and 5 of L. sativa,

L. saligna, and L. seriolla, and chromosomes 5 and 7 of L. virosa. The 5S rDNA hybridized

to chromosomes 5 of L. sativa, L. saligna, and L. seriolla, and chromosomes 7 of L. virosa.

(AAG)7 microsatellite repeats were hybridized to Chromosome 1 and 2 of L. sativa and L.

serriola, respectively. Two pairs of AAG repeat signals were observed in L. saligna at the

pericentromeric of Chromosome 3 and subtelomeric signal at Chromosome 8. And, five pairs

of microsatellite probe were observed in L. virosa at Chromosome 1 to 5. These analyses

clarify that the chromosomal characteristics, while cytological features of L. saligna and L.

virosa are different from L. sativa/L. serriola as reported by Lindqvist (1960), Haque and

Godward (1985), Koopman and De Jong (1996) and Koopman et al. (1993).

In conclusion, FISH results using rDNA and SSR probes presented different in situ

hybridization patterns that provided cytogenetic landmarks for chromosome identification in

Lactuca species, also provide information that may be beneficial for future cytogenetic

studies and could contribute the physical assembly of the Lactuca genome. The development

of cytogenetic markers should facilitate physical mapping of the genome. In future studies,

other probes prepared from different kinds of repetitive sequences, single copy genes and

65

DNA markers would be localized for the construction of precise physical chromosome maps

of lettuce.

66

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SUMMARY

Cytological studies using fluorescence in situ hybridization (FISH) technique

provide phylogenetical information in closely related taxa and have been widely applied for

karyotyping and studying chromosomal organization and evolution in many plant species.

In the present study, FISH and chromosome image analysis system IV (CHIAS IV) were

applied to obtain detailed information of the characteristics of lettuce chromosomes. FISH

using 45S and 5S rRNA genes and (AAG)n of Lactuca species was performed in order to

identify the distribution of the repetitive sequences. One lettuce cultivar (L. sativa L.) and

three wild species L. serriola, L. saligna and L. virosa were used to analyze chromosome

lengths, arm ratios and degrees of chromosomal condensation at mitotic prometaphase stage.

Different distribution patterns of rRNA genes and (AAG)n signals were shown on the

chromosomes in the four Lactuca species studied, suggesting that these signals could be used

as cytogenetic landmarks for chromosome identification in Lactuca species. Also, FISH was

conducted to identify the regions of repetitive sequences on the chromosomes at mitotic

prometaphase, and meiotic diakinesis and pachytene stages of Lactuca saligna using nine

microsatellites and 45S and 5S rDNA sequences as the probes. FISH using 45S and 5S

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rDNAs as the probes provided high resolution FISH images. Insertion of 5S rDNA into the

region of 45S rDNA was also found on Chromosome 5. This is the first report of

high-resolution FISH with 45S and 5S rDNAs on pachytene chromosomes of L. saligna. Of

the nine microsatellite probes tested, (AAC)7, (ACG)7, (CAC)7, and (CG)10 yielded no signals.

The other five microsatellite probes, (AG)10, (AC)10, (GCC)7, (ACT)7, and (AAG)7

successfully produced FISH signals. The signals of (AG)n, (AC)n, and (GCC)n were found to

be distributed across the prometaphase chromosomes. In contrast, the signals of (ACT)n and

(AAG)n showed chromosome specific patterns, indicating these signals could be used as

chromosomal markers of L. saligna. Our FISH images using rDNAs and microsatellites as

the probes provided valuable information for discrimination of chromosomes in L. saligna,

and it would be beneficial for future cytogenetic studies on Lactuca genomes.

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ACKNOWLEDGMENTS

I came to Japan in 2013, I started my school life as an exchange student at Chiba

University. It was my first experience of staying away far from my family for a long time and

facing the consequences of being in an entire new place alone. But I enjoyed my life in Japan

a lot. I am grateful I could continue my study in Chiba University as a Ph.D. student.

First of all, I would like to send my great appreciation to the support,

encouragement, and guidance from my supervisor Prof. Takato Koba. Without him, none of

the work would have been possible. His guidance helped me in all the time of research and

writing of this thesis. I could not have imagined having a better advisor and mentor for my

Ph.D. study. I would also like to express my sincere gratitude to Prof. Hidenori Sassa and

Assoc. Prof. Shinji Kikuchi for their help and guidance throughout the project. Thanks very

much for your support and help. I would like to thank to my thesis committee members Prof.

Nakamura and Prof. Sakamoto for their insightful comments and encouragement.

I gratefully acknowledge the funding from the Ministry of Education, Culture,

Sports, Science and Technology Japan, during my Ph.D. course, and the tuition exemption

from Chiba University.

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I also would like to thank many people in the lab who have contributed to my

progress on my projects, Chino San, Tang San, Duc San, Yamashita San, Naomi San, Iku San,

Fukuhara San and Agus San.

Lastly but not the least, I would like to thank my family: my parents and to my

husband and my children for their patience and all their support. Everything will come to you

at the perfect time. I love you Mama and Ayah.

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