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2019

Validation of Candidate Loci for Maize Regrowability and Selection of Near Isogenic Lines for the Loci

Tajbir Raihan South Dakota State University

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Recommended Citation Raihan, Tajbir, "Validation of Candidate Loci for Maize Regrowability and Selection of Near Isogenic Lines for the Loci" (2019). Electronic Theses and Dissertations. 3521. https://openprairie.sdstate.edu/etd/3521

This Thesis - Open Access is brought to you for free and open access by Open PRAIRIE: Open Public Research Access Institutional Repository and Information Exchange. It has been accepted for inclusion in Electronic Theses and Dissertations by an authorized administrator of Open PRAIRIE: Open Public Research Access Institutional Repository and Information Exchange. For more information, please contact [email protected]. VALIDATION OF CANDIDATE LOCI FOR MAIZE REGROWABILITY AND

SELECTION OF NEAR ISOGENIC LINES FOR THE LOCI

BY

TAJBIR RAIHAN

A thesis submitted in partial fulfillment of the requirements for the

Master of Science

Major in Biological Sciences

Specialization in Biology

South Dakota State University

2019

iii

ACKNOWLEDGEMENTS

In the beginning, I would like to express my sincere thanks and gratitude to my advisor Dr.

Yang Yen for his supervision, support, and care for the last three years. I also would thank my other committee members: Dr. Yajun Wu, Dr. Donald Auger, and Dr. Padmanaban

Krishnan for their critical evaluation and suggestion in case of designing and execution of my experiments, data analysis, and manuscript preparation.

I am also thankful to my current lab mate Bimal Paudel and Ex lab mates Subha Dahal,

Anjun Ma for their help with the lab work and helpful discussions. Most importantly, I would like to show my gratitude to my beloved parents, especially to my mother; even after her death, the memory of her love and care encourages me to get over my distress and keep me on the road of success.

iv

TABLE OF CONTENTS

LIST OF TABLES ...... v LIST OF FIGURES ...... vi ABBREVIATIONS ...... vii ABSTRACT ...... viii 1 CHAPTER 1: LITERATURE REVIEW ...... 1 1.1 Introduction ...... 1 1.2 Perennialism in woody perennials...... 3 1.3 Types of perennating structures in herbs and grasses...... 6 1.4 Progress in overall perennialism research in various crops ...... 15 1.5 Summary and concluding remarks: ...... 24 References ...... 26 2 CHAPTER 2: VALIDATION OF CANDIDATE LOCI FOR REGROWABILITY AND SELECTION OF THEIR NEAR ISOGENIC LINES IN MAIZE...... 37 2.1 Introduction ...... 37 2.2 Materials and methods ...... 39 2.2.1 Formation of the population for marker study ...... 39 2.2.2 DNA extraction ...... 39 2.2.3 PCR assay ...... 39 2.3 Results and discussions ...... 40 2.3.1 The study of gt1, id1, and tb1 for their contribution to regrowability ...... 40

2.3.2. Validation of the candidate SNPs by genotyping the F2 population ...... 42 2.3.3 Genes found in peak locations ...... 43 2.3.4 Selection of the Near Isogenic lines for reg1 and reg2 ...... 44 2.4 Prospects and future direction of maize perennialism research ...... 45 References ...... 47

v

LIST OF TABLES

Table 1. List of primers used in this study ...... 49

Table 2. The phenotype for regrowability and marker genotypes ...... 51

Table 3. Detection of polymorphic marker between Z. diploperennis and B73 parent .... 56

Table 4. Result of chi square test of independent segregation ...... 57

Table 5. Genes located around peak position ...... 58

Table 6. The results of F3 screening using allele-specific markers for reg1 and reg2 ..... 59

Table 7. The results of F4 screening using allele-specific markers for reg1 and reg2 ..... 60

vi

LIST OF FIGURES

Figure 1. Satellite image of the Gulf of Mexico ...... 61 Figure 2. Perennial Wild Maize ...... 62

Figure 3. Z. diploperennis, Mo17- Z F1 and B73- Z F1 ...... 63

Figure 4. Loci in maize inbred B73 genome that are related to regrowth ...... 64 Figure 5. Process of designing allele specific primers ...... 65

Figure 6. Results of genotyping the B73-Zd F2s using S2 primers...... 66

Figure 7. Results of genotyping the B73-Zd F2s using S7 primers ...... 68 Figure 9. The polypeptide sequence of “Chromatin remodeling protein 24” gene ...... 70 Figure 9. The coding sequence of “Chromatin remodeling protein 24” gene ...... 71 Figure 10. The polypeptide sequence of “Flocculation protein FLO11” gene ...... 73 Figure 11. The coding sequence of “Flocculation protein FLO11” gene ...... 74 Figure 12. F4 NIL screening using both chromosome 2 and 7 primers ...... 76

vii

ABBREVIATIONS

AS-PCR Allele-specific polymerase chain reaction

bp Base pair

CO CONSTANS

CTAB Cetyl trimethylammonium bromide

DNA Deoxyribonucleic acid

FT FLOWERING LOCUS T

GBS Genotype-by-sequencing

gt1 grassy tiller 1

IAA Indole acetic acid

id1 Indeterminate 1

LOD Logarithm of odds

ng Nanogram

NIL Near-isogenic line

PCR Polymerase chain reaction

pe Perennialism

QTL Quantitative trait loci

SNP Single-nucleotide-polymorphism

SOC1 SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1

SSR Simple-sequencing-repeat tb1 teosinte branched 1

TNT Tiller number at tasseling stage

viii

ABSTRACT

VALIDATION OF CANDIDATE LOCI FOR MAIZE REGROWABILITY AND

SELECTION OF NEAR ISOGENIC LINES FOR THE LOCI

TAJBIR RAIHAN

2019

Developing perennial grain crops is an effective way for sustainable agriculture and it can be a tool for the betterment of the overall ecosystem. To understand how perenniality works in perennial species, we made a cross between Zea diploperennis and maize (Z. mays) inbred line B73. F1 hybrids produced from this cross were selfed to get the F2 population and later the F2 population underwent Genotyping-By-

Sequencing (GBS) analysis. Based on the candidate loci identified on chromosome 2 and chromosome 7 from GBS, SNP-specific markers were developed from the

2 candidate locus interval and used to genotype the F2 population.  test for independence showed that the tested SNPs are associated with regrowth. Additionally, gene-specific markers (gt1, tb1, id1) were also used to genotype the F2 population and the 2 test result indicates that none of these genes is significantly associated with regrowth. This result is inconsistent with the previous works conducted on these genes.

Later the SNP-specific markers were used to select near-isogenic lines for the two regrowth loci.

Key words: maize, perennialism, GBS, SNP, chi-square test, near isogenic lines. 1

1 CHAPTER 1: LITERATURE REVIEW

1.1 Introduction

In a wider sense, perennialism refers to the phenomenon of a plant to live for more than two years (Ma et al. 2019). However, in the narrow sense, perennialism is the ability of a plant to regrow on the same body after reproduction and senescence (Ma et al. 2019). Most of the domesticated crops are annuals that reproduce, senesce and die during the same growing season, and they only have one cycle of growth and reproduction. Perennial crops are capable to overwinter or over-summer (depending on the locations) and stay alive for several consecutive years. The key to this long life is their ability to maintain some juvenile meristematic tissues even after their senescence which allow them to regrow when the condition becomes favorable again (Ma et al. 2019)

It was proposed by Wagner (1990) that the perfect utilization of perennial crop system can reduce the pressure on soil and help to protect the agriculture. There are several benefits associated with growing perennial crops such as the reduction in tillage system and lower energy input, stronger resistance to diseases and comparatively longer growing season

(Cox et al. 2002). Perennial crops can be a tool for protection against soil erosion (Figure

1). The deeper root system of perennial plants gives more access to the underground water, mineral resources and also allows them to increase in soil carbon sequestration (Cox, et al.

2006). Perennial crops provide vegetative cover on lands over a longer periods of time and amplify photosynthetic assimilation with the reduction of planting costs. Some perennial crops are already established and developed as food, fruits, horticulture, forage and hay worldwide. Besides, perennial crops can produce more biomass which is also a raw 2 material for biofuel production (Jungers et al. 2013).

All the annual plants reproduce and spread only via seeds, whereas perennials reproduce and spread through seeds or vegetative organs like rhizomes. Sometimes, they use both seeds and vegetative organs. Some perennials do not even have seeds and relying only on the vegetative organs. That is why vegetative organs like rhizomes, tuber, and bulbs are often indicated as the key for their perennialism in many research (Cox et al. 2016,

Friedman et al. 2015). For instance, Cox et al. (2006) mentioned that rhizomes allow perennial plants to over-winter and help them grow at the starting of the next growing season. However, having these vegetative organs are not an absolute requirement as many of the perennials show regrowth without even these organs (Ma et al. 2019).

Alfalfa, fennel, garlic are some examples of herbaceous perennials. Each year these herbaceous perennials die back to the ground, however as their roots remain alive, they can reappear when the condition becomes suitable (Dehaam et al. 2005). Woody perennials differ hugely from herbaceous perennials in terms of perennialism. The stems of woody perennials do not die back; they just cease to grow at the end of a growing season leaving the dead tissues inwards as the supporting structure of the body. Woody perennials like maple, pine, apple lose their leaves with their spectacular fall foliage colors. Later in Spring the same stem and branches which held the foliage last season start to grow again with fresh newborn leaves (Wahid et al. 2007).

Most of the perennial plants are polycarpic as they flower and set seeds many times throughout their life cycles (Friedman et al. 2015). However, there are several exceptions of it and one of these exceptions is bamboo. Bamboo is a monocarpic perennial which flowers, produces fruits and seeds only once in their lifetime and then dies afterward 3

(Tian 1987).

1.2 Perennialism in woody perennials.

Through the course of woody plants’ growth and development, meristematic tissue cycles between active and dormant states. The dormant state is the temporary cessation of noticeable growth (Lang, 1987). Plants have a synchronized growth and dormancy cycle triggered by specific environmental conditions. It is important for the survival of woody perennials to stop the activity of meristem and create a dormant state that the meristem becomes insensitive to growth-promoting signals. This scenario can be observed in perennial trees during autumn. Later, to release dormancy and resume growth, plants need specific environmental signals as well (Rhode and Bhalerao 2007). The conversion from an actively growing state to dormancy is a step-by-step procedure (Champagnat, 1983), and one of the first visible indicators is the cessation of meristematic cell division.

Gradually, dormancy establishes right after the growth cessation and then finally causes the total inhibition of the response of meristem. There is not enough information about what happens after growth cessation and before the establishment of dormancy, because partly this phase is masked by simultaneous bud formation. Buds are not required for dormancy but are required for the survival of the plant (Rhode and Bhalerao 2007). Forest trees exhibit perennial growth behavior characterized by their long juvenile phase which can last for many years before the appearance of the first flower (Böhlenius et al. 2006).

Tree populations belong to higher latitude ceased growth at longer day time, while tree population from lower latitude requires a shorter day for their growth cessation (Pauley et 4 al. 1954). The induction of dormancy is an important adaption for plants, as without it plants may end up dying because of the frost damage. (Böhlenius et al. 2006).

The regulation of annual growth and dormancy of vascular cambium in woody perennial species is not well understood. Short photoperiod and cold temperature cause the initiation of dormancy development, termination of cambial activity and frost hardiness

(Mellerowicz et al. 1992). At first, short photoperiod results in the initiation of dormancy and then chilling temperature (0 to 10°C) imposes the dormancy. The stimulation and inhibition of cambial activity are controlled by various concentrations of indole-3-acetic acid (IAA); however, this transition from active growth to dormancy is not IAA controlled.

(Little and Savidge 1987; Sundberg et al. 1991). Perhaps the sensitivity of cambial cells toward IAA may play the role in this transition (Little and Wareing 1981)

Physiologically, dormancy can be divided into two different phases; ecodormancy and endodormancy. They are different in their response toward growth‐promotive signals by their arrested cell division machinery (Champagnat, 1983; Lang, 1987). Actively growing trees transform toward ecodormancy as an adaptive response toward short day time and low temperature. If the trees are moved into an environment favorable for growth during the stage of ecodormancy, they are capable to reverse the dormancy process. However, it becomes irreversible once it is transitioned to endodormancy. Plants remain endodormant until they accumulate several "chilling units". "Chilling unit" means the exposure of the plant to a very low temperature for a while. After this period, trees become ecodormant again and later it moves naturally to an actively growing stage with the increase of temperature (Espinosa‐Ruiz et al. 2004). 5

FLOWERING LOCUS T (FT) and CONSTANS (CO) act as mediators of growth cessation by short day signals (Böhlenius et al. 2006). The overexpression of FT and CO homologs of poplar (Populus trichocarpa) in transgenic aspen (Populus tremula x P. tremuloides) causes the growth to continue even in the exposure of short daylight (Böhlenius et al. 2006).

