Heterosis and combining ability in rice as influencedby introgressions from wild species Oryza rufipogonincluding qyld2.1 sub-QTL into the restorer line KMR3

Sudhakar Thalapati • Haritha Guttikonda • Naga Deepthi Nannapaneni •

Prasad Babu Adari • C. Surendhar Reddy • B. P. Mallikarjuna Swamy •

Anil K. Batchu • Ramana Kumari Basava • B. C. Viraktamath •

Sarla Neelamraju

Received: 7 January 2014 / Accepted: 23 July 2014 / Published online: 5 August 2014

� Springer Science+Business Media Dordrecht 2014

Abstract Parental line improvement is a prerequisite

for developing high yielding rice hybrids. Wild rice are

reservoirs of useful genes for yield traits and introgres-

sion lines (ILs) with higher yield have been reported. The

present experiment was carried out according to line 9 -

tester mating design. 36 hybrids were phenotyped for

yield and its components along with parental lines [6

cytoplasmic male sterile (CMS) lines, 5 KMR3/Oryza

rufipogon derived ILs and KMR3 as control]. The

performance of hybrids was estimated based on relative

heterosis, heterobeltiosis and standard heterosis for yield

and its contributing characters along with analysis of

variance for combining ability. We identified a set of

good general combiners for yield traits (testers: IL50-7,

IL86-18, IL50-12 and CMS lines: IR79156A,

APMS6A). Of the 36 hybrids tested, four hybrids

IR79156A/IL50-13, IR58025A/IL50-12, APMS6A/

IL86-18 and APMS10A/IL363-5 showed significantly

high specific combining ability and standard heterosis

and APMS6A/IL86-18 showed highest yield. Heterosis

for yield/plant ranged from 17.6 to 84.9 % and hetero-

beltiosis from 18.5 to 77.4 % at P \0.05. Standard

heterosis ranged from 20 to 63 % in hybrids derived

from ILs compared to hybrid check Karnataka Rice

Hybrid-2. Only 7 out of 36 hybrids showed significantly

higher yield than their respective controls. O. rufipogon

allele of QS15 within sub-QTL3 of qyld2.1 was common

in these seven hybrids. These results demonstrate that

introgressions from wild species have the potential to

enhance yield significantly in popular rice hybrids.

Keywords Wild rice � Oryza rufipogon �Introgression lines � Hybrid rice � Heterosis

Abbreviations

SSR Simple sequence repeat

QTL Quantitative trait loci

CMS Cytoplasmic male sterile

IL Introgression line

GCA General combining ability

SCA Specific combining ability

Introduction

It is imperative to increase rice yield per unit area in all

rice-growing ecosystems to ensure food security.

Exploiting heterosis and good combining ability is

advantageous as hybrids can yield substantially more

than varieties. Though about 70 rice hybrids (http://

drd.dacnet.nic.in/Downloads/Hybrid-Varieties-of-Rice-

in-India-(2014).pdf) have been released in India, only

15–20 are widely grown. Currently, only about 5 % of

rice growing area in India is under hybrid rice (Hari

S. Thalapati � H. Guttikonda � N. D. Nannapaneni �P. B. Adari � C. Surendhar Reddy �B. P. M. Swamy � A. K. Batchu � R. K. Basava �B. C. Viraktamath � S. Neelamraju (&)

Directorate of Rice Research (ICAR), Rajendranagar,

Hyderabad 500030, Andhra Pradesh, India

e-mail: sarla_neelamraju@yahoo.com; nsarla@drricar.org

123

Euphytica (2015) 202:81–95

DOI 10.1007/s10681-014-1222-1

Prasad et al. 2013). Other important issues such as

technical challenges, market opportunities, and policy

constraints for the spread of hybrid rice in India have

been recently examined (Spielman et al. 2013). In

China, hybrids are cultivated in 57 % of rice growing

area (Yuan 2014). One of the reasons for the slow

spread of hybrid rice in India was the low yield

advantage (5–10 %) over best varieties. If hybrids

show 20–30 % yield increase over popular varieties,

they become economically more beneficial for the

farmers (Virmani 1996; Mahadevappa 2004; Wanjari

et al. 2006).

It is generally recognized that increased genetic

diversity leads to increased heterosis. Thus

indica 9 japonica hybrids yield more than indi-

ca 9 indica or japonica 9 japonica hybrids. The

highest yielding rice hybrids in the world are derived

from such inter sub specific crosses between indica

and japonica. For example, the highest yielding

Chinese two-line rice hybrid Liangyoupeijiu is a

hybrid between japonica Peiai64S and indica 9311

(Yuan 2014). The identification of wild-rice derived

yield enhancing quantitative trait loci (QTLs)/genes

offers the possibility of further increase in diversity

and yield of rice hybrids but has not been put to

practice yet. Genetic diversity can be further increased

if favorable yield enhancing QTL linked markers or

genes from wild species are included in such parental

lines (Liang et al. 2004; Thalapati et al. 2012). Hence,

improvement of elite parental lines using wild species

is worth exploring for continued yield enhancement of

hybrids.

There are only few reports on improvement of

parental lines [cytoplasmic male sterile (CMS)/main-

tainer or restorer lines] of three-line hybrids using

genes from wild species. Liang et al. (2004) used wild

rice Oryza rufipogon to improve popular indica

restorer line 9311. The same restorer line 9311 was

used in a cross with O. rufipogon to map yield QTLs

and it was shown that 38.5 % of wild rice-derived

QTLs had beneficial effect for yield-related traits (Fu

et al. 2010). Whether these improved ‘‘9311’’ intro-

gression lines (ILs) were subsequently used as parents

to develop hybrids for commercial release is not

reported. Yuan et al. (1994) developed the hybrid,

J23A/Q611 which out yielded the check hybrid by

35 %, using R line Q611 carrying one of the yield

QTLs, qyld1.1 or qyld2.1 through marker assisted

backcrossing (MABC) and field selection. This shows

that such improved restorer lines with new dominant

gene sources identified from O. rufipogon are impor-

tant in developing hybrids with higher yield.

There are previous reports of introgressions from

wild species into CMS lines also but they were

restricted to QTL mapping only (Xiao et al. 1998;

Marri et al. 2005) and the QTLs were later transferred

to restorer lines. These studies provided the proof that

the mean performance of individuals having a hetero-

zygous genotype with one allele from O. rufipogon

and the other from restorers Ce64 or KMR3 was better

for all the yield traits than that of individuals having

the standard elite hybrid combination of one allele

from CMS lines V20 or IR58025A and the other from

Ce64 or KMR3, respectively (Xiao et al. 1998; Marri

et al. 2005). KMR3 is a fertility restorer line used in

the production of the popular Karnataka Rice Hybrid-

2 (KRH2). This was identified as the stable and best

hybrid and is being used as hybrid check in irrigated

medium duration hybrid yield trials of All India

Coordinated Rice Improvement Programme

(AICRIP).

QTLs for yield traits have been mapped from

several wild species of rice using varieties other than

parental lines of hybrids. It is interesting to note that

almost half of the QTLs derived from wild species

were trait enhancing. Xiao et al. (1998) reported 68

QTLs from O. rufipogon, of which 35 were trait

enhancing. O. rufipogon (IRGC 105491), contained

two yield-enhancing QTLs, qyld1.1 and qyld2.1

associated with an 18 and 17 % increase in grain

yield, respectively (Xiao et al. 1998). Similarly,

Septiningsih et al. (2003) and Yoon et al. (2006)

reported 14 and 18 beneficial QTLs, respectively. In

addition, QTLs qGn1a, qgw9.1 and qph1 for grain

number, grain weight and plant height were also

identified in O. rufipogon (Ashikari et al. 2005; Xie

et al. 2006, 2008). Several other reports revealed that

candidate genes identified from O. rufipogon could

improve rice yield. For example, leucine rich repeat

receptor like kinase (LRK) gene (He et al. 2007),

cytokinin oxidase/dehydrogenase (OsCKX2) (Ashik-

ari et al. 2005), sucrose phosphate synthase (Ishimaru

2003), glycogen synthase kinase (Os11Gsk) (Thala-

pati et al. 2012) were reported to be likely candidates

from O. rufipogon for yield improvement. Luo et al.

