J. Basic. Appl. Sci. Res.,8(6)1-10,2018 | ISSN2090-4304 |
JournalofBasicandApplied | |
©2018,TextRoadPublication | ScientificResearch www.textroad.com |
OusmaneSeyniDiakité1,MamadouAissata1,SissokoAliou2,SanogoSekouba2,MamoutouKouressy2, VaksmannMichel3,4 ,DanielK.Dzidzienyo5,DanquahEric5,TongoonaPangirayi5, KarimTraoré6,NiabaTeme2
1InstitutNationaldelaRechercheAgronomiqueduNiger(INRAN)Niger.BP429Niamey 2Institutd’EconomieRurale(IER)BP258RueMohamedV.BamakoMali.
3CIRAD, UMR AGAP, BP 1813,Bamako, Mali 4 AGAP, Univ. Montpellier, CIRAD, INRA, Montpellier SupAgro, Montpellier, France.
5 UniversityofGhana,WestAfricaCentreforCropImprovement(WACCI).PMBLG30,LegonAccraGhana 6 AfricaRiceCenter,St.Louis,BP96,Sénégal.
Received: August 26, 2018 Accepted: October 12, 2018
ABSTRACT
Introgressionofbmr genesfromlessadapteddonorparenttowelladaptedhighyieldingbiomassvarietieswithpoor nutritional value is very important for sustainable cattle feeding during pasture scare time in the Sahel. The main objective of this work was to introgress bmr6 and bmr12 genes in Nigerien and Malian sorghum varieties background for dual purpose grain and biomass potential. The plant material was composed of two improved sorghumvarieties(Sepon82andKallaKéné) andElmotaafarmerpreferredvarietyasrecurrentparents.bmr donor parents were redlan bmr6, Tx630 bmr12 and Wheatland bmr12. The hand emasculation technique was used to introgressbmr genesinrecurrent parentsto produceF3 and BC1F3populationsatSotubaresearch Station inMali fromJanuary2016 toJune 2017.Anthocyanin pigment andheterosiseffectswere keyphenotypic traits to identify F1 andBC1F1 plantsduring thepopulationdevelopment. Anthocyanin allowed theidentification ofF1 plants in a crossinvolvinganthocyanin(purpleplant)andtanplants,whileforbothtanplantscross,heterosiseffectwasmajor keytodiscriminateF1 fromparentallinesandbmr segregationinF2toascertainsuccessfulcrosses.Theχ2 testwas used to analyzebmr segregation ration. Segregation ratios ofbmr plants in F2 and BC1F2 showed agood fit of a singlerecessivegene(3:1).bmr 6 and12 genesweresuccessfullytransferredtothreerecurrentparentsvarietieswhich areatF4andBC1F3generationforgrainandbiomassyieldspotentialtestsinNigerduringthe2017croppingseason. KEYWORDS:brown midrib genes,phenotypicmarker,segregatingpopulations.
INTRODUCTION
Thebrownmidrid(bmr)traitisaphenotypicmarkerlinkedtoageneticmutation.Thebmrplanttypehasbeen identifiedinmaize,sorghumandmillet.[1]reportedthatbmr mutantswerefirstdiscoveredinmaize,withatotalof fourgenesanddescribedeachgenesegregatingasasimpleMendelianrecessivecharacter.Insorghum,[2]claimed thatthebmr abbreviationwasadoptedtodistinguishitfrombm,alreadyinuseforthesorghumbloomlessmutants.
[3] produced 19 bmr sorghum mutants by chemically treating seed from two grain sorghum lines. These mutantswerenumberedbmr 1tobmr 19.[3]suggestedthatthebmr-6,bmr-12,andbmr-18 should beselected for furtherevaluation.However,thebmr-6andbmr-12genotypesaremoreprevalentthanthebmr-18 [4].
[5] reported four allelic groups (bmr19, bmr2, bmr6 and bmr12) of which the latter mentioned here are involved inimprovingforagequality.[6]statedthatmutationsin4-coumarate:coenzymealigase(4CL),cinnamyl alcohol dehydrogenase (CAD2) and caffeic O-methyltransferase (COMT) genes contribute to the phenotype of bmr2,bmr6 andbmr12 groupsrespectively.[7]assertedthatthebmr19 appearstobeoflimitedvalueforforageand bioenergy applications and is not publicly available. [8] demonstrated that the bmr phenotype is the result of a recessive mutation in the lignin biosynthesis pathway. While for ([9]; [10], the effect ofbmr mutations on forage qualityvariesdependingonthegeneticbackgroundofrecipientinwhichthemutationisintroduced.Moreover,[11] emphasized on the importance of identifying a suitable genetic background to allow an optimal impact of the mutation.