The downregulation of FT through interference RNA (RNAi) causes the growth cessation irrespective of the day length. Various cytological studies on cambium confirmed that the transition of growth to dormancy is associated with the increase in the size of the nuclear genome, cytoplasmic RNA, protein and carbohydrate staining intensity. On the contrary, reactivation of cambium from dormancy is related with the decrease in the size of the nuclear genome, cytoplasmic RNA, protein and carbohydrate staining intensity (Riding and Little 1984; Mellerowicz et al. 1989).

The insulation of meristematic cells from growth-promoting signals (gibberellins) is a way to maintain dormancy. After the transition to endodormancy, neighboring meristematic cells become symplasmically isolated through the disconnection of plasmodesmatal circuitry (Rinne et al. 2001). Sometimes, callose plugs the plasmodesmatal connection and prevent the transportation of gibberellin in this way. However, the transportation of auxin differs from gibberellin and it requires a special carrier. During dormancy, the level of auxin does not change in cambial cells but auxin responsiveness changes (Little and Bonga

1974).

It requires the chilling temperature to release the dormancy. Pierre Chouard (1960) critically tested and noted that there are similarities between dormancy release and vernalization. Both vernalization and dormancy release require long-term exposure to the low temperature. This chilling exposure only restores growing ability but cannot promote 6 overall growth and development. Some differences come along with the similarities as well; such as vernalization takes place in actively dividing cells only (Wellensiek 1962), whereas dormancy release happens after the termination of cell division. Although very few reports suggested a very slow growth rate during dormancy but there is no cell division at all (Samish 1954; Chouard 1960).

1.3 Types of perennating structures in herbs and grasses.

Perennial plants need to preserve their roots for having a long life. The importance of root is not only limited to nutrient and water uptake but also shoot reproduction, hormone production, and many others. From germination to senescence, roots bear the central core of plant growth and development in the form of the root meristem. Root apical meristem grows into the soil vertically, whereas lateral meristems create new roots from the pericycle and give access to nutrients and water laterally (Richter et al., 2009).

Beside roots, there is another structure called rhizome that grows underground and is a key to the survival of some perennial herb and grass plants. Rhizomes are one kind of modified subterranean stems that grow underground in a horizontal direction. Rhizomes can asexually propagate and enable overwinter/over-summer, and rapid growth in the following season. Therefore, it is no surprise that many of the perennial herb and grass species are rhizomatous (Navarro et al. 2011, Lee et al. 2013). Plants also use rhizomes as a long-term storage organ for storing proteins, starches and various other nutrients which become beneficial during the new shoot formation or for surviving the winter (Jang et al.

2006). 7

In short, rhizomes can tolerate unfavorable conditions and arise when the condition becomes favorable for growth again. Rhizomes grow from their buds either underground as a new rhizome or aboveground as an aerial shoot (Navarro et al. 2011). Although both aerial shoot and rhizome are entirely different from each other and each has a unique developmental pattern, however interestingly both are formed from auxiliary buds

(Yoshida et al., 2016). Rhizomes more likely form from down-ward axillary buds (Brock et al. 1997).

Auxiliary buds also give rise of tillers in grasses; A tiller is a structure that comprises the main stem with solid nodes and often it carries hollow internodes. Tillers remain in the vegetative state unless they get a flowering signal. Depending on the species, sometimes plants may carry only reproductive tiller, or they may carry both vegetative and reproductive tiller. Besides main shoots, sometimes they can appear from other tillers during vegetative growth and still can grow independent of each other using their adventitious roots. Tillering can be divided into two stages; the formation of an auxiliary bud from the leaf axil and their growth afterward (Bryant et al. 2015). It was reported by

Yoshida (1973) that a higher temperature is correlated with the tiller number increase.

Additionally, tiller number and size are also influenced by inter-plant competition and environmental conditions, which follows a pattern of quantitative inheritance (Krishnan et al., 2011).

Flowering and development of seeds are the sign of tiller senescence in grasses. In C4 perennials, a similar sign can also cause the induction of rhizome dormancy. In various temperate regions of the world, the first killing frost of the winter causes the death of all the above-ground tissues of the plant. Rhizomes, crowns or tiller buds of the plant remain 8 dormant until spring comes. All the requirement of photosynthate is achieved through shoots for the growth below ground. The nutrient reserves that are stored in rhizomes and/or crowns, are also responsible for the growth in spring. Appropriate allocation of the reserve in both crown and rhizome area is very important for the maintenance of cellular integrity during dormancy, enforcing the elongation of preexisting tiller after growth initiation, and development of new meristem tissues, roots, rhizomes, etc. Various C4, perennial, warm-season grasses like switchgrass, miscanthus or prairie cordgrass carry both above ground tiller and below-ground tissues like rhizomes and roots (Poirier et al.

2012, Ebrahimiyan et al. 2013).

Stolon is another rhizome-like structure; but instead of growing underground horizontally, they form a horizontal branch from the base of the existing stem on the ground. Stolons create a horizontal connection between plants. Stolon and rhizome share substantial anatomical similarities. The development of rhizome is the elongation of internodes to create shoots underneath the surface of the soil horizontally. On the other hand, stolons form from the elongation of basal internode as new tillers next to the parent plant, without previous horizontal growth. For the significant anatomical similarity between rhizome and stolon, both of them are called true stems (Mathew et al. 2000).

Tubers are also modified roots like rhizomes and a mean for asexual reproduction. Tubers act as a subterranean storehouse. They supply nutrients and energy for regrowth in the next growing season. Tubers can use their buds as a mean to produce new shoots (Munné-Bosch

2014). There are two types of tubers: root tuber, and stem tuber. Root tuber is a modified lateral root while the stem tuber is a thickened rhizome or stolon. The enlarged area of the root tuber can be in the end or in the middle or throughout the whole root. There are no 9 reduced leaves, nodes or internodes on root tubers. The proximal end of the root tuber is attached to the old plant which carries the crown tissue. Crown tissue is responsible for the formation of buds and later these buds grow into foliage and new stems. The distal end of the root tuber forms unmodified roots. However, in the case of stem tubers, it is opposite as stems are produced by distal ends (Kyte et al. 1996). The upper portion of the stem tuber form shoots that grow into a normal stem and leaves while the lower portion of the stem produces roots. Stem tuber forms near the surface of the soil, closer to their parent plants.

Despite having different origins, root and stem tubers are almost similar in appearance and function.

Additionally, there are some other perennating structures that can be found in some perennials with a similar function of nutrient storage and vegetative reproduction known as corms and true bulbs. The corm is vertical, small and fleshy underground stem carrying buds and scaly leaves. They also carry one type of protective leaves modified into a fibrous covering called tunic (Dyer 1975). A normal corm is mostly made of parenchyma cells and is very rich in starch. The corm produces two types of roots. The type of roots that are produced from the bottom of the corm is called fibrous roots. Contractile roots are thicker and layered roots that grow with the new corm and pull the corm deep underneath the soil

(Manning 2008). They also produce offshoots called cormels responsible for their vegetative reproduction (Manning 2008). True bulbs are also short stems (Bell 1997) and they comprise a base, shoot, lateral bud, storage organ known as fleshy scales and skin- like protective organ called tunic. At the center of the bulb, there is a vegetative growing point known as base and this base is formed by a reduced stem. This basal plate is responsible for the growth of the plant. Leaves and stems emerge from the upper side of 10 this base while root emerges from the lower side of the base. (Mishra 2005). True bulbs can be divided into tunicate bulbs and imbricate bulbs (Mishra 2005). There are secondary bulbs that are formed between a stem and a leaf or sometimes in the place of flowers known as bulbils or bulblet (Bell 1997). These small secondary bulbs can become large bulbs and, occasionally may replace original bulbs (renewal bulbs) (Bell 1997). As they share considerable external similarities, sometimes corms are confused with true bulbs.

However, unlike bulbs, they are structured internally by solid tissues. Bulbs consist of layered fleshy scales that forms from modified leaves (DeMunk 1993).

Perennating structures in different perennials.

Perennating structures in perennial plants vary depending on the species. Grain sorghum can generate tillers like other grasses that sprout from the plant base near the soil surface.

In a tropical climate, a new sorghum plant can regrow from basal nodes of the mother plant and produce a rattoon (Paterson et al., 1995). Perennial sorghum produces rhizomes along with tillers. Rhizomes are the most important structure for perennialism in sorghum and each new growing season starts for sorghum when the rhizome buds sprout by the influence of specific temperature, moisture, or other types of environmental stimuli. The reserve energy in rhizomes helps the formation of new shoots named ramet. Ramets emerge and grow while the rhizome degrades and dies gradually. Plants that emerge from rhizomes create vigorous new root systems and grow quicker and bigger comparing the tillers developing from the aboveground nodes (Paterson et al., 1995). 11

Another rhizomatous perennial is bamboo which is a unique group of arborescent grasses and one of the largest members of the grass family Poaceae. There are almost 1500 species of bamboo that can be found in various places all over the globe (Wang et al. 2010). They are herbaceous or sometimes woody and they are not capable of secondary thickening.

Most importantly, bamboos are rhizomatous (Calderon et al. 1980). Interestingly, most of the monocarpic plants exhibit an annual or biennial lifestyle, however, bamboos are long- lived perennial and show monocarpic characteristics. Bamboos are also called semelparous, because of its unusually long clonal growth phase which is followed by a mass synchronous flowering and the end of their lifecycle (Wang et al. 2016).

Most of the bamboos cultured worldwide are woody evergreen perennials. They use rhizomes for reproduction. The overall developmental process of the bamboo rhizomes is complex and hugely different from other grasses. According to the rhizomes they have, bamboos are divided into three different groups. The sympodial rhizome can be found in caespitose bamboos whereas monopodial rhizome can be found in scattered bamboos and both monopodial and sympodial rhizome can be found in pluricaespitose bamboos. In bamboos, a rhizome bud shows two types of developmental strategy. Firstly, it can develop itself into a bamboo shoot which will grow ultimately into a bamboo culm. Secondly, it can develop into a new rhizome helping sustainable bamboo grove production (Wang et al. 2016).

The perennial relatives of rice possess rhizomes and, occasionally stolons as well. Oryza sativa is one of the biggest food-producing crops exceeding millions of hectors every year in production in the southeast Asiatic region (IRRI). Unfortunately, the crop itself is 12 annual, and it lacks rhizome. On the other hand, O. longistaminata is its perennial relative carrying rhizomes over 8 cm long and they spread vegetatively. The main perennating structure of O. rufipogon is stolon. However, stolons are not perfectly compatible to survive drought due to the exposure of sun and dry air. On the contrary, rhizomes of O. longistaminata are protected from drought for having soil insulation (Sacks 2014). Despite having rhizome and stolon, Oryza rufipogon and O. longistaminata can also reproduce sexually through flowers and seeds. Additionally, O. officinalis, O. australiensis, and O. rhizomatis spread vegetatively through rhizomes as well (Khush, 1997).

Intermediate wheatgrass (Thinopyrum intermedium) is a perennial relative of annual wheat.

It is a sod-forming perennial grass carrying various structures like tillers and rhizomes. It can produce seeds with a yield of around 690 kg/ha (Wagoner 1990). Intermediate wheatgrass uses tillers, rhizomes, and seeds for their propagation (Wilkins and Humphreys

2003).

Helianthus tuberosus is a perennial relative of common annual sunflower Helianthus annuus; They have tuber which resembles vaguely with ginger root. Most of the time the structure of their root is elongated and little uneven (Huxley et al. 1992).

The regrowability in perennial teosinte might not only come from rhizomes but it can also come from the formation of juvenile basal auxiliary buds, robust root system, evergreen stalk, basal shoot development, and bulbils (Ma et al. 2019).

Tubers are a signature perennial organ of geophytes and they always remain underground during their entire lifespan and seasonally create above-ground shoots (Garcia et al., 2011).

Yams are perennial vines cultivated in the temperate and tropical region of the world for 13 their starchy tubers as food. White yams (Dioscorea rotundata Poir) produce both tubers and bulbils, which also has economic value as a food (Omonigho and Ikenebomeh, 2000).

Potatoes and yams are examples of tubers, while sweet potatoes and cassava are examples of storage roots. Sweet potato tubers are the massive enlargement of the secondary roots although they possess the same cellular structure as the normal root. Unlike potato tubers which are derived from an aerial shoot, some plants can produce true root tubers.

Smallanthus spp., Mirabilis spp., and Borderea spp. are some examples of the species that produce true root tubers (Garcia et al., 2015).

