全 文 ：Received date: 2008- 03- 10
Foundation item: Na tiona l Natur e Science Founda tion ( 30460064) ; Inte rna tiona l Cooperation Item ( IPGRI APO 04 /068)
Biography: L IANG Yun-tao ( 1975- ) , ma le, Assistant Pr ofesso r o f Rice Research Institute, Guangxi Academy of Ag ricul-
tural Sciences. Major in gerplasm resour ces and mo lecula r biolog y; * Autho r fo r co r respondence, E-mail:
wildrice@ 163. com.
Studies on Job s tears genetic resources in East Asia
LIANG Yun-tao1 , CHEN Cheng-bin1* , X U Zhi-jian1 , HUANG Juan1 , ZEN G Hua-zhong1 ,
LAI Qun-zhen1 , L IAN G Shi-chun1 , LUO Yu-xi1 , HUANG Ren-jian2
( 1. Rice Research Institute, Guangxi Academy o f Ag ricultural Sciences, Nanning 530007, China;
2. Fumian Ag ricultura l Bureau of Yulin City , Yulin, Guangxi 537023, China )
Abstract: The germplasm resour ces of Job s tea rs w er e investig ated in Nanning , Yulin, Baise cities from 2004
to 2006, and 117 accessions of cultiva tion and wild Job s tear s we re collec ted and planted in test field. The ag ro-
nomic tr aits w ere observ ed and reco rded, and the varieties with excellent ag r onomic traits w er e selected fo r breed-
ing. Up to today , 3 varieties w ere go t, which could be used in ag ricultural pr oduc tion. In order to know the genet-
ic div e rsity of Job s tear s in Guangxi o f China , Japan and Ko rea mo re clea rly, SSR analysis w as car ried out fo r 77
accessions from the 3 ci ties. The r esults show ed that 77 accessions were so r ted into tw o g roups acco rding to their
g enetic r elation. Three accessions of Job s tear s from Ko rea composed a g roup because th eir genetic backg r ound
w as very similar. Othe r Job s tea rs composed ano the r g roup. Mo reover , it was found tha t g enetic dive rsity of
Job s tears r esources wa sn t associa ted w ith th eir o rigin obv iously.
Key words: Job s tea rs; explora tion; collection; g enetic div e rsity
CLC number: S519. 024　 Document code: A　　　　　 Ar ticle: 1002- 8161( 2008) 04- 0413- 06
东亚薏苡遗传资源研究
梁云涛1 ,　陈成斌1* ,　徐志健1 ,　黄　娟1 ,　曾华忠1 ,　赖群珍1 ,　梁世春1 ,
罗毓喜 1 ,　黄仁健 2
( 1.广西农业科学院水稻研究所 ,　南宁　 530007;　 2.玉林市福绵区农业局 ,　广西玉林　 537023)
摘要: 2004～ 2006年 ,通过对南宁、玉林、百色等广西部分地区薏苡种质资源进行考察 ,共收集到 117份栽培和
野生薏苡资源 ,并种植在试验地保存。通过对这些种质资源的农艺性状进行观察鉴定 ,并选择农艺特性好的材料进
行育种 ,目前已育成 3个品种 ,可应用于农业生产。 为了了解来源于中国广西、日本、韩国的薏苡的多样性 ,对 77份
薏苡资源进行 SSR分析 ,结果表明 ,按亲缘关系可将 77份薏苡资源分为两大类群。其中 3份来自韩国的薏苡资源遗
传背景非常相似 ,组成一个大类群 ;其余薏苡资源则构成另一个大类群。 研究还发现 ,种质材料的遗传差异与其地
域来源没有明显的关系。
关键词: 薏苡 ;考察 ;收集 ;遗传多样性
　　 The genusCoix , af ter Tripsacum, is the clo s-
est g roup to maize. It belongs to the t ribe
Androgoneae , sub-tribeCoicineae f rom the fami ly
Gramineae . It i s composed o f fiv e species w hich
a re morpho logically distinct in rela tion to the shape
o f leaves and seeds. The fiv e species a re C.
lacrymajobi L. , C . gigantea K. , C. aquat ica R. ,
C. puel larum and C . pai lanei M.
[ 1, 2]
. Unlike
teo sinte, Tripsacum and maize are na tiv e to Ameri-
ca, Coix is origin in Asia, mainly lo cated in the
no rther n region of India, Burma, and the Fa r
East
[3, 4 ]
. The speciesCoix lacryma -jobi is popula r-
ly known as the Job s tears. Some selected st rains
are cul tivated fo r thei r edible g rains, and it show s
·413·Guangx i Ag ricultura l Sciences Vo l. 39 No . 4 2008
g rea t potential in the areas of animal and human
nutri tion
[5 ] .