(2011) identified heterotic loci for six yield related

traits from the ILs of O. rufipogon, and reported the

heterotic loci hgw2 and RM263 as positively

82 Euphytica (2015) 202:81–95

123

associated with 1,000 grain weight. Marri et al. (2005)

also reported that RM263 flanked a major yield QTL

qyld2.1. Interestingly, three ILs (IL50-7, IL86-18 and

IL363-5) of KMR3/O. rufipogon, used in this study are

homozygous for O. rufipogon allele of RM263. IL50-

12 was heterozygous and IL50-13 was homozygous

for the O. sativa (KMR3) allele. Though RM263 is

linked to yield there may be other loci in the

background which also affect yield. Three ILs IET

21943 (RPBio 4919-50-13), IET 22626 (RPBio

4919-50-7), IET 22632 (RPBio 4919-363-5) were

entered in multilocation yield trials for coastal salinity

and alkalinity and all showed significant (5.4, 29, and

63 %, respectively) yield superiority over the best

check (Annual Progress Report, AICRIP 2012). IL50-

13 was recommended for West Bengal based on four

continuous years of yield superiority over checks

(Annual Progress Report, AICRIP 2013).

The current study was designed to test if high

yielding restorer KMR3 with introgressions from O.

rufipogon at qyld2.1 will give higher yielding hybrids

with six CMS lines. This paper reports heterosis and

combining ability in 36 hybrids developed using six

CMS lines, 5 selected high yielding KMR3/O. rufipo-

gon ILs and the control restorer KMR3. The 30

hybrids using KMR3 ILs were compared with the

respective 6 hybrids obtained using KMR3.

Materials and methods

The experimental material comprised of six CMS lines

and six restorer lines (five high yielding KMR3-O.

rufipogon ILs and control KMR3-Karnataka Mandya

Restorer 3). The restorer lines viz., IL50-7, IL50-12,

IL50-13, IL363-5, IL86-18 and KMR3 were crossed

with CMS lines viz., IR58025A, IR79156A,

APMS6A, APMS10A, CRMS32A and PUSA5A for

hybrid development using line 9 tester mating design.

The 6 hybrids with KMR3 were considered as controls

for the respective 30 hybrids developed using KMR3

ILs.

Development of experimental population

Restorer lines in the present study were selected from

the cross IR58025A/O. rufipogon//IR58025B///

IR58025B////KMR3 developed by Marri et al.

(2005). Two plants were selected from 251 BC2

testcross progeny which had the segment of yield QTL

qyld2.1 flanked by simple sequence repeat (SSR)

markers RM262 and RM263 from O. rufipogon. These

plants were selected and crossed with KMR3 as the

female recurrent parent for three generations selecting

for qyld2.1 flanking markers to obtain BC3F1 popula-

tion. These plants were selfed to obtain BC3F2 and

were genotyped with two flanking markers and eight

markers within qyld2.1 (Prasad Babu 2009). The

selected high yielding lines and KMR3 were field

evaluated in randomized block design for yield and

related traits and same maturity duration checks for

four seasons.

The 36 hybrids along with corresponding parents

[B lines (maintainer) were used for yield evaluation

since the CMS-A lines are male sterile] were field

evaluated in three replications during kharif,

2007–2008 at DRR. Five popular hybrids, KRH2,

DRRH2, DRRH3, PA6201 and PA6444 were used as

standard checks in the experiment. Twenty five days

old seedlings were transplanted and each entry was

planted in two rows with 15 9 20 cm spacing. Single

seedling was transplanted per hill and all recom-

mended packages of practices were followed.

DNA isolation and genotyping of ILs

DNA was isolated from leaf tissues of selected

genotypes through CTAB (cetyl trimethyl ammonium

bromide) method as described by Rogers and Bendich

(1988) and used for polymerase chain reaction ampli-

fication following the protocol of Chen et al. (1997).

After amplification, the products were checked for

polymorphism or marker segregation on horizontal

agarose gel and scored for the polymorphic/segregat-

ing bands. The QTL qyld2.1 linked SSR markers,

RM262 and RM263 and eight polymorphic SSR

markers, RM3666, RM1303, RM3688, RM3762,

RM3874, RM3515, RM6318 and RM1920 within

the QTL region were used for marker analysis (Fig. 1).

11 polymorphic markers viz., QS2B, QS7, QS10B,

QS15, QS26, QS34, QS35, QS40A, KFM3, KFM6,

KFM10 within sub-QTL3 region were also used to

precisely locate wild introgressed segments for this

study. Details of the markers are given in Table 1. In

addition, two markers RM6100 for Rf4 on chromo-

some 10, and RM10313 on chromosome 1 for Rf3

were used to confirm that the major fertility restorer

genes of the popular restorer KMR3 were not altered

Euphytica (2015) 202:81–95 83

123

due to introgressions from O. rufipogon, and Os11Gsk

specific marker Sha3 was used for further confirmation

of O. rufipogon segment on chromosome 11 in the

selected ILs (Thalapati et al. 2012).

Phenotyping

Five plants were randomly selected from every replica-

tion and biometrical observations were recorded for the

following 14 traits, days to 50 % flowering (on plot

basis), plant height (cm), leaf length (cm), leaf width

(cm), number of tillers per plant, number of productive

tillers per plant, panicle length (cm), panicle weight (g),

number of spikelets per panicle, spikelet fertility

percent, 1,000 grain weight (g) and grain yield per plant

(g). The panicles that emerged from the primary tiller

were bagged before anthesis, the number of filled grains

and sterile spikelets in the panicle were counted at the

time of maturity. The ratio of filled grains to the total

number of spikelets was expressed as spikelet fertility.

Fig. 1 Eight markers

(located between RM262–

RM263, flanking yld 2.1)

polymorphic between

KMR3 and O. rufipogon

Table 1 Details of 13 primers (1–13) within qyld2.1 and SHA3 on chromosome 11 showing polymorphism between KMR3 and O.

rufipogon

S. nos. Markers Forward primer (50–30) Reverse primer (50–30)

1 RM3688 GTTGAATCAAGCTGTGCAGC AGCTAGGCAAAGCATGCATG

2 QS2B TTTCGCCTTTTATTAATTCGTTG TTTTTGAGGGATTCGGCTATT

3 QS7 ATATGGAACCTGGGAAGTG ACCCAAGCCAACAAGTAAT

4 QS10B TAGTCTCGCAGTGGTTGCAC GACAACCACAATGTATGAAAAGAT

5 QS15 GCAGAGTTTTGTGATGTGC TGCTGTAGGAAAAGGCTTAC

6 QS26 ACAACCCGAAACCAACTAC CTCGGTCCTAGTTGCACTT

7 QS34 CCGATTCAGTTGGGTACTT AATGCAACATCATCTTATTTTC

8 QS35 TCATTATCCATTTCCACTCA TCCACAAATTACTTCCAGCTA

9 QS40A GCCATCCCCTGCTGCTAC CTACGACGGCCATAGAGGAG

10 KFM3 CACAAACAAGAATGTGGAAA TGCAAAAGTGTGATGCTAAA

11 KFM6 CGGAAATCTGAAAATTCTGT GGATAGGAGAGGAGGAGGA

12 KFM10 AGCTGTGCAATAAATTCGAT GCAACTTGTGTTGTAAGCAA

13 RM3762 TACCGTAAGGCGCTGGATT ACGAGGTCCCCCCTCTAAA

14 SHA3 CATGAACGATCCCCACTACC ACCCCACCATTAAGCATTTG

84 Euphytica (2015) 202:81–95

123

Data analysis

The mean data was analyzed for combining ability

following the standard method of Kempthorne (1957).

Combining ability was determined for selecting supe-

rior parents with good general combining ability

(GCA) and hybrids with good specific combining

ability (SCA) effects. Genotype means were used for

analysis of variance; heterosis was calculated accord-

ing to Singh and Chaudhary (1999). Indostat statistical

analysis software was used for calculating analysis of

variance and heterosis (Indostat Services, Hyderabad,

India). Mid-parent heterosis and better-parent hetero-

sis or heterobeltiosis were determined as outlined by

Falconer and Mackay (1996). Standard heterosis was

estimated as percent gain in yield per plant of hybrid

over the standard hybrid checks.