CorrespondingAuthor:OusmaneSeyniDiakité,InstitutNationaldelaRechercheAgronomiqueduNiger(INRAN)Niger.BP 429Niamey.E-mail:dseyni@wacci.ug.edu.gh
Citation: OusmaneSeyniDiakité,MamadouAissata,SissokoAliou,SanogoSekouba,MamoutouKouressy,VaksmannMichel,DanielK.Dzidzienyo,DanquahEric,
TongoonaPangirayi,KarimTraoré,NiabaTeme,2018,Brown midrib 6 and12 Genes IntrogressioninTwoNigerienandOneMalianSorghumVarieties:A
PracticalGuidetoYoungScientistswithLimitedMolecularFacility;JournalofBasicandAppliedScientificResearch,8(6)1-10.
Thebmr plantsexhibitareddish brownpigmentation (Photo1)onits leafmidrib and stalk pith in sorghum associatedwithlignifiedtissuessincetheplantshaveaboutfiveexpandedleaves[12].[13]suggestedthatbmr plants havelignin which is lesspolymerized and containsless phenolicmonomers thatcan affectdigestion. [14] showed that thebmr mutantofsorghumhavesignificantlylower levelsoflignin contentand estimated around51%lessin theirstemsand25%lessintheirleaves; furthermore,bmr sorghumsilagehas17%lessligninthanregularsorghum silagemakingitmoredigestible.Thispropertymakesbmr stoversveryattractiveforcattlefeedinginmanycountries toimprovemilkandmeatproduction.
In South Saharan African (SSA) countries particularly in the Sahel region, cereal stovers constitute an importantsourceof feed for livestockduring feed scarcitytime. Unfortunately, stover from cereals is often of low nutritional quality. [15] affirmed that the potential of cereal crop residues as animal feed is enormous if all the different types of cereal crops are considered and if appropriate methods of improving their nutritive value are employed.
In Niger and Mali research to improve sorghum stover feed value in recent years often emphasized postharvest treatment of crop residues ammonization. This method involves a good control of the technique; additionally, it can also be expensive due to the cost of fertilizers. On the other hand, stay green trait has been incorporated into new lines to enhance cereal stovers quality after harvest. Adding bmr trait to local cultivars and newlydevelopedlineswillbemoresustainableandwillbringasignificantimpactonlivestockproductivityinthese twocountries.
To date no studies have been reported on the bmr sorghumintrogression in local cultivars in Niger and in Mali.Themainobjectivesofthispopulationbreedingweretointrogressbmr6 andbmr12 genesintwoNigerienand one Malian sorghum varieties using phenotypic markers and to validate a dominant Mendelian segregation ratio usingχ2 .
Biologyandmethodsofintrogressinganewtraitinsorghum
Sorghum has a perfect-flower and is a self-pollinated crop. Its level of outcrossing is contingent on the specificgenotypebeinggrownandtheenvironmentalconditionsencounteredpriortoandduringanthesis[16].[17] estimated theoutcrossing from1to 10% and can also reach 30-60%depending to the panicle compactness,while [18]claimedsorghumoutcrossingisestablishedfromnoneto30%.
Sorghumfloweringstartsfewdaysafterpanicleexsertionfromtop tobottom.Basedonthefloweringhabits, sorghumbreedershave developed several methods ofhybridization and outcrossing elimination (Table2).Indeed, fertile linepanicles’ earlyselfing ensurepurelinemaintenancewhereasseveral crossing methodswereestablished forsegregatingpopulations’development.[16]reportedfourdifferentmethodsofcrosses:
Hand emasculation (HE)
Panicle chosen for HE is prepared 1-2 days before emasculation. Flowers are emasculated the day before anthesis.Suchfloretsoccurbelowandwithinabout3cmofopenedfloretsinasorghumpanicle.Allopenspikelets areremovedwithscissors.Inaddition,allfloretsexceptthosethataretobeemasculatedareremoved,leavingonly thefloretsthatareexpectedtoopenthenextday.Threeanthersarecoaxedoutoftheenclosinglemmaandpaleaby inserting a sharpened pencil or similar pointed instrument such as forceps. Care must be taken not to break the anthers,andiftheanther isbreached,thatflower shouldberemovedandinstrumentsrinsedtoavoidcontaminating thenextfloret.Everyanthermustberemovedbeforethesetoffloretsiscompletelyemasculated[18].