Job s tears is dist ributed in most prov inces of
China widely. Wild Job s tears mostly g row s in
w et v alley, and sides riv er or brook. Cultiv ar is
planted in the areas w here alti tude is f rom 30 to
2500 m. Because of di fference o f geog raphy cir-
cumstance, climate and cul tiv ation condi tion a-
mong dif ferent areas, Job s tears resource is v ery
plentiful in our country and many local v arieties
g row in many a reas. Climate is mi ld and rain is
abundant in Guangxi tha t lo cates in South China,
w hich are benefi t fo r the g row th o f Job s tears.
Job s tears are dist ributed widely in Guangxi ,
w hich genetic diversi ty is prolific. Up to today ,
Guangxi province is the only area where C.
aquatica was found in China based on publica tion.
Al though the co llection, conserv ation and eva lua-
tion of Job s tears germplasm have been car ried out
and advance has been achiev ed in our country, the
evaluation on the germplasm is confined most ly to
the partial t rait s including mo rpholog y, g rain yield
and nutri tion qua li ty. Some researches on genome
o f Job s tears w ere conducted. Han et al
[ 6, 7]
mapped the maize gene mir1 on ch romosome of
Job s tears using f luo rescence in situ hybridiza tion
method, and constructed a quanti ta tive chromo-
some map for the fi rst time in C . lacryma-jobi ,
w hich w ould provide the foundation for genome
analysis of C. lacryma -jobi . Dia set al
[8 ]
cloned and
sequenced the t rnS /pseudo-tRN A /nad3 /rps12
gene cluster inCoix lacryma-jobi L. , and analy zed
i ts expression.
The know ledge of genetic div ersi ty and genetic
rela tionships among conserv ed germplasm of any
crop is o f cri tical impo rtance fo r ensuring long-
term success of i ts breeding pro g rams, lack of
molecular characterization and evaluation da ta
w ould limi t utilization of conserved Job s tea rs
g ermpla sm in breeding prog rams. The present
study employed the col lections of Job s tea rs
g ermpla sm in Guangxi and evalua ted the ag ronom-
ic trait s and genetic div ersity of germplasm from
Guangxi of China, Japan and Ko rea, fo r learning
genetic rela tionship among st g ermplasm and breed-
ing new varieties.
1　Materials and methods
1. 1　 Areas explored and investigation method of
Job s tears germplasm
Job s tears g ermplasm exploration had been
conduct a t tw o stag es in Guangxi of China f rom
November of 2004 to September o f 2006. At first
stage, f rom 2004 to 2005, w e explo red 25 counties
of 7 ci ties including Baise, Yulin, Hezhuo,
Chong zuo and Fangcheng , Beihai and Nanning,
w here there is plenty of Job s tears g ermpla sm. At
second stage, Guilin, Laibin and Liuzhou ci ties
covering 18 counties loca ting in centre and north of
Guangxi w ere explo red in 2006. The sta tus of
Job s tears in these a reas could reflect the si tuation
of Job s tears in Guangxi. The location, al titude
and a rea of nativ e lands of wild Job s tears w ere
surveyed and recorded by using GPS method. At
the same time, envi ronment , clima te and soil con-
ditions of na tiv e lands w ere investiga ted, too. Seed
or stem of representativ e plants of Job s tears
g rowing in dif ferent na tiv e lands w ere co llected.
The collected materials w ere planted in test
field of Rice Research Insti tute of GX AAS
( Guangxi Academy o f Ag ricul tural Sciences) . At
the same time, thei r seeds w ere conserv ed in
Genebank of GX AAS. The C . aquatica couldn t
produce g rain and was conserv ed in germplasm
nursery. All collected materials w ere documented
fo llow ing by the standard published by IPGRI.