Results

Marker analysis

Out of 40 markers tested in between the flanking

markers of subQTL3 (RM3688 and RM3762) of

qyld2.1 only 11 were polymorphic between KMR3

and O. rufipogon. The genotyping data of five ILs

(IL50-7, IL50-12, IL50-13, IL86-18 and IL363-5)

with these 11 markers within sub-QTL3 and the 2

flanking markers of subQTL3 of qyld2.1 is given in

Table 2. IL50-7 showed all homozygous O. rufipo-

gon alleles, IL50-12 showed all heterozygous alleles

except QS26 and IL50-13 showed all homozygous

O. sativa alleles except QS15. IL363-5 showed

homozygous O. rufipogon allele at eight loci and

IL86-18 at two loci. O. rufipogon allele for sha3

was present in all five lines but absent in KMR3.

All five ILs showed the KMR3 allele for Rf4 and

Rf3.

Combining ability analysis

The analysis of variance for combining ability of yield

and its components revealed significant difference

among the parents and crosses. The combining ability

variance in the six CMS lines exhibited differences

among themselves for panicle weight, panicle length,

spikelets per panicle, spikelet fertility, 1,000 grain

weight and yield per plant. Likewise testers also

showed significant differences for days to 50 %

flowering, flag leaf length, flag leaf width, spikelets

per panicle and 1,000 grain weight. The interaction

between lines (CMS) and testers (restorers) was

significant (\0.05) for all the 12 traits except tillers

per plant and panicles per plant indicating the

importance of dominance or non additive variance in

their expression (Table 3). GCA and SCA results

revealed the predominance of SCA variance in

relation to GCA variance for 11 of the 14 traits except

for leaf width, spikelet fertility and 1,000 grain weight

(Tables 4, 5). GCA of all ILs for yield/plant was

increased by the introgressions from O. rufipogon and

that of IL86-18 was the highest.

Per se yield of IL-derived hybrids versus KMR3-

derived control hybrid

Of the 30 hybrid combinations with introgressions

from O. rufipogon, only 7 hybrids showed signif-

icantly higher yield and 6 hybrids showed significantly

lower yield than their respective controls (CMS line 9

KMR3). The remaining 17 hybrids were not signifi-

cantly affected by introgressions from the wild

species. All CMS lines except Pusa5A figured in the

Table 2 Haplotype of five KMR3/O. rufipogon introgression lines for 13 loci within sub QTL3 of qyld2.1 on chromosome 2

ILs Locus

RM3688 QS2B QS7 QS10B QS15 QS26 QS34 QS35 QS40A KFM3 KFM6 KFM10 RM3762

IL50-7 R R R R R R R R R R R R R

IL50-12 H H H H H R H H H H H H H

IL50-13 S S S S H S S S S S S S S

IL363-5 S R R R R R R R R H H S S

IL86-18 H S S S H S S S S H S S S

S O. sativa (KMR3) allele, R O. rufipogon (WR120) allele, H heterozygous

Euphytica (2015) 202:81–95 85

123

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10

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91

-0.1

96

-0.1

96

-0.7

24

-0.0

58*

0.1

04

0.0

28

0.1

83

0.4

11

2.4

04

-0.7

59

-0.3

15

0.1

42

2.5

20**

11

IL363-5

-4.7

91***

0.3

15

0.2

93

-2.3

13*

-0.0

21

-0.1

09

0.0

06

0.2

06

-0.8

8.7

81

-0.8

14

1.7

96***

-1.1

90***

-1.7

24*

12

KM

R3

3.5

09*

-0.2

07

-0.1

07

-0.7

35

-0.0

62*

-0.2

62**

-0.8

61***

-0.5

72***

-3.6

67***

-13.2

74**

-0.2

63

3.7

41***

-0.7

56***

-2.5

13**

ILK

MR

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ne,

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esper

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icle

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spik

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sper

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icle

,SF

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fert

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86 Euphytica (2015) 202:81–95

123

Table 5 Estimation of specific combining ability effects of 36 hybrids for 14 yield traits

S. nos. Parent/cross Plant

height (1)

Tillers/

plant (2)

Panicles/

plant (3)

Leaf

length (4)

Leaf

width (5)

Panicle

weight (6)

Panicle

length (7)

1 IR58025A/50-7 -2.765 0.074 0.219 2.746 0.064 -0.261 -0.428

2 IR58025A/50-13 -5.487 -0.593 -0.626 1.869 0.003 -0.019 0.239

3 IR58025A/50-12 1.28 0.263 0.43 0.28 -0.055 0.433* -0.006

4 IR58025A/86-18 -1.709 -0.248 -0.281 1.435 -0.086 0.332 1.117**

5 IR58025A/363-5 10.857** 1.141** 0.63 -5.576* 0.017 -0.569** -1.994***

6 IR58025A/KMR3 -2.176 -0.537 -0.37 -0.754 0.057 0.084 1.072*

7 APMS6A/50-7 4.057 0.03 0.241 1.435 0.052 0.158 -0.217

8 APMS6A/50-13 -4.598 0.096 0.063 -1.376 0.037 0.106 0.183

9 APMS6A/50-12 -2.498 -0.181 -0.415 0.502 0.006 -0.088 -0.061

10 APMS6A/86-18 0.18 0.207 0.074 0.209 0.022 -0.056 -1.072*

11 APMS6A/363-5 -3.454 -0.304 0.119 0.113 -0.089 -0.09 0.683

12 APMS6A/KMR3 6.313 0.152 -0.081 -0.465 -0.028 -0.03 0.483

13 CRMS32A/50-7 -2.298 -0.004 -0.093 1.98 0.04 0.49* 0.161

14 CRMS32A/50-13 2.513 0.596 0.663 1.835 -0.015 -0.135 -0.106

15 CRMS32A/50-12 2.613 0.719 0.652 -2.22 -0.033 -0.329 0.05

16 CRMS32A/86-18 -3.176 -0.159 -0.193 -1.731 -0.004 -0.404 0.239

17 CRMS32A/363-5 1.791 -0.404 -0.481 -0.143 -0.008 0.342 0.128

18 CRMS32A/KMR3 -1.443 -0.748 -0.548 0.28 0.02 0.035 -0.472

19 IR79156A/50-7 0.591 0.507 0.352 1.346 -0.007 -0.118 -0.117

20 IR79156A/50-13 6.402 0.441 -0.093 -2.398 -0.015 -0.169 -0.917*

21 IR79156A/50-12 -0.365 -0.104 0.03 1.213 0.107 0.876*** 1.239**

22 IR79156A/86-18 10.980** -0.448 -0.481 -2.218 -0.004 -0.258 -0.172

23 IR79156A/363-5 13.654*** -0.426 -0.17 3.557 0.026 0.014 1.183**

24 IR79156A/KMR3 -3.954 0.03 0.363 -1.42 -0.107 -0.346 -1.217**

25 PUSA5A/50-7 0.798 -0.304 -0.359 -3.431 0.001 -0.089 -0.017

26 PUSA5A/50-13 2.48 -0.17 -0.07 -1.576 0.036 -0.067 0.783

27 PUSA5A/50-12 0.513 -0.115 0.119 -1.231 -0.068 -0.301 -0.794

28 PUSA5A/86-18 -5.343 0.074 0.074 1.391 0.001 -0.202 -0.739

29 PUSA5A/363-5 0.691 -0.504 -0.615 -0.354 0.07 0.223 -0.183

30 PUSA5A/KMR3 2.457 1.019* 0.852 5.202* -0.049 0.436* 0.950*

31 APMS10A/50-7 1.213 -0.304 -0.359 -4.076 -0.159* -0.179 0.617

32 APMS10A/50-13 -1.309 -0.37 0.063 1.646 -0.047 0.283 -0.183

33 APMS10A/50-12 -1.543 -0.581 -0.815 1.457 0.042 -0.591** -0.428

34 APMS10A/86-18 -0.931 0.674 0.807 1.413 0.071 0.588** 0.628

35 APMS10A/363-5 3.769 0.496 0.519 2.402 -0.016 0.08 0.183

36 APMS10A/KMR3 -1.198 0.085 -0.215 -2.843 0.108 -0.18 -0.817

S. nos. Parent/cross Primary

branches/

panicle (8)

Secondary

branches/

panicle (9)

Spikelets/

panicle

(10)

Spikelet

fertility

(%, 11)

Days to 50

% flowering

(12)

1,000 grain

weight

(13)

Yield/

plant

(14)

1 IR58025A/50-7 -0.306 -0.644 4.063 3.461 -0.574 0.093 0.357

2 IR58025A/50-13 0.261 0.533 4.441 1.614 -4.519*** 0.753* -0.931

3 IR58025A/50-12 -0.472 4.222 13.041 -4.244 0.648 0.343 4.291*

4 IR58025A/86-18 0.706 -0.089 1.207 1.41 1.648 -0.56 -2.82

Euphytica (2015) 202:81–95 87

123

seven hybrids showing significantly higher yield.