[19]reportedthataseriesofnuclearrecessivemalesterilitygenes,designatedasms1 throughms7,havebeen characterized insorghum.Thesemutations,intherecessiveconditionresultinamale-sterileplantthatcanbeused for hybridization. For [20], the most commonly used is the ms3 due to its stable expression over different environments. These mutations in the recessive condition result in a male-sterile plant that can be used for hybridization.
CMSoccursinsorghum.TheCMSsystemwasidentifiedandcharacterizedby[21].Therearemanydifferent CMSsystems,eachcausedbyadifferentmutationinthecytoplasmandeachiscomplementedbydifferentnuclear restoration loci. [18] reportedthat formostCMS systems,theinteractionofcytoplasmic andnuclear genesdefines whether all specific lines are fertile or sterile. TheCMS system(Table1) iswidely used in sorghumhybrid seeds production and the following designations were conventionally adopted: A-line=Male sterile line; B-line=Male fertileandanAsterilitymaintainerline;R-line=MalefertileandisA-linefertilityRestorerline.Themostcommonly used CMS system is the A1 system. In theCMS system, line that has [A] cytoplasm musthave adominant allele
J. Basic. Appl. Sci. Res.,8(6)1-10,2018
presentinthenucleargenometorestoremalefertility.Ifthelinelacksthedominantalleleforfertilityrestoration,the plant will be male sterile. Table1 contains information on genotypes and their corresponding phenotypes types in sorghumhybridseedsproduction.
Table1:TheCMSsysteminsorghum
Line | Cytoplasm | Genotype | Phenotype |
---|---|---|---|
A-line | [A] | rfrf | Malesterile |
B-line | [N] | rfrf | Malefertile |
R-line | [A]or[N] | RFRF | Malefertile |
Hybrid | [A] | RFrf | Malefertile |
Source:[20].
Hot-wateremasculation
This method of emasculation was developed by [22]. In practice, open florets of the selected panicle are removed and the entire panicle is enclosed in a waterproof sleeve of rubber or plastic tied securely around the peduncle. Thepanicle is immersed in water heated to 42-48°C for 10 min. Thistreatment kills the majorityofthe pollengrainsbutdoesnotdamagetheovary[18].Thepanicleisallowedtodryandthencoveredwithapaperbag. Pollenfromtheselectedmaleparentisdustedontothesterilizedpanicle3-4daysafterthehotwatertreatment[18].
Antherdehiscencecontrolbyuseofhumidity
Thismethodwasdevelopedby[23]tocontrolantherdehiscenceusingthehumiditycreatedfromcoveringthe paniclewithaplasticbagpriorto flowering.Thismethod,isalsoknownasplasticbagemasculationand/orpoured crossing. Itallowsabreedertomakelargenumbersofcrossesinashortamountoftime.Thisemasculationmethod isdescribedby[18]asfollow:plantsselectedforuseasfemalesinpouredcrosseshavefloweredapproximately2.55cmfromthepanicleapex.Theportionsof thepaniclethathaveflowered areremoved.Thebottom floretsin the panicle arealsoremoved,sothat3-5 cmofthepanicleremains.Thispanicleiscoveredwithaplasticbag andtied firmlyonitspedunclewhilecoveringitwithapollinatingbagtoshadeandtoreducethetemperatureofthepanicle undertheplasticbag.Thebagremainsontheplantfor2-3daysduringwhichthepaniclecompletesanthesis.These bagscreateahighlyhumidatmosphereinwhich themoisturecontentinhibitsantherdehiscence.Becauseallofthe anthersarenotremoved,acertainlevelofself-pollinationwilloccurinseedfromapouredcross.
Table2:advantagesanddisadvantagesofemasculationtechniquesinsorghumfordiversitydevelopment.