From the family Alliaceae, garlic is a perennial but grown as an annual crop mostly found in cold regions. It has been used for many culinary and medicinal purposes throughout the world over the centuries. On the above-ground, garlic plants have the vegetative part carrying leaves and flowers, while in the underground they have bulbs. They have a fibrous rooting system and their bulbs carry tiny bulbils known as cloves. Garlic is sterile completely and they use asexual reproduction for propagation. They do not produce flowers but a scape. Scape cannot produce seeds and known as a fake flower. However, scape produces bulbils on top, which aid in vegetative reproduction (Shemesh, 2008;

Kamenetsky, 2005, 2007a, b). Like potatoes, garlic also reproduces through vegetative reproduction and their cloves are responsible for this vegetative propagation. Garlic cloves can be planted individually and later these cloves will produce a bulb where all the cloves have the same genotype as the mother clove.

A perennial plant is only dead when both of its aboveground and underground meristems has died. For example, a tree will remain alive as long as one of the apical shoot meristems 14 and one of the root meristems are alive and connected through an active vascular system

(Munné-Bosch 2014). All the tissues play an altruistic role in serving the meristems throughout the plant development. The duration of the lifetime of cohorts of roots in perennial plants can be diverse; ranging from weeks (e.g. strawberries and apple) to several decades (e.g. sugar maple) (Eissenstat and Yanai, 1997). The development of the root system is unique structurally and temporally according to the species. With woody perennials, the completion of the vertical expansion of the structural root system might need many years. As an example, for Scots Pine, it takes almost 70 years (Fogel 1983).

Borderea spp. is also well-known for its unusually extreme longevity. There are two relict species. Borderea pyrenaica and Borderea chouardii, that can be found in the Central

Pyrenees in an isolated population (Garcia et al., 2011). Their roots are a tuber with spiral growth which holds the secret of their long life and they can live up to 300 years. Even under the extreme condition like 2100 meters above sea level, each season tuber forms above-ground structures like stems, flowers, and leaves and they reproduce and senescence during summer. However, tubers remain alive and continue the whole cycle repeatedly

(Morales et al., 2013). Tubers use alternative meristematic points to decrease mutation associated damage and remain dormant most of the time of the year. Tubers also have a reduced growth rate. These reasons might be the key to their long life (Morales et al., 2013).

Their fertility also increases with their age, which is a sign of negative senescence (Garcia et al., 2011; Morales et al., 2013).

For remarkably long-living perennial plants, sometimes perenniality might be achieved through asexual reproduction like clonal propagation (Koskela, 2002). Sometimes perenniality requires the allocation of a significant part of their energy toward asexual 15 reproduction (i.e. clonal propagation) and it can create whole new clonal plants (Koskela,

2002). Plant species that use clonal propagation as a mean of reproduction are known to have very huge lifespans comparing others. Sometimes the lifetime may reach almost a thousand years. One of the examples is King's Lomatia (Lomatia tasmanica), which is almost 43,600 years old (Lynch et al., 1998). The only viable reproduction method of

King’s Lomatia is clonal propagation. It is an ancient clone and only can be propagated through fallen branches. Fallen branches create roots and turn into a new plant identical to the parent plant (Lynch et al., 1998). Another interesting example is bristlecone pine (Pinus longaeva), with the highest lifespan of 5,062 years (Earle, 2013).

1.4 Progress in overall perennialism research in various crops

Glover et al. (2010) proposed three strategies to create perennial grain crops: direct domestication of perennial relatives of crop plants, transgenic modification of annual plants, and genetic introgression of perennial habit from wild relatives into domesticated crops through wide hybridization. Here in this section, we shall discuss how researchers have been trying to create various perennial grain crops by mainly using these three strategies over the years.

Rice

There is some notable progress in the development of perennial rice. The perennial ancestor of rice, Oryza rufipogon, can be crossed with Oryza sativa (Cheng et al. 2003). Initially, various weed characteristics like shattering and tilling were found in the hybrids but later it was eliminated through target gene selection (Lin, et al. 2007). 16

It is believed that propagation through rhizomes make the plant theoretically immortal. Hu and colleagues (2011) have identified that the initiation and the development of the trait for rhizomatousness in O. longistaminata are controlled through many complex gene networks. These gene networks include various plant hormones and regulatory genes. Two dominant QTLs were identified for the rhizomatousness. Simple-Sequence Repeat markers were used for the genetic analysis of the rhizome expression. A PCR-based molecular genetic map of chromosome 3 and chromosome 4 in rice indicates, there are two dominant complementary genes for rhizome expression known as Rhz2 and Rhz3, respectively (Hu et al 2001; Hu et al 2003). Rhz3 is located in chromosome 4 and also co-located with many other associated root traits including root number, length, branching number, branching density, internode length, internode number, and dry weight, etc. Similarly, Rhz2 is also associated with traits like root branching density, internode length, and tiller number. The strong connection between rhizome and root QTL is very significant, as rhizome formation results in changing of root structure (Hu et al 2003).

In the southeast Asiatic region, upland rice Oryza sativa has a huge negative impact on soil erosion for being an annual crop growing in hilly regions. The perennial alternative of

Oryza sativa can be very effective for reducing soil erosion if it can fulfill the other requirement of farmers. Here, O. longistaminata, with the similar genome to O. sativa, would be the best fit candidate for transferring genes of rhizome expression to develop a perennial rice cultivar (Majumder et al. 1997; Sacks et al. 2001). Donor traits like rhizome and stolon are crucial for resulting perenniality.

Some other progress in developing perennial rice cultivar is: 1) Rhizome gene fine- mapping through genomic library construction confirmed rhizome is controlled by two 17 pairs of dominant complementary genes (Rhz2 and Rhz3). Fifteen other rhizome loci and other candidate functional genes were identified. 2) Some breeding lines already hold the gene for rhizomatousness. 3) There are five perennial rice bred already developed; they are

PR23, PR57, PR129, PR137 and PR139 (Zhang et al. 2013). However, there are several obstacles researchers faced when they were developing cultivars from O. longistaminata:

1) the requirement to pyramid in the background of O. sativa multiple rhizome QTL for getting the powerful expression of rhizome, and 2) Getting rid of low pollen fertility QTLs without losing the QTL for rhizomatousness (Hu et al. 2003, Sacks et al. 2001).

Wheat

Annual wheat underwent hybridization with its perennial relative rhizomatous intermediate wheatgrass (Thinopyrum intermedium, 2n=42). Although the approach was successful, however, its agronomic traits and stability require further improvement (Dewey 1984; Cai, et al. 2001). During meiosis, the wheat and wheatgrass chromosomes do not pair with each other. So, it produces a sterile hybrid which often undergoes spontaneous chromosome number doubling and causing the formation of an amphiploid (Cox et al. 2006). The amphidiploid produced was polycarpic and perennial in nature. To determine the chromosomal basis of amphiploids, Lamer and associates examined life history characteristics with complete Thinopyrum elongatum (2n= 2x= 14) chromosome addition series on a background of Chinese Spring. After disomic and monosomic substitution or addition, chromosome 4E of T. elongatum conferred polycarpic characteristics to the annual Chinese Spring wheat (Lammer et al. 2004).

There are some studies reported on the direct domestication of intermediate wheatgrass

(Wagoner 1990). Without any sub-genome donor, the domestication of intermediate 18 wheatgrass is limited. However, recently a research group from the University of

Minnesota successfully identified the candidate sub-genome donor of wheatgrass using genome-wide markers. Genotyping-by-Sequencing also was used to create some SNP markers for high-density mapping of the candidate loci of agronomic traits (Zhang, et al.

2016). This high-density mapping can speed up the domestication of intermediate wheatgrass for developing a maximum yield perennial cultivar within a very limited selection time (Wilkins and Humphreys 2003; Hayes, et al. 2012, Zhang, et al. 2016).

Rye

For developing a perennial rye variety, annual rye (Secale cereal) was crossed with its perennial relative S. montanum (Cox, et al. 2002). No germplasm for grain production is released yet because of the limitations in grain yield by sterility. In addition, the perennial feature is also weak. As the genetic map of rye is already established, so the marker-assisted selection has been used also for perennial rye breeding (Cox, et al. 2002).

Sunflower

There has been a moderate progress in the development of perennial sunflower from the existing annual sunflower (Helianthus annuus, 2n=2x=34). The hybridization between the annual sunflower and its perennial relative H. tuberous (2n=6x=102) created perennial interspecific hybrids (Kantar, et al. 2014). However, as the parental lines had a different number of chromosomes, it resulted in abnormal chromosome pairing during meiosis and caused fertility reduction (Kantar et al. 2014). The F1 individuals were intermated and showed an increase in the head size around 20% compared to not intermated F1 individuals.

There was no correlation between tuber traits (required for perenniality) and seed yield traits. Additionally, the improvement in phenotype only after one generation of intermating 19 indicates that the best strategy for improvement of perennial sunflower should be a recurrent selection program focusing on yield (Kantar et al. 2014). Later, the perennial hybrids were assessed for agronomic traits during breeding selection. However, as the hybrid genome was unbalanced, further research about it has been paused after the F1 generation. Nevertheless, the intermated F1 generation was used to generate a genetic map of sunflower for future research on their perennial gene mapping and domestication.

(Kantar et al. 2014)

The backcrossing of perennial hybrids with the annual sunflower was not effective. When

F1 plants (tetraploid) underwent crossing with H. annuus plants (diploid); a weak annual triploid plant was generated. The same thing can be observed in various other species where backcrossing results in the loss of perennialism. Therefore, the goal of breeding perennial sunflowers should be focused on the domestication of interspecific hybrids or wild relatives

(Cox et al., 2010).

Maize

Researchers have been trying to investigate how perennialism works in teosinte for decades. Teosinte and maize can be crossed to create fertile progeny. Emerson and Beadle

(1929) were the first to document such a cross between maize and tetraploid perennial teosinte Zea perennis. Reeves and Mangelsdorf (1939) crossed between Z. perennis and eastern gamagrass to test their hypothesis that maize is a hybrid between teosinte and

Gamagrass.

Shaver (1967) proposed that rhizome is the key for regrowth and perenniality and it is controlled by a minimum of three recessive genes id1 (indeterminate1), gt1 (grassy tiller 20

1) and pe (perennialism). Id1 factor controls floral transition using an autonomous pathway

(Coneva, et al. 2007) and its loss-of-function results in absence of ears (Galiant and Naylor

1951). Gene gt1 encodes a class I homeodomain leucine zipper protein responsible for lateral bud dormancy and suppression of elongation of lateral ear branches (Whipple et al.

2011). Doebley et al. (1997) introgressed specific chromosomal segment containing teosinte branched 1 (tb1) maize allele into teosinte and discovered that the introgression alters the overall plant structure from the candelabra-shape to the pole-shape. As a result, they concluded, gene tb1 controls the apical dominance of the plants. Additionally, tb1 affects the branch development as well. Shaver (1967) mentioned that not only genetics but also the environment plays a vital role in shaping the perennial trait.

Iltis et al. (1979) discovered a perennial diploid teosinte in Mexico in 1979 known as Zea diploperennis. At that time, it was being used as a food locally. Later Camara-Hernandez and Mangelsdorf (1981) used Z. diploperennis to make a cross with a primitive Mexican popcorn. They hypothesized perennialism is a recessive trait because they found annual growth habit dominant to the perennial with a segregation ratio around 3:1. Galiant (1980) scored evergreen stalks and analyzed its relationship with the development of the basal shoot. He suggested that perennialism trait is at least partially controlled by two dominant complementary genes showing a 9:7 F2 segregation ratio. Ting and Yu (1982) suggested that perenniality is controlled by dominant genes. They performed an experiment using

152 F1 hybrids of maize and Z. diploperennis and found all F1 plants were regrowable.

Kelly Dawe’s group at the University of Georgia used pollens from Z. diploperennis to pollinate the maize inbred line B73. They generated a F2 population by inter-crossing F1 plants and concluded that perennialism is a recessive trait (Coatney 2011). Recently, Yang 21

Yen and his group at South Dakota State University reciprocally crossed Z. diploperennis with maize inbred lines Mo 17 and B73 and a landrace Rhee Flint and found that all the

F1s from these crosses were regrowable (Ma et al. 2019). They also concluded that regrowability (the key trait of perennialism) in Z. diploperennis is controlled by two dominant, complementary genes. Galiant (1980) explained that the factor that causes differences among various studies is the corn background. It is also responsible for the reversal of dominance for the expression of perennialism.

Westerbergh and Doebley (2004) crossed annual Zea mays ssp. Parviglumis with perennial

Z. diploperennis and formed a hybrid population and studied quantitative trait loci (QTL) for perenniality. They detected 38 QTL for eight different morphological traits such as number of tillers (NO-TL), number of side branches (NO-SB), withered stems (WT-ST), rhizomes (RHIZ), loss of tillers (LO-TL), presence of thin roots (TH-RO), presence of slender tiller (SL-TL) and elongated underground stems other than rhizome (EL-ST) from

425 F2 individuals. Their QTL showed chromosome 2 and 6 regions played an important role in the evolution of perennial habit. Although none of the QTL they identified showed a singular large effect on the regrowth.

