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

INTRODUCTION Rice (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 type  awns  leaf colour  Days 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 linesExcept Line:

Plate 1: Anthocyanin colouration on stem in L2Plate 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 L2Plate 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 LINELEAF COLOURTILLER TYPEAWNS1ST FLOWERING ControlMedium greenErect tillersLong, brown(2.5cm)95 days L1Medium greenErect tillersLong, brown(2.0cm)98 days L2Medium greenErect tillersLong, brown(2.3cm)89 days L3Medium greenErect tillersLong, brown(2.1cm)97 days L4Medium greenErect tillersLong,brown(2.4cm)96 days L5Medium greenErect tillersShort,brown(1.2cm)95 days L6Medium greenErect tillersLong,brown(2.0cm)93 days L7Light greenErect tillersLong,brown(1.8cm)96 days L8Medium greenErect tillersLong,brown(1.8cm)94 days L9Medium greenErect tillersLong,brown(2.2cm)95 days L10Medium greenErect tillersLong,brown(2.0cm)96 days L11Medium greenErect tillersLong,brown(1.8cm)95 days L12Dark greenErect tillersLong,whitish(3.0cm)96 days L13Medium greenOpen tillersNo awns97 days L14Medium greenErect tillersLong,brown(1.6cm)99 days

Quantitative parameters SRIPADDY Taller ( cm) More tillers ( tillers) Less productive tillers ( %) Longer panicles ( cm) More grains per panicle ( grains) More filled grains per panicle ( %) Heavier grains ( grams for 1000 grains) More yield ( kg/ha) Shorter ( cm) Less tillers ( tillers) More productive tillers ( %) Shorter panicles ( cm) Less grains per panicle ( grains) Less filled grains per panicle( %) Lighter grains( grams for 1000 grains) Less yield ( kg/ha)

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 Marker Chromosome location Number of alleles Number f rare alleles Size range (bp) Highest frequency allelePIC value RM RM , RM , RM RM Total3312 Mean

RM 16 primer LINESBand positions due to primers (bps) RM 16 primer CONTROL NON BASMATI++++ L1+ L2+++ L3+++ L4+++ L5+++ L6+ L7+ L L9+ L L L L13+ L14+++

RM 161 primer LINESBand positions due to primers (bps) RM 161 primer CONTROL+++++ NON BASMATI L1++ L L L L5++++ L L L L L L L L13+++ L14+++

LINESBand positions due to primers (bps) RM 223 primer CONTROL+++++ NON BASMATI++++ L1+ L L L4+++ L L6+++ L7+++ L L9+++ L L11+++ L L13+ L14+ RM 223 primer

RM 72 primer LINESBand positions due to primers (bps) RM 72 primer CONTROL++ NON BASMATI++ L1+ L2+ L3+ L4+ L5++ L6++ L7++ L8++ L9+ L10+ L11+ L12++ L13++ L14++

RM 171 primer LINESBand positions due to primers (bps) RM 171 primer 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

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