Method | Advantages | Disadvantages |
---|---|---|
Handemasculation-HE | Requiresimpletools[17]. | Varietiesdifferineaseofemasculation[17]. Producesmallquantitiesofseeds[16].Takesspecialskillandtime consuming[16]. |
Geneticmalesterility-GMS | Simpleandbreederscanmakealarge numberofcrossesinashortamount oftime[18]. | Cannotbeusedforcommercialhybridsseedsproduction.Request thepriordevelopmentofms3lines. |
Cytoplasmicmalesterility-CSM | Possibilityofcommercialhybrid seedsproduction[17]. Simpleandbreederscanmakealarge numberofcrossesinashortamount oftime[18]. | RequestthepriordevelopmentofAandBlines.Femaleparent limitedtoAlinesandthusreductionofavailablecytoplasms |
Hot-wateremasculation | Simpletouseingreenhouse[18]. | Highlevelofseedlingsfromself-fertilizationinthenurseries. |
Antherdehiscencecontrolwith humidity | Simpleandbreederscanmakealarge numberofcrossesinashortamount oftime[18]. | Acertainlevelofself-pollinationcanoccurinseedfromapoured cross[18]. Inmostcases,theproportionofprogenythatareF1hybridswill varybasedonthespecificgenotypeusedasafemaleparent,the fecundityofthepollenparent,andtheenvironmentalconditions duringtheprocess[18]. |
Sources:[17];[18];[16]
MATERIALANDMETHODOLOGY
Material
The plant material was composed of six Caudatum types (Table 3): Sepon82 (Niger); Kalla Kéné fromthe MaliannationalcatalogueandElmota(Nigerienfarmervariety)asrecurrentparents(RP).Threebmr donorparents (DP), originated from Purdue University, Indianapolis (USA), composed of Redlan bmr6, Wheatland bmr12, and Tx630bmr12, wereused[24].
Citation: OusmaneSeyniDiakité,MamadouAissata,SissokoAliou,SanogoSekouba,MamoutouKouressy,VaksmannMichel,DanielK.Dzidzienyo,DanquahEric, TongoonaPangirayi,KarimTraoré,NiabaTeme,2018,Brown midrib 6 and12 Genes IntrogressioninTwoNigerienandOneMalianSorghumVarieties:A PracticalGuidetoYoungScientistswithLimitedMolecularFacility;JournalofBasicandAppliedScientificResearch,8(6)1-10.
TheRPs(Sepon82andElmota)arelargelygrownbyNigerienfarmersfortheirstoverandgrainyieldpotential.RPs stoveryieldpotentialdatawerecollectedfromsorghumbreedersofInstitutNationaldelaRechercheAgronomique duNiger(INRAN)andfarmersduringthe2016ParticipatoryRuralAppraisal(PRA)surveyinNiger.SelectedRPs werethereforeverygoodcandidatesforthedualpurposesorghumbreeding.KallaKénéisastaygreenlatematuring improvedvarietyfromMaliansorghumbreedingprogram.KallaKénéisadwarfandwellappreciatedforitsstover quality.TwentyF1selfedseeds(F2)andsixty(60)BC1F1selfedseeds(BC1F2)wereusedforχ²test.
Table3:Origins,agronomiccharacteristicsandenvironmentalconditionsofrecurrentanddonorparents involvedinthebmr sorghumpopulationdevelopmentinNigerandMali.[25]
Plant material | Agronomiccharacteristicsofrecurrentparents | ||||||||
---|---|---|---|---|---|---|---|---|---|
Origin | Race | Planttype | Midrib color | Plant Height (cm) | Days Maturity (day) | Grain yield (t/ha) | Biomass yield(kg/ha) | Lodging | |
Sepon82 | ICRISAT | Caudatum | Tan | Green | 150-170 | 90-105 | 2-2.5 | Good | Resistant |
Elmota | Landrace | Caudatum | Anthocyanin | White | 200-250 | 80-90 | 0.8-1 | Acceptable | Resistant |
Kallakene | IER | Caudatum | Tan | White | 150-190 | 130-140 | 2.8 | 11553 | Resistant |
DonorParents | |||||||||
Redlan bmr6 | Purdue Univ. | Caudatum | Anthocyanin | Brown | 130-135 | 95-100 | 1.1-1.4 | Poor | Resistant |
Tx630 bmr12 | Purdue Univ. | Caudatum | Tan | Brown | 90-100 | 95-100 | 1-1.5 | Poor | Resistant |
Wheatland bmr12 | Purdue Univ. | Caudatum | Anthocyanin | Brown | 90-100 | 95-100 | 1-1.5 | Poor | Resistant |
Photo1showsdifferenttypesofmidribofRPandDPusedinthebmrpopulationdevelopment.