Sorghum

One of the main focuses of perennial research was developing crop systems that are more resilient to extreme environmental conditions. Sorghum (Sorghum bicolor) is one of the most important crops worldwide and a key staple crop across most of Sub-Saharan Africa.

Sorghum has been used as a grain, forage or sugar crop. It is equally efficient in the conversion of solar energy and the use of water and often denoted as high-energy, drought- tolerant crop (Harlan 1971). It could decrease the costs, efforts, and risks of seed sowing, 22 solve the problem of plant establishment and take the full advantage of water and nutrients.

Multiple harvesting from perennial sorghum is another key advantage (Michigan state literature review and topic modeling).

Rhizomes are necessary for having a continuous harvest for many seasons from a single sowing in sorghum. However, rhizome genes are not suitable for agricultural purpose because it produces a big number of thin, flexible stems and small hulled seeds that drop after ripening. As a result, the ideal perennial sorghum should have large, thresh-able seeds on big panicles supported by sturdy columns. It can be achieved by incorporating genes that confer good grain-production traits into a common pool with genes from the wild perennial species. This hybridization produces populations of plants carrying genes from both parents in different combination (Cox et al. 2018).

The process of perennial sorghum breeding starts with the controlled hybridization between annual grain sorghum and one of the two wild perennial grasses S. halepense and S. propinquum. Both diploid (2n = 20) species S. bicolor and S. propinquum diverged from a common ancestor approximately 1 to 2 million years ago (Sezan et al. 2016). However, S. halepense resulted from natural hybridization between S. bicolor and S. propinquum causing a doubling in the number of chromosomes to 40. Two QTL for rhizomatousness were identified in recent studies from the hybrid between this annual sorghum and perennial wild sorghum (Washburn et al. 2013). However, despite the perennial growth habit of those hybrids, the reduced winter-hardiness has limited their great potential for only the tropics or subtropics areas.

Hu et al (2003) conducted comparative mapping between sorghum and rice. They found that genes controlling rice rhizomatousness (Rhz2 and Rhz3) closely correspond to major 23 quantitative trait loci controlling rhizomatousness in wild S. propinquum.

For producing a perennial hybrid with overwintering ability, a cross was made between

Johnsongrass (S. halapense, 2n=40) and S. bicolor. Although these hybrids showed increased winter-hardiness but decreased rhizome mass (Piper and Kulakow 1994). Even though there is a homology between the chromosomes of sorghum and Johnsongrass, however, the multivalent chromosome association at meiosis results in the poor hybrid seed setting (Luo et al. 1992). Because of its poor agronomic performance, the perennial hybrid created in this study cannot be used.

Non-crop perennial

Perennial plants have evolved multiple times from their annual ancestors throughout evolution (Soltis et al. 2013). However, the evidence of perennials evolving from annual is very rare. Melzerand at al. (2008) studied two genes (SUPPRESSOR OF

OVEREXPRESSION OF CONSTANS 1 gene and FRUITFULL gene) and argued that silencing these two genes suffices for converting Arabidopsis (Arabidopsis thaliana) to its perennial growth habit; although the woody mutant produced in this process was sterile.

Heidel et al. (2016) found five families of genes in the perennial Arabidopsis lyrata and

Arabidopsis alpina which are absent in each of the annual Arabidopsis species. These five candidate families encode an oxidoreductase, a lactoylglutathione lyase, an F-box protein, a kinase, and a zinc finger protein. 24

1.5 Summary and concluding remarks:

The mechanism of perennialism and the perennating structures differ greatly depending on the species. There are various traits associated with perennialism; such as regrowability, winter-hardiness, rhizome formation, etc. Pinpointing genes or loci associated with these traits related to perennialism is important. Researchers need to identify whether these traits are controlled by the same genetic network in every perennial species or it is different depending on the species. Comparative analysis (comparative mapping) among various perennial species can be a very effective tool here. After successful identification of the genes, then the next step should be transferring these traits successfully to an annual cultivar using modern molecular genetic techniques. Several researchers reported about such attempts to show how they genetically transfer perennial traits into annual cultivar.

Development of sustainable perennial crops through the wide hybridization between annual and wild perennials was described with sorghum (Sorghum bicolor × S. halepense)

(Cox et al. 2018), wheat (Triticum spp. × Thinopyrum spp.) (Hayes et al. 2018), rice (Oryza sativa × O. longistaminata) (Huang et al. 2018), and buckwheat (Fagopyrum spp. ×

Fagopyrum spp.) (Chen et al. 2018). Successful new hybrids would mostly inherit important traits, for example, end-use quality and yield, from the domesticated annual parent. However, the challenge is, when the parents have a different chromosome number

(ploidy) and chromosome sets, they cannot match or recombine during meiosis (Cox et al.

2018 and Hayes et al. 2018). Crops undergoing domestication or hybridization for the development of perennialism mostly rely on conventional breeding strategies like single seed descent, mass selection, bulk population, pedigree, backcrossing, multinet, and 25 composite techniques. However, early farmers and breeders lacked the contemporary toolbox we have now. This toolbox includes a very sophisticated molecular genetics and analytic approach that can produce viable perennial grain crops. Breeders should use the latest molecular and cytogenetic techniques to transform conventional breeding approaches.

26

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37

2 CHAPTER 2: VALIDATION OF CANDIDATE LOCI FOR

REGROWABILITY AND SELECTION OF THEIR NEAR ISOGENIC LINES

IN MAIZE.

2.1 Introduction

Maize is the most important cereal crops worldwide (FAOSTAT, 2013). In its genus maize has two perennial relatives; a tetraploid Zea perennis and a diploid Zea diploperennis

(Figure 2). These two perennial relatives are also known as teosinte, which is a common name of four different groups of plants endemic to Mexico and Central America (Doebley

1990; Wilkes 1967). Teosinte is represented by annual diploid species (Zea mays ssp. mexicana and Zea mays ssp. parviglumis) and by perennial diploid (Zea diploperennis) and tetraploid (Zea perennis) species.

Teosinte and maize (Z. mays L. spp. mays) do not look similar; although these two are very similar at the genetic level. Teosinte is capable to crossbreed with various maize varieties and form maize-teosinte hybrids. These hybrids are also capable to reproduce naturally on their own. These crosses have been documented in the last several decades. For example,

Emerson and Beadle (1929) crossed maize and tetraploid perennial teosinte Zea perennis.

Kelly Dawe’s group at the University of Georgia crossed Z. diploperennis and maize inbred line B73 and generated a population of inter-crossing F1 plants (Coatney 2015); Camara-

Hernandez and Mangelsdorf (1981) used the diploid Zea diploperennis to make a cross with primitive Mexican popcorn; and Westerberg and Doebley (2004) crossed annual Z. mays ssp. parviglumis and Z. diploperennis. 38

In our previous work, we crossed Z. diploperennis Iltis, Doebley & R. Guzman with three maize inbred lines or variety and successfully created regrowable maize hybrids.

Based on the segregation ratio of F2 and F3 generations derived from the B73-Z, diploperennis cross, we hypothesized that regrowability in Z. diploperennis seems to be controlled by two dominant complementary genes reg1 (regrowth 1) and reg2 (Ma et al.

2019). However, some researchers hypothesized the perennialism as a recessive trait

(Camara-Hernandez and Mangelsdorf 1981). According to Shaver (1967), rhizome is the key for regrowth and three recessive genes known as id1 (indeterminate1), gt1 (grassy tiller 1) and pe (perennialism) control the perennialism in Z. diploperennis. However, based on our phenotypic data, rhizome and tuber formation are not necessary for perennialism in Zea. Our previous correlation study of the F2 and F3 populations from the

Z. diploperennis-maize variety Rhee Flint cross also suggested that gt1 and id1 display a positive, yet insignificant association with regrowability (Qiu 2016).

To identify candidate genes responsible for regrowability in Zea, we previously conducted genotyping by sequencing (GBS) analysis on 94 B73-Zd F2 plants and called 10,431 SNPs after filtering. Among them 946 SNPs were found to be associated with regrowth by a chi- square goodness-of-fit test with P (χ 2) > 0.05. Two chromosomal locations with high LOD scores (P > 0.05) on chromosome 2 and chromosome 7, respectively, are considered as the candidate loci for regrowability (Ma et al. 2019).

The main goal of this research here is to validate the two candidate loci identified by GBS by genotyping the whole F2 population by PCR with allele-specific markers. We would like to select near isogenic lines (NIL) for those loci with the same markers as well. 39

2.2 Materials and methods

2.2.1 Formation of the population for marker study

Z. diploperennis and Z. mays inbred line B73 were crossed for making a segregating population at the greenhouse in the winter of 2014 and later in a field at Brookings, SD in the summer of 2015 with the help from Dr. Donald Auger. The F1 seeds produced in the field were grown in Hawaii over the 2015/2016 winter and produced F2 seeds. The F2 and

F3 generations were derived from these F1s by selfing. A total of 134 F2 plants were randomly selected for genetic study and QTL mapping. A F3 plant that is heterozygous for both the regrowth loci was selected and selfed to produce the F4 plants, from which NILs of the loci were identified, respectively.

2.2.2 DNA extraction

Leaf samples were collected from Z. diploperennis, B73 and the F1, F2, F3 and F4 plants for DNA extraction. DNA extraction was done using the CTAB method established by

Doyle and Doyle (1987). The DNA samples were dissolved in TE buffer, quantified using

Nanodrop ND-1000 UV-Vis Spectrophotometer and stored at 1.5 ml tube at -20℃ until being used. It was further diluted using nuclease-free water up to a concentration at 40 ng/μl and used for PCR and other purposes.

2.2.3 PCR assay

PCR amplification was used for genotype identification, development of allele-specific markers and NIL selection. The primers for gt1, tb1, id1 reported in Qiu (2016) were used.

For designing the allele-specific markers for reg1 and reg2, the improved SNP specific

PCR primer designing method described by Liu et al. (2012) was followed with necessary 40 modifications. Basically, an artificial mismatch of a single nucleotide was introduced within the last three bases closer to the 3' end (the SNP site). The primer annealing temperature was kept below or around 60 °C, with around 50% GC content. Each primer set consists of a common reverse primer for both alleles of the SNP and an allele-specific forward primer. The allele-specific markers generated by these primer sets are dominant.

Our GBS analysis has found that reg1 is in the candidate interval from S2_24,244,192 to

S2_28,975,747 on chromosome 2 with a peak at S2_27,934,739 and reg2 is in the candidate interval from S7_2,862,253 to S7_6,681,861 on chromosome 7 with a peak at

S7_5, 06739 (Ma et al. (2019). Several primer sets were produced for each candidate interval (Table 1).

For the PCR assay, GoTaq Green Master Mix (Catalog# M7505, Promega, Madison, WI) was used. And for most of the reactions, PCR was done in following conditions: denaturation at 95°C, 35 cycles of 95°C for 45s, annealing at 55∼62°C (primer dependent) for 1 min, 72°C for 1 min, and a final extension at 72° for 10 min. As the primers are allele specific so determining the annealing temperature based on the primer melting temperature is very crucial. Therefore, to identify the right annealing temperature, touchdown PCR was practiced with the initial annealing temperature set at 62°C with 1°C lower in each subsequent cycle until 55°C was reached. The primers used in this study are listed in Table

1.

2.3 Results and discussions

2.3.1 The study of gt1, id1, and tb1 for their contribution to regrowability

The gene for rice rhizomatousness also known as Rhz2 has been mapped in rice 41 chromosome 3 and sorghum chromosome 1 (Hu et al. 2003, Paterson et al. 1995).

Interestingly, both are homologous to maize chromosome 1 (Nguyen et al. 2012).

Previously, gt1 and id1 were associated with the perenniality in Zea (Shaver, 1967). Gene tb1 (teosinte branched 1) was found to control gt1 (Whipple et al. 2011). All these genes are located in chromosome 1 of Zea (Whipple et al. 2011). However, no significant correlation between any of the three genes and regrowth was observed in our previous study of the hybrid derivatives of the Z. diploperennis-Rhee Flint cross (Qiu 2016).

Nevertheless, no significant regrowth QTL was mapped to this chromosome by our GBS analysis though two insignificant peaks were identified (Ma et al. 2019; Fig. 4). Therefore, it is necessary to investigate if these three genes are indeed associated with regrowability in Zea.

The B73-Zd F2 population was genotyped with gene-specific markers for these three genes.

As shown in Table 2, of the 131 F2 B73-Zd F2 plants, only 33, 5, 115 showed homozygosity for the maize tb1, gt1 and id1 alleles, respectively. With a degree of freedom 5, primer gt1and tb1 have given χ2 value 52.104 and 36.979 (the χ2 value for id1 can`t be calculated)

(Table 3). All of them have shown p-value less than or equal to 0.0001 (p≤0.0001).

Therefore, this result indicates that gt1 and id1 are highly unlikely in control of regrowth and thus perenniality, which confirms the conclusion by Qiu (2016) but conflicts with the conclusion by Shaver (1967).