1. 2　 Investigation of agronomic traits of partial
Job s tears
From 2004 to 2006, the ag ronomic characters
of 77 accessions of Job s tears w ere investiga ted.
The materials w ere planted in the test field of Rice
Research Insti tute of GX AAS where soi l condition
is simi lar. Minimum descriptors fo r Job s tears
published by IPGRI w ere applied fo r characteriza-
tion in the field. Night kinds o f ag ronomic trait s,
including plant height , number of ti llers, diameter
of stem, leng th and width of leaf , g rain set ting
·414· Guangx i Ag ricultura l Sciences Vo l. 39 No . 4 2008
lay er, g rain shape, g rain colo r and g row th dura-
tion w ere investig ated, and the obtained data wa s
analy zed using SAS so ftw are package. The da ta on
the materials f rom three countries w ere used fo r
g enetic div ersity analysis.
Tw enty accessions including cul tiv ar and wild
species w ere picked out for breeding prog ram due
to thei r ex cellent perfo rmance of ag ronomic charac-
ters.
1. 3　 Analysis genetic diversity of Job s tears
Seventy-seven accessions of Job s tea rs includ-
ing 30 accessions f rom Guangxi of China , 17 acces-
sions f rom Japan and 30 accessions f rom Ko rea
w ere planted fo r investig ation o f ag ronomic charac-
ters. M olecular analy sis of g enetic div ersi ty of the
77 accessions w as ca rried out using SSR method.
One hundred and ninety-three pairs o f SSR
primers w ere developed referring to the primers in-
forma tion of maize at internet w ebsi te www.
maizegdb. com. Primers w ere synthesized by
Shanghai Sangon Bio-technique Limi ted Company.
1. 3. 1　 DN A ex tract and SSR analy sis
DN A o f Job s tears w as ex tracted f rom leaves
o f 3-w eek-old plants using CTAB method w ith mi-
nor modification. The ex t racted DN A samples
w ere disso lv ed in TE buf fer ( 10 mmo l Tris base,
0. 1 mmo l /L EDTA) and test fo r qualitativ e and
quanti tativ e characteristics using MBA2000 UV /
V IS Spect ropho tometer. The samples w ere di luted
into 100 ng /μl wi th T E buf fer for further analysis.
The PCR condi tion of each pair o f primer of
SSR markers w as optimized. PCR reaction sy stem
w as as fo llow ed: 20μl to tal v olume o f reaction so-
lution contained 50ng DN A template, 0. 6 uni ts
Taq DN A Po lymerase, 400 mol /L dN TP, 2. 5
mol /L M gCl2 , 1× PCR buffer ( 10 mmol /L Tris-
HCl , 50 mmol /L KCl , 100 mg /mL gel, pH8. 3) ,
100 ng SSR primer. The reaction so lution w as cov-
ered wi th mineral oil. PCR ampli fication reactions
w ere perfo rmed in a PTC-100 PCR sy stem.
Af ter amplification, product wa s added wi th
fo rmamide dena turation dye to dena ture fo r 8 min
a t 94℃ , elect ropho resis w as performed on a 6%
po lyacrylamide denaturation gel in TBE fo r one
hour. At the end, the gel w as v isualized using si l-
ver dye method.
1. 3. 2　 Diversity analy sis
Basic statistics w ere calculated using the ge-
netic analy sis package PO PGEN E version 1. 31
[9 ]
fo r diversi ty measurements among 77 Job s tears
accessions at each microsatellite locus.
The genetic simi lari ty among accessions w as
determined w ith similari ty matrix. Unequivocally
reproducible bands w ere scored and entered into a
binary cha racter matrix ( 1 for presence and 0 fo r
absence) . The unweighted pai r g roup method us-
ing ari thmetic averages ( U PGM A ) algo ri thm
based on simi lari ty matrix da ta was used to con-
struct a dendrog ram w ith the help of SAHN-clus-
tering and TREE prog rams from N TSYS-pc.
V. 2.
[10 ]
.
2　Results and analysis
2. 1　 Investigation and col lection of Job s tears
germplasm in Guangxi of China
It spent about 3 years to explo re ten ci ties of
Guangxi Prov ince o f China including Nanning , Li-
uzhou, Gui lin ci ties and o ther places, w here the
clima te and env ironmental conditions are very fi t
fo r g row th of Job s tea rs.