Photo1:differenttypesofsorghumleavesmidribcolortakenatSotubain2016:A=whitemidrib(Elmota); B=brown midrib (Wheatlandbmr12,Redlanbmr6 andTx630bmr12)andC=dullgreenmidrib(Sepon82)
J. Basic. Appl. Sci. Res.,8(6)1-10,2018
Methodology
bmr populations development
Populationdevelopmentactivities for theintrogressionofbmr traitinto threerecurrentparentstookplacein MalifromJanuary2016untilMay2017atSotubaResearchStation.ThegeographiccoordinatesofSotuba:12°39’ N;07°56’Oat320maltitude.
January2016-June2016:F1production
Crossing blocks were installed in early January2016 off season under drip irrigation system (Photo 1). As sorghum is a short-day plant, its cultivation in January, when photoperiod is short, causes early flowering thus allowing crossing synchronization between the two parents. Nevertheless, two planting dates (27/01/2016 and 9/02/2016) for the DP parents and one date of planting for the RP (27/01/2016) were used to ensure flowering synchronizationrequiredforcrossings.Furthermore,tooearlyparentswereprunedduringvegetativeperiodtoallow newshootsemergence for floweringsynchronization betweenparents.Therewere10 hillsplanted for expected10 plantsperDPorRPparentalline.
At floweringstage,RPswerehand emasculated using tweezers/forceps(Photo2A).Thehandemasculation techniqueusingtweezerswaschosentominimizetheamountofseedlingsfromself-fertilizationinF1plants. Fifty to sixty flowers of each RP per cross were chosen for each crossing. Flowers from the top of each panicle were emasculated.Antherswereremovedineachfemaleandmaleflower.Twotothreedaysafteremasculationrecurrent parentsweredusted with donor parentpollen.Flowersfromthebottomofthesamefemalepaniclewerebaggedin order to produce identical recurrent parents’ seeds (Photo 2 B). Other flowers on the same female plant were completelydiscarded to avoid seed pollution. Likewise, each donorparentwas crossed to each recurrentparent to produceF1 seed for each population.Maturedcrosseswereharvested andkeptseparately. Donor parentplantseed wasalso selfed.RP andF1 seedswerethreshed independentlyto avoid anyseed mixtureandstored at4°C for the nextF2orBC1populationdevelopment.
June2016-October2016:BC1F1production
F1sseeds fromall successfulcrosses and their respective parental line seedswere treated with aCaïman Rouge P (Permethrine 25 g/kg + Thirame 250 g/kg) an insecticide fungicide, pre-germinate in Petri-dishes, and then were transferred in plastic pots containing 1 kg of compost for BC1F1 seed production to secure seed. After 20 days period, F1 were transplanted in one row of 3 m. Parental seed was planted next to F1 for conformity control. Heterosisandpresenceofanthocyanin wereusedtodifferentiateF1 fromtheirparentalphenotypes.Hybridvigoris easy to distinguish from parental lines. Anthocyanin (purple plant) is dominant over tan plant. If male parent is purpleandthefemaleparenttan,thentheF1derivingfromthesetwoparentsmustbepurple.Conversely,ifF1istan plants, this is considered a failure. These two techniques allowed the identification of few F1s plants. From F1 derivingfromtwopurpleortanplants,segregationofbmr plantsinF2orBC1B2wasasuremeanofverificationof trueF1cross.
Citation: Ousmane Seyni Diakité, Mamadou Aissata, Sissoko Aliou, Sanogo Sekouba, Mamoutou Kouressy, Vaksmann Michel, Daniel K. Dzidzienyo, Danquah Eric,
Tongoona Pangirayi, Karim Traoré, Niaba Teme, 2018, Brown midrib6 and 12 Genes Introgression in Two Nigerien and One Malian Sorghum Varieties: A
Practical Guide to Young Scientists with Limited Molecular Facility; Journal of Basic and Applied Scientific Research, 8(6)1-10.
October 2016-June 2017: BC1F2 and F2 segregation
BC1F1 seeds obtained during 2016 cropping season crosses between F1s and recurrent parents were planted for segregation study and generation advancement to obtain BC1F2. Each BC1F1 plant seeds were germinated in Petri dish (21-31 October), transplanted in plastic pots containing 1kg of compost when primary root appeared, then transplanted in nursery field for BC1F2 plant identification. As the RP (El-mota) population heights were heterogeneous, each crossing within this population crosses was kept separate for population identity and further DNA analysis. Harvest was done in early January for the first day short cool off season period.
