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Advances in Biochemical Engineering/Biotechnology 164
Series Editor: T. Scheper
Rajeev K. Varshney · Manish K. Pandey

Annapurna Chitikineni Editors
Plant
Genetics and
Molecular
Biology
164
Advances in Biochemical
Engineering/Biotechnology
Series editor
T. Scheper, Hannover, Germany
Editorial Board
S. Belkin, Jerusalem, Israel
T. Bley, Dresden, Germany
J. Bohlmann, Vancouver, Canada
M.B. Gu, Seoul, Korea (Republic of)
W.-S. Hu, Minneapolis, Minnesota, USA
B. Mattiasson, Lund, Sweden
J. Nielsen, Gothenburg, Sweden
H. Seitz, Potsdam, Germany
R. Ulber, Kaiserslautern, Germany
A.-P. Zeng, Hamburg, Germany
J.-J. Zhong, Shanghai, Minhang, China
W. Zhou, Shanghai, China
Aims and Scope
This book series reviews current trends in modern biotechnology and biochemical
engineering. Its aim is to cover all aspects of these interdisciplinary disciplines,
where knowledge, methods and expertise are required from chemistry, biochemis-
try, microbiology, molecular biology, chemical engineering and computer science.
Volumes are organized topically and provide a comprehensive discussion of devel-

opments in the field over the past 3–5 years. The series also discusses new
discoveries and applications. Special volumes are dedicated to selected topics
which focus on new biotechnological products and new processes for their synthe-
sis and purification.
In general, volumes are edited by well-known guest editors. The series editor and
publisher will, however, always be pleased to receive suggestions and supplemen-
tary information. Manuscripts are accepted in English.
In references, Advances in Biochemical Engineering/Biotechnology is abbreviated

as Adv. Biochem. Engin./Biotechnol. and cited as a journal.
More information about this series at http://www.springer.com/series/10

Rajeev K. Varshney • Manish K. Pandey •
Annapurna Chitikineni
Editors
Plant Genetics and

Molecular Biology
With contributions by
V. Anil Kumar J. Batley P. Chaturvedi A. Chitikineni
J. Cockram R. R. Das S. Datta D. Edwards A. Ghatak
J. Jankowicz-Cieslak Y. Jia K. Jiang P. L. Kulwal
I. Mackay N. Mantri P. R. Marri S. Mazicioglu
M. Muthamilarasan N. Nejat I. Ocsoy G. Pandey
M. K. Pandey S. K. Pandey A. Parveen M. Prasad
A. Ramalingam C. S. Rao A. Rathore S. D. Rounsley
J. K. Roy M. Saba Rahim A. Scheben H. Sharma
V. K. Singh W. Tan D. Tasdemir V. Thakur B. J. Till
R. K. Varshney W. Weckwerth C. B. Yadav L. Ye
Editors
Rajeev K. Varshney Manish K. Pandey
International Crops Research Institute International Crops Research Institute
for the Semi-Arid Tropics (ICRISAT) for the Semi-Arid Tropics (ICRISAT)
Hyderabad, India Hyderabad, India
International Crops Research Institute
for the Semi-Arid Tropics (ICRISAT)
Hyderabad, India
ISSN 0724-6145 ISSN 1616-8542 (electronic)

Advances in Biochemical Engineering/Biotechnology
ISBN 978-3-319-91312-4 ISBN 978-3-319-91313-1 (eBook)
DOI 10.1007/978-3-319-91313-1
Library of Congress Control Number: 2018948681
© Springer International Publishing AG, part of Springer Nature 2018

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Preface
The elimination of hunger and malnutrition from society is a key challenge of all
agricultural stakeholders around the world. Feeding the global population has never
been so challenging, especially in the context of diminishing land and water
resources, an ever-increasing global population, and climate change. The only
solution may be to develop climate-smart plant varieties that are produced with
appropriate agricultural management practices. Today, agriculture is facing an
acute shortage of advanced germplasms to replace inferior varieties in farmers’
fields. A “game-changer” strategy for the development of improved germplasms
and cultivation practices needs to be implemented quickly and precisely to tackle
both current and future adverse environmental conditions.
Fast-evolving technologies can serve as a potential growth engine in agriculture
because many of these technologies have revolutionized other industries in the
recent past. The tremendous advancements in biotechnology methods, cost-effec-
tive sequencing technology, refinement of genomic tools, standardization of mod-
ern genomics-assisted breeding methods, and digitalization of the entire breeding
process and value chain hold great promise for taking global agriculture to the next
level through the development of improved climate-smart seeds. These technolo-
gies can dramatically increase our capacity for understanding the molecular basis of
traits and utilizing the available resources for accelerated development of stable,
high-yield, nutritious, efficient, and climate-smart crop varieties. These improved
crop varieties and agricultural practices will help us to address global food security
issues in an equitable and sustainable manner.
For these reasons, this book aims to explore and discuss future plans in the
key areas of plant genetics and molecular biology. It contains 12 chapters written
by 42 authors from Australia, Austria, India, Turkey, the United Kingdom,
and the United States (see List of Contributors). The editors are grateful to all of
the authors for contributing high-quality chapters with information from their areas
of expertise. The editors also would like to thank the reviewers (see List of
Reviewers) for their help in providing constructive suggestions and corrections,
which helped the authors to improve the quality of the chapters. The editors are also
v
vi Preface
grateful to Dr. David Bergvinson (Director General, ICRISAT) and Dr. Peter
Carberry (Deputy Director General–Research, ICRISAT) for their encouragement
and support. The editors thank the series editors (T. Scheper, S. Belkin, T. Bley, J.
Bohlmann, M.B. Gu, W.-S. Hu, B. Mattiasson, J. Nielsen, H. Seitz, R. Ulber, A.-P.
Zeng, J.-J. Zhong and W. Zhou) of the Springer publication Advances in Biochem-
ical Engineering/Biotechnology (http://www.springer.com/series/10) for giving us
this opportunity to compile such a wealth of information on plant genetics and
molecular biology for the research and academic community. The assistance
received from Springer—in particular, Judith Hinterberg, Elizabeth Hawkins,
Arun Manoj, and Alamelu Damodharan—has been a great help in completing
this book. The cooperation and encouragement of the publisher are gratefully
acknowledged.
We also appreciate the cooperation and moral support from our family members,
especially when the precious time we should have spent with them was taken up by
editorial work. R.K.V. acknowledges the help and support of his wife Monika, son
Prakhar, and daughter Preksha, who allowed their time to be taken away to fulfill R.
K.V.’s editorial responsibilities in addition to research and other administrative
duties at ICRISAT. Similarly, M.K.P. is grateful to his wife Seema for her help and
moral support during the evenings and weekends of editorial responsibilities in
addition to research duties at ICRISAT, with special thanks to his brave daughter,
the late Tanisha, who was alive for only a short period of time (3 months) after birth.
A.C. thanks her husband Sudhakar and daughter Shruti for their cooperation and
understanding during the fulfillment of her editorial commitments.
We hope that our efforts in compiling the information herein on the different
aspects of plant genetics and molecular biology will help researchers to develop a
better understanding of the subject and frame future research strategies. In addition,
we hope that this book will also benefit students, academicians, and policymakers in
updating their knowledge on recent advances in plant genetics and molecular
biology research.
Hyderabad, India Rajeev K. Varshney

Manish K. Pandey
Contents
Plant Genetics and Molecular Biology: An Introduction . . . . . . . . . . . . 1

Rajeev K. Varshney, Manish K. Pandey, and Annapurna Chitikineni
Advances in Sequencing and Resequencing in Crop Plants . . . . . . . . . . 11
Pradeep R. Marri, Liang Ye, Yi Jia, Ke Jiang, and Steven D. Rounsley
Revolution in Genotyping Platforms for Crop Improvement . . . . . . . . . 37
Armin Scheben, Jacqueline Batley, and David Edwards
Trait Mapping Approaches Through Linkage Mapping in Plants . . . . . 53
Pawan L. Kulwal
Trait Mapping Approaches Through Association Analysis in Plants . . . 83
M. Saba Rahim, Himanshu Sharma, Afsana Parveen, and Joy K. Roy
Genetic Mapping Populations for Conducting High-Resolution Trait
Mapping in Plants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 109
James Cockram and Ian Mackay
TILLING: The Next Generation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 139
Bradley J. Till, Sneha Datta, and Joanna Jankowicz-Cieslak
Advances in Transcriptomics of Plants . . . . . . . . . . . . . . . . . . . . . . . . . 161
Naghmeh Nejat, Abirami Ramalingam, and Nitin Mantri
Metabolomics in Plant Stress Physiology . . . . . . . . . . . . . . . . . . . . . . . . 187
Arindam Ghatak, Palak Chaturvedi, and Wolfram Weckwerth
Epigenetics and Epigenomics of Plants . . . . . . . . . . . . . . . . . . . . . . . . . . 237
Chandra Bhan Yadav, Garima Pandey, Mehanathan Muthamilarasan,
and Manoj Prasad
Nanotechnology in Plants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 263
Ismail Ocsoy, Didar Tasdemir, Sumeyye Mazicioglu, and Weihong Tan
vii
viii Contents
Current Status and Future Prospects of Next-Generation Data

Management and Analytical Decision Support Tools for Enhancing
Genetic Gains in Crops . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 277
Abhishek Rathore, Vikas K. Singh, Sarita K. Pandey, Chukka Srinivasa Rao,
Vivek Thakur, Manish K. Pandey, V. Anil Kumar, and Roma Rani Das
Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 293
List of Contributors
V. AnilKumar International Crops Research Institute for the Semi-Arid Tropics

