Molecular Analysis of Oryza latifolia Desv. (CCDD Genome)-Derived Introgression Lines and...

Journal of Hereditydoi:10.1093/jhered/esu032

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Molecular Analysis of Oryza latifolia Desv. (CCDD Genome)-Derived Introgression Lines and Identification of Value-Added Traits for Rice (O. sativa L.) ImprovementRosalyn B. angeles-shim, Ricky B. VinaRao, BalRam maRathi, and kshiRod k. Jena

From the Novel Gene Resources Laboratory, Plant Breeding, Genetics, and Biotechnology Division, International Rice Research Institute, DAPO Box 7777, Metro Manila, Philippines (Angeles-Shim, Vinarao, Marathi, and Jena); and the Bioscience and Biotechnology Center, Nagoya University, Chikusa, Nagoya, Aichi 464-8601, Japan (Angeles-Shim).

Address correspondence to Kshirod K. Jena at the address above, or e-mail: [email protected].

AbstractOryza latifolia is a tetraploid wild Oryza species with a CCDD genome that has been reported to harbor resistance to bacterial blight (BB), brown planthopper, and whitebacked planthopper. Aside from these traits, O. latifolia is also being tapped as a new source of resistance to lodging and high biomass production. To explore the genetic potential of O. latifolia as a novel genetic resource for the improvement of existing O. sativa cultivars, 27 disomic derivatives of O. latifolia monosomic alien addition lines (MAAL) were characterized for alien chromosome segment introgressions and evaluated for yield components, BB resistance, and strong stem characteristics. A total of 167 simple sequence repeat, sequence tagged site, and single nucleotide polymor-phism markers, along with newly developed indel markers that were specifically designed to detect O. latifolia chromosome seg-ment introgressions in an O. sativa background, were used to define alien introgressions in 27 disomics derived from O. latifolia MAALs. Genotype data showed that 32 unique introgressions spanning 0.31–22.73 Mb were introgressed in different combina-tions in each of the 27 disomic derivatives. Evaluation of the disomic derivatives for agronomic traits identified lines with puta-tive QTLs for resistance to Philippine races 3A, 4, 9A, and 9D of BB. Putative quantitative trait loci (QTLs) conferring strong stem in 19 out of the 27 disomic derivatives studied were also identified from O. latifolia introgressions on chromosome 6.Subject areas: Genomics and gene mapping; Conservation genetics and biodiversity

Key words: disomics, DNA markers, lodging resistance, monosomic alien addition lines, wild introgression, Xanthomonas oryzae pv. oryzae

The genus Oryza is relatively small but complex, consisting of 2 cultivated species, Oryza sativa and O. glaberrima, and 22 well-recognized wild relatives (Ge et al. 1999; Zeigler 2013). Based on classical taxonomy and isozyme, restric-tion fragment length polymorphism (RFLP) and sequence analysis, the members of this genus are grouped into 4 species complexes, namely, O. sativa, O. officinalis, O. ridleyi, and O. granulata. Species belonging to the O. sativa complex, including both cultivated species, are diploids (2n = 24) with AA genome. The more distantly related species are either diploid or allotetraploid (2n = 48) with BB, CC, EE, FF, GG, BBCC, CCDD, HHJJ, and KKLL genomes (Khush 1997; Ge et al. 1999; Vaughan et al. 2003; Lu et al. 2009; Sanchez et al. 2013).

The wild relatives of rice have long been recognized as a rich source of novel genes underlying traits of agricul-tural importance (Jena and Khush 1990). Earlier attempts to transfer useful genes from wild to cultivated rice have been confined to using only the wild relatives with AA genome via conventional breeding methods. The F1 hybrids that are derived from such crosses exhibit regular chromosome pair-ing and recombination, thus facilitating an easy transfer of genes into cultivated rice (Khush et al. 1990; Brar and Khush 1997; McCouch et al. 2007). In contrast, F1 hybrids between cultivated rice and wild species with genomes other than AA are often difficult to produce. Incompatibility barriers and nonhomologous chromosome pairing result in low crossabil-ity and abortion of hybrid embryos. However, male-sterile

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interspecific F1 hybrids could be produced by embryo rescue (Jena and Khush 1984; Brar and Singh 2011). These hybrids were used as female parents in subsequent backcrossing to produce fertile progenies with normal diploid chromosome complements (2n = 24 or 2n = 24 + 1) (Jena 2010).

Rice plants carrying the normal chromosome comple-ment of O. sativa and an extra chromosome from any wild species (2n + 1) are known as monosomic alien addition lines (MAALs). MAALs are important genetic stocks for broad-ening the existing gene pool of rice. They are useful in the assignment of alien traits to specific chromosomes of the wild relatives and in transferring genes of economic impor-tance from wild species to cultivated rice (Brar and Khush 1997; Jena 2010). MAALs can also provide a convenient way of dissecting wild rice genomes into individual chromosome entities in a functional genomic background. To date, MAALs of O. sativa, trisomic plants carrying single additional chro-mosome, from 1 of 6 different wild rice species have been established. These sets of MAALs include a complete set of MAALs for chromosomes 1–12 of O. officinalis (Jena and Khush 1989) and O. australiensis (Jena et al. 1991); MAALs for 8 chromosomes of O. minuta (Amante-Bordeos et al. 1992); and MAALs for 6, 7, and 11 chromosomes of O. granulata, O. brachyantha, and O. latifolia, respectively (Multani et al. 2003).

The disomics (2n) segregate in the progenies of MAALs. The disomics are fertile backcross progenies carrying the normal diploid chromosome complement of O. sativa and limited chromosome introgressions from the wild parent. These progenies mostly resemble the O. sativa parent and are usually recovered in selfed progenies of MAALs. The extremely limited homeologous pairing between the chro-mosomes of cultivated rice and its wild relatives has been proposed to facilitate the rapid recovery of the recurrent phenotype (Stephens 1949; Rick 1971; Jena et al. 1992). Like the MAALs, disomics constitute a valuable genetic resource for the identification of novel genes that control important agronomic traits. Continuous selfing of these disomics pro-duces advanced introgression lines that carry short chromo-somal segments from the wild relative. The uniform genetic background of these introgression lines allows for easy association of a phenotype with the introgressed chromo-some segment from the wild relative. Because of this, QTLs, even those with only minor effects, are easily detected. The uniform genetic background of MAAL-derived disomic introgression lines also provides an advantage in identifying genes/QTLs using simple statistical analysis.

O. latifolia is a tetraploid (CCDD) wild Oryza species endemic to South and Central America. The species is char-acteristically perennial, with tall, erect plants having broad foliage and high biomass, hard stem, and long, lax panicles with a large number of spikelets. It can grow in a wide range of ecosystems, including swamps, savannas, woodlands, hill slopes, and coastal belts (Ying and Song 2003). O. latifolia has been reported to be an important source of resistance to bac-terial leaf blight (BB), brown planthopper, and whitebacked planthopper (Multani et al. 2003) and is being explored for high biomass production and lodging resistance (Jena KK, unpublished data).