At harvest, each bmr BC1F2 panicle and F3 panicles was threshed separately. Seeds were pre-germinated in petri-dish first, then transferred in pots after the appearance of their primary root and finally transplanted in the field in late January to early February. Photoperiod sensitive sorghum cultivation in long days (starting March) delays flowering until September avoiding harvest in May or early June. To speed up panicle initiation, seedlings after 21 days of transplantation in open field, were covered (Photo 2 A) with cages every day from 5PM to 8AM, during 30 days. This artificial shortening of day length induces reproductive phase and seed for day length sensitive plants. There is a black plastic layer under each white cotton tissue for firm shade to cover each sorghum plot. All panicles were selfed during all generation advancement. BC1F2 and F3 populations were harvested for adaptation, grain yield and stover potential under rain-fed conditions in Niger in 2017 cropping season.
A
B
Photo 2: shows the cage technique (A) and breeding population selfing (B) for generation advancement
Figure 1: General scheme of the bmr breeding population development in Sotuba research Station, Mali. |
J. Basic. Appl. Sci. Res.,8(6)1-10,2018
All BC1F1 plantswereplanted and non bmr segregating BC1F2 populations werediscarded.Bydiscarding nonbmr populationinBC1F2,wehad1/4(3:1)bmr plantsinasegregatingpopulation insteadof1/8fromthetotal numberofBC1F2 population planted. Itisworthnotifying thatsegregationratiowas1:1 inBC1F2 population for bmr andnonpopulations.Identificationofwildtypes(nonbmr plants)couldhavebeendoneonF1orBC1F1plants usingparentalpolymorphicmarkerclosetobmr 6 andbmr 12 duringparentalsurvey.Anotheroptionistoplant1020seedsfromBC1F1inplasticpotfilledwithcompostandtransplantseedlingtoopenfieldonlygenotypesshowing bmr traittosavetime,moneyandspace.
Dataanalysis
Dataforbmr segregatingphenotypeswerecollected in thefieldat BC1F2 andF2 generation for Chi-square (X2)test on every population. Chi-Square test is used in plant breeding for goodness of fit between expected and observedvalues.Itisastatisticaltermwhichreferstohowwellnumbersofwhatwasexpectedandnumbersofwhat isobservedarethesame.Inthiscase,thenumberofexpectedbmr plantswillbecomparedtoobservedonesaswell asthoseexpectednonbmr toobservednonbmr plants.Thistestacceptsorrejectsthesegregationratiobasedonone recessiveallele(bmr)inourcase.ThefollowingformulawasusedfortheChi-squaretest.
forsum;d=observed-expected;drepresentsthedeviationfromeortheexpectedvalue, e=expected
RESULTS
F1production
Atotal of 38 seeds were obtained as F1s (Table 4) in April 2016.During the following cropping season (June to September),theF1sandtheirparentsweregrown for F2 andBC1F1 seedproductionandgenerationadvancement. AlltheF1sexhibitedwhitemidribsconfirmingthedominanceofwhitemidriboverthebrownmidribtype.Selection basedontheanthocyanininheritancecombinedwithheterosiswasdoneforprogeniesobtainedintanxanthocyanin crosses.Anytanplantwasdiscardedduetoself-fertilizationseeds.Thesemethodsallowedaneasyidentificationof 9trueF1samong35crosses(Table4).Thislownumberof9F1seedsoutof35seedsindicatesthatcertainselfing occurredduringcrossings.Allprogenyofpossiblecrosseswereexamined:
• Sepon82(tan plant)xRedlanbmr6 (purple, anthocyanin):amongthenine(9)F1splants,only1plant(F11-3) was anthocyanin, therefore it was considered true F1 plant.The remaining seedlingswere tan plants andwerediscardedfromthebreedingpopulations’development.
• Sepon82(tan plant)xWheatlandbmr12 (purple, anthocyanin plant): two(2)F1plantswereanthocyanin, thuswereconsideredasF1s.
werealldiscardedfromthebreedingpopulationdevelopment. Table4indicatesthenumber ofsuccessfulcrossesandTable5reportsonthesegregationratiosofbmr plantinF2 populationandrespectiveKhisquarevalueofeachpopulation.
Table4:quantitiesofF1seedproducedandF1seedlingselectedpercrossatSotubaApril2016.