(ICRISAT), Hyderabad, India
Jacqueline Bately University of Western Australia, Crawley, WA, Australia
Palak Chaturvedi University of Vienna, Vienna, Austria
Annapurna Chitikineni International Crops Research Institute for the Semi-Arid
Tropics (ICRISAT), Hyderabad, India
James Cockram National Institute of Agricultural Botany (NIAB), Cambridge,
UK
Roma Rani Das International Crops Research Institute for the Semi-Arid Tropics
Sneha Datta International Atomic Energy Agency (IAEA), Vienna, Austria
David Edwards University of Western Australia, Crawley, WA, Australia
Arindam Ghatak University of Vienna, Vienna, Austria
Joanna Jankowicz-Cieslak International Atomic Energy Agency (IAEA),
Vienna, Austria
Yi Jia Dow Agrosciences, Indianapolis, IN, USA
Ke Jiang Dow Agrosciences, Indianapolis, IN, USA
Pawan L. Kulwal Mahatma Phule Agricultural University, Rahuri, India
Ian Mackay National Institute of Agricultural Botany (NIAB), Cambridge, UK
Pradeep R. Marri Dow Agrosciences, Indianapolis, IN, USA
Sumeyye Mazicioglu Erciyes University, Kayseri, Turkey
Mehanathan Muthamilarasan National Institute of Plant Genome Research
(NIPGR), New Delhi, India
ix
x List of Contributors
Naghmeh Nejat RMIT University, Melbourne, VIC, Australia

Ismail Ocsoy Erciyes University, Kayseri, Turkey
Garima Pandey National Institute of Plant Genome Research (NIPGR),
New Delhi, India
Manish K. Pandey International Crops Research Institute for the Semi-Arid
Sarita K. Pandey International Crops Research Institute for the Semi-Arid
Afsana Parveen National Agri-Food Biotechnology Institute (NABI), Mohali,
India
Manoj Prasad National Institute of Plant Genome Research (NIPGR), New Delhi,
India
M. Saba Rahim National Agri-Food Biotechnology Institute (NABI), Mohali,
India
Chukka Srinivasa Rao International Crops Research Institute for the Semi-Arid
Abhishek Rathore International Crops Research Institute for the Semi-Arid
Steve D. Rounsley Genus plc, De Forest, WI, USA
Joy K. Roy National Agri-Food Biotechnology Institute (NABI), Mohali, India
Armin Scheben University of Western Australia, Crawley, WA, Australia
Himanshu Sharma National Agri-Food Biotechnology Institute (NABI), Mohali,
India
Vikas K. Singh International Crops Research Institute for the Semi-Arid Tropics
Weihong Tan University of Florida, Gainesville, FL, USA
Didar Tasdemir Erciyes University, Kayseri, Turkey
Vivek Thakur International Crops Research Institute for the Semi-Arid Tropics
Bradley J. Till International Atomic Energy Agency, Vienna, Austria
Rajeev K. Varshney International Crops Research Institute for the Semi-Arid
Wolfram Weckwerth University of Vienna, Vienna, Austria
List of Contributors xi
Chandra Bhan Yadav National Institute of Plant Genome Research (NIPGR),

New Delhi, India
Liang Ye Dow Agrosciences, Indianapolis, IN, USA
List of Reviewers
Harsha Gowda Institute of Bioinformatics (IoB), Bangalore, India

Himabindu Kudapa International Crops research Institute for the Semi-Arid
Chikelu Mba Food and Agriculture Organization (FAO), Rome, Italy
Reyazul Rouf Mir Sher-e-Kashmir University of Agricultural Sciences &
Technology of Kashmir (SKUAST-K), Sopore, India
Manish K. Pandey International Crops research Institute for the Semi-Arid
Lekha Pazhamala International Crops research Institute for the Semi-Arid
Samir Sawant CSIR-National Botanical Research Institute (NBRI), Lucknow,
India
Vikas Singh International Rice Research Institute (IRRI) -South Asia Hub,
Hyderabad, India
Mahendar Thudi International Crops research Institute for the Semi-Arid Tropics
xiii
Adv Biochem Eng Biotechnol (2018) 164: 1–10
DOI: 10.1007/10_2017_45
© Springer International Publishing AG 2018
Published online: 16 February 2018
Plant Genetics and Molecular Biology: An

Introduction
Rajeev K. Varshney, Manish K. Pandey, and Annapurna Chitikineni
Abstract The rapidly evolving technologies can serve as a potential growth engine
in agriculture as many of these technologies have revolutionized several industries in
the recent past. The tremendous advancements in biotechnology methods, cost-
effective sequencing technology, refinement of genomic tools, and standardization
of modern genomics-assisted breeding methods hold great promise in taking the
global agriculture to the next level through development of improved climate-smart
seeds. These technologies can dramatically increase our capacity to understand the
molecular basis of traits and utilize the available resources for accelerated develop-
ment of stable high-yielding, nutritious, input-use efficient, and climate-smart crop
varieties. This book aimed to document the monumental advances witnessed during
the last decade in multiple fields of plant biotechnology such as genetics, structural
and functional genomics, trait and gene discovery, transcriptomics, proteomics,
metabolomics, epigenomics, nanotechnology, and analytical tools. This book will
serve to update the scientific community, academicians, and other stakeholders in
global agriculture on the rapid progress in various areas of agricultural biotechnol-
ogy. This chapter provides a summary of the book, “Plant Genetics and Molecular
Biology.”
R. K. Varshney (*), M. K. Pandey, and A. Chitikineni

International Crops Research Institute for the Semi-Arid Tropics (ICRISAT), Hyderabad, India
e-mail: r.k.varshney@cgiar.org
2 R. K. Varshney et al.
Graphical Abstract
Keywords Decision support tools, Epigenomics, Genomics, Metabolomics,

Nanotechnology, Plant biotechnology, Proteomics, Transcriptomics
Contents
1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
2 High-Throughput Genotyping Platforms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
3 Trait Dissection and Gene Discovery . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
4 Beyond Genomics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
5 Data Management and Analytical Decision Supporting Tools . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
6 Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
1 Introduction
Making society hunger-free and malnutrition-free is the main goal for the stake-
holders in world agriculture. Feeding the global population has never been so
challenging, especially in the context of diminishing land and water resources
together with an ever-increasing global population and climate changes. One of
the possible solutions is to develop climate-smart varieties of plants complimented
with appropriate agricultural management practices. Today world agriculture is
facing an acute shortage in developing improved germplasm to replace the old
varieties existing in farmers’ fields. The global agriculture needs a “game-changer”
strategy to be implemented with high priority in order to develop improved
Plant Genetics and Molecular Biology: An Introduction 3
germplasm and cultivation practices rapidly and with high precision to tackle the
current and future adverse environmental conditions. Improved crop varieties
together with improved agricultural practices will be able to address the global
food security issue in an equitable and sustainable manner.
A recent survey on hunger and malnutrition has identified 52 of 119 countries as
having a serious, alarming, or extremely alarming situation. Even today, 13% of the
global population is undernourished and 27.8% of children under 5 years of age are
stunted (http://www.globalhungerindex.org/pdf/en/2017.pdf). Despite the availabil-
ity of sufficient food production, these problems still exist as a large number of
people do not have access to nutritious food. The quality and nutrition of food
products define the physical and mental health of the global population, not the
quantity. In this context, agricultural research on developing nutrition-rich crops
should be given equal importance to the major objective of increasing productivity.
The genetic gains achieved over the decades in several crop species have been able
to feed starving populations and have saved the lives of millions of people. Food and
nutritional security in the coming years can only be made possible by achieving
rapid and higher genetic gains in food crops with enhanced quality, nutrition, and
adaptation to adverse climatic conditions. This goal can be achieved by integrating
available biotechnological interventions with ongoing efforts. Not only agriculture
but also biotechnology has been a great support in boosting several sectors such as
the pharmaceutical, medical, and food processing sectors. In fact, the biotechnology
interventions have already produced game-changing contributions in agriculture and
the future contributions from biotechnology for society depend on strong policy,
commitment, and the investment made in biotechnology research in coming years.
The rapid advances in biotechnological processes, approaches, and technologies
have revolutionized agricultural research by developing a better understanding of
plant genomes, gene discovery, genomic variations, and manipulation of desired
traits in plant species. Additionally, these approaches also help researchers in
developing a better understanding beyond genomes such as plant-pathogen and
plant-environment interactions. The advanced technology support has helped to
track the entire journey from genomes to phenotype using different “omics”
approaches such as genomics (DNA/genome/genes), epigenomics (epigenetic
modifications on the genetic material), transcriptomics (transcripts/RNA),
proteiomics (proteins), metabolomics (metabolites), interactomics (protein interac-
tions), and phenomics (phenotype) (Fig. 1). The other important intervention is
nanobiotechnoloy (a combination of nanotechnology and biology), which provides
very sophisticated technical approach/devices for tracking, understanding, and solv-
ing biological problems. This book aimed to document current updates and advances
in these frontier areas of biotechnology research. This chapter provides an overview
of the different chapters included in the book.
Fig. 1 Plant genetics and molecular biology for trait dissection and crop improvement
2 High-Throughput Genotyping Platforms
The tremendous advances in sequencing technologies have made it possible to

sequence complete genomes of plant species for better understanding of the genome
architecture evolution including whole genome duplications, dynamics of transpos-
able elements, and several other components of the genome that define and control
genome function leading to a particular phenotype [1]. Chapter 2 on “Advances in
Sequencing and Resequencing in Crop Plants,” authored by SD Rounsley and other
colleagues from Dow Agrosciences, USA and Genus plc, UK, provides updates on
advancements in different sequencing technologies over the last two decades and
their impact on plant genomics research. Cost-effective sequencing technologies
have facilitated sequencing of a large number of plant genomes, which have
impacted greatly on developing better understanding of plant genomes and their
evolution [1, 2]. These advances have further helped in faster gene discovery,
characterization, and deployment in plant improvement [3]. In addition to this, this
chapter discusses the current challenges and future opportunities in further
exploiting genomics information for plant improvement.
The reference genome of any plant species provides the foundation for genomics
research, but mere sequencing of only one genome is not enough for harnessing the
wealth of genetic diversity available within and across plant species. Therefore,
sooner or later genome sequences will eventually be available for all the germplasm
and exist in different genebanks for capturing the sequence variations followed by
their manipulations using appropriate genetic improvement approaches such as
molecular breeding, genetic engineering (transgenics), genome editing, and any