MAALs of O. latifolia and their disomic derivatives have been established at the International Rice Research Institute (IRRI) since 2000. However, because of the lack of available DNA markers that are specific for O. latifolia, molecular char-acterization of alien introgressions in disomic lines has not been possible and therefore they cannot be used in breeding programs.

This study aims to define alien introgressions in disomic derivatives of O. latifolia in the genetic background of O. sativa. The simple sequence repeats (SSRs), single nucleotide poly-morphisms (SNPs), and newly developed indel markers that amplify distinctive regions in both O. sativa and O. latifolia genomes were used to identify the wild chromosome segment introgressions in the disomic derivatives. Agronomic evalua-tion of these introgression lines identified value-added traits for yield and yield components and disease resistance for the improvement of existing rice cultivars. Experimental validation of culm strength in the disomic derivatives also identified lines with potential to improve lodging resistance in cultivated rice.

Materials and MethodsPlant Materials

Twenty-seven introgression lines derived from the MAALs 4, 5, 6, 7, 8, 9, 10, 11, and 12 of O. latifolia were selected from a total of 216 plants and used in this study (Table 1). No MAALs for chromosomes 1, 2, and 3 of O. latifolia were available. The MAALs were developed by successive back-crossing of the allotriploid F1 hybrid derived from a cross between an O. sativa elite breeding line (IR31917-45-3-2) and O. latifolia (IRGC 100914) to the O. sativa parent. All intro-gression lines have 2n = 24 and carry a limited number of chromosome segments from O. latifolia (Multani et al. 2003). The breeding scheme used for the development of the intro-gression lines from the O. latifolia MAALs is presented in Figure 1.

Development of O. latifolia-Specific Primers and Detection of Alien Introgression in Disomics

A total of 843 O. sativa-based markers that include 293 SSRs, 166 sequence tagged site (STS), and 384 SNPs were used to determine the extent and location of O. latifolia chromosome segment introgressions in 27 disomic derivatives of O. latifo-lia. The 384-plex SNP set used was a GoldenGate VeraCode oligo pool assay (OPA; VC0013033 OPA) designed for the Illumina BeadXpress Reader (Illumina Inc., San Diego, CA) to differentiate among indica/indica populations (Thomson et al. 2012). For a fuller coverage of the genome, additional indel-based markers were designed based on the existing sequence data for O. sativa and the CCDD genome species O. alta (www.ncbi.nlm.nih.gov; 5 February 2012). Sequence information for O. latifolia is still unavailable. Since O. alta shares the same CCDD genome as O. latifolia, the available bacterial artificial chromosome (BAC) sequences for O. alta were used. Sequence alignment of a total of 127 784 BAC end sequences of O. alta (IRGC 105143) to v7 of the genomic

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sequence of O. sativa subsp. japonica cv. Nipponbare from TIGR-MSU was kindly provided by Professor R. Wing of the Oryza Map Alignment Project (OMAP) of the Arizona Genomics Institute of the University of Arizona, Tuscon, AZ (Ammiraju et al. 2010; Jacquemin et al. 2013). The align-ments were generated using MEGABLAST, an alignment

tool specifically designed to find long alignments between similar sequences, and hence it is the best tool in finding an identical match to any query sequences. Using these align-ments, 35 indel-based markers were designed and validated for their usefulness in differentiating between O. latifolia and O. sativa genomes.

Table 1 Classification of the 27 introgression lines derived from Oryza latifolia based on alien introgression patterns

O. latifolia MAAL source Disomic derivativeIntrogressed chromosome fragment from O. latifolia Introgression type

MAAL 4 WH12-2236 1, 2, 3, 4, 6, 9, 11 MAAL4IWH12-2237 1, 2, 3, 4, 6, 9, 11 MAAL4IWH12-2238 1, 2, 3, 4, 6, 9, 11 MAAL4IWH12-2239 1, 2, 3, 4, 6, 9, 11 MAAL4I

MAAL 5 WH12-2240 4, 5, 6, 8, 12 MAAL5IWH12-2241 4, 5, 6, 8, 12 MAAL5IWH12-2242 4, 5, 6, 8, 12 MAAL5IWH12-2243 4, 5, 6, 8, 12 MAAL5IWH12-2244 4, 5, 6, 8, 12 MAAL5IWH12-2245 4, 5, 6, 8, 12 MAAL5IWH12-2246 4, 5, 6, 8, 12 MAAL5IWH12-2247 4, 5, 6, 8, 12 MAAL5I

MAAL 6 WH12-2248 6, 8 MAAL6IWH12-2249 6, 8 MAAL6IWH12-2250 6, 8, 10 MAAL6IIWH12-2251 6, 8, 10 MAAL6II

MAAL 7 WH12-2252 1, 2, 3, 4, 5, 6, 8, 9 MAAL7IWH12-2253 1, 3, 4, 6, 8, 9, 10, 11 MAAL7IIWH12-2254 1, 3, 4, 6, 8, 9, 10, 11 MAAL7II

MAAL8 WH12-2255 1, 2, 6, 8, 9, 10, 12 MAAL8IWH12-2256 1, 2, 4, 6, 8, 9, 10, 12 MAAL8II

MAAL 9 WH12-2257 4, 6, 8, 10 MAAL9IMAAL 10 WH12-2258 1, 6, 8, 10 MAAL10I

WH12-2259 6, 8, 10 MAAL10IIMAAL 11 WH12-2260 6, 8, 10 MAAL11IMAAL 12 WH12-2261 6, 8, 10 MAAL12I

WH12-2262 6, 8, 10 MAAL12I

Figure 1. Breeding scheme for the production of Oryza latifolia introgression lines.



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Agronomic Evaluation of the Disomic Derivatives of O. latifolia for Value-Added Traits

Phenotypic evaluation of the 27 introgression lines was car-ried out during the dry and wet seasons of 2012 at the IRRI in Los Baños, Laguna, Philippines. Twenty-four seedlings of each line, along with the O. sativa elite breeding line IR31917-45-3-2 as a control, were grown to maturity in double-row plots in the field. The seedlings were first germinated in seed-beds and 21-day-old seedlings were transplanted in field plots at 20 × 20-cm spacing.

At maturity, 5 healthy plants were selected from each of the introgression lines and phenotyped for plant height, pani-cle length, number of primary branches per panicle, grain number per panicle, grain length and width, 100-grain weight, and fertility. The number of days to heading was determined from the time of sowing up to the actual flowering of 50% of the plants for each line. Data on 100-grain weight were recorded after air drying of seeds in the glasshouse for 7 days to achieve a moisture content of 13–14%.