Crosses | ‘F1s’seedscount Seedlingscount TrueF1scount | |||
---|---|---|---|---|
Sepon82xRedlanbmr6 | 10 | 9 | 1 | |
Sepon82xWheatlanbmr12 | 2 | 2 | 2 | |
ElmotaxTx630bmr12 | 14 | 12 | 3 | |
ElmotaxRedlanbmr6 | 3 | 3 | 2 | |
KallaKénéxRedlanbmr6 | 2 | 2 | 1 | |
KallaKénéxRedlanbmr12 | 7 | 7 | 0 | |
Total | 38 | 35 | 9 |
Citation: OusmaneSeyniDiakité,MamadouAissata,SissokoAliou,SanogoSekouba,MamoutouKouressy,VaksmannMichel,DanielK.Dzidzienyo,DanquahEric, TongoonaPangirayi,KarimTraoré,NiabaTeme,2018,Brown midrib 6 and12 Genes IntrogressioninTwoNigerienandOneMalianSorghumVarieties:A PracticalGuidetoYoungScientistswithLimitedMolecularFacility;JournalofBasicandAppliedScientificResearch,8(6)1-10.
F2productionandsegregationratios
Table5:segregationintheF2families,SotubaDecember2016.
Population | Phenotype | Obs | Exp | df | Deviatio n(d) | d2 | d2/e | Cal X2 | X2Critical value | |
---|---|---|---|---|---|---|---|---|---|---|
Sepon82//Sepon82/Redla nbmr6 | Dullgreenmidrib | 13 | 14 | 1 | 1 | 1 | 0.071 | 0.271 | 3.840 | |
Bmr | 6 | 5 | 1 | 1 | 1 | 0.200 | ||||
Total | 19 | 19 | - | - | - | - | - | |||
Sepon82//Sepon82/Whea tlanbmr12 | Dullgreenmidrib | 14 | 15 | 1 | 1 | 1 | 0.066 | 0.266 | 3.840 | |
Bmr | 6 | 5 | 1 | 1 | 1 | 0.200 | ||||
Total | 20 | 20 | - | - | - | - | - | |||
Elmota//Elmota/Tx 630bmr12 | Whitemidrib | 15 | 15 | 1 | 1 | 1 | 0.066 | 0.266 | 3.840 | |
Bmr | 5 | 5 | 1 | 1 | 1 | 0.200 | 3.840 | |||
Total | 20 | 20 | - | - | ||||||
KallaKéné//KallaKéné /Redlanbmr6 | Whitemidrib | 15 | 15 | 1 | 1 | 1 | 0.066 | 0.266 | 3.840 | |
Bmr | 5 | 5 | 1 | 1 | 1 | 0.200 | 3.840 | |||
Total | 20 | 20 | - | - |
Foratestwith1df(degreeoffreedom),the"critical"valueofthechi-squarestatisticis3.84:whenX2>tothe criticalvalue,thenthedatadidnotfittheX2modelordeviatedfromtheexpectedvalue.Obs:numberofbmr plants counted in the field out of atotal of20 plants.Exp:number ofbmr plants derived fromthe single dominantgene segregation.
BC1F1populations
Tables6givesthenumberofBC1F1seedsproducedpercrossatSotubainSeptember2016.
PopulationBC1F1 | Numberof seedsproduced | |
---|---|---|
Elmota//Elmota/Tx630bmr12 | 15 | |
Sepon82//Sepon82/wheatlandbmr12 | 12 | |
Sepon82//Sepon82/Redlanbmr6 | 15 | |
KallaKéné//KallaKéné/Redlanbmr6 | 74 | |
Total | 116 |
BC1F2andBC1F3development
Four(4)BC1F2 bmr populationswereobtained(Table7)andadvancedtoBC1F3.
Table7:SegregationratiosintheBC1F2families
BC1F2Populations | Phenotypes | Total plants | Obs | Exp | df | Deviation(d) | d2 | d2/e | X2 | X2Critical value |
---|---|---|---|---|---|---|---|---|---|---|
Sepon82//Sepon82/Redlan bmr6 | bmr | 60 | 18 | 15 | 1 | 3 | 9 | 0.60 | 0.80 | 3.84 |
Dullgreen | 42 | 45 | 1 | 3 | 0.20 | |||||
Sepon82//Sepon82/Wheatlan dbmr12 | bmr | 60 | 13 | 15 | 1 | 2 | 4 | 0.26 | 0.35 | |
Dullgreen | 47 | 45 | 1 | 2 | 0.09 | |||||
Elmota//Elmota/Tx630 bmr12 | bmr | 60 | 17 | 15 | 1 | 2 | 4 | 0.26 | 0.35 | |
White midrib | 43 | 45 | 1 | 2 | 0.09 | |||||
KallaKéné//Kalla Kéné/Redlanbmr6 | bmr | 60 | 15 | 15 | 1 | 0 | 0 | 0 | 0 | |
White midrib | 45 | 45 | 1 | 0 | 0 | 0 | 0 |
Foratestwith1df(degreeoffreedom),the"critical"valueofthechi-squarestatisticis3.84.