other such technology developed in future. Sequence variations in different genomes
of the same species have been exploited as genetic markers for conducting different
genetics and breeding studies.
Chapter 3 on “Revolution in Genotyping Platforms for Crop Improvement,”
authored by David Edwards and his colleagues from the University of Western
Australia (UWA), Australia, describes how different types of genetic variations can
be used in genetics research and breeding applications through different genotyping
platforms. Similar to sequencing, genotyping platforms have also gone through a
rapid evolution and played an important role in advancing crop genetics and
breeding. These genotyping platforms have been deployed in a range of genetic
and breeding applications in most of the plant species. This chapter not only provides
details on the evolution of different genotyping platforms over the decades, but also
compares different genotyping platforms and predicts the future of genotyping in
plants. This chapter clearly advocates the sequencing of entire genetic and breeding
populations in future crop improvement programs for more precise and efficient
plant selection in field.
3 Trait Dissection and Gene Discovery
The availability of genetic diversity is crucial for further improving the existing
cultivars, which can sustain higher productivity under ever-challenging environ-
ments by acting as a buffer for adaptation and fighting climate change [4]. The
development of improved cultivars using the diverse germplasm has helped farmers
to replace these cultivars with older released or local varieties. The faster replace-
ment of improved cultivars in the farmer’s field will help in achieving higher
productivity under changing environments. Genomics-assisted breeding (GAB)
holds great promise for accelerated development of improved cultivars; however,
information on genes and diagnostic markers is required for deployment in any plant
species. There are three major approaches of trait mapping, namely linkage mapping,
linkage disequilibrium mapping/genome-wide association study (GWAS), and joint-
linkage association mapping (JLAM).
Linkage mapping uses bi-parental genetic populations for traits with high vari-
ability between the parental genotypes. Chapter 4 on “Trait Mapping Approaches
through Linkage Mapping in Plants,” authored by Pawan Kulwal from Mahatma
Phule Agricultural University (MPAU), India, discusses different types of
bi-parental populations and software for genetic mapping and quantitative trait
locus (QTL) analysis in several plant species. Detailed information on key factors
affecting the precision and accuracy of QTL discovery is presented. This mapping
approach has been the most successful as diagnostic markers could be developed and
deployed in breeding in several crop plants and many of these improved cultivars are
grown in farmers’ fields.
In contrast to linkage mapping, the second trait mapping approach, genome-wide

association study/linkage disequilibrium mapping, uses the diverse set of germplasm
(natural population) and, therefore, no time is spent on development of genetic
populations. The other advantage is that the association mapping panel can be
used for mapping for several traits, while linkage mapping is possible for a couple
of traits in a single bi-parental population. Furthermore, in many of the plant species,
the development of bi-parental populations is not feasible or possible.
Chapter 5 on “Trait Mapping Approaches through Association Analysis in
Plants,” authored by Joy Roy and his colleagues from the National Agri-Food
Biotechnology Institute (NABI), India, provides greater insights different technical
and applied aspects of GWAS analysis, advantages, and disadvantages of different
software, and key factors affecting the precision and accuracy of results. This
mapping approach has been deployed in many plant species.
The above two trait-mapping approaches have certain limitations and, therefore,
the joint linkage association mapping approach came into existence; this approach
can harness the advantages of both trait-mapping approaches. In this context, the
shift now has moved from bi-parental to multi-parental populations, which allow
high recombination leading to greater resolution for trait dissection. James Cockram
and Ian Mackay from the National Institute of Agricultural Botany (NIAB), UK, in
chapter 6 on “Genetic Mapping Populations for Conducting High Resolution Trait
Mapping in Plants” summarize in-depth information on development and deploy-
ment of multi-parent populations such as multi-parent advanced generation inter-
cross (MAGIC) and nested association mapping (NAM). This chapter also provides
examples that showed better results in trait mapping in larger population size than in
smaller ones.
All three above trait-mapping methods for trait mapping are forward genetics
approaches, while Targeting Induced Local Lesions IN Genomes (TILLING) is a
reverse genetics approach [5]. The TILLING approach involves creation of genetic
variation through mutagenesis and then identification of genomic variation causing a
change in phenotype. Chapter 7 on “TILLING: The Next Generation,” authored by
Bradley Till and his colleagues from International Atomic Energy Agency (IAEA),
Austria, describes the entire process of developing and deploying TILLING popu-
lation for trait dissection and gene discovery. The chapter also discusses how
integration of NGS technologies with TILLING have greatly accelerated the process
of gene discovery. These populations also serve as a very good source for breeding
and functional genomics studies.
4 Beyond Genomics
Genome sequencing greatly helped in understanding of genome organization and

gene(s) structure that determines the basic features of each species. Nevertheless,
just having genes in its genome does not provide certainty about the expected
phenotype, which depends hugely upon other aspects of gene regulation. The
journey of a gene to a particular phenotype is very complicated, depending on as and

when the DNA passes through different levels of regulation following the central
dogma. It is, therefore, very essential to see beyond genomics for better clarity on
gene function, networks, and interactions. In this context, the other “omics”
approaches such as transcriptomics, proteomics, metabolomics, and interactomics
play important roles in gene function and phenotype development. The phenotype is
also affected by non-genomic elements, which bring epigenetic modifications to the
genetic material, called as epigenomics. The epigenomic compounds modify the
function of DNA without changing the sequence, thereby deviating from following
the instruction of the genome. The interesting part is that these epigenetic features
are being passed down over generations.
Transcriptomics plays an important role in gene discovery and functional char-
acterization of the gene and its network. Chapter 8, authored by Nitin Mantri and his
colleagues from RMIT University, Australia, on “Advances in Transcriptomics of
Plants” discusses in detail discovery of transcriptional regulatory elements and
deciphering mechanisms underlying transcriptional regulation. This chapter also
covers related important aspects of gene regulation such as RNA splicing,
microRNAs, small interfering RNAs (siRNAs), and long non-coding RNAs in
plant development and response to biotic and abiotic stresses.
Metabolomics is very complex to understand due to development and interaction
of the large number of metabolites produced during attaining metabolic homeostasis
and biological balance in response to multiple cellular and extra-cellular factors.
Wolfram Weckwerth and his colleagues from the University of Vienna, Austria, in
chapter 9 on “Metabolomics in Plant Stress Physiology,” describe the importance of
the study of metabolomics for functional genomics and system biology research
leading to functional annotation of genes and better understanding of cellular
responses for different biotic and abiotic stresses in plants. This chapter also pro-
vides details on different modern techniques that play a key role in developing more
precise and high throughput data for comprehensive analysis. In addition to the
above, this chapter also describes the complete processes involved in metabolomics
study and lists the limitations faced by this scientific stream.
The epigenetic marks modifying the function of the gene can pass on over
generations, making epigenomics an important component in better understanding
the phenotype development. In other words, mere genome sequence is not respon-
sible for phenotype development, and the epigenetic modifications play a key role by
altering the chromatin structure and forcing deviation from the instructions
contained in the genome. Detailed information on the types of epigenetic changes
and their impact on phenotype development in plants is provided in chapter 10, enti-
tled “Epigenetics and Epigenomics of Plants,” authored by Manoj Prasad and his
colleagues from the National Institute of Plant Genome Research (NIPGR), India.
This chapter also discusses the key role of NGS technologies and improved analyt-
ical software in better understanding the role of epigenomics in plant development
and defense. Further information is also provided on different types of studies
conducted in plants for identifying epigenetic factors and their potential role in
plant improvement.
Nanotechnology has emerged recently as a very useful approach for plants and
has already demonstrated its potential in the development of several nanomaterials in
the pharmaceutical industry and in improving human health. Plants are the best
source for developing such nanomaterials due to their large-scale availability and
ease of production. Chapter 11 on “Nanotechnology in Plants,” authored by Ismail
Ocsoy and Weihong Tan and their colleagues from Erciyes University, Turkey and
University of Florida, USA, explains the importance of nanotechnology in plants by
citing several successful examples in medicine and industrial applications. The
chapter mentions several advantages of plant extract over other biomolecules such
as protein, enzyme, peptide, and DNA followed by their use in food, medicine,
nanomaterial synthesis, and biosensing. This chapter also provides information on
different extract preparation techniques, their use in the synthesis of nanoparticles,
and demonstration of their antimicrobial properties against pathogenic and plant-
based bacteria.
5 Data Management and Analytical Decision Supporting