The selection of the 27 introgression lines for inclu-sion in this study was based on field evaluation for strong stem. To verify the physical strength of the stems, the sec-tion modulus (SM), a parameter that measures the physical strength of the culm and that is directly influenced by culm morphology (i.e., diameter and wall thickness), was calcu-lated for each of the test materials. Twenty days after head-ing, 5 main culms from each line were sampled and fixed in 1:1:18 FAA (35% formalin:glacial acetic acid:70% alcohol). After a series of washes, tissue samples were sectioned and stained for 4 min with phloroglucinol-hydrochloric acid. Cross-sections of the third internodes of each culm were viewed under an Axioplan stereomicroscope and images were captured using an Olympus DP71 camera. Processing of the captured images for dimensional measurements was carried out using the ImagePro (ver. 7) software. SM was calculated using the formula SM = π/32 × (a1

3b1 − a2

3b2)/a1, where a1 is the outer diameter of the minor axis in an oval cross-section, b1 is the outer diameter of the major axis in an oval cross-section, a2 is the inner diameter of the minor axis in an oval cross-section and b2 is the inner diam-eter of the major axis in an oval cross-section following Ookawa et al. (2010).

Each of the introgression lines, along with IR31917-45-3-2 as a control, was screened for reaction to BB caused by Xanthomonas oryzae pv. oryzae (Xoo) in the screenhouse during the wet season of 2012. The resistance reaction of selected lines during the wet season screening was con-firmed during the dry season of 2013. The inocula of the 10 Philippine races of the pathogen, PXO61 (race 1), PXO86 (race 2), PXO79 (race 3A), PXO340 (race 3B), PXO71 (race 4), PXO112 (race 5), PXO99 (race 6), PXO145 (race 7), PXO280 (race 8), PXO339 (race 9A), PXO349 (race 9B), PXO347 (race 9C), PXO363 (race 9D), and PXO341 (race 10), were prepared by suspending the bacterial mass in sterile water at approximately 109 cells/ml. Inoculation was carried out during the maximum tillering stage of the plants fol-lowing the leaf clipping method of Kauffman et al. (1973).

Scoring for the disease reaction of the test materials 14 days after inoculation was based on lesion length: <3 cm = resist-ant, 3.1–5 cm = moderately resistant, and >5 cm = suscepti-ble (Jeung et al. 2006; Suh et al. 2013).

Statistical Analysis and Detection of Quantitative Trait Loci for Agronomic Traits

A one-way Anova and comparison of means using Dunnett’s multiple comparisons test at 95% confidence interval (P < 0.05) were performed. A probability of 0.005 was used as the threshold for the detection of putative QTLs control-ling any of the traits examined. If the calculated mean value for a trait in any introgression line was significantly different from that of IR31917-45-3-2, the existence of a QTL was considered and assigned in the introgressed segment from the wild donor.

ResultsMolecular Characterization of O. latifolia Introgression in an O. sativa Background

Of the 878 molecular markers used to genotype the 27 intro-gression lines, 169 (38 SSRs, 17 STS, 85 SNPs, and 29 indels) markers detected polymorphism between O. sativa and O. lati-folia. These markers were distributed across the 12 chromo-somes, providing genomic coverage at an average interval of 2.25 Mb. Markers targeting an approximately 12-Mb region spanning the short arm up to the distal part of the long arm of chromosome 10 of O. sativa were not informative when used to amplify targets in both IR31917-45-3-2 and O. latifo-lia, leaving this region on chromosome 10 uncharacterized for O. latifolia introgression (Figure 2).

Genotyping of the disomic derivatives identified a total of 32 unique introgressions of O. latifolia chromosome seg-ments ranging from 0.31 Mb on chromosome 3 to 22.73 Mb on chromosome 6. Based on the different combinations of these wild chromosome segment introgressions, 12 distinct patterns of O. latifolia introgression were observed. Two types of introgression patterns were observed for the lines derived from MAALs 6, 7, 8, and 10, whereas only a single introgres-sion pattern each was observed for the lines derived from MAALs 4, 5, 9, 11, and 12. Introgression lines from MAAL 4 recorded the highest number (11) of introgressions, whereas the derivatives of MAAL 6I recorded the lowest number (2) of wild introgressions (Table 1, Figure 3). Lines belong-ing to MAAL 10II and MAAL 11I had the same introgres-sion patterns. All introgression lines carried different sizes of O. latifolia chromosome segment on chromosome 6, and the introgression lines belonging to MAAL 4I and MAAL 12I harbored O. latifolia segments measuring up to 9.42 Mb. Introgression lines belonging to MAAL 7I carried O. latifolia introgressions covering 22.73 Mb on the same chromosome, whereas the rest of the lines harbored an O. latifolia segment that spanned 22.23 Mb. There was no O. latifolia chromosome segment introgression on the chromosome 7 of the recur-rent parent genome (Figure 3).



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Phenotypic Evaluation of the Introgression Lines

Examination of gross plant morphology showed a close resemblance of the 27 introgression lines to the O. sativa par-ent. None of the undesirable traits of the O. latifolia parent, particularly the tall stature and spreading growth habit, were observed in any of the test materials (Figure 3). Data on the morphometric measurements for different agronomic traits of the 27 introgression lines are presented in Table 2.

Heading date during the dry season was significantly earlier in the introgression lines (80–97 days) than in IR31917-45-3-2 (99 days), except for the 5 lines (WH12-2241, WH12-2243, WH12-2244, WH12-2245, and WH12-2246) that recorded increased number of days to heading (98–100 days) than the control. In the wet season, no significant differences were observed in the heading date of the introgression lines and IR31917-45-3-2.

On average, the introgression lines had a significantly shorter stature (92.9 cm) during the dry season compared with IR31917-45-3-2 with a recorded mean plant height of

99.00 cm. The mean values for panicle length were higher in lines of introgression types MAAL 5I, MAAL 71, MAAL 7II, MAAL 8I, MAAL 8II, MAAL 9I, MAAL 10I, MAAL 10II, and MAAL 12I than in IR31917-45-3-2 during both the dry and wet seasons except for line WH12-2259 of the introgression type MAAL 10II, which recorded shorter pani-cles than IR31917-45-3-2. Significantly longer panicles were observed in lines WH12-2241, WH12-2244, WH12-2245, and WH12-2252 during the dry season (25.71–25.85 cm) and line WH12-2242 during the wet season (26.20 cm).

Alien introgressions from O. latifolia had no significant effects on the number of primary branches per panicle of the test materials during the wet season. However, during the dry season, the lines WH12-2241, WH12-2257, WH12-2259, and WH12-2262 recorded 20% more primary branches than IR31917-45-3-2.

The spikelet fertility of the introgression lines and IR31917-45-3-2 did not show significant differences except the line, WH12-2238 of the introgression type MAAL 4I and the line WH12-2246 of the introgression type MAAL

Figure 2. Molecular markers used for the whole-genome survey of the 27 introgression lines derived from O. latifolia MAAL disomics. Numbers on the left indicate the physical distance (Mb) of each marker (right) along the length of each chromosome. Arrowheads represent the position of the centromere on each chromosome. Marker identification is as follows: rice microsatellite (RM) = SSRs, id and fd = SNPs (blue), Lat = indel-based markers (green) and S = STS.



