Zhizhou HE,Zhenyu XIE,Jiankai SHEN,Yue WANG

1.Tropical Crops Genetic Resources Institute of Chinese Academy of Tropical Agricultural Sciences,Danzhou571737,China;

2.College of Agronomy,Hunan Agricultural University,Changsha 410128,China

Breeding of Indica Rice CMS Line Renong 1A with Virescent-yellow Leaf

Zhizhou HE1*,Zhenyu XIE1,Jiankai SHEN1,Yue WANG2

1.Tropical Crops Genetic Resources Institute of Chinese Academy of Tropical Agricultural Sciences,Danzhou571737,China;

2.College of Agronomy,Hunan Agricultural University,Changsha 410128,China

Renong 1Ais a new indica CMS line with leaf color marker,developed by testcrossing and continuous backcrossing,using a spontaneous mutant with virescent-yellow leaf isolated from the progenies of T98B/Jin 23B×Yixiang 1B as the male parent,and Fengyuan A as the female parent.All the chlorotic leaves of Renong 1A turn green in later developmental stages from leaf tip to base.This is the reason why Renong 1A is a new ideal leaf color marker material in indica rice. It shows apparent and stable leaf marker trait,good agronomic traits,high outcrossing rate,complete pollen sterility,fine grain quality and high combining ability. Renong 1A has already been technically identified and certified by Hainan Provincial Crop Variety Appraisal Committee in 2013.

Indica CMS line;Renong 1A;Leaf color marker;Breeding

H igh seed quality is important to promote the steady development in hybrid rice.During sensitive period,the fertility of two-line sterile lines and some excellent threeline sterile lines is easily affected by light and temperature.This problem will seriously affect the original seed purity in sterile line,or the hybrid seed purity,thus bringing risks in seed and field production[1-2].

The advantage of leaf color mutant is its clear and easily recognized characteristic.As a marker trait,it has drawn wide attention due to its potential application on seed production of hybrid rice and purity identification of F1seeds.However,the variation of leaf color is often directly or indirectly responsible for chlorophyll synthesis and degradation,then lowering the photosynthetic rate,and finally causing plant abnormal growth[3-4].This is the reason why the majority of leaf color mutants in rice can not be used in breeding application.Based on the expression period of leaf color maker gene,leaf-color marker materials in current breeding programs can be divided into two major types[5].Type one expresses different leaf color at a certain growth stage,mainly in seedling stage,and the other type expresses throughout the whole growth period. As the former is invalid in other stages and the latter may affect plant photosynthesis,the two types of leaf color marker materials have many limits and shortages in breeding.

Renong 1A is a new three-line sterile line with leaf color marker,developed by testcrossing and continuous backcrossing,using a spontaneous mutant with virescent-yellow leaf isolated from the progenies of indica rice maintenance lines as the male parent and Fengyuan A as the female parent.Different from the two types of materials with leaf-color marker described above,Renong 1A has more characteristics and higher utilization values,and the dynamic process of all

Breeding Process

In Sanya City of Hainan Province during the spring of 2007,a spontaneous mutant with virescent-yellow leaf was found in the progenies of T98B/Jin23b×Yixiang 1B,and a stable mutant with leaf color marker was obtained by self-crossing the spontaneous mutant for generations.Then, testcross was conducted between the stable mutant as the male parent and Fengyuan A as the female parent. From their progenies,excellent sterile single plants with leaf color marker were selected and backcrossed for eight generations in Hainan Province during 2009-2013.Meanwhile,the fertility,grain quality,resistance and combining ability of the progenies were measured and evaluated.Finally, the indica sterile line with virescent-yellow leaf Renong 1A and maintenance line Renong 1B were obtained with uniform population characteristics, good comprehensive traits and stable fertility.Renong 1A cultivar was approved by the Crop Cultivar Registration Committees of Hainan(Qiongshendao2013024)in 2013,and applied for new varieties protection in agricultural plant(CNA010797E).

Main Characteristics of Renong 1A

Characteristics in leaf color of Renong 1A

During the whole growth period of Renong 1A,the new leaves(rolled leaves and young leaves)are yellow at first,then gradually turn to green from leaf tip to base over time,this phenotype was observed from all leaves of main stem and tillers.The yellowing degree and greening process of etiolated leaves are regulated by temperature.For example,the leaves yellow more obviously and turn green more slowly at a low temperature(≤25℃), while the leaves yellow slightly and green faster at a high temperature(≥35℃).As the specific expression of leaf color marker in Renong 1A is different from that of other leaf color marker materials reported previously, it is considered as a new type of leaf color marker material in rice.

Growth duration and morphological characteristics of Renong 1A

Renong 1A is a three-line sterile line of indica rice.In Sanya City of Hainan Province,Renong 1A sown on January 3 of 2013 began to head on March 27,e.g.it took 83 d from sowing to initial heading date.Renong 1A sown on January 22 began to head on April 7,e.g.it was 75 d from sowing to initial heading date.In Danzhou, Renong 1A sown on May 24,2013 began to head on August 2,taking 70 d from sowing to initial heading date. Renong 1A sown on September 7 began to head on November 12,taking 66 d from sowing to initial heading date.Renong 1A plant has 12-14 leaves on the main stem,a height of 66-70 cm,moderate plant shape, strong tillering capacity,purple sheath, purple apiculus,purple stigma,no awns,flag leave of 32 cm long and 2.1 cm wide on average.Renong 1A planted spacing 20 cm×20 cm in Sanya City of Hainan Province yielded 15 panicles per plant,and 126 grains per panicle.The 1 000-grain weight was 25.5 g,and the panicle was 21.9 cm long on average.