The Z. diploperennis gt1 allele might have a little association with regrowth as most of the plants that showed regrowth phenotype carried one or two copies of it. However, as some plants regrew without it, so it cannot be called as essential.

Interestingly, id1 didn`t show any heterozygosity and tb1 showed very few (less than 42

expected) heterozygosity in all the examined F2 plants, regardless of their regrowth phenotype. Only 16 F2 plants had the Zea diploperennis id1 allele (Table 2). On the other hand, maize id1 allele showed too much homozygosity which dictates one type of selection.

It is possible that the maize chromosome fragment carrying id1 transmits preferentially into the F2s. Any deficiency or rearrangement issues next to the Z. diploperennis id1 allele might cause this excessive maize homozygosity and make the transmission inefficient, or perhaps the Z. diploperennis id1 allele does not let the hybrids grow or flower in South

Dakota.

2.3.2 Validation of the candidate SNPs by genotyping the F2 population

Our GBS analysis identified reg1 and reg2 on chromosomes 2 and 7, respectively (Fig. 4).

This analysis was conducted in 94 F2 plants. To validate the relationship between regrowability and the candidate loci, SNPs located in the proximity of peak areas of chromosome 2 and chromosome 7 were used for developing allele-specific PCR markers according to the protocol described by Liu et al. (2012) (Figure 5A). The allele-specific markers so designed were used to identify their polymorphism between Z. diploperennis and B73 (Table 4). These polymorphic markers were used to screen the 134 B73-Zd F2 plants. Figures 6 and 7 show the results along with the phenotype of the F2 plants. The chi- square goodness-of-fit test was used to identify whether the regrowth and the SNP segregate independently or not. The chi-square test of independence tested the null hypothesis that the regrowth trait and the Z. diploperennis allele of the tested SNP are segregated independently. With a degree of freedom 5, primer S2-1, S2-2, and S7-1/S7-2 have given an χ2 value 36.161, 25.358 and 27.220 respectively (Table 3). All of them have shown p-value less than or equal to 0.0001 (p≤0.0001) (Table 3). So, the test results of 43 significance rejected the null hypothesis and showed all the SNPs that were tested are associated with regrowth. As the result, the candidacy of the SNPs for the two regrowth loci are confirmed.

2.3.3 Genes found in peak locations

The GBS analysis has located reg1 to the interval from S2_24,244,192 to S2_28,975,747 on chromosome 2 with the peak at S2_27,934,739 and reg2 to the interval from

S7_2,862,253 to S7_6,681,861 on chromosome 7 with the peak at S7_5, 060739 (Figure

4). Therefore, MaizeGDB was used for further analysis of these candidate genes in these intervals. The result shows that there are approximately 162 genes found in the reg1 interval. Most of the genes are protein-coding without any known functions. Some genes yet to have any ID. However, there is a gene identified in the peak location of chromosome

2, spanning from 27, 929, 335 bp to 27, 238, 858 bp. So, the reg1 peak SNP is within this gene. The name of the gene is Zm00001d002950. In the previous assembly version, it was called GRMZM2G138125. According to the NCBI annotation release, the gene is called

LOC100384583. This protein is also known as "Chromatin Remodeling Protein 24". It is a protein-coding gene with a protein of 1,048 amino acids (Fig. 8) and 3,552 bp of coding sequence (Fig. 9). This gene is located in the short arm of chromosome 2. The gene was first mentioned by Pang et al. (2018) for its association with kernel size. It is very interesting considering Z. diploperennis, B73 and the B73-Zd hybrids are very different in kernel shape and size.

For chromosome 7, there are approximately 125 probable genes found within the reg2 candidate interval. Most of the genes are protein-coding without any known functions.

Some genes yet to have any ID. However, there is a gene found near the peak location of 44 chromosome 7, spanning from 5,058,282 bp to 5,060,333 bp. So, the reg2 peak SNP is very close to this gene (5,060,739 bp). The name of the gene is Zm00001d018780. In the previous assembly version, it was called GRMZM2G048172. According to the NCBI annotation release, the gene is called LOC103631938. This gene is also known as flocculation protein 11 (FLO11). It is a cell wall protein that contributes directly in adhesive cell-cell interactions. This gene has 688 amino acids (Fig. 10) and 2337 bp of coding sequence (Fig. 11).

2.3.4 Selection of the Near Isogenic lines for reg1 and reg2

With the allele-specific markers in hand, it is possible to select the NILs for the two regrowth loci. For this purpose, F2 plants BZ2002-19 and BZ2002-22 were chosen because they are double heterozygous for both the loci (Figs. 6 and 7; Table 2). Twenty-six selfed seeds were collected from BZ2002-19 and five selfed seeds were collected from BZ2002-

22. They were grown in the East Greenhouse. Their genotypes were identified by PCR using our allele-specific markers (Table 6). Six out of 19 plants derived from BZ2002-19 showed double heterozygosity and none of those from BZ2002-22 showed double heterozygosity. The six-double heterozygous F3 plants were grown in the East Greenhouse and showed regrowth phenotype at the end of their lifecycle (Table 6). Selfed F4 seeds were harvested from each of the plants. The selfed F4 seeds from one of the six F3 plants were used in the production of the NILs. A total of 90 F4 plants were genotyped with the allele- specific markers for the two regrowth loci. Of the 90 F4 plants, five different genotypes were selected: NIL1 (Reg1Reg1 reg2reg2), NIL2 (reg1reg1 Reg2Reg2), NIL3 (Re1reg1

Reg2reg2), NIL4 (Reg1Reg1 Reg2Reg2) and NIL5 (reg1reg1 reg2reg2). Only the plants having these genotypes were grown further and treated with an appropriate light cycle/dark 45 cycle to induce flowering development. Later, they were all selfed. Testcrosses were made to verify the recovery regrowability (Table 7).

2.4 Prospects and future direction of maize perennialism research

In order to select near isogenic lines, our strategy is to combine marker-assisted selection with conventional phenotypic identification. The reason we tried to select NILs is that they are a very valuable resource for investigating regulation and function of a single gene.

Therefore, NIL can be very effective for the isolation of regrowth genes.

Although we have the marker, however marker generally doesn`t give any idea about the function or molecular mechanism of the candidate genes. Additionally, how far the markers are away from our candidate gene is still unknown. To obtain the information it is necessary to isolate the gene. If possible, we need to fine map the gene to a much higher resolution position using more markers. If we have markers which are inside the gene or very tightly linked, then it will be very easy to isolate the gene. Once we have the sequence of the gene, searching for the homologous genes whose function is already known can be a way to predict our gene function. Gene replace is also a way to predict the function; however, the process is very laborious. Identifying gene function through gene silencing using sense and antisense suppression has been a very popular method for the last decade. Once the function is identified, we have to clone the gene into annual maize and observe the gene expression.

Here agrobacterium mediated transformation and gene gun (microparticle -bombardment) are the two most commonly used methods. Additionally, as we found " Chromatin remodeling protein 24 and flocculation protein 11 (FLO11) within or near the peak 46 positions of our candidate locus intervals, respectively; so, identifying their functions is also necessary.

47

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Liu, J., Huang, S., Sun, M., Liu, S., Liu, Y., Wang, W. and Zhang, X. et al. (2012). An improved allele‐specific PCR primer design method for SNP marker analysis and its application. Plant Methods, 8, 34. 48

Ma, A., Qiu, Y., Raihan, T., Paudel, B., Dahal, S., Zhuang, Y., Galla, A., Auger, D. L., and Yen, Y. (2019). The Genetics and Genome-Wide Screening of Regrowth Loci, a Key Component of Perennialism in Zea diploperennis. G3.

Nguyen, T. D., Lawn, R. J., Bielig, L. M. (2012). Expression and inheritance of perenniality and other qualitative traits in hybrids between mungbean cultivars and Australian wild accessions. Crop Pasture Sci. 63: 619–634. doi:10.1071/CP12263.

Pang, J., Fu, J., Zong, N., Wang, J., Song, D., Zhang, X., He, C., Fang, T., Zhang, H., Fan, Y., Wang, G. and Zhao, J. (2019). Kernel size‐related genes revealed by an integrated eQTL analysis during early maize kernel development. Plant J, 98: 19-32. doi:10.1111/tpj.14193.

Paterson, A. H., Schertz, K. F., Lin, Y. R., Liu, S. C., and Chang, Y. L. (1995). The Weediness of Wild Plants - Molecular Analysis of Genes Influencing Dispersal and Persistence of Johnsongrass, Sorghum-Halepense (L) Pers. Proc. Natl. Acad. Sci. USA 92: 6127–6131. 10.1073/pnas.92.13.6127.

Shaver, D. L. (1967). Perennial maize. J. Hered. 58, 271-273.

Westerbergh, A. and Doebley, J. (2004). Quantitative trait loci controlling phenotypes related to the perennial versus annual habit in wild relatives of maize. Theoretical and Applied Genetics. 109: 1544-1553.

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Wilkes, H. G. (1967). Teosinte: the closest relative of maize. Bussey Inst., Harvard Univ., Cambridge, MA.

49

Table 1: List of primers used in this study

Primer name Sequences tb1MF 5’-AGTAGGCCATAGTACGTAC-3’ tb1MR 5’-CTCTTTACCGAGCCCCTACA-3’ tb1ZF 5’-ACTCAACGGCAGCAGCTACCTA-3’ tb1ZR 5’-CGTGTGTGTGATCGAATGGT-3’ tga1cF 5’-AATAAAATAGAGGAACGTCA-3’ tga1cR 5’-TGCTGCAAAGGATTACTGAT-3’ id1cF 5’-ACCGGACGGATCGAGAGAAA-3’ id1cR 5’-CCGTACTCACTCGCAGATCG-3’ bnlg2162F 5’-GTCTGCTGCTAGTGGTGGTG-3’ bnlg2162R 5’-CACCGGCATTCGATATCTTT-3’ mmc0381F 5’-GTGGCCCTGTTGATGAG-3’ mmc0381R 5’-CGACGAGTACCAGGCAT-3’ bnlg1194F 5’-GCGTTATTAAGGCAAGCTGC-3’ bnlg1194R 5’-CACCGGCATTCGATATCTTT-3’ gt1-ZF 5’-TCGCCTACATGACCGAGTAC-3’ gt1-ZR 5’-ATACTCTCAGCTGCTACGCG-3’ gt1-MF 5’-GAGACCGAGCTGCTGAAGAT-3’ gt1-MR 5’-TGTAGCTGTTGTAGGCGTACT-3’

S2-1MF 5’-CTCTTCGCCTACTGCTAT-3’

S2-1ZF 5’-CTCTTCGCCTACTGCTAC-3’

S2-1R 5’-AATGTCAATGCAGACAAGCCT-3’ 50

S2-5MF 5’-CGATGGTGAACATGATAAACGGA-3’

S2-5MR 5’-TATGGCTTGATTCGCTCTCTT-3’

S2-5ZF 5’-CGATGGTGAACATGATAAATAGG-3’

S2-5ZR 5’-ACGCAAAAAGTATGGCTTGAT-3’

S7-1MF 5’-CGTATCATCATACGAGCATG-3’

S7-1MR 5’-TGAATGAGCTCGATTGTGCC-3’

S7-2ZF 5’-GTGCCTACGCTCCATCCGAA-3’

S7-2ZR 5’-GTCGCTACCACTGTATCGCA-3’

51

Table 2: The phenotype for regrowability and the gt1, id1, tb1, S2-1, S2-5, S7-1 and S7-2 marker genotypes of 134 B73-Z hybrid plants are shown in the table here.