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Figure 3. Morphology and genotype of the plant materials. Gross morphology and graphical genotype of the O. sativa elite breeding line IR31917-43-2, O. latifolia (IRGC 100914), and representative plants from each of the 12 introgression types identified from the 27 introgression lines derived from O. latifolia disomics. Red circles indicate the 32 unique O. latifolia chromosome segments carried by the introgression lines.



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Tabl

e 2

Mor

phom

etric

mea

sure

men

ts fo

r yie

ld a

nd y

ield

com

pone

nts o

f 27

intro

gres

sion

lines

der

ived

from

O. l

atifo

lia

Intr

ogre

ssio

n

line

nam

eIn

trog

ress

ion

ty

pe

Hea

ding

da

te (

days

)Pl

ant

he

ight

(cm

)Pa

nicl

e

leng

th (

cm)

Num

ber

of p

rim

ary

bran

ches

Fert

ility

(%

)10

0-gr

ain

w

eigh

t (g

)G

rain

le

ngth

(m

m)

Gra

in

wid

th (

mm

)

Num

ber

of

grai

ns p

er

pani

cle

DS

WS

DS

WS

DS

WS

DS

WS

DS

WS

DS

WS

DS

WS

DS

WS

DS

WS

IR31

917-

45-3

-299

102

113.

8010

5.60

23.4

623

.40

1012

90.7

589

.87

2.84

2.91

9.80

9.01

2.95

2.62

100

130

WH

12-2

236

MA

AL4

I91

**10

287

.87*

*10

1.20

21.7

122

.00

1112

92.3

381

.39

2.23

**2.

40**

8.83

8.93

2.67

**2.

5511

015

0W

H12

-223

7M

AA

L4I

92**

102

90.8

0**

99.4

022

.40

22.2

011

1294

.76

90.1

62.

32**

2.35

**8.

709.

032.

69**

2.53

123

157

WH

12-2

238

MA

AL4

I97

**10

290

.20*

*98

.80

22.7

321

.00

1112

75.1

2**

83.1

72.

33**

2.33

**7.

439.

062.

70**

2.60

128

129

WH

12-2

239

MA

AL4

I95

**10

290

.20*

*96

.00

22.5

621

.90

1111

94.0

789

.63

2.34

**2.

29**

8.90

9.43

2.63

**2.

5613

2*14

9W

H12

-224

0M

AA

L5I

93**

102

89.0

0**

97.2

522

.62

21.8

811

1292

.90

88.6

82.

24**

2.34

**8.

979.

062.

63**

2.46

125

123

WH

12-2

241

MA

AL5

I99

106

96.8

0**

100.

2025

.71*

25.7

012

*13

90.4

681

.01

2.66

**2.

60**

8.77

8.91

2.87

2.77

120

151

WH

12-2

242

MA

AL5

I97

**10

694

.90*

*10

2.80

24.6

526

.20*

1113

88.9

386

.26

2.64

**2.

58**

8.77

9.04

2.89

2.88

119

162

WH

12-2

243

MA

AL5

I98

108

95.9

0**

102.

6024

.66

25.6

011

1390

.49

89.1

82.

62**

2.58

**9.

039.

242.

842.

8710

914

4W

H12

-224

4M

AA

L5I

9910

398

.50*

*10

0.80

26.3

2*25

.30

1212

92.7

186

.25

2.66

**2.

62**

9.20

9.31

3.01

2.82

131

140

WH

12-2

245

MA

AL5

I10

010

696

.30*

*10

5.60

25.8

5*25

.30

1113

91.9

779

.12

2.68

**2.

60**

9.08

0.00

2.90

0.00

121

148

WH

12-2

246

MA

AL5

I10

010

397

.40*

*10

3.00

25.5

425

.40

1112

89.5

074

.26*

*2.

712.

62**

9.03

9.39

2.85

2.81

120

146

WH

12-2

247

MA

AL5

I10

010

895

.70*

*99

.60

26.1

725

.50

1112

92.8

980

.14

2.63

**2.

67**

9.07

9.32

2.91

2.86

123

134

WH

12-2

248

MA

AL6

I93

**10

289

.90*

*10

1.20

23.7

624

.10

1112

94.4

288

.08

2.76

2.74

**9.

579.

88*

2.82

**2.

7211

513

8W

H12

-224

9M

AA

L6I

93**

102

93.0

0**

100.

0023

.30

24.1

011

1192

.83

90.4

12.

772.

72**

9.73

9.92

*2.

78**

2.76

9613

4W

H12

-225

0M

AA

L6II

96**

102

91.2

0**

105.

4024

.08

24.5

011

1391

.38

90.9

92.

762.

72**

9.67

9.87

*2.

842.

7610

514

3W

H12

-225

1M

AA

L6II

94**

102

93.4

0**

100.

4022

.23

22.6

011

1293

.21

87.4

42.

63**

2.76

**9.

409.

74*

2.83

2.75

106

112

WH

12-2

252

MA

AL7

I91

**10

286

.00*

*10

7.20

25.7

1*25

.50

1113

85.1

689

.92

2.89

2.82

9.70

10.0

9*2.

822.

7410

816

8W

H12

-225

3M

AA

L7II

88**

102

93.4

0**

109.

2024

.47

25.5

011

1293

.53

90.2

52.

702.

819.

4810

.12*

2.67

**2.

5610

715

1W

H12

-225

4M

AA

L7II

88**

102

91.8

0**

106.

6024

.55

25.1

011

1191

.39

92.1

52.

722.

849.

8010

.07*

2.68

**2.

5710

912

6W

H12

-225

5M

AA

L8I

94**

102

99.1

3**

104.

8024

.50

25.1

011

1293

.44

89.8

72.

732.

76**

9.83

9.95

*2.

76**

2.61

120

140

WH

12-2

256

MA

AL8

II94

**10

295

.80*

*10

6.00

23.9

324

.60

1111

93.0

389

.26

2.64

**2.

74**

9.40

10.2

3*2.

77**

2.54

118

127

WH

12-2

257

MA

AL9

I97

**10

294

.60*

*10

3.60

23.9

523

.40

12*

1287

.27

85.6

62.

59**

2.61

**9.

439.

342.

82**

2.69

114

132

WH

12-2

258

MA

AL1

0I80

**10

187

.00*

*92

.00

24.6

123

.60

1112

89.4

980

.93

2.56

**2.

63**

9.00

9.02

2.79

**2.

5912

013

0W

H12

-225

9M

AA

L10I

I80

**10

186

.40*

*78

.00*

*24

.68

22.6

012

*11

90.6

181

.94

2.57

**2.

77**

9.38

9.37

2.78

**2.

6011

811

6W

H12

-226

0M

AA

L11I

91**

102

95.0

0**

106.

4022

.91

23.7

011

1292

.11

85.9

32.

702.

75**

9.03

9.69

*2.