In the investigation process, 117 accessions of
Job s tea rs covering wi ld species and cultiva rs w ere
co llected. The most of co llected materials w ere
wi ld species including the C. aquatica . Amongst
tho se, 46 accessions came from Baise Ci ty and 24,
10, 5, 3, 9, 18 and 4 accessions w ere collected in
Hezhuo , Yulin, Beihai, Nanning, Guilin, Laibin
and Liuzhou ci ties, respectiv ely. Wild Job s tears
w as found a t ov er 50 si tes o f 8 ci ties, which mostly
g rew in w et v alley and sides o f brook o r pool w here
enough w ater could be supplied to Job s tears
plant. How ever, there w ere few si tes w here a
larg e a rea of Job s tears g rew .
The collection o f cultiv ars most ly focused on
local v arieties nea r to ex tinction o r po ssessing ex-
cellent ag ronomic t rai t. The cultiv ars w ere mostly
planted in mountainous areas o f no rthw estw ard
·415·LIANG Yun-tao et al : Studies on Job s tears g ene tic resources in Ea st Asia
and central section of Guangxi Prov ince. How ever,
the area o f land planted cul tiv ars w as regulated
constantly based on the selling amount of g rain of
Job s tea rs in market. The a rea increased when
selling amount increased, o therwise i t decreased.
2. 2　 Characterization and evaluation of agronomic
traits of Job s tears
The characterization investig ation of 77 acces-
sions o f Job s tears w ere conducted f rom 2004 to
2006. Nine ag ronomic trait s, including plant
height , number of tillers and so on, w ere investi-
ga ted and analy zed
[11 ] . There w as obvious dif fer-
ence in the ag ronomic t rai ts among the materials
f rom three a reas. The mean value of th e plant
height , diameter of stem , leng th and width of
leaf , g rain setting layer of Job s tears f rom
Guangxi o f China was bigg er than those o f Japan
and Korea. In particular, the plant height , diame-
ter o f stem and g rain set ting layer o f Job s tea rs
f rom Guangxi o f China w as about double as those
o f Japan and Ko rea , while the number o f tiller of
Job s tears f rom Guangxi was few er than those
f rom Japan and Korea. It w as po ssible that the
materials f rom Japan and Korea w ere no t adapted
to local env ironmental condition due to dif ferences
in climate condi tions betw een the tw o counties and
Nanning , Guangx i. But field perfo rmance o f Job s
tears f rom Korea w as similar to those f rom Japan,
w hich suggest that relationship betw een them
might be clo ser.
2. 3　 Breeding program
Tw enty accessions including cultiva rs and wild
species w ere chosen for breeding prog ram acco rd-
ing to the perfo rmance o f field cha racteriza tion.
Aiming at sho rtcomings that cultiva rs ex isted, w e
improved some ag ronomic t rait s of those by w ay of
crossing and back-crossing betw een cultiv ars need
to be improved and parents possessing excellent
cha racters. Af ter cro ssing fo r sev eral g enerations,
materials w ith good comprehensiv e ag ronomic
cha racters w ere selected. At the end, w e succeed-
ed in get ting 3 accessions, which perfo rmed very
good agronomic trai t s. Therefore, the Job s tea rs
v arieties in Guangxi w ould be improved af ter these
accessions could be used in production.
2. 4　Molecular analysis for genetic diversity
Up to now , there has been no repo rt about the
development o f micro satel li te ma rkers of Job s
tea rs. Maize and Job s tea rs belong to Ma ydeae
tribe o f the g rass family , so there is simila ri ty in
genomic composi tion betw een them. Mo re studies
on genome o f maize have been conducted so far.
Many SSR markers of maize have been developed,
w hich covered all ch romosomes of maize almost.
M oreover, the info rmation about them was easy to
be obtained from publica tions, internets and so on.
In addi tion, th ere has been successful ex ample that
SSR markers o f maize w ere applied for mo lecula r
analy sis of Job s tears befo re. Therefore, the
mo lecula r ana lysis o f genetic div ersi ty of Job s
tea rs w as conducted using SSR markers of maize in
this study.