Tools
Large-scale data are generated at each step of the plant experiment related to
understanding of the genome, gene discovery, functional characterization of gene,
marker discovery, and deployment of diagnostic markers in the breeding program in
addition to phenotyping data. All these data sets require efficient and effective
database management systems, and analytical and decision support tools for storing
and retrieving useful information that impacts the genetic improvement efforts.
Chapter 12 on “Current Status and Future Prospects of Next-generation Data
Management and Analytical Decision Support Tools for Enhancing Genetic Gains
in Crops,” authored by Abhishek Rathore and his colleagues from ICRISAT, India,
provides details on data management and analysis and decision support tools
(DMAST) for plant improvement. The chapter also provides examples of how
DMAST has simplified and empowered researchers in data storage, data retrieval,
data analytics, data visualization, and sharing.
6 Summary
Ensuring food and nutritional security for an ever-increasing global population

under the changing global climate is a top priority for policy makers across the
globe. The existing conventional research efforts and traditional technologies will
not be able to provide adequately nutritious food for the global population, neces-
sitating the incorporation of modern science into the current genetic improvement
programs. Biotechnology has great potential in bridging the supply-demand gap in
food through developing improved agricultural technologies. All the scientific

streams are witnessing a rapid pace of development due to integration of new
technologies such as robotics, automation, etc. Theses advancements have improved
our understanding of genome architecture and its complexity: gene structure, func-
tion, and interactions, and improved methodologies for modification of the genome/
gene to achieve a desired phenotype. The plant-pathogen and plant-environment
interactions complicate the expression of scripts in the plant genome. This book
covers these important research areas pertaining to plant biotechnology, which are
key for achieving higher genetic gains. This wealth of information will be a great
value for students, researchers, academicians, and policymakers.
References
1. Wendel JF, Jackson SA, Meyers BC, Wing RA (2016) Evolution of plant genome architecture.
Genome Biol 17:37
2. Michael TP, Jackson S (2013) The first 50 plant genome. Plant Genome 6(2). https://doi.org/10.
3835/plantgenome2013.03.0001in
3. Varshney RK, Nayak SN, Jackson S, May G (2009) Next-generation sequencing technologies
and their implications for crop genetics and breeding. Trends Biotechnol 27(9):522–530
4. Buchanan-Wollaston V, Wilson Z, Tardieu F, Beynon J, Denby K (2017) Harnessing diversity
from ecosystem to crop to genes. Food Energy Secur 6(1):19–25
5. Henikoff S, Till BJ, Comai L (2004) TILLING: traditional mutagenesis meets functional
genomics. Plant Physiol 135(2):630–636
Adv Biochem Eng Biotechnol (2018) 164: 11–36
DOI: 10.1007/10_2017_46
© Springer International Publishing AG 2018
Published online: 8 March 2018
Advances in Sequencing and Resequencing

in Crop Plants
Pradeep R. Marri, Liang Ye, Yi Jia, Ke Jiang, and Steven D. Rounsley
Abstract DNA sequencing technologies have changed the face of biological

research over the last 20 years. From reference genomes to population level
resequencing studies, these technologies have made significant contributions to
our understanding of plant biology and evolution. As the technologies have
increased in power, the breadth and complexity of the questions that can be asked
has increased. Along with this, the challenges of managing unprecedented quantities
of sequence data are mounting. This chapter describes a few aspects of the journey so
far and looks forward to what may lie ahead.
Graphical Abstract
Cost ($/Mbp) Read length (bp)
0.52
Oxford Nanopore
MinION
commercially available
10,000 ~ 30,000bp
Sanger & 454
sequencing Introduction of
500 ~ 800bp Illumina read length PacBio RS II
gradual increase 5,000bp
Introduction of ~ 300bp
Illumina
36 ~ 50bp
0.014
Jan Apr Jun Oct

2010 2013 2015 2015
P. R. Marri, L. Ye, Y. Jia, and K. Jiang

Dow AgroSciences, Indianapolis, IN, USA
S. D. Rounsley (*)
Genus plc, De Forest, WI, USA
e-mail: steve.rounsley@gmail.com
12 P. R. Marri et al.
Keywords Assembly, Crops, NGS, Sequencing
Contents
1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
2 Current Technologies, Standards, and Strategies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14
2.1 Sequencing Technologies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14
2.2 Assembly Technologies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
2.3 Reference Genome Project Strategies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
2.4 Resequencing Strategies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
2.5 Data Management and Visualization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
3 Trends, Advanced Technologies, and Strategies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27
3.1 Sequencing Technologies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27
3.2 Assembly Strategies/Technologies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29
3.3 Genome Project Strategies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29
3.4 Resequencing Strategies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30
3.5 Data Management, Visualization, and Storage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30
3.6 Beyond Individual Variants: Alleles, Haplotypes, LD Blocks, and Pan-Genomes . . . 30
4 Conclusion and Outlook . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32
Abbreviations
ABYSS Assembly by Short Sequences

AGI Arabidopsis Genome Initiative
API Application Programming Interface
BAC Bacterial Artificial Chromosome
CCD Charge Coupled Device
CIGAR Concise Idiosyncratic Gapped Alignment Report
CNV Copy Number Variation
CRT Cyclic Reversible Termination
DBG de Bruijn Graph
ddNTPs Dideoxynucleotides
DNA Deoxyribonucleic acid
dNTPs Deoxynucleotides
GB Giga-basepairs
GMOD Generic Model Organism Database
GWAS Genome Wide Association Mapping
HapMap Haplotype Map
IGV Integrative Genomics Viewer
InDel Insertion-Deletion
Kb Kilo-basepairs
LD Linkage Disequilibrium
MAGIC Multiparent Advanced Generation InterCross
Mb Mega-basepairs
Advances in Sequencing and Resequencing in Crop Plants 13
MTP Minimum Tiling Path

NAM Nested Association Mapping
NGS Next-Generation Sequencing
OLC Overlap Layout Consensus
ONT Oxford Nanopore
PacBio Pacific Biosciences
PAV Presence-Absence Variation
PCAP Parallel Contig Assembly Program
PCR Polymerase Chain Reaction
PHRAP Phil’s Revised Assembly Program
PHRED Phil’s Read Editor
SBL Sequencing by Ligation
SBS Sequencing by Synthesis
SMRT Single Molecule Real Time
SNA Single Nucleotide Addition
SNP Single Nucleotide Polymorphism
SOLiD Sequencing by Oligonucleotide Ligation and Detection
Tb Tera-basepairs
TIGR The Institute for Genomic Research
UCSC University of California at Santa Cruz
VCF Variant Call Format
VEP Variant Effect Predictor
WGS Whole Genome Shotgun
ZMV Zero Mode Waveguide
1 Introduction
When History of Science books are written in the future, there seems to be a more-
than-reasonable chance that DNA sequencing and the birth of genomics will feature
prominently. It is hard to think of a technology that has had a more dramatic effect on
the study of biology than DNA sequencing. For those active in research today, with
all the data and technology available, it is also hard to remember how little we knew
about genomes before the mid 1990s. And despite the huge gulf in technology and
knowledge between then and now, the field may still be in its infancy – in the first
stages of a journey with a double helix as its guide. This chapter describes a few
aspects of the journey so far and looks forward to what may lie ahead.
14 P. R. Marri et al.
2 Current Technologies, Standards, and Strategies
2.1 Sequencing Technologies
2.1.1 Sanger Sequencing
In 1977, Frederick Sanger published a DNA sequencing technique that became the
base technology for the field of genomics [1]. Sanger sequencing relies on the chain
terminating properties of dideoxynucleotide triphosphates (ddNTPs), which were
added to a mix of the four standard deoxynucleotides (dNTPs). When a comple-
mentary strand of sequence is synthesized using these reagents (the sequencing
reaction), the result is a mixture of DNA fragments each terminated at different
lengths. These fragments must then be separated by size (via electrophoresis),
detected, and then recorded. Initially, slab polyacrylamide gels, radioactivity, and
typing in sequence were integral to the standard (very manual) technique. Auto-
mated DNA sequencers were later developed, which automated the detection and
capture of the resulting DNA sequence. Improvements such as fluorescently-labeled
terminating nucleotides and capillary electrophoresis were incorporated into the ABI
line of DNA sequencers. Hundreds of these instruments were sold to large genome
centers working on genome projects in the 1990s and early 2000s – including
bacteria, yeast, Arabidopsis, mouse, and human genomes [2].
2.1.2 Next-Generation Sequencing (NGS) Technologies
Over the last decade, sequencing technologies have evolved rapidly and led to a
significant increase in throughput and reduction in cost, thereby enabling large-scale
sequencing of genomes. They have done so by removing a limitation of Sanger
sequencing of having to separate DNA fragments by size. In Sanger sequencing, the
sequencing reaction occurs outside of the instrument, and the instrument simply
separates and detects fragments. For most NGS technologies, the sequencing reac-
tion is occurring on the instrument, and each base addition onto a growing DNA
molecule is detected and recorded. The first generation of NGS technologies have
relied largely on two approaches for sequencing, sequencing by ligation (SBL) and
sequencing by synthesis (SBS) [3]. Both approaches rely on spatially constrained,
clonal amplification of DNA and facilitate massive parallelization of sequencing
reactions, each with its own clonal DNA template, resulting in the sequencing of
millions of sequences in parallel.
SBL involves hybridization and ligation of fluorophore-labelled probes and
anchor sequences to a DNA strand and capturing the emission spectrum to identify
the DNA base, whereas SBS relies on strand extension using a DNA polymerase and
uses changes in color or changes in ionic concentration to identify the incorporated
nucleotide [3]. SBL is used in platforms such as SOLiD and Complete Genomics,
whereas 454, Ion Torrent and Illumina use the SBS approach.
The SBS technologies can be classified into two approaches: the first, single
nucleotide addition (SNA), used in 454 and Ion Torrent sequencers. This approach
adds four nucleotides iteratively and scans for a signal after each to record an
incorporated nucleotide. In the case of 454, which sold the first NGS instrument
(the GS20), template-bound beads are distributed into a PicoTiterPlate and emulsion
PCR is performed to clonally amplify a single DNA fragment within a water-in-oil
microreactor. The addition of dNTPs triggers an enzymatic reaction that results in a
fluorescent signal that is captured by a charge-coupled device (CCD) camera and is
indicative of incorporated nucleotide [4]. The SNA method as implemented in Ion
Torrent relies on ion sensing rather than fluorescence and detects the H+ ions that are
released after the incorporation of each dNTP and the resulting shift in pH is used to
determine the incorporated nucleotide. Both 454 and Ion Torrent methods have
limitations in accurately measuring the homopolymer lengths, because all nucleo-
tides in a homopolymer are incorporated at the same time, and the magnitude of the
signal must be used to estimate the homopolymer’s length.
The other SBS approach is found in the NGS instruments that have come to
dominate the market – those manufactured by Illumina. This technology, which was
developed by Solexa before they were acquired by Illumina, uses terminating
nucleotides similar to Sanger, except the termination is reversible. Cyclic reversible
termination (CRT) uses a mixture of four reversible terminators each with a distinct
fluorescence. Each template is extended by a single base only using the appropriate
terminator and the resulting labeled templates are imaged recording which nucleo-
tide was added to each template. The terminators are then cleaved off, and the cycle
continues with the addition and imaging of the next nucleotide. An additional key to
Illumina’s success is the massive number of templates the technology can sequence
in parallel – approaching three billion on a single flow cell in the HiSeq-X instru-
ment. They achieve this through the immobilization of a DNA library onto a glass
flow cell coated with adapter oligos. Clonal clusters of each DNA fragment are
synthesized using bridge amplification on the flow cell resulting in a very large
number of sequence-ready templates. Illumina currently has the largest market share
for sequencing instruments and offers a wide variety of sequencing systems, read
lengths, and throughput to cater to a wider range of applications (Table 1).
2.2 Assembly Technologies
The developments in automated, higher throughput sequencing technologies have

been matched by concomitant development of algorithms and tools to use the
resulting data in various applications. For projects where the goal is the generation
of a reference genome, assembly algorithms have been a key area of development.
The selection of an appropriate algorithm depends on the sequencing strategy being
used (see next section), but here we will describe the main classes available.
Assembly algorithms can be broadly divided into two classes: overlap-layout-
consensus (OLC) and De-Bruijn-graph (DBG) [5]. The OLC approach identifies
16
Table 1 Illumina sequencing systems