Significantly, three of these seven hybrids involved

IL50-7, three involved IL86-18 and only one involved

IL50-13 as restorer. APMS6A/IL86-18, CRMS32A/

IL50-7 and IR79156A/IL86-18 were the top three

hybrids showing more than 40 g yield/plant (Table 6).

Among the six maintainer lines APMS6B exhibited

significantly higher yield (27.3 g/plant). It is impor-

tant to note that hybrids derived from PUSA5A with

all five ILs gave significantly lower yield when

compared with its control hybrid PUSA5A/KMR3.

Heterosis in derived hybrids

Overall heterosis (mid parent) and heterobeltiosis

(better parent) in 36 hybrids for 14 traits ranged from

17.36 (Pusa5A/IL50-13) to 79.97 (APMS10A/IL50-

12) and 18.51 (Pusa5A/IL86-18) to 77.44 %

Table 5 continued

S. nos. Parent/cross Primary

branches/

panicle (8)

Secondary

branches/

panicle (9)

Spikelets/

panicle

(10)

Spikelet

fertility

(%, 11)

Days to 50

% flowering

(12)

1,000 grain

weight

(13)

Yield/

plant

(14)

5 IR58025A/363-5 -0.45 -6.744** -15.037 -0.942 1.537 -0.495 -2.109

6 IR58025A/KMR3 0.261 2.722 -7.715 -1.3 1.259 -0.135 1.213

7 APMS6A/50-7 -0.006 -1.867 13.341 2.669 2.259* 0.14 -1.154

8 APMS6A/50-13 0.094 1.178 1.185 1.776 -3.019** 0.433 -1.043

9 APMS6A/50-12 -0.706 3 -11.481 -0.686 -0.185 0.23 -0.22

10 APMS6A/86-18 0.139 -3.111 2.285 -1.092 -3.852*** 0.166 3.935*

11 APMS6A/363-5 -0.017 -1.367 -0.026 -1.059 2.704* -1.335*** -1.02

12 APMS6A/KMR3 0.494 2.167 -5.304 -1.607 2.093 0.365 -0.498

13 CRMS32A/50-7 0.806* 2.2 14.785 -1.532 -0.407 -1.289*** 3.224

14 CRMS32A/50-13 -0.094 1.311 2.763 -0.905 2.315* -0.022 -0.465

15 CRMS32A/50-12 -0.094 -1.533 -9.17 0.776 1.481 -0.392 -2.576

16 CRMS32A/86-18 -0.45 -3.578 -15.537 -2.375 0.148 0.484 -3.287

17 CRMS32A/363-5 0.394 1.9 -1.315 2.897 -2.963** 1.336*** 1.624

18 CRMS32A/KMR3 -0.561 -0.3 8.474 1.138 -0.574 -0.117 1.48

19 IR79156A/50-7 0.239 3.644 -17.026 -1.161 0.315 0.034 -0.131

20 IR79156A/50-13 0.006 -4.578 -4.048 0.469 -2.630* -0.412 6.046**

21 IR79156A/50-12 0.139 5.044* 6.619 0.65 -3.796*** 0.778* -1.065

22 IR79156A/86-18 -0.017 0.667 2.119 1.954 3.87*** -0.239 2.024

23 IR79156A/363-5 0.028 1.078 15.407 0.584 2.759* -1.134*** -4.531*

24 IR79156A/KMR3 -0.394 -5.856* -3.07 -2.496 -0.519 0.973** -2.343

25 PUSA5A/50-7 -0.261 -0.444 -2.559 -0.323 0.926 0.231 -1.92

26 PUSA5A/50-13 -0.228 -1.6 -9.381 -4.467* 3.315** 0.451 -0.476

27 PUSA5A/50-12 0.306 -3.711 6.152 0.866 -0.185 -0.599* -1.587

28 PUSA5A/86-18 -0.517 0.044 7.519 0.367 -0.852 0.451 0.769

29 PUSA5A/363-5 -0.272 2.522 -8.793 -0.264 -2.963** 0.536 2.146

30 PUSA5A/KMR3 0.972** 3.189 7.063 3.82 -0.241 -1.070*** 1.069

31 APMS10A/50-7 -0.472 -2.889 -12.604 -3.113 -2.519* 0.791** -0.376

32 APMS10A/50-13 -0.039 3.156 5.041 1.512 4.537*** -1.202*** -3.131

33 APMS10A/50-12 0.828* -7.022** -5.159 2.638 2.037 -0.359 1.157

34 APMS10A/86-18 0.139 6.067* 2.407 -0.266 -0.963 -0.302 -0.62

35 APMS10A/363-5 0.317 2.611 9.763 -1.217 -1.074 1.09*** 3.891*

36 APMS10A/KMR3 0.139 -1.922 0.552 0.445 -2.019 -0.017 -0.92

* P \ 0.05, ** P \ 0.01, *** P \ 0.001

88 Euphytica (2015) 202:81–95

123

(IR58025A/IL50-12), respectively (Table 7). For

yield per plant, 23 hybrids out of 36 showed signif-

icantly positive heterosis and heterobeltiosis but only

7 showed significantly higher yield per se (Table 6).

Standard heterosis ranged from 19.6 (PUSA5A/IL363-

5) to 63 (APMS6A/IL86-18) over KRH2 and from

13.9 (IR58025A/IL50-12) to 17.5 % (APMS6A/IL86-

18) over the recently released hybrid DRRH3. Each of

the 36 hybrids showed significant positive mid parent

heterosis except Pusa5A 9 IL50-7. Better parent

heterosis was significant in 22 hybrids. Both mid and

better parent heterosis was highest in IR58025A/IL50-

12. It is noteworthy that IL50-12 was the only line

which showed highly significant mid parent heterosis

(31–77 %) and better parent heterosis (35–84 %). It

was also heterozygous for 12 of the 13 loci in subQTL3

of yld2.1. However, hybrids derived from IL50-12 did

not yield higher than those derived from KMR3 in

combination with any of the six CMS lines.

Most of the hybrids were shorter than the testers and

check hybrid PA6201. Significant positive heterobelti-

osis and standard heterosis for hybrids ranged from -7.5

(CRMS32A/IL50-7) to -21.1 (IR79156A/IL363-5) and

-2.7 (APMS6A/IL363-5) to -15.8 (IR79156A/IL363-

5), respectively. Almost all hybrids flowered earlier than

their parents. Significant negative heterobeltiosis ranged

from -6.6 (IR79156A/KMR3) to -21.4 (IR58025A/

IL50-13) %. 18 hybrids flowered earlier than the earliest

check DRRH2. Days to 50 % flowering showed signif-

icant negative standard heterosis [-4.3 (IR58025A/

IL86-18) to -13.7 (IR58025A/IL50-13) %].

Heterosis for flag leaf length ranged from 18.4

(IR58025A/IL50-7) to 25.5 (CRMS32A/IL50-7) %

and standard heterosis from 20.7 (IR58025A/IL50-13)

to 30.7 (CRMS32A/IL50-13) %. Seven hybrids

showed significant higher heterosis for flag leaf length

over the best check DRRH3. Nine hybrids showed

significant and positive mid parent heterosis but non-

significant positive heterobeltiosis indicating partial

dominant type of gene action. Heterobeltiosis for leaf

width ranged from 14 (IR79156A/IL50-7) to 21.7

(IR58025A/IL50-7). Five hybrids showed significant

mid parent heterosis which ranged from 13.1

(CRMS32A/IL50-7) to 22.6 % (IR58025A/IL50-7).