S7- S7-1/ Plant PT gt1 tb1 id1 S2-1 S2-5 Plant PT gt1 tb1 id1 S2-1 S2-5 1/ S7-2

S7-2

Zea diploperenis R 1 1 1 1 1 1 BZ2-007-17 NR 1 - 1 2 - -

B73 NR 2 2 2 2 2 2 BZ2-007-18 R 1 1 2 2 3 1

BZ2-001-1 R 1 3 2 1 1 1 BZ2-007-19 R 3 2 2 3 3 3

BZ2-001-2 R 3 3 1 3 3 2 BZ2-007-20 R 3 2 2 3 3 1

BZ2-001-3 R 1 3 2 1 3 3 BZ2-007-21 NR 3 2 2 2 1 2

BZ2-001-4 R 1 3 2 3 3 2 BZ2-008-1 R 1 1 2 3 3 2

BZ2-001-5 R 1 1 2 1 1 1 BZ2-008-2 R 3 1 2 3 3 3

BZ2-002-1 NR 1 3 2 1 1 3 BZ2-008-3 R 3 1 2 1 3 3

BZ2-002-2 NR 1 2 2 3 1 2 BZ2-008-4 NR 3 2 2 2 3 1

BZ2-002-3 R 3 3 2 1 3 3 BZ2-008-5 R 3 3 2 2 2 3

BZ2-002-4 R 3 2 1 1 3 3 BZ2-008-6 NR 3 2 2 3 2 3

BZ2-002-5 NR 3 3 2 2 2 3 BZ2-008-7 NR 3 3 2 2 2 2

BZ2-002-6 NR 3 2 2 2 2 3 BZ2-008-8 R 3 2 2 3 1 1

BZ2-002-7 NR 3 2 2 3 1 2 BZ2-008-9 NR 3 2 2 3 3 3

BZ2-002-8 R 1 - 2 - - 2 BZ2-008-10 R 1 1 2 3 3 1

BZ2-002-9 R 3 2 2 1 1 1 BZ2-008-11 NR 3 2 2 2 1 2

BZ2-002-10 R 3 3 2 3 - 3 BZ2-008-12 R 3 3 2 1 3 3

BZ2-002-11 NR 1 2 1 2 - 2 BZ2-008-13 R 1 2 2 3 3 3

BZ2-002-12 R 1 2 2 2 3 2 BZ2-008-14 NR 1 3 2 2 2 2

BZ2-002-13 R 3 3 2 3 3 - BZ2-009-1 NR 3 3 2 3 3 3

BZ2-002-14 NR 1 1 2 1 1 3 BZ2-009-2 R 1 3 1 3 3 3

BZ2-002-15 R 1 3 1 3 3 2 BZ2-009-3 NR 3 2 2 2 2 2

BZ2-002-16 R 3 3 1 1 1 3 BZ2-009-4 R 1 3 2 1 3 1

BZ2-002-17 NR 3 2 2 3 - - BZ2-009-5 NR 3 3 2 3 3 2

BZ2-002-18 R 1 2 2 1 1 1 BZ2-009-6 R 1 1 2 1 3 3

BZ2-002-19 R 1 2 2 3 3 3 BZ2-009-7 NR 1 - 2 2 2 3

BZ2-002-20 R 1 2 2 3 3 2 BZ2-009-8 NR 3 2 2 1 1 2 52

BZ2-002-21 R 1 2 1 1 - - BZ2-009-9 R 3 1 2 2 3 2

BZ2-002-22 R 3 2 2 3 3 3 BZ2-009-10 NR 1 3 2 3 2 2

BZ2-002-23 R 3 2 1 3 1 1 BZ2-009-11 R 1 3 2 2 3 3

BZ2-002-24 R 3 2 2 3 3 3 BZ2-009-12 R 3 3 2 3 1 3

BZ2-002-25 NR 1 2 2 2 2 2 BZ2-010-1 R 3 2 2 2 3 3

BZ2-004-1 R 3 1 2 3 3 - BZ2-010-2 NR - 3 2 2 2 3

BZ2-004-2 R 1 3 2 3 1 1 BZ2-010-3 R 3 1 2 2 3 1

BZ2-004-3 NR 3 2 2 3 1 - BZ2-010-4 R 3 2 1 2 3 1

BZ2-004-4 R 1 3 2 3 3 3 BZ2-010-5 NR 3 1 2 2 2 3

BZ2-004-5 R 3 2 2 3 3 1 BZ2-010-6 R 3 2 2 3 3 3

BZ2-004-6 R 3 1 2 - 1 2 BZ2-010-7 NR 3 1 2 2 2 2

BZ2-006-1 NR 3 1 2 3 3 - BZ2-010-8 R 3 2 2 3 3 3

BZ2-006-2 NR 1 1 2 2 2 3 BZ2-010-9 R 1 3 2 1 1 3

BZ2-006-3 R 3 3 2 3 3 3 BZ2-010-10 R 3 2 2 3 2 3

BZ2-006-4 R 3 2 2 3 1 1 BZ2-010-11 R 2 3 2 3 3 3

BZ2-006-5 R 3 3 2 3 3 3 BZ2-010-12 NR 2 3 2 3 3 3

BZ2-006-6 NR 3 3 2 3 1 3 BZ2-010-13 R 3 3 2 1 3 2

BZ2-006-7 R 3 1 2 1 3 1 BZ2-010-14 R 1 2 2 3 1 1

BZ2-006-8 R 3 1 2 2 2 3 BZ2-010-15 NR 3 3 2 2 1 3

BZ2-006-9 R 1 1 2 2 3 1 BZ2-010-16 NR 1 3 2 3 2 3

BZ2-006-10 R 1 3 2 1 1 1 BZ2-010-17 R 1 3 2 3 2 3

BZ2-006-11 NR 3 2 2 3 3 3 BZ2-010-18 NR 3 3 2 3 3 3

BZ2-006-12 NR 3 3 2 3 2 3 BZ2-010-19 R 2 2 1 1 1 1

BZ2-006-13 NR 3 2 1 - 2 1 BZ2-010-20 NR 1 2 1 2 - -

BZ2-006-14 R 3 2 2 3 3 3 BZ2-010-21 NR 3 3 2 3 3 3

BZ2-007-1 NR 3 2 2 3 3 2 BZ2-011-1 NR 1 - 1 - 1 1

BZ2-007-2 NR 3 2 2 2 3 3 BZ2-011-2 R 3 2 2 1 1 3

BZ2-007-3 R 1 2 2 3 3 3 BZ2-011-3 R 2 3 2 3 3 3

BZ2-007-4 R 3 2 2 2 2 3 BZ2-011-4 R 3 1 2 3 3 3

BZ2-007-5 R 3 3 2 2 2 2 BZ2-011-5 R 1 - 1 - 1 1

BZ2-007-6 NR 3 3 2 3 3 3 BZ2-011-6 NR 3 2 2 3 3 2

BZ2-007-7 R 1 3 2 3 3 1 BZ2-011-7 NR 1 2 2 3 2 3 53

BZ2-007-8 R 3 1 2 2 2 1 BZ2-011-8 NR 2 1 1 2 - 1

BZ2-007-9 R 3 1 2 3 3 3 BZ2-011-9 R 3 2 2 3 3 1

BZ2-007-10 NR 1 3 2 3 3 3 BZ2-011-10 R 3 1 2 1 1 3

BZ2-007-11 NR 3 1 2 2 3 2 BZ2-011-11 R 1 2 2 3 3 3

BZ2-007-12 R 1 2 2 3 3 3 BZ2-011-12 R 1 3 2 3 3 2

BZ2-007-13 R 3 1 2 3 3 1 BZ2-011-13 R 3 1 2 3 3 1

BZ2-007-14 NR 1 3 2 3 3 2 BZ2-011-14 NR 3 1 2 3 3 3

BZ2-007-15 NR 3 1 2 3 3 2 BZ2-011-15 NR 3 3 2 1 1 3

BZ2-007-16 R 1 3 2 3 3 3 BZ2-011-16 NR 1 2 2 2 - 3

BZ2-002-1 NR 1 3 2 1 1 3 BZ2-008-3 R 3 1 2 1 3 3

BZ2-002-2 NR 1 2 2 3 1 2 BZ2-008-4 NR 3 2 2 2 3 1

BZ2-002-3 R 3 3 2 1 3 3 BZ2-008-5 R 3 3 2 2 2 3

BZ2-002-4 R 3 2 1 1 3 3 BZ2-008-6 NR 3 2 2 3 2 3

BZ2-002-5 NR 3 3 2 2 2 3 BZ2-008-7 NR 3 3 2 2 2 2

BZ2-002-6 NR 3 2 2 2 2 3 BZ2-008-8 R 3 2 2 3 1 1

BZ2-002-7 NR 3 2 2 3 1 2 BZ2-008-9 NR 3 2 2 3 3 3

BZ2-002-8 R 1 - 2 - - 2 BZ2-008-10 R 1 1 2 3 3 1

BZ2-002-9 R 3 2 2 1 1 1 BZ2-008-11 NR 3 2 2 2 1 2

BZ2-002-10 R 3 3 2 3 - 3 BZ2-008-12 R 3 3 2 1 3 3

BZ2-002-11 NR 1 2 1 2 - 2 BZ2-008-13 R 1 2 2 3 3 3

BZ2-002-12 R 1 2 2 2 3 2 BZ2-008-14 NR 1 3 2 2 2 2

BZ2-002-13 R 3 3 2 3 3 - BZ2-009-1 NR 3 3 2 3 3 3

BZ2-002-14 NR 1 1 2 1 1 3 BZ2-009-2 R 1 3 1 3 3 3

BZ2-002-15 R 1 3 1 3 3 2 BZ2-009-3 NR 3 2 2 2 2 2

BZ2-002-16 R 3 3 1 1 1 3 BZ2-009-4 R 1 3 2 1 3 1

BZ2-002-17 NR 3 2 2 3 - - BZ2-009-5 NR 3 3 2 3 3 2

BZ2-002-18 R 1 2 2 1 1 1 BZ2-009-6 R 1 1 2 1 3 3

BZ2-002-19 R 1 2 2 3 3 3 BZ2-009-7 NR 1 - 2 2 2 3

BZ2-002-20 R 1 2 2 3 3 2 BZ2-009-8 NR 3 2 2 1 1 2

BZ2-002-21 R 1 2 1 1 - - BZ2-009-9 R 3 1 2 2 3 2

BZ2-002-22 R 3 2 2 3 3 3 BZ2-009-10 NR 1 3 2 3 2 2

BZ2-002-23 R 3 2 1 3 1 1 BZ2-009-11 R 1 3 2 2 3 3 54

BZ2-002-24 R 3 2 2 3 3 3 BZ2-009-12 R 3 3 2 3 1 3

BZ2-002-25 NR 1 2 2 2 2 2 BZ2-010-1 R 3 2 2 2 3 3

BZ2-004-1 R 3 1 2 3 3 - BZ2-010-2 NR - 3 2 2 2 3

BZ2-004-2 R 1 3 2 3 1 1 BZ2-010-3 R 3 1 2 2 3 1

BZ2-004-3 NR 3 2 2 3 1 - BZ2-010-4 R 3 2 1 2 3 1

BZ2-004-4 R 1 3 2 3 3 3 BZ2-010-5 NR 3 1 2 2 2 3

BZ2-004-5 R 3 2 2 3 3 1 BZ2-010-6 R 3 2 2 3 3 3

BZ2-004-6 R 3 1 2 - 1 2 BZ2-010-7 NR 3 1 2 2 2 2

BZ2-006-1 NR 3 1 2 3 3 - BZ2-010-8 R 3 2 2 3 3 3

BZ2-006-2 NR 1 1 2 2 2 3 BZ2-010-9 R 1 3 2 1 1 3

BZ2-006-3 R 3 3 2 3 3 3 BZ2-010-10 R 3 2 2 3 2 3

BZ2-006-4 R 3 2 2 3 1 1 BZ2-010-11 R 2 3 2 3 3 3

BZ2-006-5 R 3 3 2 3 3 3 BZ2-010-12 NR 2 3 2 3 3 3

BZ2-006-6 NR 3 3 2 3 1 3 BZ2-010-13 R 3 3 2 1 3 2

BZ2-006-7 R 3 1 2 1 3 1 BZ2-010-14 R 1 2 2 3 1 1

BZ2-006-8 R 3 1 2 2 2 3 BZ2-010-15 NR 3 3 2 2 1 3

BZ2-006-9 R 1 1 2 2 3 1 BZ2-010-16 NR 1 3 2 3 2 3

BZ2-006-10 R 1 3 2 1 1 1 BZ2-010-17 R 1 3 2 3 2 3

BZ2-006-11 NR 3 2 2 3 3 3 BZ2-010-18 NR 3 3 2 3 3 3

BZ2-006-12 NR 3 3 2 3 2 3 BZ2-010-19 R 2 2 1 1 1 1

BZ2-006-13 NR 3 2 1 - 2 1 BZ2-010-20 NR 1 2 1 2 - -

BZ2-006-14 R 3 2 2 3 3 3 BZ2-010-21 NR 3 3 2 3 3 3

BZ2-007-1 NR 3 2 2 3 3 2 BZ2-011-1 NR 1 - 1 - 1 1

BZ2-007-2 NR 3 2 2 2 3 3 BZ2-011-2 R 3 2 2 1 1 3

BZ2-007-3 R 1 2 2 3 3 3 BZ2-011-3 R 2 3 2 3 3 3

BZ2-007-4 R 3 2 2 2 2 3 BZ2-011-4 R 3 1 2 3 3 3

BZ2-007-5 R 3 3 2 2 2 2 BZ2-011-5 R 1 - 1 - 1 1

BZ2-007-6 NR 3 3 2 3 3 3 BZ2-011-6 NR 3 2 2 3 3 2

BZ2-007-7 R 1 3 2 3 3 1 BZ2-011-7 NR 1 2 2 3 2 3

BZ2-007-8 R 3 1 2 2 2 1 BZ2-011-8 NR 2 1 1 2 - 1

BZ2-007-9 R 3 1 2 3 3 3 BZ2-011-9 R 3 2 2 3 3 1

BZ2-007-10 NR 1 3 2 3 3 3 BZ2-011-10 R 3 1 2 1 1 3 55

BZ2-007-11 NR 3 1 2 2 3 2 BZ2-011-11 R 1 2 2 3 3 3

BZ2-007-12 R 1 2 2 3 3 3 BZ2-011-12 R 1 3 2 3 3 2

BZ2-007-13 R 3 1 2 3 3 1 BZ2-011-13 R 3 1 2 3 3 1

BZ2-007-14 NR 1 3 2 3 3 2 BZ2-011-14 NR 3 1 2 3 3 3

BZ2-007-15 NR 3 1 2 3 3 2 BZ2-011-15 NR 3 3 2 1 1 3

BZ2-007-16 R 1 3 2 3 3 3 BZ2-011-16 NR 1 2 2 2 - 3

Here the bolded plants were used for Genotype by sequencing (PT: phenotype, NR: non-regrowable; R: regrowable; “1”: homozygous for Z. diploperennis allele; “2”: homozygous for B73 allele; “3”: heterozygous and “- “: missing data).