Metrics MiniSeq MiSeq v3 NextSeq HiSeq2500 v4 HiSeq3000/4000 HiSeq X
Maximum output 7.5 Gb 15 Gb 120 Gb 1,000 Gb 1,500 Gb 1,800 Gb
Cluster number (millions) 25 25 400 4,000 5,000 6,000
Read length 1 75 bp 2 75 bp 1 75 bp 1 36 bp 1 50 bp 2 150 bp
2 75 bp 2 300 bp 2 75 bp 2 50 bp 2 75 bp
2 150 bp 2 150 bp 2 100 bp 2 150 bp
2 125 bp
Run time 7–24 h 21–56 h 11–29 h 1–6 days <1–3.5 days <3 days
bp basepairs, Gb gigabase pairs, PE paired-end sequencing, SE single-end sequencing
P. R. Marri et al.
overlaps between all reads, and the reads and overlap information are laid out on a
graph and consensus sequences are then inferred. This algorithm, often used with
Sanger-generated data, has been widely incorporated into assembly programs such
as Arachne [6], Celera Assembler [7], PCAP [8], and PHRAP [9]. Although this
approach provides a cheaper and faster way of utilizing Sanger sequencing for
reference genome development, with larger datasets the assemblies usually have
gaps and result in unplaced scaffolds that require more effort to verify and finish.
This heralded the era of draft genome assemblies and a subsequent change in
standards for the quality of a reference genome.
The significantly higher data volume, shorter read lengths, and platform-specific
error profiles of NGS data present challenges for algorithm developers. The higher
amounts of short-read data from the next generation sequencers furthered new
developments in assembly algorithms and a few overlap-layout-consensus assem-
blers such as Celera Assembler [7], PCAP [8], and Newbler [4] were extended from
their original versions to handle both Sanger and NGS data from 454 sequencers.
However, the increased usage of short read Illumina sequences for assembling large
complex genomes spurred the development of the second class of assembly algo-
rithms – those using the more efficient DBG-based approaches. The DBG approach
works by first chopping reads into shorter k-mers, using those k-mers to build a
graph and using the graph to infer the genome sequence. Assemblers such as ABySS
[10], ALLPATHS-LG [11], and SOAPdenovo [12, 13] rely on the DBG approach
for increased efficiency.
2.3 Reference Genome Project Strategies
2.3.1 Sanger-only Assemblies
Sequencing technologies have enabled the study of genomes across all spheres of
life. The first genomes to be sequenced were bacterial [14, 15] and employed a whole
genome shotgun approach. However, at the time, larger genomes were not consid-
ered good candidates for this approach. Consequently, a hierarchical shotgun strat-
egy was developed for the first large genomes, including the generation of the first
plant reference genome for the model plant Arabidopsis thaliana. The Arabidopsis
Genome Initiative (AGI), an international consortium, generated comprehensive
BAC libraries and used the BAC end-sequences and fingerprints of individual
BAC clones to create a physical map. A minimum tiling path of BAC clones
along each chromosome was identified and the selected BACs were then individu-
ally shotgun-sequenced by consortium members and assembled using assemblers
such as the TIGR Assembler [16] to produce assembled contigs. The BAC ends were
later used to link contigs into scaffolds and the genetic map served as a foundation
for integrating assembled scaffolds into chromosomes [17].
The initial strategies for reference genomes relied predominantly on Sanger
sequencing and continued to make advancements through automation or
Another random document with
no related content on Scribd:
This giant's head was smaller than the Sphinx and of an antiquity more
profound. The countenance lacked majesty and was indeed malignant—not
with the demoniac intelligence of man-cut fiends, such as "Le Stryge" on
Notre Dame, but rather with the brutish, semi-human doubt and uncertainty
of a higher ape. So the Minotaur might have scowled to seaward. The
expression of the monster trembled on the verge of consciousness; it
suggested one of those vanished beings created near the end of our hundred
thousand years' journey, after man's ancestors descending from the trees set
forth on the mighty march to conscious intelligence.
The face belonged to the forefathers of the neolithic people: it

burlesqued hugely those beetle-browed, prognathous paleoliths of old time,
and for them, perchance, possessed an awe and sublimity we cannot grant it
to-day.
But it had challenged a boy and girl, who were still many thousands of
years nearer to prehistoric ancestors than their parents. For children still
move through the morning of days, and through minds ten and eleven years
old the skin-clad dreamers and stone men were again reflected and
survived.
Now Dinah heard with what force the discovery of the stone Titan had
struck upon the boy and girl.
"A new baby was coming," said Lawrence, "and sister and me were
each given a bit of food and told to run out on the moor and play till
nightfall. That pleased us very well and we made our games and wandered
and picked hurts.* And then I suddenly found yonder face and shouted to
Milly and made her see it too. It excited me a lot, and Milly always got
excited when I did. She said 'twas like father, but I said, 'No, 'tis a lot
grander and finer than father.' Then she was frighted and wanted to run
away; but I wouldn't have that. 'He'd blow out of his mouth and scat us to
shivers if we ran,' I told her. I took pleasure in giving great powers to the
monster, and wondered if he was good, or wicked. And little sister thought
he must be wicked, but I didn't see why he should be. 'Perhaps he's a good
'un,' I said; and then I decided that he might be good. Milly was for sloking
off again, but my child's wits worked, and I very soon lifted up the stone
into a great, powerful creature. 'Us'll say our prayers to him,' I told Milly,
but she feared that also. 'I never heard of nobody saying no prayers except
to Gentle Jesus,' answered Milly to me. 'The Bible's full of 'em,' I told her.
'How would it be if we offered to be his friends?'"
* Hurts—whortleberries.
"Tempted your little sister to turn heathen!" exclaimed Dinah.
"Yes, and she soon fell. I minded her how we had once prayed with all
our might to Gentle Jesus to kill father, because he wouldn't take us to a
circus as had come to Bovey. 'Gentle Jesus have got His Hands full without
us,' I said to Milly. 'He haven't got no time to think about two little squirts
like us. But this here great creature might be a good friend to us; and
nobody the wiser!'"
"You was a crafty little boy."
"No craft, only a queer twist of the brain. I smile sometimes, looking
back, to see what thoughts I'd gotten. But child's thoughts die like flowers.
We can never think 'em again when we grow up. Milly held out a bit, yet
she never withstood me very long. She was only afraid that Gentle Jesus
would hear tell about it and punish us; but I said, 'Not Him. If harm comes,
I'll take the blame. And we won't put anything very hard upon this
monstrous old rock till we know how strong he be.' We thought then what
we should pray for, and Milly had a bright idea. 'Ax him to make the new
baby a boy,' she advised, and I agreed, for we was very wishful to have a
boy home, and so was our mother. Then Milly had another thought. 'What
be us to call him?' she asked me. 'Something terrible fearful,' I said—'the
fearfullest thing we can think upon.' We strove after the most dreadful
words we knew, and they were our father's swear-words. 'Let's call him
"Bloody,"' I said; and Milly thought we ought to say 'Mr. Bloody.' But I told
her 'Mister' was a name for a gentleman, with nothing fierce or grand to it.
'We'll call him "Bloody" and chance it,' I said; and so we did. I prayed to the
stone then. I said, 'Dear Bloody, please let mother's new babby be a boy.
Amen'; and Milly done the same; and when we got home in the dimpsy
light, all was over and father eating for the first time that day. There had
come a little boy and mother was happy. Milly whispered to me, 'That's one
for him!'"
Dinah laughed with delight. Her own troubles were for the time
forgotten.
"I'll mind that story so long as I live," she said, gazing up at the iron-
black, impassive features above her.
"That's not all, though. We got terrible friendly with our great idol, and
then, a week later, the baby fell ill and seemed like to die. For the nurse that
waited on mother had come from whooping-cough and the poor child
catched it afore it was five days old. We were in a terrible upstore about
that, and I minded this rock; and when a day came and the little one was at
his last gasp, me and Milly went up and stood here, where we sit now. I said
we must bring offerings, but us hadn't nothing but my knife and Milly's pet
bunny rabbit. But such was the fearful need, we determined to sacrifice
both of 'em; and we did. Lord knows how we could, but I killed her little
rabbit for 'Bloody,' and I dropped it and my knife in that cleft below the
rocks at his feet. We used to call 'em his paws. The rabbit and my knife
went down there, and we asked for our new-born brother, and prayed the
creature to save him alive. And we wept a good bit, and I remember Milly
felt glad to see me cry as well as her. We went home a lot comforted—to
find the baby was dead."
He broke off and the listener expressed sorrow.
"You poor little things—to think of you trotting back together—to that!
I could cry for 'e now."
"We cried for ourselves I warrant you. We was terrible upset about it,
and I properly gnashed my teeth I remember. Savage I was, and loved to
hear father damn to hell the nurse that had done the mischief. 'Douglas
Champernowne' the poor child was called. My mother doted on high-
sounding names. And the day he was buried, my sister and me roamed on
the moor again in our black after the funeral, bewailing our loss; and it was
Milly that called my mind to our stone god, for I'd forgot all about him just
then. 'There he is—aglaring and agrinning!' she said, and I looked up and
saw we'd come to him without thinking. It had been raining all day, and his
face was wet and agleam in evening sunlight. We liked him that way, but
now I turned my hate on him and cursed him for a hard-hearted, cruel devil.
'Beast—hookem-snivey beast!' I yelled up at the tor; 'and I wish to God I
was strong enough to pull you down and smash your face in!' Milly
trembled with fear and put her arms around me, to save me, or die with me
if need be. But I told her the idol couldn't hurt us. 'He can only kill babbies,'
I yelled at him. Then I worked myself up into a proper passion and flung
stones and mud at the rock, and Milly, finding our god helpless, egged me
on. We made faces and spat on the earth and did everything our wits could
hit on to insult him. Then, tired out, we turned our backs on him, and the
last he heard was my little sister giving him the nastiest cut of all. 'We be
going back to Gentle Jesus now,' screamed Milly."
Maynard ceased and lighted his pipe.
"It's a sad, lovely story. I don't wonder you come and have a look at the
face sometimes. So shall I now. May I tell it again?" asked Dinah.
"No, miss, don't do that—I'd rather none heard it for the present. I've my
reasons for not wishing to be linked up with these parts."
"Call me 'Dinah,' and let me call you 'Lawrence,'" she said. From her
this was not a startling suggestion. Indeed she had already called him
"Lawrence" sometimes.
"If you like," he answered. "It's easier. We see a good many things the
same."
"I suppose we do. And did you and Milly go back to 'Gentle Jesus'?"
"Certainly we did; and I'll make bold to say she never left Him no
more."
"But you—you ain't exactly a Christian man, are you? When did you
change?"
He looked into the past and did not answer for a moment.
"I don't know," he said at last. "It's hard to tell sometimes when we
change. Them that come to the penitent bench, or what not, know to an hour
when they was 'saved,' as they call it; but them that have gone the other
way, and heaved up anchor, and let their reason steer the ship and their faith
go astern—such men can't always answer exactly when the change came.
Sometimes it's just the mind getting bigger and the inner instinct dropping
the earlier teaching; and sometimes things happen to shake a man for ever
out of his hope and trust."
"A very sad thought," she said.
"It's always sad to see a thing fall down—whether it's a god or a tree.
The sound of the woodman's axe be sad to some minds."
"It is to me," said Dinah.
He looked up at the features above them, carved on the mass of the tor.
Beyond swung out Rippon's granite crown against the sky, and nearer
stretched miles of wild and ragged heath. Then, in long, stone-broken
curves the moor rose and fell across the western light to Honeybag and
Chinkwell and huge Hameldown bathed in faint gold. The sun kneaded
earth with its waning lustres until matter seemed imponderable and the wild
land rolled in planes of immaterial radiance folding upon each other. The
great passages of the hills and dales melted together under this ambient
illumination and the stony foreground shone clear, where, through the
hazes, a pool glinted among the lengthening shadows and reflected the sky.
Quartz crystals glittered where the falling rays touched the rocks, and as the
sun descended, great tracts of misty purple spread in the hollows and flung
smooth carpets for the feet of night.
For a moment "Bloody" seemed to relax his brutal features in the glow.
Sunset lit a smile upon the crag, and nature's monstrous sculpture appeared
to close its eyes and bask in the fading warmth.
"It would be a pity if ever Hey Tor were thrown down, as I wanted to
throw it, when I was that little angry boy," said Lawrence.
She put out her hand to him.
"Don't you fling over God," she said very earnestly.
"I hope never," he answered.
After they had talked awhile longer he looked at his watch.
"Half after seven," he exclaimed.
They set off for home and she asked for another tale.
"Tell me what happened to you when you went out into the world," she
begged; but this he would not do. He made no mystery, but definitely
declined.
"You've heard enough about me, I reckon. Speak of yourself a bit."
She obeyed and described her life in childhood, while he listened to the
simple story, interested enough.
He reminded her of his desire as their walk ended and they reached the
door of Falcon Farm.
"Don't say nothing of my past in this, or tell the tale of the rock again,
Dinah. I'm not wishful for the people to know anything about me."
She promised.
"I can keep secrets," she assured him.
CHAPTER XV
BEN BAMSEY'S DOUBTS