Mid-parent and high-parent heterosis ranged from

10.8 to 15.6 and 10.3 %, respectively, for tillers per

plant. Three hybrids showed significant positive heter-

osis and heterobeltiosis indicating overdominance type

of gene action. For panicles per plant, minimum mid-

parent heterosis of 8.6 % was observed in IR58025A/

IL363-5 and maximum of 14.5 % in PUSA5A/KMR3.

Only one hybrid (PUSA5A/KMR3) showed high-parent

heterosis i.e., 12.9 %. For panicle weight, ten hybrids

showed significant positive heterosis over their better

parents and it ranged from 19 (IR79156A/IL50-13) to

72.3 (IR79156A/IL50-12) %. 21 hybrids showed posi-

tive heterosis over mid parent and it ranged from 17.2

(APMS6A/IL50-12) to 82.2 (IR79156A/IL50-13) %.

One hybrid, IR79156A/IL50-12 showed 15.4 % heter-

osis over the better check DRRH3. 23 hybrids exhibited

significant positive heterosis from 4.6 (IR79156A/IL86-

18) to 15.4 (IR79156A/IL50-12) % over their better

Table 6 Mean yield/plant (g) of parents, control hybrid and hybrids derived from introgression lines

CMS/R line B line mean yield (L) ; KMR3 IL50-7 IL50-13 IL50-12 IL86-18 IL363-5 Mean

R line mean yield (T) ? 28.270 30.400 29.930 23.930 29.530 28.800 28.480

IR58025A 22 35.033 36.480 35.823 36.347 38.400* 30.530 35.436

APMS6A 27.33 31.987 35.186** 34.350 31.603 43.470*** 30.547 34.524

CRMS32A 23.07 37.607 41.483** 37.340 32.753 38.070 31.470 36.454

IR79156A 20.93 36.617 36.277 40.443 33.320 42.220* 35.000 37.313

PUSA5A 22.67 38.023 32.186*** 33.676** 33.770*** 31.353*** 32.086*** 33.516

APMS10A 18.67 32.920 35.506* 34.240* 33.487 32.620 30.180* 33.159

Mean 22.44 35.364 36.186 35.979 33.547 37.689 31.635 35.067

KRH2 yield in bold, only underlined values represent significant increase over hybrids with KMR3, other significant values show

decrease

L line/CMS line, Isogenic Maintainer lines (B) of respective CMS lines were used for measuring yield since A lines are sterile,

T tester/restorer line R

* P \ 0.05, ** P \ 0.01, *** P \ 0.001

Euphytica (2015) 202:81–95 89

123

parents and 35 hybrids exhibited positive heterosis over

mid parent with a maximum of 21.7 (IR79156A/IL50-

12) %. Heterosis range of hybrids over the check

DRRH2 was from 5.2 (APMS10A/IL363-5) to 11.3

(CRMS32A/IL50-7) % and 15 hybrids showed signif-

icantly high heterosis for panicle length.

Table 7 Estimation of heterosis, heterobeltiosis and standard heterosis for yield/plant in 36 crosses

S. nos. Cross Heterosis Standard heterosis

Mid Better KRH2 DRRH3 DRRH2 PA6201 PA6444

1 IR58025A/50-7 44.78** 24.78** 41.19** 1.79 31.11** 25.88** 41.54**

2 IR58025A/50-13 36.84** 18.71* 32.26** -4.65 22.81* 17.92* 32.59**

3 IR58025A/50-12 84.91** 77.44** 58.06** 13.95* 46.77** 40.93** 58.46**

4 IR58025A/86-18 41.79** 23.7** 35.98** -1.97 26.27** 21.24* 36.32**

5 IR58025A/363-5 29.92** 14.58 22.83* -11.45 14.06 9.51 23.13*

6 IR58025A/KMR3 41.38** 25.71** 13.36 -4.65 22.81* 17.92* 32.59**

7 APMS6A/50-7 27.94** 21.49* 37.47** -0.89 27.65** 22.57* 37.81**

8 APMS6A/50-13 25.49** 20.04* 33.75** -3.58 24.19** 19.25* 34.08**

9 APMS6A/50-12 50.07** 40.73** 43.18** 3.22 32.95** 27.65** 43.53**

10 APMS6A/86-18 54.04** 48.31** 63.03** 17.53* 51.38** 45.35** 63.43**

11 APMS6A/363-5 23.28** 20.14* 28.78** -7.16 19.59* 14.82 29.1**

12 APMS6A/KMR3 23.5** 21.46* 27.79** -7.87 18.66* 13.94 28.11**

13 CRMS32A/50-7 40.65** 23.68** 39.95** 0.89 29.95** 24.78** 40.3**

14 CRMS32A/50-13 23.77** 9.58 22.08* -11.99 13.36 8.85 22.39*

15 CRMS32A/50-12 37.87** 35.38** 20.6* -13.06 11.98 7.52 20.9*

16 CRMS32A/86-18 24.97** 11.29 22.33* -11.81 13.59 9.07 22.64*

17 CRMS32A/363-5 29.31** 16.44 24.81* -10.02 15.9 11.28 25.12*

18 CRMS32A/KMR3 27.01** 15.33 21.34* -12.52 12.67 8.19 21.64*

19 IR79156A/50-7 49.61** 26.32** 42.93** 3.04 32.72** 27.43** 43.28**

20 IR79156A/50-13 70.9** 45.21** 61.79** 16.64* 50.23** 44.25** 62.19**

21 IR79156A/50-12 69.69** 59.05** 41.69** 2.15 31.57** 26.33** 42.04**

22 IR79156A/86-18 67.77** 43.34** 57.57** 13.6 46.31** 40.49** 57.96**

23 IR79156A/363-5 26.81** 9.49 17.37 -15.38* 8.99 4.65 17.66

24 IR79156A/KMR3 33.88** 16.51 22.58* -11.63 13.82 9.29 22.89*

25 PUSA5A/50-7 15.08 0.44 13.65 -18.07* 5.53 1.33 13.93

26 PUSA5A/50-13 17.36* 3.12 14.89 -17.17* 6.68 2.43 15.17

27 PUSA5A/50-12 35.05** 31.48** 17.12 -15.56* 8.76 4.42 17.41

28 PUSA5A/86-18 34.1** 18.51* 30.27** -6.08 20.97* 16.15 30.6**

29 PUSA5A/363-5 24.87** 11.57 19.6* -13.77* 11.06 6.64 19.9*

30 PUSA5A/KMR3 18.85* 7.08 12.66 -18.78** 4.61 0.44 12.94

31 APMS10A/50-7 47.55** 19.08* 34.74** -2.86 25.12** 20.13* 35.07**

32 APMS10A/50-13 33.06** 8.02 20.35* -13.24 11.75 7.3 20.65*

33 APMS10A/50-12 79.97** 60.17** 42.68** 2.86 32.49** 27.21** 43.03**

34 APMS10A/86-18 56.57** 27.77** 40.45** 1.25 30.41** 25.22** 40.8**

35 APMS10A/363-5 60.11** 31.94** 41.44** 1.97 31.34** 26.11** 41.79**

36 APMS10A/KMR3 38.07** 14.62 20.6* -13.06 11.98 7.52 20.9*

Control crosses are underlined. Standard heterosis is with reference to five popular hybrids

* P \ 0.05, ** P \ 0.01, *** P \ 0.001

90 Euphytica (2015) 202:81–95

123

The estimates of mid parent heterosis and hetero-

beltiosis for primary branches per panicle were in the

range of 8 (IR79156A/IL50-12) to 23.9 (APMS10A/

IL50-10) % and 8.7 (CRMS32A/IL50-7) to 15

(APMS10A/IL50-12) %, respectively. Seven hybrids

showed higher mean performance and high heterosis

and heterobeltiosis for primary branches per panicle.