:

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Table 3: Detection of polymorphic marker between Z. diploperennis and B73

POSITION PRIMER NAME PRESENCE OF BANDS

MAIZE ALLELE ZEA ALLELE

S7. SNP1 MF1 ✓

MF2 ✓

ZF1

ZF2

ZF3

ZF4

S7. SNP2 MF1 ✓

MF2 ✓

ZF1

ZF2

ZF3 ✓

ZF4 ✓

ZF5 ✓

S2. SNP5 MF1 ✓

MF2 ✓

ZF1 ✓

ZF2 ✓

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Table 4: It is the result of chi square test of independent segregation between target SNPs and regrowth (Here, P: Zea diploperennis allele; p: Z. mays allele, R: regrowth allele, r : non-regrowth allele).

Phenotypes Genotypes gt1 tb1 Id1 S2-1 S2-2 S7-1/S7-2

(Ratio) Obs Exp Obs Exp Obs Exp Obs Exp Obs Exp Obs Exp

R_PP (3/16) 33 25 20 24 10 25 20 24 18 23 26 24

Regrowth R_Pp (6/16) 45 50 27 48 0 50 45 48 53 47 40 47

R_pp (3/16) 3 25 30 24 0 25 13 24 7 23 12 23

rrPP (1/16) 17 8 9 8 6 8 4 8 12 8 4 8 Non- rrPp (2/16) 33 17 20 16 0 16 25 17 18 16 26 16 regrowth rrpp (1/16) 2 8 22 8 118 8 22 8 17 8 18 8

χ 2, df=5 52.104 36.979 - 36.161 25.358 27.220

P(χ2) 0.0001 0.0001 - 0.0001 0.0001 0.0001

* R: regrowth allele, r: non-regrowth allele; P: Zea diploperennis allele; p : Z. mays allele.

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Table 5: Genes located around the peak position.

The peak 2 2  P ( ) Chr SNP position LOD Gene Gene position Annotation

27, 929, 335 bp 2 27,934,739 4.944 4.097 0.393 Protein chromatin Zm00001d002950 to 27, 238, 858 remodeling 24

Zm00001d018780 5,058,282 bp to Flocculation protein

7 5,060,739 4.764 7.029 0.134 5,060,333 b FLO1

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Table 6: The results of F3 screening using the allele-specific markers for reg1 and reg2

Family Plant ID Double heterozygotes

for the markers

BZ2002-19 (1-26) F3-NIL 1

F3-NIL 6

F3-NIL 18

F3-NIL 19

F3-NIL 25

F3-NIL 26

BZ2002-22 1-5 None of them

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Table 7: The results of F4 screening using allele-specific markers for reg1 and reg2. B: presence of the particular marker.

Plant ID S2.SNP5.MF1 S2.SNP5.ZF1 S7.SNP1.MF2 S7.SNP2.ZF4 F4 NIL 46 B B B B F4 NIL 47 B B F4 NIL 48 B B F4 NIL 49 B B

F4 NIL 50 B B F4 NIL 51 B B F4 NIL 53 B B F4 NIL 55 B B B B F4 NIL 56 B B F4 NIL 57 B B F4 NIL 58 B B F4 NIL 59 B B B B

F4 NIL 60 B B F4 NIL 61 B B F4 NIL 62 B B B F4 NIL 63 B B B B F4 NIL 64 B B B F4 NIL 65 B B B B F4 NIL 66 B B F4 NIL 67 B B F4 NIL 68 B B B F4 NIL 69 B B F4 NIL 70 B B F4 NIL 71 B B B B F4 NIL 72 B B F4 NIL 73 B B F4 NIL 74 B B F4 NIL 75 B B

61

Figure 1: A satellite photo showing nutrient pollution in Gulf of Mexico. Here, the whitish colored water indicates pollution by nutrient-rich sediment. This sediment water is concurrently flowing into the deeper ocean. As a result, this nutrient rich sediment allows more and more growth of phytoplankton blooms and eventually it leads to very dangerous hypoxic conditions.

62

Figure 2: Photos showing perennial wild maize (Teosinte) species Zea diploperennis

Iltis, Doebley & R. Guzman (left) and Z. perennis [Hitchc.] Reeves and Mangelsdorf

(right).

63

Figure 3. A photo showing the growth of Zea diploperennis, Mo-17- Z. diploperennis F1 and B73- Z. diploperennis F1 plants in the field.

64

Figure 4: Loci in maize inbred B73 genome that are related to regrowth . A graphic showing the result of mapping regrowth QTL with SNP revealed by GBS analysis. A total of 16 SNPs on Chromosome 2 (two region) and Chromosome 7 (1 region) pass the 90% threshold. The peak positions of the QTL intervals are indicated in the picture by red circles. For chromosome 2; position S2_24,244,192 to S2_28,975,747 is the candidate interval of reg1 with a peak at S2_27,934,739. For chromosome 7; position S7_2,862,253 to S7_6,681,861 is the candidate interval for reg2 with a peak at S7_5, 06739 (Ma 2017).

65

Figure 5: An illustration of the process to design allele specific primers for a SNP as described by Liu et al. (2012) 66

Figure 6: Photos showing the results of genotyping the B73-Zd F2 population using allele- specific primers S2.SNP5.MF1 and S2.SNP5.ZF1 for the reg1 interval peak on B73 chromosome 2. The primer set is also known as S2-2 and was used as that in the table 1, 2, and 4 for analysis. At the top of the picture parents screening is shown. (Z: Z. diploperennis,

B73: Z. mays inbred line B73, X-Y numbers: individual B73-Zd F2 hybrid plants, 67

S2.SNP5. ZF1: the Z-specific marker, S2.SNP5. MF1: the B73-specific marker, phenotypes were scored as “1” for regrowth and “2” for non-regrowth, Genotypes were scored as “1”, “3” and “2” for homologous for Z allele, heterozygous and homozygous for

B73 allele, respectively).

68

Figure 7: Photos showing the results of genotyping the B73-Zd F2 population using allele- specific primers S7.SNP1.MF2 and S7.SNP2.ZF4 for the reg2 interval peak on B73 chromosome 7. The primer sets also known as S7-1/S7-2 and were used as that in the tables 69

1, 2 and 4 for analysis. At the top of the picture parents screening is shown. (Z: Z. diploperennis, B73: Z. mays inbred line B73, X-Y numbers: individual B73-Zd F2 hybrid plants, S7.SNP2. ZF4: the Z-specific marker, S7.SNP1. MF2: the B73-specific marker, phenotypes were scored as “1” for regrowth and “2” for nonregrowth, Genotypes were scored as “1”, “3” and “2” for homologous for Z allele, heterozygous and homozygous for

B73 allele, respectively).

70

>NP_001334119.1. Chromatin remodeling protein 24

MASPPPFDLCGDLDDDDIDEPVTAVPKDRHLPAAAPTPNGLNDHLLRFTRSLQRPTQNPNPSPNPPPPPP

PAAAEEQVEKVKLAGRRRLCKLATTTTATQRVDEEEAEWDNQDDGESILDILDDLTTRFDSLSVQKPSTA

ARSRTQQLTPLPCAITVDDDLDDHSPDDVDAHAGASSPLQISSSDEARAPTRRSEVKIETDLVSSACTHY

ACDDVRGKGKNKGTTKDVGRLNRVSKASSFVDSYSDSDYDDCEEDQGTRTDYAVKQLRSKGFTRRPPNTP

TFRNHGVSDDELGQEKENLGAVENNAEDVGWEKTEDFKMDPTGTAATSKPYKLPGKIFKMLFAHQREGLR

WLWVLHCRGTGGILGDDMGLGKTMQVAAFLAGLFHSRLVKRVLIVAPKTLLAHWTKELSIVGLKEKIRDY

SGPSTNIRNYELQYAFKEGGILITTYDIVRNNYKLIRGNSYNNSNDDDDEEGTLWNYVILDEGHLIKNNK

TQRAQSLYEIPCAHRIVISGTPIQNNLKEMWTLFNFCCPDVLGDKQQFKIRYETAILRGNDKNATAREKH

VGSNVAKELRERIKPYFLRRLKSEVVFDTGASEEKTLAKKNELIVWLKLTPCQRKLYEAFLNSELVHLAL

QPKASPLAAITILKKICDHPLLLTKKGAEGVLEGMGEMLNDQDIGMVEKMAMNLADMAHDDNALEVGQDV

SCKLSFIMSLLRNLVGEGHHVLIFSQTRKMLNLIQEAIILEGYAFLRIDGTTKVSDRERIVKDFQEGCGA

PVFLLTTQVGGLGLTLTKATRVIVVDPAWNPSTDNQSVDRAYRIGQTKNVIVYRLMTSATIEEKIYKLQV

LKGALFRTATEQKEQTRYFSKSEIQELFSLPQQGFDVSLTHKQLQEEHGQQVVLDESLRKHIQFLEQQGI

AGVSHHSLLFSKTATLPTLSENDALDSKPRGMPMMPQQYYKGSSSDYVANGASFALKPKDESFTVRNYIP

SNRSAESPEEIKARINRLSQTLSNAVLLSKLPDGGEKIRRQINELDEKLTSAEKGLKEGGTEVISLDD

Figure 8. The polypeptide sequence encoded by “Chromatin remodeling protein 24” gene which hosts the reg1 peak position on chromosome 2.