The primers o f SSR markers w ere determined
referring to related publications and the resul ts of
our previous studies on Job s tea rs. To tal 193
pairs of primers were uti li zed in the evaluation of
po lymo rphism of Job s tea rs g ermplasm. They dis-
tributed through al l chromosomes of maize. The
results of the present study show ed that there w ere
32 primer pai rs that could amplify bands f rom
DNA of representativ e Job s tea rs selected a-
mongst 77 accessions. It suggested tha t genetic
backg round betw een Job s tears and maize w as no t
so similar as their o rigins. Out of them , 4 primer
pairs produced reproducible polymorphic bands,
w hich w ere selected fo r further mo lecular analy sis.
Thei r number w as p-bnlg1037, p-umc1284,
p-umc1078 and p-umc1335, respectiv ely. Detailed
info rmation about them could be obtained from
www. maizegdb. o rg.
The 4 polymorphic microsatellite ma rkers
w ere used for genetic div ersi ty analysis among the
77 accessions. By using the 4 SSRmarkers, a to tal
of 48 alleles w as detected. For each SSR marker,
the number of alleles ranged from 2 to 22 wi th an
average o f 12.
·416· Guangx i Ag ricultura l Sciences Vo l. 39 No . 4 2008
The results show ed that the 77 accessions
w ere classified into tw o main g roups according to
g enetic relation among them ( Fig. 1) . One g roup
included 3 accessions f rom Korea and the o ther in-
cluded the remaining 74 accessions. There w as ge-
netic dif ference among the 77 accessions f rom Chi-
na, Japan and Ko rea. However, th e genetic div er-
si ty amongst Job s tears resources f rom Guangxi of
China, Japan and Ko rea w asn t so plentiful as be-
fo re. In this study, i t w as dif ficult to judge how
much genetic backg round of the 77 accessions w as
dif ferent due to too small mo lecula r markers used
fo r div ersity analysis. So i t is necessa ry to develop
mo re molecular ma rkers of Job s tears fo r further
research on genetic diversi ty in the future. M ore-
over , the genetic di fference amongst accessions
·417·LIANG Yun-tao et al : Studies on Job s tears g ene tic resources in Ea st Asia
wasn t associated with their origins obviously.
3　 Discussion
In the present study, to tal 117 accessions of
Job s tea rs germplasm w ere collected f rom 10 ci ties
o f Guangxi Province, China, and the ag ronomic
cha racters of 77 accessions of Job s tea rs f rom Chi-
na, Japan and Ko rea w ere investig ated. The sur-
vey on habi tat of wild Job s tea rs w ould be helpful
fo r set ting down st rateg y o f in-si tu pro tection of
Job s tea rs g ermplasm. The collection o f Job s
tears g ermplasm could contribute to the conserv a-
tion of g ermplasm near to ex tinction. In order to
bet ter understand the genetic div ersi ty of the Job s
tears g ermplasm, the diversi ty analysis of 77 ac-
cessions f rom Guangxi of China, Japan and Ko rea
w as conducted using SSR markers tech nolog y. Al-
though the achiev ements w ere obtained, there
w ere several aspects needed to be improved and re-
info rced in future. Fi rstly , the surv ey on Job s
tears germplasm should be continued to carry out.
There is plenti ful Job s tears g ermplasm in
Guangxi. Mo re area s should be investig ated,
w hich would be helpful for comprehensively know-
ing about the status of Job s tears g ermplasm in
Guangxi. Secondly , the breeding pro gram of them
should be continued to carry out , because the se-
lected ma terials couldn t be direct ly applied in com-
mercia l production. In particular, new breeding
too l like mo lecular assistant selection ( M AS )
should be used to sho rten the breeding dura tion.
Thirdly , molecular analysis of Job s tea rs
g ermpla sm should be reinforced. SSR markers
technolog y w as an ef fectiv e too l fo r studying ge-
netic div ersi ty of g ermplasm. But the resul ts of
molecular analysis in this study w ere no t ideal e-
nough, which w as mostly caused by lacking proper
primers of SSR markers in Job s tears. Therefore,
the concerned study needed to be reinforced fur-
ther.
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(责任编辑　韦莉萍 ;　审校　李杨瑞 )
·418· Guangx i Ag ricultura l Sciences Vo l. 39 No . 4 2008