75**

2.59

106

131

WH

12-2

261

MA

AL1

2I97

**10

295

.40*

*10

4.40

24.0

124

.70

1111

92.6

881

.84

2.82

2.80

**9.

839.

68*

2.86

2.59

9813

2W

H12

-226

2M

AA

L12I

94**

101

93.8

0**

101.

2024

.73

23.5

012

*11

81.6

187

.48

2.73

2.89

9.50

9.82

*2.

892.

6910

111

7

DS,

dry

seas

on; W

S, w

et se

ason

. Val

ues p

rese

nted

are

mea

ns o

f 5

sam

ples

. Sig

nific

ance

cal

cula

ted

at P

< 0

.05.

*Mea

n va

lue

signi

fican

tly h

ighe

r tha

n IR

3191

7-45

-3-2

.**

Mea

n va

lue

signi

fican

tly lo

wer

than

IR31

917-

45-3

-2.



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Journal of Heredity

8

5I which recorded 17.2% and 17.4% lower fertility than the recurrent parent during the dry and wet season, respectively.

One hundred grain weight in all the introgression lines was lower than that of IR31917-45-3-2 during both the sea-sons except for the line WH12-2252 of the introgression type MAAL 7I, which recorded a higher mean 100-grain weight than IR31917-45-3-2 (2.84 g) during the dry season (Table 2).

Differences in the mean values for grain length during the dry season and grain width during the wet season were not significant between the introgression lines and IR31917-45-3-2. However, lines of the introgression types MAAL 6I, MAAL 6II, MAAL 7I, MAAL 7II, MAAL 8I, MAAL 8II, MAAL 11I, and MAAL 12I recorded significantly longer grains (9.74–10.23 mm) than IR31917-45-3-2 (9.01 mm) dur-ing the wet season. The mean values for grain width of the introgression lines during the dry season were lower than that of IR31917-45-3-2 (2.95 mm), except for line WH12-2244 of the introgression type MAAL 5I, which recorded an average grain width of 3.01 mm.

The number of grains per panicle for the introgression lines was not significantly different from that of IR31917-45-3-2 during both the dry and wet seasons except for line WH12-2239, which registered 32% more grains than the recurrent parent during the dry season.

The introgression lines used in this study were selected based on field evaluation for strong stem. SM analysis showed that all introgression lines had a higher SM than IR31917-45-3-2. Measurements were not taken for WH12-2259 due to the poor condition of the fixed samples. Of the remaining 26 introgression lines, 19 exhibited significant increases in SM ranging from 59.5% to 116.5% compared with the mean SM value observed for IR31917-45-3-2 (Figure 4a). Examination of the fourth internode cross-sections showed that all the 19 lines had a wider culm diameter than IR31917-45-3-2 (Figure 4b).

BB Resistance of the O. latifolia Introgression Lines

The disease reaction of IR31917-45-3-2 to different races of Xoo used in this study ranged from resistant to moder-ately resistant to susceptible based on the length of lesions in the leaf caused by the pathogen 14 days after inoculation (Table 3). IR31917-45-3-2 showed resistance to races 1, 5, 7, 8, and 10, with lesion lengths ranging from 1.17 to 2.83 cm, and moderate resistance to races 2 and 6, with lesion lengths of 3.67–4.00 cm. IR31917-45-3-2 was susceptible to races 3A, 3B, 4, 9A, 9B, 9C, and 9D, which caused 6.17–13.93-cm lesion length on the leaves. A wide range of reactions to the disease was similarly observed among the introgres-sion lines. Like IR31917-45-3-2, all the test materials were susceptible to races 3B, 9B, and 9C of BB but resistant to moderately resistant to races 1, 5, 7, and 8 of the pathogen. Most of the introgression lines also shared the suscepti-ble reaction of IR31917-43-2 to races 3A, 4, 9A, and 9D. However, some introgression lines showed resistance to these races. In particular, lines WH12-2255 and WH12-2256 showed strong BB resistance, with lesion lengths of only 1.83 and 1.67 cm, respectively, compared with the 12.00-cm

lesions in IR31917-45-3-2 inoculated with race 9A of the pathogen. Both lines have unique introgressions of chromo-some segments from O. latifolia on chromosome 12 span-ning 13.96 Mb. Introgression line WH12-2246 also exhibited resistance to race 9D, recording an average lesion length of 4.00 cm, whereas the rest of the introgression lines remained susceptible like the recurrent parent, O. sativa. Additionally, introgression lines WH12-2236, WH12-2237, WH12-2238, WH12-2240, WH12-2241, WH12-2247, WH12-2254, and WH12-2255 showed moderate resistance to race 3A, whereas lines WH12-2236, WH12-2237, WH12-2239, WH12-2240, WH12-2241, WH12-2244, WH12-2246, WH12-2247, and WH12-2259 showed moderate resistance to race 4 of the pathogen.

DiscussionTargeted Markers for Evaluating Wild Rice Derivatives

MAALs and MAAL-derived disomic introgression lines rep-resent an important genetic resource for both genetic and genomic studies. Molecular characterization of alien intro-gressions in these lines is particularly essential in the study of both single gene and quantitative trait inheritance from wild Oryza species. Isozymes, RFLPs, and genome-specific clones developed via representational difference analysis have been used to characterize alien introgressions in MAALs and their disomic derivatives (Huang and Kochert 1994; Jena et al. 1994; Multani et al 1994; Shim et al. 2010). However, the inherent limitations of each marker had restricted the char-acterization of wild introgressions in these materials, and consequently their application in breeding programs.

A genomic survey using 843 O. sativa-based markers in the form of SSRs, STS, and SNPs was carried out to iden-tify markers that can differentiate between O. latifolia and O. sativa alleles. Of these markers, only 16.5% (139) detected polymorphic targets in O. latifolia and O. sativa, with SNPs being the most informative (21.88% polymorphic), followed by the SSRs (12.97% polymorphic) and the STS (10.24% polymorphic). In comparison, indel-based markers that were designed based on sequence alignments between O. sativa cv. Nipponbare and O. alta exhibited 83% polymorphism.