As the summer advanced, Jane Bamsey let it be known that she
proposed to wed Enoch Withycombe's son, Jerry. For some time her parents
refused to believe it, but as Jane persisted and brought Jerry to see them,
they began to accept the fact. Benjamin felt hopeful of the match, while
Jane's mother did not. In the first place she was disappointed, for while a
fine and amiable man, of good repute, not lacking in respect, Jerry could
not be considered a very promising husband. He was too old; he was only a
woodman and would always remain a woodman. Mrs. Bamsey held that a
daughter of hers should have looked higher.
Jane, however, now declared her undying love, and, for the moment, it
was undoubtedly true that she did love and desire the son of the huntsman
better than anything in the world. She did not share her parents' estimate of
him but perceived possibilities and believed that, with her help and
supported by the dowry she expected to bring him on their marriage, Jerry
would prove—not her head, but her right hand. For Jane had her own
private ambitions, and though they staggered Jerry when he heard them,
such was his devotion that he agreed to propositions for the future
inevitably destined to upset his own life and plunge it into a wrong
environment. Their absurdity and futility were not as yet apparent to him,
though Jane's ideas from anybody else had been greeted with contempt.
They embraced a radical change in his own existence that had been
unbearable to contemplate save in one light. But that light his sweetheart
created; and when she described her ambitions to leave Dart Vale and set up
a little shop in town, Jerry, after some wondering protests, found that the
choice might actually lay between this enterprise and Jane herself.
Therefore, he did not hesitate. He stated his case, however, when she agreed
to marriage on her own conditions.
"Away from trees, I'm much afraid I should be but a lost man," declared
Jerry.
"And in the country, I'm but a lost woman," replied Jane. "I'm sick of
trees, and fields too."
She had never hinted at the possibility of accepting him at all before the
occasion of these speeches; and it was natural that no stipulation could long
daunt Jerry in the glorious hour of success. Jane was actually prepared to
accept him at last, and since life with Jane in a dungeon had been better
than life without her under any conditions whatsoever, Jerry, after a display
of argument that lasted not five minutes, agreed to her terms, and found his
sweetheart on his knees, his arms round her, the astounding softness of her
cheek against the roughness of his own.
How far Jane might be looking ahead, neither her future husband, nor
anybody else knew; but one guessed; and since it was Enoch Withycombe
who received this spark of divination, he kept it to himself for the present.
Now the invalid spoke to Jane's father, who came to see him upon the
subject; but both old men considered the situation without knowledge of the
facts, because Jane, with greater insight than Jerry and shrewd convictions
that her terms would meet with very hearty protests from her lover's family,
if not her own, had counselled Jerry, indeed commanded him, to say
nothing of their future intentions until the marriage day was fixed.
"And how's yourself, Enoch?" asked Benjamin, as he smiled and took

Mr. Withycombe's hand.
"Middling, but slipping down, Ben. The end's getting nearer and the bad
days getting thicker sprinkled in the pudding. I shan't be sorry to go."
"Well, well, if you ban't, there's a cruel lot will be when you do," said
Mr. Bamsey. "And often and often I catch myself asking if the deaders do
really go at all. Married to Faith, as I am, I can't help but feel we've got a
cloud of witnesses round about. How it may be in other places, of course, I
can't say, but there's no doubt that the people who drop around here, hang
about after; and if you've got Faith's amazing gift, they ban't hidden. She
see widow Nosworthy last week, down by the stile in 'five acre,' where
there's a right of way. She was standing there, just like she used to stand
time without count waiting for her drunken son of a night, to steer him past
the pond to his home. I say naught, however, whatever I may think."
Mr. Withycombe showed a little impatience.
"'Tis no good prattling about ghosts to a man who'll damn soon be one
himself," he said. "As you very well know I don't believe in 'em, Ben; and if
us understood better, we'd be able to prove, no doubt, that your wife don't
see nothing at all, and that the ghosts be in her own mind's eye and nowhere
else. Not a word against her, of course. I respect her very much. But' second
sight,' so to call it, be just a thing like gout, or bad teeth—handed down, and
well inside nature, like everything else. I don't believe in no future life
myself, but I don't quarrel with them that do. I'm like my old master—large-
minded, I hope. And if another life there is, then this I will swear, that the
people as be called home have got their senses, and the next world have its
duties and its upper ghosts set over the unknown country to rule and direct
it. You can't suppose that everybody's on his own there, to moon about and
poke about, like a lot of birds, with no law and order. When the men and
women go out of this world, they've done with this world, and I never will
believe they be allowed back, to waste our time and fright the silly ones and
talk twaddle to people in dark rooms and play senseless tricks we'd whip a
child for."
"Leave it," said Ben. "I go largely along with you, and for that matter
my wife herself thinks no more of it than her power to make butter."
"How's Johnny?"
"Got a lot more silenter than he was. Comes and goes; and he's civil to
Dinah now, but don't see her alone. Us be a bit hopefuller about him, but
not her. In fact Dinah's one of the things I be come to tell about. I'm a bit
afeared in that quarter. I might see a ray of light where she's concerned; but
John, being what he is, the light, even if there is any, looks doubtful."
"Leave him then. You want to talk of this here match between my Jerry
and your Jane."
"I do. I'm very wishful to hear you speak out on the subject, Enoch. For
myself, being a great believer that marriages are made in heaven and 'tis
only our human weakness mars 'em on earth, I'm always willing to hope the
best and trust true love. And true love they've gotten for each other I'm very
sure indeed, though I wish they was nearer of an age."
"What does Faith Bamsey say?"