DISCUSSION
Afterhandemasculationcrosses38F1seedswereobtainedfromalmost2000expectedseeds.Thislowlevel ofsuccessofcrossesismainlyduetohightemperaturesoccurringatthisperiodoftheyear(almost45°C)atSotuba. Inthisview,[17]demonstratedthat crossing sorghumatatemperatureof40°C(withlowhumidity)usuallyresults infailureofseedset.Regardingthelowsuccessofcrosses(only9 trueF1sover38seedlings),breakageofanthers and pollen pollution on tweezers tips during the emasculation process can be responsible. This hypothesis was
J. Basic. Appl. Sci. Res.,8(6)1-10,2018
supported by[17] whoprevented about anther ruptureand pollen sticking to the emasculation tool combined with heat. In addition, [16] reported that the success of hybridization varies with personal skill, the amount of injuries sustainedbythefloretpartduringtheemasculation.
Themidrib trait segregation in the breeding populationswasverified using thechi-squaretest and heterosis vigor.Everybreedingpopulationwasconstitutedoftwotypesofphenotypes(bmr plantsandnonbmr plants).Fora testwith1degreeoffreedom,thecriticalvalueofthechi-squarestatisticwas3.84(chi-squaretable).Consequently, averysmall chi-squarestatisticvalue obtained here means that theobserved data fits the expected data extremely well.Inthisorder,ourresultsshowednostatisticdeviationfromtheratioof3:1inthesegregatingpopulations(F2s andBC1F2).Indeed,twosituationswereobserved fromour dataanalysis:(1)agood fitofthesegregation;(2)the Chi-squarevalueswere inferior to the criticalvalues. Our results are in harmonywith those found by[3].For the sametrait,[26]alsofoundsimilarresultin F2bmr segregatingpopulations.Furthermore,inacrossinvolvingarice wild typeandamutant,[27]observed inthe followingF2populationaratioof3:1 andsuggestedthateach mutant phenotypewascausedbyarecessivemutationinasinglelocus.
Nighttimeof15hoursimposedonKallaKénéusingcagetechniquewascriticalforinducingitsreproductive stage.Thiscageislocallymanufactureandiseasymanipulated.
CONCLUSION-PERSPECTIVES
Conventional hand emasculation (HE) technique used to create diversity in self pollinated crop such as sorghumis still averypractical and worth technique in the absence of molecular makers for young breederswith resources limited to use molecular techniques facility. Mali and recently Niger breeders have been using HE techniquewhich needs skill field technicians to pursuetheir breeding activities while out sourcing sometime their crossingswhenfinancialopportunityisavailable.Opportunitytotrainotherinstitutiontechnicianwithlimitedaccess tomolecularlaboratoryfacilityisavailableatSotubain Maliforhandemasculationtraining.Thistechniqueisstill very useful in genes introgression for sorghum segregating populations development. However, in such circumstance, the environmental conditions such as temperature and humidityare ofgreat importance in sorghum hybridization. This was clearly observed during the bmr genes transfer. The bmr6 and bmr12 genes were successfullyintrogressedinNigerienandMaliansorghumvarietiesbackground.
HE genetic transfer of thebmr geneslaid out thedualpurposesorghumvarieties with potentialhigh stover quality populations for the two countries. Large populations bmr populations are developed and are under field evaluationforgrainandyieldpotentialinNigerandMali,twoSaheliancountriesfacinglivestockfeedingissuewith quality stover during dry season. Furthermore, introgression of bmr trait in late maturing photoperiod sensitive varietiesincreasebothbiomassproductivityandqualityduetotheirlowerlignincontents.
Identification of productive and quality stover lines, experimented in pilot study with livestock holders at farmerlevel,willsetuptheopportunityforintensivemilkandbeefproductiontopavetheroad forstabilizedcattle husbandryandtoendlongtermfreegrazingconflicts.
Acknowledgements
Iamgratefulto SMIL/USAID for myscholarship,WACCI for PhD training. IERMalifor the facilities and implicationoffieldtechniciansthroughoutthiswork.
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