SSRs have long been used as a marker of choice for appli-cations in plant breeding and genetic studies not only in rice but in other crops, including wheat (Röder et al. 1995), barley (Saghai Maroof et al. 1994), soybean (Maughan et al. 1995), tomato (Smulders et al. 1997), and corn (Senior and Heun 1993). However, a significant number of SSRs have rarely been transferred across species and used in alien introgres-sion studies (Röder et al. 1995). At best, the interspecific transferability of SSRs had been unpredictable. Studies on Hordeum chilense introgression in wheat showed that SSRs were suitable in identifying alien loci (Hernández et al. 2002), whereas reports on the introgression of Lophopyrum elon-gatum in wheat showed the genome specificity of SSRs and, consequently, their low transferability across related species (Mullan et al. 2005). In rice, diversity studies have shown that a high proportion of SSRs designed for O. sativa were



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9

successfully amplified in O. glaberrima (Lorieux et al. 2000) as well as 5 other wild rice relatives with AA genome (Wu and Tanksley 1993; Panaud et al. 1996; Chen et al. 2002b). However, characterization of alien addition from O. officinalis (CC genome) in an O. sativa background using SSRs showed low efficiency of the markers in detecting the alien loci (Jin et al. 2006). SSR markers are based on tandemly arranged repetitive DNA sequences that represent a significant part of a eukaryotic genome (Temnykh et al. 2001). Throughout the course of evolution and speciation, the hypervariable SSR loci would have naturally undergone extensive expan-sion/contraction, while simultaneously affecting changes in the sequences flanking the SSRs (Chen et al. 2002b). In O. latifolia, such nuclear changes, combined with insertion/

deletion and base substitution events, transposon amplifica-tion, chromosomal rearrangements, gene duplications, and polyploidization, would have generated genomic regions that are highly diverged from those of the cultivated rice. Huang and Kochert (1994) have explained such chromosomal rear-rangements between AA and CCDD genomes by compara-tive RFLP analysis. This would explain the limited number of SSR markers that were amplified in O. latifolia-derived intro-gression lines in this study.

Similarly, the highly diverged genomic regions in O. lati-folia would greatly affect how existing SNP assays would perform in differentiating between O. sativa and O. latifolia alleles. Low-, medium-, and high-resolution SNP assays are currently available for different applications in rice research,

Figure 4. (a) Section modulus of the third internodes of each of the 27 introgression lines derived from O. latifolia measured 20 days after heading. Each column represent means ± standard deviation for 5 replicates. Significance was calculated at P < 0.05 (**) and P < 0.01 (*). (b) Graphical representation of a rice culm section indicating the different axes measured to determine SM. SM was calculated using the formula SM = π/32 × (a1

3b1 − a23b2)/a1, where a1 is the outer diameter of the minor axis in an oval

cross-section, b1 is the outer diameter of the major axis in an oval cross-section, a2 is the inner diameter of the minor axis in an oval cross-section and b2 is the inner diameter of the major axis in an oval cross-section (Ookawa et al. 2010). (c) Cross-section of the third internodes of disomic derivatives with significantly higher SM than IR31917-45-3-2; bar = 1 mm.



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Journal of Heredity

10

Tabl

e 3

Bact

eria

l lea

f bl

ight

reac

tion

of th

e 27

intro

gres

sion

lines

14

days

afte

r ino

cula

tion

with

Phi

lippi

ne ra

ces o

f X

anth

omon

as or

yzae

pv.

oryz

ae.

Intr

ogre

ssio

n

line

nam

eIn

trog

ress

ion

type

Bact

eria

l blig

ht s

trai

n

Rac

e 1

PXO

61R

ace

2 PX

O86

Rac

e 3A

PX

O79

Rac

e 3B

PX

O34

0R

ace

4 PX

O71

Rac

e 5

PXO

112

Rac

e 6

PXO

99R

ace

7 PX

O14

5R

ace

8 PX

O28

0R

ace

9A

PXO

339

Rac

e 9B

PX

O34

9R

ace

9C

PXO

0347

Rac

e 9D

PX

O36

3R

ace

10

PXO

341

IR31

917-

45-3

-22.

433.

676.

178.

336.

502.

674.

001.

171.

0712

.00

13.9

312

.17

8.67

2.83

WH

12-2

236

MA

AL4

I2.

903.

803.

679.

175.

003.

104.

731.

501.

2712

.67

11.6

713

.33

6.50

2.50

WH

12-2

237

MA

AL4

I2.

673.

834.

509.

504.

002.

004.

331.

101.

5011

.17

15.0

013

.83

11.1

72.

17W

H12

-223

8M

AA

L4I

3.43

4.13

5.00

10.1

75.

602.

674.

001.

931.

0010

.33

16.5

010

.33

11.0

02.

67W

H12

-223

9M

AA

L4I

2.50

4.03

8.43

13.5

05.

003.

675.

430.

501.

109.

5016

.33

12.5

07.

001.

17W

H12

-224

0M

AA

L5I

3.00

3.50

3.33

11.0

04.

172.

276.

670.

671.

3311

.33

13.6

713

.33

8.67

1.83

WH

12-2

241

MA

AL5

I3.

703.

673.

009.

174.

402.

173.

771.

831.

0012

.17

13.6

710

.17

10.3

35.

67W

H12

-224

2M

AA

L5I

2.17

4.73

6.67

8.33

7.77

ND

2.83

1.17

0.83

16.8

39.

5012

.17

9.50

2.17

WH

12-2

243

MA

AL5

I4.

004.

604.

5013

.67

5.67

1.67

9.17

1.17

0.83

13.8

317

.83

12.6

717

.17

1.17

WH

12-2

244

MA

AL5

I1.

175.

835.

5017

.17

4.17

1.33

9.33

1.33

0.50

13.0

0N

D11

.33

13.1

71.

33W

H12

-224

5M

AA

L5I

4.00

3.50

5.83

10.3

36.

171.

675.

170.

831.

1710

.00

12.8

312

.17

12.6

71.

00W

H12

-224

6M

AA

L5I

1.50

3.43

5.50

12.3

33.

671.

337.

171.

170.

6712

.00

18.0

07.

335.

001.

33W

H12

-224

7M

AA

L5I

1.50

2.50

4.33

11.0

03.

832.

174.

671.

330.

379.

5012

.00

8.67

11.3

31.

17W

H12

-224

8M

AA

L6I

3.17

4.83

7.00

12.6

76.

201.

675.

501.

001.

0012

.50

14.0

013

.33

13.0

01.

33W

H12

-224

9M

AA

L6I

2.67

3.33

6.17

13.8

35.

672.

8311

.00

1.17

1.00

14.5

014

.50

11.1

711

.33

2.17

WH

12-2

250

MA

AL6

II1.

673.

508.

1713

.00

5.50

2.33

9.17

1.17

1.33

15.6

712

.50

13.8

314

.33

2.17

WH

12-2

251

MA

AL6

II1.

832.

837.

3315

.33

5.17

2.17

10.3

30.

831.

3314

.67

21.3

314

.67

11.1

72.

50W

H12

-225

2M

AA

L7I

1.83

5.67

10.3

320

.33

8.67

3.17

10.8

30.

671.

3317

.67

22.0

015

.33

19.3

30.

67W

H12

-225

3M

AA

L7II

2.33

2.07

5.83

13.5

06.

171.

176.

670.

830.

5010

.83

19.3

312

.17

16.3

34.

33W

H12

-225

4M

AA

L7II

1.50

6.33

4.43

16.1

79.

000.

679.

170.

830.

2711

.17

12.8

39.

839.

502.

20W

H12

-225

5M

AA

L8I

2.73

5.17

3.50

12.8

35.

832.

5710

.67

1.33

1.17

1.83

15.6

712

.50

10.8

31.