Hybrids exhibited significant positive heterosis over

their better parents for secondary branches, and it

ranged from 15.2 (IR79156A/IL86-18) to 30

(IR79156A/IL363-5) %. 14 hybrids exhibited mid

parent heterosis which ranged from 14.9 (IR58025A/

IL50-12) to 42.6 (IR79156A/IL50-10) %.

Number of spikelets per panicle, spikelet fertility

and 1,000 grain weight are three most important traits

for yield. For total number of spikelets per panicle, 11

hybrids showed heterobeltiosis ranging from 16.6

(APMS10A/IL50-13) to 34.1 (IR79156A/IL50-

12) %. 19 hybrids showed positive mid parent heter-

osis ranging from 14.6 (CRMS32A/IL50-7) to 40.2

(IR79156A/IL50-12) %. Hybrid IR58025A/IL50-7

showed 11.4 % significant positive mid parent heter-

osis for spikelet fertility but heterobeltiosis was not

significant indicating partial dominance. 13 hybrids

revealed heterobeltiosis for 1,000 grain weight and it

ranged from 4.1 (IR58025A/IL50-7) to 12.6

(PUSA5A/IL50-7) % and 28 hybrids showed signif-

icant positive mid parent heterosis ranging from 5.8

(APMS10A/IL50-13) to 20.7 (APMS10A/IL50-7) %.

Standard heterosis ranged from 4.6 (CRMS32A/IL50-

7) to 16.4 (PUSA5A/IL50-7) % over KRH2 the best

hybrid check for 1,000 grain weight.

In all, seven hybrids with introgressions from O.

rufipogon showed significantly higher yield than their

controls without introgressions. Three of these had

IL50-7, three had IL86-18 and one hybrid had IL50-13

as restorer. These three ILs which gave the seven best

hybrids for yield had only one qyld2.1 subQTL3

marker allele i.e., QS15 in common among themselves

from O. rufipogon. IL50-7 was homozygous for this

allele and the other two were heterozygous. Six of these

seven hybrids showed significant heterosis over both

mid and better parent and also standard heterosis over

KRH2. It is interesting to note that these six hybrids

involved only two restorers IL50-7 and IL86-18. The

only common O. rufipogon introgressions studied in

these two ILs in qyld2.1 subQTL3 region were

RM3688, QS15 and KFM3, all the three loci were

homozygous in IL50-7 and heterozygous in 86-18.

Discussion

The commercial exploitation of hybrid vigor depends

upon per se yield of hybrids which is also contributed

by combining ability and degree of heterosis. The

mean performance of lines, testers and their hybrids

ultimately indicates worth of genetic variability for the

improvement of yield traits. In our 6 line 9 6 tester

study when yield of 30 hybrids with introgressions

from wild was compared with that of their 6 respective

controls without introgressions, only 13 hybrids were

significantly influenced (7 positively and 6 negatively)

by introgressions from wild species. The maximum

yield obtained was 43 g/plant in APMS6A 9 IL86-18.

Theoretically, it translates to a yield of 14 t/ha

considering a planting density of 35 plants/sqm. Only

a large scale evaluation of these seven hybrids can

validate if such yields are achievable. One can draw

comparison with multilocation trials conducted by

AICRIP during 2013 using 21 released and 31 new

hybrids tested at 37 locations, the maximum overall

mean yield was 6.5 t/ha but at individual locations

some medium duration hybrids did yield 10–11 t/ha

(Annual Progress Report, AICRIP 2013). The hybrid

pair of APMS6A/IL86-18 with positive influence on

yield and Pusa5A/IL86-18 with negative influence on

yield can be compared for basic studies on yield, GCA

and effect of cytoplasmic line on yield. On the other

hand, effect of wild introgressions in these two hybrids

is opposite compared with their respective control

hybrids without introgressions from wild. These are

interesting hybrids for studying complexity of yield at

gene expression or molecular level.

Wild progenitors have emerged as important reser-

voirs for improving yield and related traits in several

crop plants and QTLs for yield have been mapped

using wild species (Tanksley and McCouch 1997;

Swamy and Sarla 2008). These deal with varietal

improvement programs but in hybrid improvement

programs, the wild accessions have largely been used

only as source of cytoplasmic male sterility. There are

very few reports of using wild rice species for

improving yield of CMS/maintainer or restorer paren-

tal lines in three-line hybrid rice development or of

parental lines used in two-line hybrids (Liang et al.

2004; Sudhakar et al. 2012; Varma et al. 2012).

Inter-specific breeding offers a way of expanding

the gene pool of cultivated rice and enlarging the range

of genetic variation available for plant improvement.

Euphytica (2015) 202:81–95 91

123

Other approaches to develop hybrid rice involve

improvement of new plant type, exploitation of inter

sub specific heterosis and pyramiding of heterosis

genes (Jiang et al. 2002; Wu 2009). The lines derived

from the cross 9311/O. rufipogon had very high yield

potential (Liang et al. 2004). Once an elite restorer line

or male-sterile line is developed, it can be used to

breed a series of hybrids with strong heterosis that can

be applied in rice production. Yuan et al. (1994)

developed the hybrid, J23A/Q611 which out yielded

the check hybrid by 35 %, using R line Q611 carrying

one of the yield QTLs, qyld1.1 or qyld2.1 through

MABC and field selection. O. rufipogon introgressions

have been reported to increase yield across several

genetic backgrounds and environments (Xiao et al.

1998; Moncada et al. 2001; Septiningsih et al. 2003;

Thomson et al. 2003; Lee et al. 2005; Marri et al. 2005;

McCouch et al. 2007; Thalapati et al. 2012).

The selection of parents with good GCA is a prime

requisite for hybrid breeding (Rahimi et al. 2010). In

the present study, three CMS lines (APMS6A,

IR79156A and CRMS32A) and three testers (IL86-

18, IL50-12 and IL50-7) were found to be good

general combiners for yield/plant and 1,000 grain

weight. Two crosses with these lines (IR58025A/

IL50-12 and APMS6A/IL86-18) showed significantly

high SCA as well. Qu et al. (2012) demonstrated that a

large number of additive effect QTLs are associated

with performance per se of back cross recombinant

inbred lines and GCA and dominant effect QTLs are

associated with SCA and heterosis. Xiao et al. (2012)

showed additive effect of QTLs and dominant gene

actions operated independently, and identified 38

(90.5 %) over-dominant genes, and 4 dominant genes

out of 42 QTLs for heterotic loci in test cross F1

population. The usefulness of a particular cross in the

exploitation of heterosis is also judged by SCA effects

(Chakraborty et al. 1994; Swain et al. 2003). The cross

IR79156A/IL50-13 showed highest SCA (6.04) for

yield followed by IR58025A/IL50-12 (4.2),

APMS10A/IL363-5 (3.9) and APMS6A/IL86-18

(3.9) but only APMS6A/IL86-18 gave significantly

higher yield (36 %) over its control APMS6A/KMR3.

High yield in ILs was associated with early

flowering, high number of tillers, panicles, grains,

panicle weight and 1,000 grain weight (Sudhakar et al.

2012). Negative heterosis for plant height and days to

50 % flowering is desirable for breeding short and

early hybrids (Young and Virmani 1990; Mishra and

Pandey 1998). In the present study 16 hybrids showed

significantly negative mid parent and high parent

heterosis for plant height and all these hybrids were

also high yielding. For plant height, Mishra and

Pandey (1998) reported -26.5 to 15.20 % standard

heterosis, while we observed -2.7 to -15.8 % over

better check hybrid PA6201. Peng and Virmani (1991)

reported -27 to 19 % improvement for days to 50 %

flowering. We also observed a desirable decrease of

days to flowering by 6.6–21.4 % in most of the tested

hybrids when compared with their control hybrids.

This is useful as early duration hybrids can fit into

more cropping systems.

In general, the number of productive tillers per

plant is associated with high yield and longer panicle is

associated with more number of spikelets per panicle

(Peng and Virmani 1991). Our observations also

support these reports as there was linear positive

correlation among number of tillers per plant, panicle

length, number of spikelets per panicle and grain yield.