"It don't so much matter as to her. She's a right to her opinions, and
seldom we differ, but in this affair, to be honest, we don't see eye to eye. In
marriage, the woman be more practical than the man."
"I'll tell you what she says, Ben. She don't like it. All her reasons I
cannot tell: one I'm dead sure about. It's a come-down for her Jane to marry
Jerry. I grant that. I've told Jerry so too."
"Perfect love casteth out any such thought," said Mr. Bamsey.
"It may cast it out, but it will come back. In some girls it wouldn't. In
Jane it will. Jane's on powerful good terms with herself, as you'll grant. The
toad knows she's a beauty, and she knows a lot else—a lot more than Jerry
knows for that matter."
"You don't like her," said Mr. Bamsey.
"I do not; and she don't like me."
Ben was silent.
"She came up to tea Sunday, and I seed 'em side by side. She's sly and
she's making Jerry sly. How the devil she's larned such an open sort of
creature as Jerry to keep secrets I don't know. But secrets they've got. I dare
say they'll be married. But I agree with Faith Bamsey that it won't come to
overmuch good. I don't think Jane is a very likely pattern of wife."
"She's young and there's bright points to her. She wasn't saucy nor
anything like that I hope?"
"Oh no—butter wouldn't melt in her mouth."
"What does Melinda think about her?"
"Melinda thinks better of her than what I do. She didn't before the
match, but now, womanlike, she's all for it."
"That's to the good then. They talk of late autumn."

"Have you asked your girl where she thinks to live?"
"We haven't raised that yet."
"I did, and she put me off. There's things hidden. What are you going to
give her?"
"Five hundred pound, Enoch."
"Don't, Ben. Keep it against their future. That's a hugeous lot of money
and Lord knows what she'd do with it. I hope you haven't told her no such
thing."
"My only daughter must have a good start. It's none too much; she
knows about it."
Mr. Withycombe considered.
"Then I'll tell you what she's doing, Ben. She's marrying for money!
Yes, she is. Not Jerry's, because he won't have a penny till I die, and then he
can only have one-third of the lot, which ain't much. But Jane's marrying
for your money, and I'll lay my life that she's going to spend it, thinking that
there'll be plenty more where that comes from."
"You mustn't say such things. She knows the value of money better than
most."
"Then have it out and learn what's in their minds. They've no right to
secrets if you're going to pay the piper and start 'em in that generous way.
Keep her money and let her have the interest on it and no more, till you see
how they get on."
"Secrets there didn't ought to be I grant," said Benjamin. "But, mind

you, I'm not allowing there are any. You may be mistook."
"Then find out where they be going to live and how Jane thinks to
handle that dollop of cash," warned Mr. Withycombe. "You've a right to
know: it's your duty to know, and your wife will tell you the same. And
don't let Jane throw dust in your eyes. She's a tricky piece, like most of
them beauties. I wish she'd took after the pattern of Orphan Dinah."
"So do I," admitted the other. "And now, to leave this, I'd like to speak a
word about Dinah, because there's some things you may know bearing upon
her that I do not. I'm struck with doubt, and I've been keeping her on along
with me for two reasons. Firstly, because the right sort of place in my
opinion don't offer."
"It never will," said Enoch. "Truth's truth, and the truth is you can't part
from her."
"No, you mustn't say that. She's a very great deal to me, but I ban't
selfish about her. I'm thinking of her future, and I don't want her placed
where her future will be made dark and difficult. There's a lot to consider.
And while I've been considering and withstanding Faith, here and there, and
Dinah also for that matter, there's drifted into my mind the second point
about Dinah. And that's in my mind still. But I begin to feel doubtful,
Enoch, whether it's very much use for us old people to worry our heads as
we do about the young ones. This generation will go its own way."
"Pretty much as ours did before them no doubt," answered the sick man.
"We ancient folk love to bide in the middle of the picture so long as we can,
and when I was a lot younger than now, yet not too young to mark it, I often
thought it was rather a sad sight to see the old hanging on, and giving their
opinions, and thinking anybody still cared a damn for what they said, or
thought."
"Yes, only the little children really believe in us," confessed Mr.
Bamsey; "and that's why I say we squander a good bit of wisdom upon the
rising race, for they'll go on rising, for good or evil, without much troubling
their heads as to whether we approve, or don't. They put education before
experience, so, of course, what we've got to offer don't appeal to 'em. But
Dinah—she does heed me, and now there's come a shadow of a suspicion in
my mind about another man for her. That's why I'm so content to mark time
a little and get hard words for doing it. She don't know herself, I believe,
and very like the man don't know yet. Or he may know, and be biding his
time. But more than once or twice—more, in fact, than she guesses—Dinah,
when she's been talking to me, have named a certain person. I very near
warned her against it, for women's ears are quicker than ours, and if Jane
and my wife had marked it, evil might have risen out of it at once. But
Dinah says a lot more in my hearing than in theirs and they don't know so
far. And, as I tell you, Dinah herself don't know. But the name echoes along.
In a word, what do you think of Maynard at Falcon Farm? You know him
better than I do, though the little I've seen of the man leads me to like him.
He's a sensible soul and none too cheerful—rather a twilight sort of man
you might say; but that's a way life have with some of the thinking sort. It
turns 'em into their shells a bit."
"He's a kindly, well-meaning chap and old for his years," said
Withycombe. "I should say he's not for a wife."
"How d'you know?"
"I don't know. We never talk about himself, nor yet myself. He gives me
his company sometimes and we tell about pretty high matters, not people.
He's got a mind, Ben."
Melinda Honeysett joined them at this moment and entered their

conversation.
"He's got a mind, no doubt," said Mr. Bamsey. "But he's also got a body,
and it would be unnatural in a young, hearty man of his years, prosperous
too I suppose, because, with his sense, he's sure to have put by a bit—it
would be unnatural, I say, if he'd never turned his thoughts to a home of his
own."
Enoch spoke to his daughter.
"Here's Ben trying to hatch up a match for Dinah," he said.
"No, no—too wise, I'm sure," answered Melinda.
"Far too wise," declared the visitor. "But if such a thing was possible in
fulness of time, it would cut a good many knots, Melindy."
"Dinah likes freedom now she's got it, I believe, and I wouldn't say she
was too fond of children neither," answered Enoch's daughter.
"They never like children, till they meet the man they can see as father
of their own children," answered Ben; "and if Dinah ever said she didn't
want 'em, that's only another proof that she never loved poor John."
"The love of childer be knit up with other things no doubt," admitted

Enoch.
"There's lots love childer as never had none, like myself," answered
Melinda.
"True, my dear. There's lots love game as never shot it. But the snipe
you brought down yourself be always the one that tastes best. A mother
may love her own children, or she may not; but it depends often enough on
the husband."
It was Mrs. Honeysett's father who spoke.
"There's some child-lovers who only wed because that's the way to get
'em," he continued. "Such women don't think no more of the husband than
the doctor—I've known such. But perfect love of childer did ought to begin
with the perfect love of the man that got 'em. Take me. I had but three, but
they were the apple of my wife's eye, because they was mine as well as
hers."
"My brother, Robert, be coming home presently," said Melinda. "My

sailor brother, Mr. Bamsey. How would you like him for Dinah? I'm sure
she'd make a proper wife for him. He's like Jerry, only quicker in the
uptake. But not so clever as father."
"Wouldn't like Dinah to marry a sailor man," confessed Ben. "I know
Robert is a fine chap; but they've got a wife in every port. A sailor sees a lot
more than the wonders of the deep."
Mr. Withycombe laughed.

"He ain't that sort, I promise you," he said.
"The point is, in strictest confidence, Melindy," explained Ben, "that I

believe, though she scarce knows it herself, Dinah's interested in the Falcon
Farm cowman. She's seen him off and on and, in my ear alone, speaks of
the man. And your father here has nought but good to say of him."
"He's not for a wife, so Joe tells me. He was naming him a bit agone,"
answered Melinda, "and he said that the most comforting thing about him,
and Mr. Palk also, was that they were cut out for the bachelor state for
evermore. Perhaps you'd best to name that to Dinah. Though, for my part, I
should hope it would be years after her last adventure afore she ever dared
to think upon a man again."
"So it would be—so it would be in the course of nature," granted Ben.

"No doubt you're right; and yet—there it is. He seems to attract her—
against her own reason I dare say."
"When that happens, it means love," declared Enoch.
Melinda spoke like a woman. She was fond of Dinah, but had been
exceedingly sorry for Johnny.
"Queer—sure enough," she said. "If Dinah, now, was to feel drawn to a
man as hadn't any use for her, it would be fair justice in a manner of
speaking, wouldn't it, Mr. Bamsey?"
"In a manner of speaking I dare say it might, Melindy," he admitted.

"But I'll not hear Dinah tongue-handled over that no more."
"I'll sound Lawrence on his next visit," promised Enoch. "But he's very
shy where his own affairs are concerned."
"And another thing be certain," added Melinda, "Joe Stockman would

be terrible put about if he thought any such doings as that was in the wind."
Then Mr. Bamsey went his way, as doubtful as when he came.