71

>NM_001347190.1 mRNA for CHROMATIN REMODELING PROTEIN 24

GGAACTGCAACTACAACCGCAATGTTGGTGGAGGTAGAGTCGTAGAGACTAGAGAGTCACACACGCTCGC

TGCTGCCTCTCCTTCCTCTCCTCGGCAAGTGAGAGAGGTGTGAACCGACGAGGAAGAGCACCCATCCCCA

CTGCGCATCCTCGATTCGATTCCGTCCCCTTGCCTCTTGCCTCTTGGGCGTCTCCGCGGCTAGATGGCGT

CTCCCCCTCCCTTCGACCTCTGCGGCGATCTCGACGACGACGACATCGATGAACCCGTCACCGCCGTCCC

CAAGGATCGCCATCTCCCTGCCGCCGCCCCTACGCCGAACGGCCTCAACGACCACCTCCTCCGCTTCACC

CGCAGCCTCCAGCGCCCCACCCAAAACCCTAACCCTAGCCCCAATCCGCCACCGCCTCCGCCCCCAGCCG

CCGCCGAAGAACAAGTCGAGAAGGTCAAGCTCGCCGGCCGCCGCCGCCTATGCAAGCTTGCCACCACCAC

CACCGCCACTCAGCGAGTGGATGAAGAGGAGGCGGAGTGGGACAACCAGGACGACGGTGAAAGCATACTC

GACATCCTAGACGACCTCACCACACGATTTGACTCTCTATCCGTCCAGAAGCCCAGCACCGCCGCGAGGT

CCAGGACACAACAGCTCACCCCTTTGCCGTGCGCCATCACCGTGGACGACGACCTAGATGACCATAGCCC

AGATGATGTGGATGCTCACGCCGGTGCCTCCTCACCCCTTCAAATTTCTAGCTCTGATGAAGCTAGGGCT

CCCACCAGACGCTCCGAGGTCAAGATCGAAACTGATTTAGTCTCCTCAGCCTGTACCCATTATGCCTGTG

ATGACGTCCGTGGCAAGGGGAAGAACAAAGGGACCACCAAGGATGTTGGGAGGCTAAATAGGGTATCAAA

GGCCTCATCCTTTGTTGATTCTTATTCCGATTCTGATTATGACGACTGCGAGGAGGACCAAGGAACAAGA

ACAGATTATGCTGTTAAGCAGCTAAGAAGCAAGGGATTCACAAGGAGACCACCCAACACCCCAACATTCA

GGAACCATGGTGTCAGCGACGATGAGCTGGGTCAGGAGAAGGAGAACCTTGGAGCTGTGGAGAACAATGC

TGAGGATGTTGGATGGGAGAAGACAGAGGACTTCAAGATGGATCCAACTGGAACTGCTGCAACATCCAAG

CCATACAAGCTCCCAGGAAAGATATTCAAGATGCTTTTCGCCCACCAGCGCGAGGGCCTCCGATGGCTCT

GGGTTCTGCACTGCAGGGGAACAGGAGGAATCCTAGGGGATGACATGGGTCTTGGCAAGACGATGCAGGT

TGCTGCATTTTTGGCTGGACTGTTTCATTCTCGTCTAGTCAAGAGGGTGCTCATTGTTGCTCCAAAGACA

CTTCTGGCCCATTGGACAAAGGAGCTTTCAATTGTTGGCCTTAAAGAAAAGATCAGAGACTACTCTGGCC

CCAGCACAAATATTCGCAATTATGAACTCCAATATGCCTTCAAGGAGGGTGGTATCCTCATAACCACCTA

TGACATTGTCAGGAACAACTACAAGCTCATAAGAGGCAACTCCTACAACAACAGCAATGATGATGATGAT

GAGGAAGGAACTTTGTGGAATTACGTAATTCTTGATGAGGGACATCTAATAAAAAATAATAAGACACAAA

GGGCCCAAAGTTTGTACGAAATACCTTGTGCCCATCGCATTGTGATCAGTGGAACACCTATTCAAAATAA

CTTGAAGGAAATGTGGACTCTGTTCAATTTCTGTTGCCCAGATGTCTTGGGTGATAAACAGCAGTTCAAA

ATAAGGTATGAAACGGCTATCCTTCGAGGAAATGACAAAAATGCTACCGCTCGAGAGAAGCACGTAGGCT 72

CAAATGTAGCAAAGGAACTAAGAGAGCGAATCAAGCCATACTTTTTGCGGCGCCTGAAAAGTGAAGTTGT

CTTTGATACTGGTGCATCAGAAGAAAAAACATTAGCCAAGAAGAATGAGCTAATTGTCTGGCTGAAGTTA

ACACCATGCCAGAGGAAACTATATGAAGCTTTTCTAAATAGTGAGCTGGTTCATTTAGCATTGCAGCCAA

AGGCATCACCGTTGGCTGCAATCACAATATTGAAGAAAATATGTGATCATCCACTGCTATTAACTAAGAA

AGGTGCTGAGGGTGTGTTGGAAGGAATGGGTGAAATGTTGAATGATCAAGACATTGGAATGGTGGAAAAA

ATGGCCATGAACCTTGCAGATATGGCTCATGATGATAATGCACTGGAAGTTGGTCAGGATGTCTCATGCA

AGCTATCATTCATCATGTCCTTGTTGCGGAACCTTGTTGGAGAGGGGCATCATGTTTTAATATTTTCACA

GACTCGTAAAATGCTAAACCTTATTCAGGAAGCTATAATATTAGAGGGCTATGCGTTTTTGCGCATTGAT

GGCACCACCAAGGTTTCTGACCGGGAAAGGATTGTGAAGGACTTCCAAGAGGGTTGTGGAGCTCCAGTTT

TTCTGCTAACCACACAAGTTGGTGGGCTTGGACTTACACTCACCAAGGCAACTCGTGTCATTGTAGTTGA

TCCTGCATGGAACCCTAGTACAGACAATCAAAGTGTTGATCGTGCTTACCGAATTGGACAGACTAAAAAT

GTGATTGTATACCGCTTGATGACATCTGCGACCATTGAAGAAAAGATATACAAATTGCAGGTTTTGAAGG

GCGCTCTGTTCAGGACAGCTACGGAGCAAAAAGAGCAAACACGTTACTTCAGCAAGAGTGAGATTCAAGA

GCTATTTAGTTTGCCACAACAAGGATTTGATGTTTCCCTCACACATAAGCAGTTGCAAGAAGAGCATGGT

CAACAAGTTGTTCTGGATGAGTCCTTGAGGAAGCATATACAGTTTCTGGAGCAACAAGGAATAGCCGGTG

TGAGTCATCACAGCCTCCTATTCTCTAAAACTGCAACCCTGCCCACTCTGAGTGAGAATGATGCACTGGA

CAGCAAACCTCGGGGCATGCCCATGATGCCCCAGCAATATTACAAGGGATCCTCATCTGACTATGTCGCC

AACGGGGCATCTTTTGCGCTGAAGCCAAAGGATGAAAGTTTCACTGTTCGAAACTACATTCCAAGTAACA

GAAGCGCAGAGAGTCCTGAAGAGATAAAGGCAAGAATCAACCGGCTTTCACAGACCCTCTCCAACGCTGT

GCTGTTGTCGAAGCTACCAGATGGTGGTGAGAAGATAAGGAGGCAGATAAATGAGCTGGACGAGAAGCTG

ACTTCTGCTGAGAAGGGGCTGAAGGAGGGGGGCACTGAAGTGATTTCCTTGGATGACTGATCCAAGACAT

GGAGAGTCTGTGCTCGGCAAAAGTAAAGTGTTTTGAATAGCTTTAGTCACTGGGTTGTGACTAGCATCAA

TCAAGTCTGCTCTTTTTGCTGCATCTCTGGGCTGGGTCTATCGTTTATGCAATACAATGCTTTTTCTGAT

GATGATTATATGAATAATATAATCCCCAGACAAAGCAGCAACCATTACAGTC

Figure 9. The coding sequence of “Chromatin remodeling protein 24” gene which hosts the reg1 peak position on chromosome 2. 73

>NP_001349598.1 mRNA for flocculation protein FLO11 [Zea mays]

MGEVTMRPLGYVTLLAIFFAIHLHLCVSSRLLAERPATPNGQESAAAAAPVPSAVGSLPENTPASNDVPV

NMPAKANAVTGAAAPVPSAAGSLSENTPASNVPVNMPANANAAIGAAAPVPPAAGSLLEYTPATNVSVTM

PANANAATGAAAPVPSTVGSLPENTPASNVPVNMPANTNTATGAAAPVPPTAGSLLEYTPATNVPVTMPA

NANAATGAAAPVPSAAGSLPENTPASNVPVNMPANANAATGAAAPVPSATGSLPENTPASNVPVNMPANA

NAATGATPPVPPPAGSLLEYTPATNVPVTMPANANPATGASAPVPPATGSLPENTPTTNVPVTMLANANV

ATGAPPPVPPAVGSLSENIPATNVPVTMPATATTGAPVPPAAGSLPENTPATNVPVTMPANTATGAPVPP

AAGSLPENTPATNVPVTMPANANANAATGAPAPVPPAVGSLPENTQTTNVPVTIPNTATGSPAAVPPATG

SLPENTPATNVPVNMPANANQPANVVPPQVVVSKLPPEVLAKLPPDVLASIPPETLANIAASQGQLQTSE

ILATIPAAQAQGQLPADLPPEVLAKLPAVQSQLPANITPEMMTSLAAVQQPAAAGQPGAAPALPADIPQI

PKMPDLSGLANISFPAMPSEPRMPQLPPDFSLYGYEVPIPKFITKMVDGNGAGGGQQN

Figure 10. The polypeptide sequence encoded by “Flocculation protein FLO11” gene which neighbors the reg2 peak position on chromosome 7.

74

>NM_001362669.1 flocculation protein FLO11

CCATTTTTTGCTGGGTCTGGAATCTCGATTCCATGGGAGAAGTCACAATGAGGCCTCTCGGCTACGTGAC

TCTCCTGGCCATTTTTTTCGCTATCCACCTACACCTCTGTGTGTCCTCGCGCCTCCTCGCCGAACGTCCG

GCGACTCCAAATGGCCAGGAATCAGCGGCGGCGGCGGCGCCGGTGCCTTCCGCCGTTGGGTCGTTGCCGG

AGAACACCCCGGCGAGCAACGACGTCCCGGTGAACATGCCCGCCAAGGCCAACGCAGTGACAGGTGCAGC

GGCGCCGGTCCCTTCCGCCGCCGGGTCGTTGTCGGAGAACACTCCGGCGAGCAACGTCCCGGTGAACATG

CCCGCCAACGCCAATGCAGCGATAGGTGCAGCGGCGCCGGTCCCTCCCGCCGCTGGGTCGTTGTTGGAAT

ACACCCCAGCGACCAACGTCTCGGTGACCATGCCCGCCAACGCCAACGCAGCGACAGGTGCAGCGGCGCC

AGTCCCTTCCACCGTCGGGTCGTTGCCGGAGAACACTCCGGCGAGCAACGTCCCGGTGAATATGCCCGCC

AACACCAACACAGCGACAGGTGCAGCGGCGCCGGTCCCTCCCACCGCCGGGTCGTTGCTAGAATACACCC

CAGCGACCAACGTCCCGGTGACCATGCCCGCCAACGCCAACGCAGCAACAGGTGCAGCGGCGCCAGTCCC

TTCCGCCGCTGGGTCGTTGCCGGAGAACACTCCGGCGAGCAACGTCCCGGTGAACATGCCCGCCAATGCC

AACGCAGCAACAGGTGCAGCGGCGCCAGTCCCTTCCGCCACCGGGTCGTTGCCGGAGAACACTCCGGCGA

GCAACGTCCCGGTGAACATGCCCGCCAACGCCAACGCAGCGACAGGTGCAACGCCGCCGGTCCCTCCCCC

TGCTGGGTCGTTGCTGGAATACACCCCAGCGACCAATGTCCCGGTGACCATGCCCGCCAATGCCAACCCA

GCGACAGGTGCATCGGCGCCGGTCCCTCCCGCCACCGGGTCGTTGCCGGAGAACACCCCGACGACCAACG

TCCCGGTGACCATGCTCGCCAACGCCAACGTAGCGACAGGTGCACCGCCACCGGTCCCTCCCGCCGTCGG

GTCGTTGTCGGAGAACATCCCGGCGACCAACGTCCCGGTGACCATGCCTGCCACCGCAACGACAGGTGCA

CCGGTCCCTCCTGCCGCTGGGTCGTTGCCGGAGAACACCCCGGCTACCAACGTCCCGGTGACCATGCCCG

CCAACACAGCGACAGGTGCACCGGTCCCTCCCGCTGCCGGGTCGTTGCCGGAGAACACCCCGGCGACCAA

CGTCCCGGTGACCATGCCCGCCAACGCCAACGCCAACGCAGCGACAGGTGCACCGGCGCCGGTCCCTCCC

GCCGTCGGGTCGTTGCCGGAGAACACCCAGACGACCAACGTCCCGGTGACCATACCCAACACAGCGACAG

GTTCACCCGCGGCGGTCCCTCCCGCCACCGGGTCGTTGCCGGAAAACACCCCGGCGACCAACGTCCCAGT

AAACATGCCCGCCAACGCCAACCAGCCGGCCAACGTCGTTCCACCGCAAGTAGTGGTGTCGAAGCTTCCG

CCGGAGGTGCTGGCCAAGCTCCCGCCCGACGTGCTGGCCAGCATCCCGCCGGAGACGCTGGCCAACATCG

CGGCGTCGCAGGGGCAGCTGCAGACGAGCGAGATCCTGGCCACCATCCCGGCGGCGCAGGCGCAGGGCCA

GCTGCCGGCGGACCTGCCGCCGGAGGTGCTGGCCAAGCTCCCGGCCGTGCAGAGCCAACTGCCGGCCAAC 75

ATTACGCCCGAGATGATGACCAGTCTCGCCGCCGTGCAGCAGCCTGCGGCTGCTGGCCAGCCTGGGGCGG

CCCCGGCTCTCCCGGCCGACATCCCTCAGATCCCCAAGATGCCCGACCTCTCCGGGCTGGCCAACATCTC

GTTCCCGGCGATGCCGTCGGAGCCCAGGATGCCGCAGCTGCCGCCCGACTTCTCGTTGTACGGGTACGAG

GTCCCCATCCCTAAGTTCATCACCAAGATGGTCGACGGCAATGGCGCCGGCGGTGGGCAACAAAACTGAG

CCGAAGCATTGTTTCGATGTTTGTGGCACCTGAGCTGTCAATTACACGTTAACTGAAGTTTCGCCATGCT

TTTCAGTTCTAATGTCGTCATTCTTTTGGTTTGGGTGATGTCTGGCAGGAAATGTGATGATGGGCTATGT

TCTTGTCATGTATATACTCTCTTTGTCCTAAATTATAAATTGTTTTATACTTTTTAAATTCAAAATATCT

AAAATCAATTAAATTTATGTTTTTGTT

Figure 11. The coding sequence of “Flocculation protein FLO11” gene which neighbors the reg2 peak position on chromosome 7.

76

Figure 12: F4 NIL screening using the allele-specific markers