Previous reports also suggest that panicle length,

number of filled grains per panicle and 1,000 seed

weight contribute to increased grain yield (Patnaik

et al. 1990; Mishra and Pandey 1998; Singh and

Maurya 1999). Half of the hybrids showed positive

hybrid vigor for panicle length.

Spikelet fertility, 1,000 grain weight, spikelets per

panicle, and grains per panicle are the most important

traits for yield. It has been suggested that increase in

the latter two traits can help to break the yield plateau

(Singh and Maurya 1999). Our results also showed an

increase of 16.6–34.1 % in spikelets per panicle. In all,

19 hybrids showed positive mid parent heterosis for

spikelets per panicle (14.6–40.2).

The 60 kb region of sub QTL3 of qyld2.1 has nine

candidate genes including five open reading frames

with known functions. QS15 was the only polymor-

phic marker derived from O. rufipogon and present in

all five ILs. QS15 marker amplifies a region which

includes the sixth coding exon of the gene PHYLLO

201 for chloroplast phylloquinone biosynthesis

(Gramene release 39, Oct 2013). LOC_ Os02g37090

for alpha beta hydrolase fold1 domain containing

protein and menaquinone synthesis is also within the

same gene. In another project we have used a large

mapping population of 750 F2 plants from IL50-

7 9 KMR3 to determine linkage between these sub-

QTL markers and yield or yield related traits. QS15

showed significant linkage with height, number of

92 Euphytica (2015) 202:81–95

123

tillers and yield/plant in that population based on

single marker analysis (Haritha et al. 2013). However

loci such as QS15 which are heterozygous in three

KMR3 ILs (50-12, 50-13 and 86-18) may segregate in

hybrids and half the hybrids are expected to be

heterozygous for QS15. The hybrids in our study were

phenotypically uniform and homogenous indicating

that the phenotype is not altered even if such

heterozygous loci in restorer segregate in the hybrid.

There are heterozygous loci reported in several,

perhaps all varieties. These are possibly maintained

as heterozygotes even after continuous selfing or

backcrossing as there may be a chromosomal segment

inversion in this region, so crossing over is inhibited or

in rare cases they may be close to gametic abortive or

other such lethal loci so that homozygotes for those

loci do not survive. A highly heterozygous locus RG2

was reported on chromosome 11 in recombinant

inbred lines of rice and its possible involvement in

heterosis was suggested (Nair et al. 1995). At the level

of single nucleotides also 70,000–76,000 heterozy-

gous SNPs were reported recently in 3 elite restorer

lines IR24, MH63 and SH527 (Li et al. 2012).

All the five check hybrids used in this study are high

yielding and grown widely in India. Most of these

hybrids are of mid early duration. KRH2 is a high

yielding and most popular hybrid since 1996, whereas

DRRH2 is an early hybrid with high yield potential.

Another hybrid DRRH3 is a medium duration hybrid

released recently from DRR. PA6201 and PA6444 are

very popular hybrids developed by private sector. All

these released hybrids are reported to show a mean

grain yield of 6–8 t/ha with 15–20 % yield increase

over best high yielding varieties of the same maturity

duration. In our study, only three hybrids APMS6A/

IL86-18, IR58025A/IL50-12 and IR79156A/IL50-13

showed significantly higher standard heterosis com-

pared to each of these popular hybrids and the control

KRH2. However only APMS6A/IL86-18 showed

significantly higher yield also which is necessary for

commercialization.

These are interesting lines for basic studies on CMS

lines, heterosis and GCA and SCA. APMS6A has

significantly high GCA showed significantly high

yield with only two ILs (IL50-7 and IL86-18) but

significantly high mid and better parent heterosis with

all ILs. The CMS line IR79156A which showed the

highest GCA for yield among all lines/testers showed

significantly high yield only with IL86-18 which itself

showed highest GCA among all ILs. This underscores

the complexity of overall yield in hybrids which

depends on GCA, SCA, heterosis as much as the

interaction of all yield components and environment.

QTLs for combining ability and heterosis have been

mapped in rice and it was shown that QTLs for

combining ability were similar with those of agro-

nomic traits and grouped in clusters (Qu et al. 2012). In

our study GCA of IL86-18 was most significantly

improved when compared to KMR3. It is possible that

there are more introgressions in chromosomal regions

other than at qyld2.1.

In conclusion, we show that some KMR3 ILs with

O. rufipogon introgressions show better combining

ability and heterosis than KMR3 and gave signifi-

cantly higher yield with specific CMS lines. Based on

our results, seven hybrids IR58025A/IL86-18,

APMS6A/IL50-7, APMS6A/IL86-18, CRMS32A/IL50-

7, IR79156A/IL86-18, APMS10A/IL50-7, APMS10A/

IL50-13 appear to be worthy for large scale field

evaluation. Other hybrids can be used for basic studies

on yield, heterosis and combining ability.

Acknowledgments Financial support of Department of

Biotechnology, Government of India (DBT No. BT/AB/FG-2

(PH-II) 2009) and (DBT No. BT/PR13357/AGR/02/695/2009)

to NS is gratefully acknowledged. We thank G Ashok Reddy for

field assistance.

References

Annual Progress Reports vol 1, 2012–13. All India Coordinated

Rice Improvement Project (AICRIP), Directorate of Rice

Research, Hyderabad

Ashikari M, Sakakibara H, Lin S, Yamamoto T, Takashi T,

Nishimura A, Angeles ER, Qian Q, Kitano H, Matsuoka M

(2005) Cytokinin oxidase regulates rice grain production.

Science 29:741–745

Chakraborty S, Hazarika MH, Hazarika GN (1994) Combining

ability in rice. Oryza 31:281–283

Chen X, Temnykh S, Xu Y, Cho YG, McCouch SR (1997)

Development of a microsatellite frame work map provid-

ing genome wide coverage in rice (Oryza sativa L.). Theor

Appl Genet 95:553–567

Falconer DS, Mackay TFC (1996) Introduction to quantitative

genetics. Chapman and Hall, London

Fu Q, Zhang P, Tan L, Zhu Z, Ma D, Fu Y, Zhan X, Cai H, Sun C

(2010) Analysis of QTLs for yield-related traits in Yu-

anjiang common wild rice (Oryza rufipogon Griff.).

J Genet Genomics 37:147–157

Hari Prasad AS, Senguttuvel P, Revathi P, Kemparaju KB,

Ramesha MS, Shobha Rani N, Viraktamath BC (2013)

Rice hybrids released in India. Technical Bulletin No. 68.

Directorate of Rice Research (ICAR), Hyderabad, p 84

Euphytica (2015) 202:81–95 93

123

Haritha G, Naga Deepthi N, Sudhakar T, Gowthami C, Krish-

nam RA, Subhasisa B, Tripura VVGN, Nisha R, Jyothi B,

Sarla N (2013) Genetic dissection of markers within QTL

yld 2.1 from O. rufipogon and their linkage with yield traits

in rice. In: 11th International Symposium on Rice Func-

tional Genomics: sustaining food and nutritional security,

20–23 November, New Delhi, p 133

He GM, Luo X, Tian F, Li K, Zhu Z, Su W (2007) Haplotype

variation in structure and expression of a gene cluster

associated with a quantitative trait locus for improved yield

in rice. Genome Res 16:618–626

Ishimaru K (2003) Identification of a locus increasing rice yield

and physiological analysis of its function. Plant Physiol

133:1083–1109

Jiang T, Li R, Sun C, Wang X (2002) Utilization of diverse rice

ecotypes in heterosis breeding. Breed Sci 52:107–113

Kempthorne O (1957) An introduction to genetic statistics.

Wiley, New York

Lee SJ, Oh CS, Suh JP, McCouch SR, Ahn SN (2005) Identi-

fication of QTLs for domestication-related and agronomic

traits in an Oryza sativa 9 O. rufipogon BC1F7 population.

Plant Breed 124:209–219

Li S, Wang S, Deng Q, Zheng A, Zhu J, Liu H, Wang L, Gao F,

Zou T, Huang B, Cao X, Xu L, Yu C, Ai P, Li P (2012)

Identification of genome-wide variations among three elite

restorer lines for hybrid rice. PLoS ONE 7(2):e30952.

doi:10.1371/journal.pone.0030952

Liang F, Deng Q, Wang Y, Xiong Y, Jin D, Li J, Wang B (2004)

Molecular marker-assisted selection for yield-enhancing

genes in the progeny of 93119 O. rufipogon using SSR.