CHAPTER XVI
SUNDAY
Jerry and his sweetheart wandered together along the lane from
Buckland on an afternoon when Jane had been visiting the Withycombes.
The sun beat down through the trees and even in the shade it was too hot to
tempt the lovers far.
"We'll climb up the Beacon a little ways and quott down in the fern,"
said Jerry, "and I'll smoke in your face and keep the flies off."
Jane, however, objected.
"I'm going to Hazel Tor I reckon, and then to Cousin Joe's for tea. We'll
meet John at Hazel Tor. Shall we tell him our secret plans, Jerry?"
"I don't care who knows it. If you feel there's no more need to hide up
what we've ordained to do, then tell everybody."
"There's every need to hide up for that matter. Only Johnny's different
from others. Me and Johnny are pretty close pals and always were. He won't
mind us having a shop in a town. I've often told him I was set on the
thought of a shop."
"The doubt will lie with me," said Jerry. "I know very well my father
and Melindy and everybody will say I ban't the sort of man to shine at a
shop."
"There's shops and shops," answered Jane, "and it's a very difficult
question indeed to decide what to sell. If we sold some things you'd be a lot
more useful than if we sold others. But there's a lot of things I wouldn't care
about selling."
They had already debated this matter many times, and never failed to
find it attractive.
"There's certain goods ruled out, I know," he said. "You don't hold with
butching, nor yet a fish shop."
"Nothing like that. I won't handle dead things," declared Jane. "It lies in
my mind between three shops now. I've brought 'em down to three. There's
a shop for children's toys, which I'd very much like, because new toys be
clean and bright and interesting to me; but you wouldn't be much use in
that."
"I should not," admitted Jerry.
"Then," continued Jane, "there's a green-grocer's; and there's a great

deal to be said for that, because we should have father behind us in a
manner of speaking, and he'd let us have tons of fruit and vegetables at a
very small price, or no price at all I dare say."
"Only if he comes round, Jenny. You grant yourself he'll little like to
hear you be going to spend your capital on a shop."
"He'll come round when he sees I'm in earnest. And it might help him to
come round if we took a green-grocer's. But I'm not saying I'd specially like
a green-grocer's myself, because I shouldn't. 'Tis always a smelly place, and
I hate smells."
"All shops have their smells," answered Jerry. "Even a linen draper to
my nose have a smell, though I couldn't describe it in words."
"They have," admitted she. "And the smell I'd like best to live in be
tobacco. If I'd only got myself to think for, it would be a tobacco shop,
because there you get all your stuff advanced on the cheap, I believe, and if
you once have a good rally to the shop, they bring their friends. And it's
quite as much a man's job as a woman's."
"My head spins when I think of it, however," confessed Jerry. "The only
shop I see myself in is the green-grocer's, and only there for the cabbages
and potatoes and such like. The higher goods, such as grapes and fine fruits,
would be your care."
Jane shook her head.
"Half the battle is to feel a call to a thing," she said, "same as I felt a call
to you. And as I'll be shopwoman most of the time, it's more important as I
shall be suited than you."
"Certainly."
"There's more money moving among men than women: you must
remember that too," continued Jane. "Men have bigger views and don't
haggle over halfpennies like women. A green-grocer's be a terrible shop for
haggling; but with tobacco and pipes and cigars, the price is marked once
for all, and only men buy 'em, and the clever shop women often just turn the
scales and sell the goods. I've watched these things when I've been in
Ashburton along with father, or Johnny; and I've seen how a pleasant, nice-
spoken woman behind the counter, especially if she's good-looking, have a
great power. Then, again, the bettermost sort of men go into a tobacconist;
but never into a green-grocer. Buying vegetables be woman's work."
"I can see you incline your heart to tobacco," said Jerry. "And so, no
doubt, it will be tobacco; but I must work, and if there's no work for me in
our shop, then I'll have to find it outside our shop. Lots of women keep a
shop and their men do something else."
"Why not?"
"Us may say the lot's pretty well cast for tobacco then. Shall us tell
Johnny to-day and get his opinion?"
"I'll see what sort of frame he's in," replied Jane. "He's been dark lately,
because he's getting slowly and surely to know that Dinah Waycott ain't
going back on her word. It makes me dance with rage sometimes to think
that John can want her still, and would forgive the woman to-morrow if she
offered to take up with him again."
"Love's like that, I dare say," guessed Jerry. "It'll sink to pretty well
anything."
"Well, I hate to see it—a fine man like my brother. He comes and goes,
and they've made it up and are going to be friends; at least, father thinks so
—as if anything could ever make up a job like that. If I was a man, and a
woman jilted me, I know when I'd make it up. I'd hate her to my dying day,
and through eternity too."
"You oughtn't to say things like that, Jane."
"It ain't over yet," she continued. "I shouldn't wonder much if there was
an upstore before long. Dinah can't keep secrets and she's shameless.
There's another in her eye as I have told you—talk of the devil!"
They were abreast of Falcon Farm and a man descended from it by a

path to the main road as Jane spoke. Maynard was on his way to Buckland.
He met them and gave them "good day" pleasantly enough. Jerry responded
and praised the weather, but his sweetheart did not speak.
"Your brother be coming up to tea at the farm," said Lawrence.
"I know that," was all Jane answered, and he went his way without more
words.
"There!" she exclaimed, when he was out of earshot.
"Why did you say 'talk of the devil'?" asked Jerry. "Surely nobody have
a quarrel with that chap? My father says he's a very proper sort and a lot
cleverer than you might think."
"I dare say he is a lot cleverer than some people," answered Jane; "but
he ain't a lot cleverer than me. He's a tricky beast, that's what he is, and us'll
know it presently."
Jerry was much astonished.
"I never! You're the first person as I've heard tell against him. Joe
Stockman thinks the world of him. What have he done to you? If you have
got any fair thing against the man, I'll damn soon be upsides with him."
"I'll tell you this," she replied. "I believe Dinah's hanging on at home
and letting father have his way, not because she cares two straws for father
really. She's a heartless thing under all her pretence. But she's on that man's
track, and she's too big a fool to hide it from me. And him that would look
at her, after what she done to Johnny, must be a beast. And I hope you see
that if you're not blind."
Jerry scratched his head and stared at her.
"I'm sure I trust you be wrong, Jenny. That would be a very ill-
convenient thing to happen, because Farmer Stockman would be thrown
very bad."
"What does he matter? Can't you see the insult to John? And can't you
see that, if they be after each other on the quiet, it must have been Maynard
that kindiddled Dinah away from John in the first place? What I believe is
that he came between Dinah and John, and got round her, and made her
give John up."
"For God's sake don't say such things," begged Jerry. "Don't you rush in
like that, or you'll very likely wish you hadn't. 'Tis too fearful, and you can't
tell what far-reaching trouble you might make if you was to tell John such a
thing. Him being what he is, you might land him—Lord knows where!"
She considered this.
"You're right so far I suppose; all the same I've had it on the tip of my
tongue to whisper this to John and bid him watch them."
"Don't then—for the Lord's love, don't," implored Withycombe. "It

would be playing with fire. If she's given over John once for all, then let
him think no more about the woman. 'Tis no good spying, nor nothing like
that. It ain't your business; and for that matter, it might be the best thing to
happen for somebody to get hold of Dinah, and marry her, and take her far
ways off. John have got to come to it, and when he found she loved

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Plant Genetics and Molecular Biology

Population Biology of Plant Pathogens Genetics Ecology

Molecular Approaches in Plant Biology and Environmental

Plant Genomic and Cytogenetic Databases Methods in

Plant Virus Interactions Molecular Biology Intra and

Human Molecular Genetics 5th Edition Strachan

Reverse Genetics of RNA Viruses Methods and Protocols

Plant Proteomics Methods and Protocols Methods in

Analytical Methods for the Assessment of Maillard

Rajeev K. Varshney · Manish K. Pandey

Volumes are organized topically and provide a comprehensive discussion of devel-

In references, Advances in Biochemical Engineering/Biotechnology is abbreviated

More information about this series at http://www.springer.com/series/10

Plant Genetics and

ISSN 0724-6145 ISSN 1616-8542 (electronic)

Library of Congress Control Number: 2018948681

© Springer International Publishing AG, part of Springer Nature 2018

Hyderabad, India Rajeev K. Varshney

Plant Genetics and Molecular Biology: An Introduction . . . . . . . . . . . . 1

Current Status and Future Prospects of Next-Generation Data

V. AnilKumar International Crops Research Institute for the Semi-Arid Tropics

Naghmeh Nejat RMIT University, Melbourne, VIC, Australia

Chandra Bhan Yadav National Institute of Plant Genome Research (NIPGR),

Harsha Gowda Institute of Bioinformatics (IoB), Bangalore, India

Plant Genetics and Molecular Biology: An

Rajeev K. Varshney, Manish K. Pandey, and Annapurna Chitikineni

R. K. Varshney (*), M. K. Pandey, and A. Chitikineni

Keywords Decision support tools, Epigenomics, Genomics, Metabolomics,

2 High-Throughput Genotyping Platforms

The tremendous advances in sequencing technologies have made it possible to

molecular breeding, genetic engineering (transgenics), genome editing, and any

3 Trait Dissection and Gene Discovery

In contrast to linkage mapping, the second trait mapping approach, genome-wide

Genome sequencing greatly helped in understanding of genome organization and

journey of a gene to a particular phenotype is very complicated, depending on as and

5 Data Management and Analytical Decision Supporting

Ensuring food and nutritional security for an ever-increasing global population

food through developing improved agricultural technologies. All the scientiﬁc

Advances in Sequencing and Resequencing

Pradeep R. Marri, Liang Ye, Yi Jia, Ke Jiang, and Steven D. Rounsley

Abstract DNA sequencing technologies have changed the face of biological

Jan Apr Jun Oct

P. R. Marri, L. Ye, Y. Jia, and K. Jiang

Keywords Assembly, Crops, NGS, Sequencing

ABYSS Assembly by Short Sequences

MTP Minimum Tiling Path

2 Current Technologies, Standards, and Strategies

2.1 Sequencing Technologies

2.1.1 Sanger Sequencing

2.1.2 Next-Generation Sequencing (NGS) Technologies

2.2 Assembly Technologies

The developments in automated, higher throughput sequencing technologies have

Table 1 Illumina sequencing systems

2.3 Reference Genome Project Strategies

2.3.1 Sanger-only Assemblies

The face belonged to the forefathers of the neolithic people: it

"Tempted your little sister to turn heathen!" exclaimed Dinah.

"You was a crafty little boy."

He broke off and the listener expressed sorrow.

Maynard ceased and lighted his pipe.

"A very sad thought," she said.

"It is to me," said Dinah.

"Don't you fling over God," she said very earnestly.