PHENOTYPIC AND MOLECULAR CHARACTERIZATION OF KENYAN

BASMATI RICE VARIANT LINES: AN APPROACH TO GERMPLASM

IMPROVEMENT AND PRESERVATION

FAITH M. MWENDWA, PROF. E. N. MAGIRI, DR. WANJOGU

INTRODUCTIONRice (Oryza sativa) cultivation is a source of

livelihood for one fifth of the world’s population.

Many families around Mwea rely on rice farming as their source of livelihood.

The most preferred varieties in Kenya are basmati (pishori).

In Kenya, the quality of basmati has been declining as reflected by reduced aroma, poor grain quality and other unique characteristics.

The basmati rice grown in Mwea rice paddies has not been characterized.

Project was carried out to characterize the basmati variant lines currently grown in Mwea and identify those with desirable traits and preserve the germplasm.

Certified rice seeds will be generated and availed to farmers in order to increase yield hence income and alleviate poverty.

General objective

Specific objectives

1. Identify and select variant lines of Kenyan basmati 370 grown at Mwea Irrigation Scheme

2. Perform phenotypic and histochemical characterization of the lines

3. Undertake molecular characterization of the lines using SSR markers

4. Select lines with desirable qualities based on phenotypic, histochemical and molecular characterization for germplasm certification and preservation

To identify and characterize the Kenyan basmati variant lines through phenotypic and genotypic analysis

METHODOLOGY

Selection of variant lines

Planting of lines-SRI

-Paddy

-Phenotypic characterization-Histochemical test for aroma compound

Molecular characterization (SSR

primers)

Selection of lines with desirable

characteristics

PHENOTYPIC CHARACTERIZATION

Qualitative data tiller typeawnsleaf colourDays to flowering

Quantitative data plant height (cm) tiller number productive tillers (%) panicle length (cm) number of grains per panicle filled grains per panicle (%) 1000 grain weight (grams) yield (kg/ha)

Unique characteristics in some lines

Erect tillers Medium green leaves

No anthocyanin colouration on stem

Awns Brown awns

Flowered 95 ± 2 days after sowing

L13 (open tillers) L7 (light green) and L12 (dark

green) L2

L13 L12 (long whitish awns) and

L3 & L14 (short purple awns in some hills) but brown when grains dry

L2 (89 days after sowing) and L1 & L14 ( 98 and 99 days after sowing)

Control and other lines Except Line:

Plate 1: Anthocyanin colouration on stem in L2 Plate 2: No anthocyanin colouration on Stem in control

L2 CONTROL

Morphological characteristics

Plate 3: Light green leaves in L7

Plate 4: Medium green leaf colour in control

Plate 5: Dark green leaves in L12

Plate 6 : Open tillers in L13

Plate 7 : Erect tillers in control

Plate 8: Early flowering in L2 Plate 9: Flowering in control and L11

Plate 10: Short purple awns and tips in some L3 and L14 hills Plate 12: Sample L13 had no