67W

H12

-225

6M

AA

L8II

3.50

3.67

5.10

14.5

05.

431.

176.

831.

332.

331.

6714

.67

13.3

314

.00

ND

WH

12-2

257

MA

AL9

I3.

677.

879.

1717

.83

9.67

2.30

7.67

1.17

1.50

14.8

317

.50

16.5

017

.50

3.00

WH

12-2

258

MA

AL1

0I3.

008.

337.

8315

.67

7.10

1.67

7.83

1.33

0.67

15.1

716

.83

19.1

714

.33

1.67

WH

12-2

259

MA

AL1

0II

1.67

4.60

8.83

14.8

34.

671.

6715

.00

0.83

1.67

15.8

316

.00

16.0

013

.25

2.43

WH

12-2

260

MA

AL1

1I2.

177.

178.

6712

.33

6.50

1.17

2.83

1.17

1.67

12.0

013

.83

15.8

312

.63

3.17

WH

12-2

261

MA

AL1

2I2.

337.

007.

5016

.00

5.33

2.33

11.1

71.

171.

0014

.17

22.0

015

.83

14.1

72.

33W

H12

-226

2 M

AA

L12I

4.50

4.50

5.50

16.0

010

.00

2.33

11.1

71.

170.

8313

.67

18.8

317

.00

14.0

02.

17

ND

, no

data

. Val

ues p

rese

nted

are

mea

ns o

f 3

sam

ples

; <3

cm =

resis

tant

, 3.1

–5 c

m =

mod

erat

ely

resis

tant

, >5

cm =

susc

eptib

le.



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11

although the SNP discovery pools used for these assays were all based on sequences of O. sativa varieties and of a few wild relatives with AA genome (O. rufipogon, O. nivara, and O. barthii) (McNally et al. 2009; Huang et al. 2010; McCouch et al. 2010; Chen et al. 2011; Thomson et al. 2012). For this study, a low-resolution 384-plex SNP assay designed to define variation within the O. sativa subspecies indica varietal group was used. The target SNPs for this assay were particu-larly selected following a SNP discovery effort that provides information about the frequency of polymorphism within an indica germplasm. Of the 384 SNPs in the assay, only 84 suc-cessfully differentiated between O. sativa and O. latifolia. SNPs are biallelic and the individual base change that is detected as a SNP is expected to have occurred only once in evolu-tionary time. Because of this, SNPs are generally informative only for a particular set of genetic materials (McCouch et al. 2010). In the current study, the SNP assay used was specifi-cally optimized for indica populations, which explains the low polymorphism rate observed between O. sativa and O. latifolia. Our study suggests that, for a SNP assay to work for a spe-cific population, the SNP set has to be optimized first for the population to be studied.

In order to fully harness the potential of genetic resources that are derived from wild species, molecular markers that are tailored to detect alien chromosome introgressions in any given background must be developed. This will not only accelerate selection in breeding programs that involve wild rice derivatives but will also advance the identification and transfer of novel genes from wild species into culti-vated rice. To this end, the progress in the end sequencing of the BAC and construction of physical maps of 12 rep-resentative genomes of rice by the OMAP project offers an unprecedented opportunity to develop unique, genome-spe-cific molecular markers that can accurately evaluate genetic resources derived from highly diverged wild rice relatives (Ammiraju et al. 2010). The highly polymorphic indel mark-ers that were developed for this study using the sequence alignment between cv. Nipponbare and the O. alta BAC ends that were generated by the OMAP project highlight the sig-nificance of wild reference genomes toward the develop-ment of reliable markers to define wild introgressions in any given genetic background.

Characterization of the MAAL Disomic-Derived Introgression Lines

Molecular characterization of the disomic derivatives of the O. latifolia MAALs demonstrated a conserved pattern of wild genome introgression into an O. sativa background, with more than one line carrying the same set of intro-gressed chromosome segments from O. latifolia. Across introgression types, transmission of common chromosome segments was also observed. In particular, varying lengths of O. latifolia chromosome segments were systematically transferred into chromosome 6 in all the disomic deriva-tives, whereas none were transmitted into chromosome 7. In cotton, systematic chromosome or chromosome seg-ment transmission has also been reported in several studies.

Genotyping of trispecies hybrids and backcross progenies used in breeding for low gossypol content in seeds and high gossypol in plants showed that 4 AFLP markers specific to Gossypium sturtianum (C genome) were systematically present in all the backcross progenies of 2 trispecies hybrids ([G. hir-sutum × G. raimondii] × G. sturtianum and [G. raimondii × G. sturtianum] × G. hirsutum) (Vroh Bi et al. 1999). Similarly, Benbouza et al. (2007) reported the conserved transmission of 3 SSR markers specific to G. sturtianum in all selected progenies of the trispecies hybrid (G. hirsutum × G. raimon-dii) × G. sturtianum. In both cases, the higher pairing affinity of the chromosome segment of G. sturtianum harboring the specific AFLP or SSR loci with the chromosomes of the other parents resulted in the preferential transmission of those particular chromosome segments. In this study, the disomic derivatives showed a similar pattern of preferential introgression of small chromosome segments, marked by the conserved transmission patterns of the wild SSR, STS, SNP, and indel loci on specific chromosomes of O. sativa. The systemic replacement of these loci with chromosome segments from the wild species demonstrates crossing over that would have been facilitated only by the limited home-ologous chromosome pairing between the 2 excessively divergent species.

The variable length of O. latifolia chromosome segment introgressions on chromosome 6 of the O. sativa parent may be associated with chromosome breakage and repairs dur-ing homeologous recombination and crossing over, similar to what has been reported in rice and cotton (Jena et al. 1994; Benbouza et al. 2007).

Single Gene Transfer and Quantitative Trait Loci Inheritance in Disomic Derivatives of O. latifolia MAALs

Yield Components

Significant divergence of the mean values for different agro-nomic traits of the disomic introgression lines from those of the control, IR31917-45-32, indicates the presence of putative QTLs in the O. latifolia introgressions. These puta-tive QTLs, alone or in combination, conferred either positive or negative effects on the traits we examined. The variable expression in heading date, plant height, number of primary branches per panicle, number of grains per panicle, and grain width and length during the wet and dry seasons indicates that these traits are highly affected by the environment (Yano et al. 2000; Cao et al. 2001; Luan et al. 2009). It may also be possible that the wide range of phenotypic variations observed are due to genetic or epigenetic variations induced by genomic shock imposed due to wild species chromosome segments in the recipient genome (Shan et al. 2005; Wang et al. 2005). In our earlier collaboration with University of Georgia, Athens, GA, USA to prove the possibility of the role of transposable elements for creating genetic variations by CC genome wild species introgression in the recipient genome due to genomic shock, it was concluded that there was no role of transposable element in creating novel genetic variation of the introgression lines (Wessler S, unpublished data).