Euphytica 139:159–165

Luo XJ, Wu S, Tian F, Za XJ, Dong XX, Fu YC, Wang XK,

Yang JS, Sun CQ (2011) Identification heterotic loci

associated with yield-related traits derived from Chinese

common wild rice (Oryza rufipogon Griff.). Plant Sci

181:14–22

Mahadevappa M (2004) Rice production in India—relevance of

hybrid and transgenic technologies. Indian J Genet Plant

Breed 64:1–4

Marri PR, Sarla N, Reddy VLN, Siddiq EA (2005) Identification

and mapping of yield and yield related QTLs from an

Indian accession of Oryza rufipogon. BMC Genet 6:33

McCouch SR, Sweeney M, Li J, Jiang H, Thomson M, Sep-

tinginsih E, Edwards J, Moncada P, Xiao J, Garris A, Tai T,

Martinez C, Tohme J, Sugiono M, McClung A, Yuan LP,

Ahn SN (2007) Through the genetic bottleneck: O. ruf-

ipogon as a source of trait-enhancing alleles for O. sativa.

Euphytica 154:317–339

Mishra M, Pandey MP (1998) Heterosis breeding in rice for

irrigated sub-humid tropics in North India. Oryza 35:8–14

Moncada P, Martinez CP, Borrero J, Chatel M, Gauch HJ,

Guimaraes E, Tohme J, McCouch SR (2001) Quantitative

trait loci for yield and yield components in an Oryza sati-

va 9 Oryza rufipogon BC2F2 population evaluated in an

upland environment. Theor Appl Genet 102:41–52

Nair S, Prasada Rao U, Bennett J, Mohan M (1995) Detection of

a highly heterozygous locus in recombinant inbred lines of

rice and its possible involvement in heterosis. Theor Appl

Genet 91:978–986

Patnaik RN, Pandey K, Ratho SN, Jchuck PJ (1990) Heterosis in

rice hybrids. Euphytica 49:243–247

Peng JY, Virmani SS (1991) Heterosis in some inter-varietal

crosses of rice. Oryza 28:31–36

Prasad Babu A (2009) Genomics of yield enhancing QTLs from

wild species of rice. PhD Thesis, Jawaharlal Nehru Tech-

nological University, Hyderabad

Qu Z, Li L, Luo J, Wang P, Yu S, Mou T, Zheng X, Hu Z (2012)

QTL mapping of combining ability and heterosis of agro-

nomic traits in rice backcross recombinant inbred lines and

hybrid crosses. PLoS ONE 7(1):e28463. doi:10.1371/

journal.pone.0028463

Rahimi M, Rabiei B, Samizadeh H, Ghasemi AK (2010)

Combining ability and heterosis in rice (Oryza sativa L.)

cultivars. J Agric Sci Technol 12:223–231

Rogers SO, Bendich AJ (1988) Extraction of DNA from plant

tissue. In: Gelvin SB, Schilperoort RA (eds) Plant biology

manual, vol A6. Kluwer Academic Publishers, Boston,

pp 1–10

Septiningsih EM, Prasetiyono J, Lubis E, Tai TH, Tjubaryat T,

Moeljopawiro S, McCouch SR (2003) Identification of

quantitative trait loci for yield and yield components in an

advanced backcross population derived from the Oryza

sativa variety IR64 and the wild relative O. rufipogon.

Theor Appl Genet 107:1419–1432

Singh RK, Chaudhary BD (1999) Biometrical methods in

quantitative genetics and analysis. Kalyani Publishers,

New Delhi

Singh M, Maurya DM (1999) Heterosis and inbreeding

depression in rice for yield and yield components using

CMS system. Oryza 36:24–37

Spielman DJ, Kolady DE, Ward PS (2013) The prospects for

hybrid rice in India. Food Sec 5:651–665

Sudhakar T, Madhusmita P, Lakshmanaik M, Prasad Babu A,

Surendhar Reddy C, Anuradha K, Swamy BPM, Sarla N

(2012) Variation and correlation of phenotypic traits con-

tributing to high yield in KMR3-Oryza rufipogon intro-

gression lines. Int J Plant Breed Genet 6:69–82

Swain B, Acharya B, Pandey K (2003) Combining ability ana-

lysis for yield and yield components in lowland rice. Oryza

40:70–72

Swamy BPM, Sarla N (2008) Yield enhancing quantitative trait

loci (QTLs) from wild species. Biotechnol Adv 26:106–

120

Tanksley SD, McCouch SR (1997) Seed banks and molecular

maps: unlocking genetic potential from the wild. Science

277:1063–1066

Thalapati S, Batchu AK, Sarla N, Ramanan R (2012) Os11GSK

gene from a wild rice, O. rufipogon improves yield in rice.

Funct Integr Genomics 12:277–289

Thomson MJ, Tai TH, Mc Clung AM, Lai XH, Hinga ME,

Lobos KB, Xu Y, Martinez CP, McCouch SR (2003)

Mapping quantitative trait loci for yield, yield components

and morphological traits in an advanced backcross popu-

lation between Oryza rufipogon and the Oryza sativa cul-

tivar Jefferson. Theor Appl Genet 107:479–493

Varma CMK, Kalmeshwer Gouda P, Saikumar S, Shenoy V,

Shashidhar HE, Neelamraju S (2012) Transgressive Segre-

gation forYield Traits in Oryza sativa IR58025B X Oryza

meridionalis Ng. Bc2F3 Population under Irrigated and

Aerobic Conditions. J Crop Sci Biotech Springer 15(3):231–

238

Virmani SS (1996) Hybrid rice. Adv Agron 57:377–462

94 Euphytica (2015) 202:81–95

123

Wanjari RH, Mandal KG, Ghosh PK, Adhikari T, Rao NH

(2006) Rice in India: present status and strategies to boost

its production through hybrids. J Sustain Agric 28:19–39

Wu X (2009) Prospects of developing hybrid rice with super

high yield. Agron J 101:688–695

Xiao J, Li J, Grandillo S, Ahn SN, Yuan L, Tanksley SD,

McCouch SR (1998) Identification of trait-improving

quantitative trait loci alleles from a wild rice relative,

Oryza rufipogon. Genetics 150:899–909

Xiao J, Xiao Y, Jin S (2012) Genetic-basis analysis of heterotic

loci in Dongxiang common wild rice (Oryza rufipogon

Griff.). Genet Res Camb 94:57–61. doi:10.1017/S00166723

12000250

Xie X, Song MHF, Jin SN, Ahn JP, Suh HG, Wang H, McCouch

SR (2006) Fine mapping a grain weight quantitative trait

loci on rice chromosome 8 using nearly-isogenic lines

derived from a cross between Oryza sativa and O. rufipo-

gon. Theor Appl Genet 113:885–894

Xie X, Jin F, Song MH, Suh JP, Hwang HG, Kim YG, McCouch

SR, Ahn SN (2008) Fine mapping a yield-enhancing QTL

cluster associated with transgression variation in an Oryza

sativa and O. rufipogon cross. Theor Appl Genet 116:613–622

Yoon DB, Kang KH, Kim HJ, Ju HG, Kwon SJ, Suh JP, Jeong

OY, Ahn SN (2006) Mapping quantitative trait loci for

yield loci for yield components and morphological traits in

an advanced backcross population between Oryza gran-

diglumis and the O. sativa japonica cultivar Hwaseongb-

yeo. Theor Appl Genet 112:1052–1062

Young J, Virmani SS (1990) Heterosis in rice over environ-

ments. Euphytica 51:87–93

Yuan L (2014) Development of hybrid rice to ensure food

security. Rice Sci 21:1–2

Yuan L, Yang Z, Yang J (1994) Hybrid rice in China. In: Vir-

mani SS (ed) Hybrid rice technology: new developments

and future prospects. International Rice Research Institute,

Los Banos, pp 143–147

Euphytica (2015) 202:81–95 95

123