awns and had short and broad grains

Plate 11: Long brown awns and tips normally

found in control

Plate 13: Long brown awns and tips normally found in control

Plate 14 and 15: Long whitish awns in L12 but brown when dry

Leaf colour, tiller type, awns and flowering dates of the lines

LINE LEAF COLOUR TILLER TYPE AWNS 1ST FLOWERING

Control Medium green Erect tillers Long, brown(2.5cm) 95 days

L1 Medium green Erect tillers Long, brown(2.0cm) 98 days

L2 Medium green Erect tillers Long, brown(2.3cm) 89 days

L3 Medium green Erect tillers Long, brown(2.1cm) 97 days

L4 Medium green Erect tillers Long ,brown(2.4cm) 96 days

L5 Medium green Erect tillers Short ,brown(1.2cm) 95 days

L6 Medium green Erect tillers Long ,brown(2.0cm) 93 days

L7 Light green Erect tillers Long ,brown(1.8cm) 96 days

L8 Medium green Erect tillers Long ,brown(1.8cm) 94 days

L9 Medium green Erect tillers Long ,brown(2.2cm) 95 days

L10 Medium green Erect tillers Long ,brown(2.0cm) 96 days

L11 Medium green Erect tillers Long ,brown(1.8cm) 95 days

L12 Dark green Erect tillers Long ,whitish(3.0cm) 96 days

L13 Medium green Open tillers No awns 97 days

L14 Medium green Erect tillers Long ,brown(1.6cm) 99 days

Control

L1 L2 L3 L4 L5 L6 L7 L8 L9 L10 L11 L12 L13 L1485

90

95

100

DAYS TO FLOWERING

DAYS TO FLOWERING

DA

YS

Con-trol

L1 L2 L3 L4 L5 L6 L7 L8 L9 L10 L11 L12 L13 L14 0

0.5

1

1.5

2

2.5

3

3.5

AWN LENGTH

AWN LENGTH

LEN

GTH

(cm

)

Quantitative parametersSRI PADDY Taller (105.97-124.42 cm) More tillers (39.43-54.67

tillers) Less productive tillers

(66.41-88.53%) Longer panicles (24.64-

29.12 cm) More grains per panicle

(97.52-124.26 grains) More filled grains per

panicle (76.25-93.33%) Heavier grains (24.65-

33.48 grams for 1000 grains)

More yield (12.67-25.42 kg/ha)

Shorter (97.50-106.51 cm) Less tillers (16.22-19.82

tillers) More productive tillers

(84.36-90.29%) Shorter panicles (19.59-

25.04 cm) Less grains per panicle

(36.88-69.19 grains) Less filled grains per

panicle(59.73-83.04%) Lighter grains(15.25-24.84

grams for 1000 grains) Less yield (0.87-3.53

kg/ha)

Con-trol

L1 L2 L3 L4 L5 L6 L7 L8 L9 L10 L11 L12 L13 L1495

100

105

110

115

120

125

130

PLANT HEIGHT (SRI AND PADDY)

SRIPADDY

LINES

PLA

NT

HEIG

HT (

cm)

Con-trol

L1 L2 L3 L4 L5 L6 L7 L8 L9 L10 L11 L12 L13 L14 10

15

20

25

30

35

40

45

50

55

60

TILLER NUMBER (SRI and PADDY)

SRIPADDY

LINES

TIL

LER

N

UM

BER

Con-trol

L1 L2 L3 L4 L5 L6 L7 L8 L9 L10 L11 L12 L13 L14 60

65

70

75

80

85

90

95

PRODUCTIVE TILLERS (SRI and PADDY)

SRIPADDY

LINES

PR

OD

UC

TIV

E

TIL

LER

S (

%)

Con-trol

L1 L2 L3 L4 L5 L6 L7 L8 L9 L10 L11 L12 L13 L14 15

20

25

30

35

PANICLE LENGTH (SRI and PADDY)

SRIPADDY

LINES

PA

NIC

LE

LEN

GTH

(cm

)

Con-trol

L1 L2 L3 L4 L5 L6 L7 L8 L9 L10 L11 L12 L13 L14 30

40

50

60

70

80

90

100

110

120

130

NUMBER OF GRAINS PER PANICLE (SRI and PADDY)

SRIPADDY

LINES

NO

. O

F G

RA

INS

PER

PA

NIC

LE

Con-trol

L1 L2 L3 L4 L5 L6 L7 L8 L9 L10 L11 L12 L13 L14 55

60

65

70

75

80

85

90

95

FILLED GRAINS PER PANICLE (SRI and PADDY)

SRIPADDY

LINES

FIL

LED

GR

AIN

S P

ER

PA

NIC

LE (

%)

Con-trol

L1 L2 L3 L4 L5 L6 L7 L8 L9 L10 L11 L12 L13 L14 10

15

20

25

30

35

1000 GRAIN WEIGHT (SRI and PADDY)

SRIPADDY

LINES

1000 G

RA

IN W

EIG

HT (

gra

ms)

Con-trol

L1 L2 L3 L4 L5 L6 L7 L8 L9 L10 L11 L12 L13 L14 5000

55000

105000

155000

205000

255000

YIELD (SRI and PADDY)

SRIPADDY

LINES

YIE

LD

(kg/M

2)

HISTOCHEMICAL TEST FOR AROMA

2 acetyl pyrolline is the main compound responsible for aroma in basmati rice (Bradbury et. al., 2005).