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BB Resistance

BB is one of the most serious diseases of rice in Asia, caus-ing yield losses of up to 50% during disease severity (Huang et al. 1997; Chen et al. 2002a). The identification of single or multiple genes of a dominant nature (Xa1, Xa2, Xa3/Xa26, Xa4, Xa7, Xa10, Xa11, Xa12, Xa14, Xa16, Xa17, Xa18, Xa21, Xa22, Xa25(t), and Xa27) and/or recessive nature (xa5, xa8, xa9, xa13, xa15, xa19, xa20, xa24, xa25/Xa25(t), xa26(t), xa28(t), xa31(t), xa33(t), and xa34(t)) for BB resistance (Khush and Kinoshita 1991; Kinoshita 1995; Lin et al. 1996; Chen et al. 2002a; Li and Wang 2013) in existing rice varieties has been the most effective and economical control of the disease. However, as in many host plant–pathogen systems, resistance of the hosts to the pathogens eventually breaks down after many years of cultivation due to shifts in patho-gen race frequency (Rao et al. 2002), hence the need for the identification of new sources of resistance to the disease.

The O. sativa elite breeding line IR31917-45-3-2 has been reported to have resistance to BB races 1 and 5 of the Philippines, whereas O. latifolia has known resistance to races 1, 2, and 3 (Multani et al. 2003). In the current study, the recurrent parent also exhibited moderate to high resistance to races 2, 6, 7, 8, and 10 of the disease, whereas it dem-onstrated susceptibility to the rest of the races tested. The O. latifolia introgressions in the background of IR31917-45-3-2 demonstrated either a positive or negative influence on the inherent reaction of the recurrent parent to the different races of BB. For example, some introgression lines exhib-ited weaker resistance to races 1, 2, 6, and 10 compared with the recurrent parent, suggesting the presence of putative QTLs on the introgressed segments coming from O. latifo-lia that have negative effects on BB resistance. Conversely, a few lines demonstrated resistance to race 9A and 9D of BB despite the susceptibility of the recurrent parent to the pathogen. In particular, lines WH12-2255 and WH12-2256 demonstrated a high resistance to race 9A of the dis-ease (PXO339), recording lesion lengths of only 1.83 and 1.67 cm, respectively, compared with the 12.48 cm average lesion length of the rest of the lines, including the recur-rent parent. Both lines share a common introgression on chromosome 12 from O. latifolia, suggesting the presence of a putative QTL for BB resistance in the chromosome seg-ment introgressed from wild rice. Previous studies identified the BB resistance gene, Xa25(t) from cv Minghui 63 in the centromeric region of chromosome 12. This gene confers resistance to Philippine race 9 of Xoo during both the seed-ling and adult stages of the rice plant. The linkage map for Xa25(t) shows that the genomic location of this gene coin-cides with the map position of the O. latifolia introgression identified in this study (Chen et al. 2002a), indicating the presence of an allelic variant of Xa25(t) in the O. latifolia genome. A survey of gene annotations showed the presence of several NBS-LRR genes containing a nucleotide binding site and a leucine-rich repeat within the map position of the introgressed O. latifolia chromosome segment. Further stud-ies are in progress to fine map the resistance gene(s) inher-ited from O. latifolia.

Strong Stem

The success of the green revolution in rice and wheat was attributed to the identification and incorporation of the semi-dwarf1 (sd1) and reduced height1 (Rht1) genes, respectively (Sakamoto and Matsuoka 2008). A reduction in plant stat-ure has been the predominant strategy in breeding for high-yielding and lodging-resistant crop varieties (Hargrove and Cabanilla 1979). However, despite the short plant stature conferred by sd1 or Rht1 genes in improved rice and wheat varieties, respectively, lodging remains a big problem. To realize higher crop production, it is imperative to identify genes that will improve resistance to lodging (Terashima et al. 1992; Berry et al. 2004). The morphology of the rice culm is one of the parameters that define its strength, which in turn is closely associated with lodging resistance (Ookawa et al. 2010).

The wild rice species O. latifolia has a plant height of 100–200 cm. At maturity, the leaf blades of the plant start to droop, while the stems remain erect up to 1 m from the plant base. Qualitative evaluation for stem strength showed that all the disomic derivatives from O. latifolia MAALs exhibit a strong stem. Verification of the physical strength of the stems showed that all introgression lines had higher SM than the recurrent parent, indicating the presence of putative QTLs for stem strength in the introgression lines we examined. Genotypic examination of the disomic derivatives showed that all lines share a common chromo-some segment of O. latifolia on chromosome 6 spanning 9.42–22.72 Mb. A 9.42 overlapping segment flanked by the SNP markers id6004481 and id6009055 is common in all the lines, suggesting that a putative QTL controlling culm diameter may lie in this introgressed region. Differences in the absolute value of SM in the disomic derivatives may be attributed to the presence of other QTLs with minor effects on the trait. Mapping of this QTL using a popula-tion generated from any of the disomic derivatives might reveal an allelic variant of SCM2 from O. latifolia (Ookawa et al. 2010). Further studies are underway to precisely iden-tify the gene controlling lodging resistance derived from O. latifolia.

Conclusions

The molecular characterization of MAALs and their dis-omic derivatives is fundamental for the development of appropriate strategies for the purposeful and efficient use of exotic genetic resources in rice breeding programs. The identification of wild species introgressions in the 27 O. lat-ifolia-derived disomic derivatives, as well as their agronomic evaluation in the field, is only the first step in tapping into the large amount of genetic diversity present in O. latifolia. The localization of O. latifolia introgressed segments in the uniform genetic background of O. sativa allowed the easy identification by association of putative QTLs controlling valuable traits from the wild species. In particular, putative QTLs exhibiting strong control of resistance to Philippine races 3A, 4, 9A, and 9D of BB as well as strong stem were



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identified. Cloning and functional validation of these QTLs in different genetic backgrounds would contribute in wid-ening the resource base for BB resistance as well as for lodging resistance in rice. The molecular markers that were identified to differentiate between O. sativa and O. latifolia genomes would not only aid in the rapid mapping and clon-ing of the genes identified from these lines but would also facilitate both genomic and genetic studies on breeding resources derived from wild Oryza species that share the same genome as O. latifolia. The evaluation of the disomic derivatives for other biotic and abiotic stresses could facili-tate the identification of several novel genes from O. latifolia that can be used to improve existing rice cultivars.

FundingGlobal Rice Science Partnership (GRiSP) program of the International Rice Research Institute (A-2012-180), Manila, Philippines.

AcknowledgementsWe would like to thank N. T. Quilloy and R. M. Malabanan for providing excellent technical help during the experiment. We are grateful to 2 anony-mous internal reviewers for their critical review of the manuscript. We are also grateful to Dr J. Jacquemin of Arizona Genomics Institute, USA for providing us the sequence alignments between O. alta and O. sativa cv Nipponbare. We also thank B. Hardy, Science Editor, IRRI for editing the manuscript.

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Received September 22, 2013; First decision January 25, 2014; Accepted April 21, 2014

Corresponding Editor: Kenneth Olsen



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