2-acetyl-1-pyrroline + 2, 4-dinitrophenylhydrazine 2-acetyl-phenyl hydrazone

(Orange-red colour)

An orange red colour on sections indicated presence of 2-acetyl-pyrolline.

Thirteen of the fourteen lines had the orange red colour in the sections which varied in intensity and distribution.

L13 did not have the colour.

POSITIVE CONTROL

Orange-red regions in section

L6

L7

Plate 16,17,18: orange red colour distributed throughout the section

L2

L8

L9

Plate 19,20,21 : Colour is found near the margin

L3

L11

L12

L14

Plate 22,23,24 : Orange red spots in regions near margins

L1

Orange-red regions on margin

L4

L5

L10

Plate 25,26,27,28 : Colour found on the margins

NEGATIVE CONTROL

L13

Plate 29,30 : No reaction

MOLECULAR CHARACTERIZATION

MarkerChromosome

locationNumber of

allelesNumber f rare alleles

Size range (bp)

Highest frequency

allele PIC value

RM16 3 9 2 175-1414 175 0.837

RM161 5 9 3 177-810 211, 277 0.831

RM223 8 7 2 170-1438 530, 1438 0.812

RM72 8 3 1 101-209 209 0.550

RM171 10 5 4 374-723 374 0.42

Total 33 12

Mean 6.6 2.4 0.690

RM 16 primer

LINES Band positions due to primers (bps)RM 16 primer

1414 1347 440 365 308 289 213 194 175CONTROL + + + + + +

NON BASMATI + + + +L1 +L2 + + +L3 + + +L4 + + +L5 + + +L6 +L7 +L8 + + + + +L9 +

L10 + + + + +L11 + + + +L12 + + + + +L13 +L14 + + +

RM 161 primer

LINES Band positions due to primers (bps)RM 161 primer

810 775 705 550 523 401 277 211 177CONTROL + + + + +

NON BASMATI + + + + + +L1 + +L2 + + + + +L3 + + + + +L4 + + + + +L5 + + + +L6 + + + + +L7 + + + + +L8 + + + + +L9 + + + + + +

L10 + + + + +L11 + + + + +L12 + + + + +L13 + + +L14 + + +

LINES Band positions due to primers (bps)RM 223 primer

1438 530 413 381 187 179 170CONTROL + + + + +

NON BASMATI + + + +L1 +L2 + + + + +L3 + + + + +L4 + + +L5 + + + + +L6 + + +L7 + + +L8 + + + + +L9 + + +

L10 + + + + +L11 + + +L12 + + + +L13 +L14 +

RM 223 primer

RM 72 primer

LINES Band positions due to primers (bps)RM 72 primer

209 192 101CONTROL + +

NON BASMATI + +L1 +L2 +L3 +L4 +L5 + +L6 + +L7 + +L8 + +L9 +

L10 +L11 +L12 + +L13 + +L14 + +

RM 171 primer

LINES Band positions due to primers (bps)

RM 171 primer723 579 425 391 374

CONTROL +NON BASMATI + +

L1 +L2 +L3 +L4 +L5 +L6 + +L7 +L8 +L9 +

L10 +L11 +L12 + +L13 + +L14 +

Figure : Phylogenetic tree of 14 variant rice lines based on UPGMA cluster analysis using allelic diversity data for 5 SSR markers (33 alleles)

CONCLUSION

Variant lines of basmati have some characteristics like normal basmati.

L13 was high yielding but lacked the characteristics of basmati 370 rice.

 L6 and L7 had much aroma, long slender half-

spindle shaped grains and high yield in SRI.

RECOMMENDATIONS

Further characterization of the variant lines be carried out based on cooking ability, measure of amount of 2-acetyl-1-pyrroline and sequencing to establish the genetic diversity.

Further work should be on L6 and L7 focusing on molecular characterization and test of the amount of aroma compound with view of developing certified seeds.

ACKNOWLEDGEMENTS

JKUAT RPE for funding the project

Collaborators MIAD Centre Staff

Thank You