Fertility of Renong 1A

The plants of Renong 1A sown in late cropping season of 2012 and early cropping season of 2013 grew well and uniformly.And the percentage of sterile plants was 100%.Fifty single panicles were sampled randomly from each population at heading stage for pollen microscope examination.The results showed that the pollens of Renong 1A were small and milk-white, of which 90%were typically abortive pollens,a small amount were round abortive pollens,a very few were stainable abortive pollens,and no normal and fertile pollens were observed. The self-fruitful rate was 0 when panicle was bagged.In September of 2013,the identification of sterile line organized by Hainan Province Crop Variety Approval Committee indicated that the para-typical plants and offtype plants had not been found in 1 000 plants or more.51 panicles sampled randomly from 51 plants were examined under a microscope,and the results showed that the abortive pollen rate was 100%.At the same time,the majority of the abortive pollen performed typically abortive.When the rice was bagged,55 samples of selfcrossed panicles were randomly selected and checked,and the self-fruitful sterile rate was 100%.All indices reached the identification standard of indica three-line sterile line in rice.

Flowering and outcrossing characteristics of Renong 1A

During the middle September in Hainan Province,a preliminary investigation of flowering characteristics of Renong 1A was conducted.The main results were as follows:①the rice started flowering on the second day after heading,and the blooming periods of single panicle and single plant were 5-7 and 7-9 d respectively;②the early flowering time was at 8:00-8:30 am,while above 70%flowers would outbreak before noon,and the peak flowering occurred at 10:30-12:30 am was easy to be distinguished;③they had long spikelet opening time,large full opening angle of spikelet and big stigma;④without the spraying of hormone gibberellin, the total stigma exsertion rate was over 65%,of which more than40% were dual exserted stigmas.The outcrossing rate of stigmas reached about 51%.As Renong 1A was sensitive to gibberellin,panicle enclosure could be eliminated after spraying gibberellin at a concentration of 150-180 g/hm2three times when 10%rice began to head.Propagation and hybrid seed production trials were conducted in the early season of 2013 in Sanya,and the yield reached 3.6 and 3.3 t/hm2respectively.

Rice quality and resistance of Renong 1A

The rice samples obtained from early rice in Sanya during 2013 were determined and analyzed by Tropical Crops Genetic Resources Institute of Chinese Academy of Tropical Agricultural Sciences.The results showed that the percentage of brown rice was 79.2%,the head milling rice rate was52.8%,grain length was 7.1 mm,the ratio of grain length to width was 2.8, chalkiness rate was 55%,chalkiness degree was 5.6%,alkali spreading value was 6.5,gel consistency was 45 mm,amylose content was 23.8%and protein content was 9.3%.In Danzhou City of Hainan Province during 2013, the resistances of Renong 1A to rice bacterial blight and blast were identified.The results turned out that its resistances to bacterial blight,seedling blast,leaf blast and panicle blast were level 5,4,5 and 7 respectively.In Sanya and Danzhou,rice sheath blight and blast did not occur in the early rice growth period;bacterial blight and hopper had little harm to rice,and the smut rate was 0 in seed production;the bacterial blight often happened to the late rice in rainy and typhoon season.

Combination ability of Renong 1A

Some good combinations were selected by testcrossing restorer linesR116,R118,R121,R124,R126, R191,R210,R231,R527,R599 and Huazhan with Renong 1A,and compared in field trials in Sanya and Danzhou,using Luyue 589 cultivar which was approved and widely promoted in Hainan Province as the control.The tests demonstrated that all the selected combinations showed normal green leaf,early maturity,good plant type and grain quality,moderate tillering ability,heading uniformity, short plant height,strong lodging resistance and so on.The combinations of Renong 1A with R116,R118,R121, R124,R126,R527,R599 and Huazhan sown in early cropping season in Sanya took 82-92 d from seeding to initial heading date,with plant height of 81-97 cm,9.2 valid panicles per plant,144 grains per panicle,seed setting rate of 92%and 1 000-grain weight of 29.1g on average.The actual yield of the combinations was up to 8.65 t/hm2which was 7.7%more than the control[5].The combinations of Renong 1A withR118,R124,R191, R210,R231 and Huazhan sown in late cropping season in Danzhou took 71-81 d from seeding to initial heading date,with plant height of 96-106 cm, 12.5 valid panicles per plant,173 grains per panicle,seed setting rate of 81%and 1 000-grain weight of 30.5 g on average respectively.The actual yield was up to 6.94 t/hm2,which was 5.2%more than the control.The results above showed that Renong 1A had good general combing ability and restorability,and the yields of some combinations(such as Renong1A/R121,Renong 1A/R124 and Renong 1A/Huazhan)were obviously increased compared with the control.New combination of hybrid rice with high application potential can be identified and screened out in regional test.

Application Prospects

In the breeding and production practice of hybrid rice,if a leaf color mutation was controlled by only one recessive nuclear gene,this gene can be easily introduced into a three-line or two-line sterile line by hybridization and backcrossing to generate a marker trait,which will help to remove the off-type plants during sterile line reproduction and hybrid seed production and to rapidly and effectively identify the purity of hybrid F1seeds.In the current period,the materials with leaf color marker,which have already been successfully applied to rice breeding programs,are divided into two expression patterns[5].One type express only at a certain stage,such as sterile lines Quanlong A[2],Baifeng A[6],Yutu S[7], Yunfeng 88A[8],whose leaves were albino at seedling stage then turn to normal green;the other type express throughout the whole stage,such as the yellowish leaf mutant Annongbiao 810S[9],CMS line Biao-A with yellowgreen leaf[10],japonica CMS line Jinhui A with yellowish leaf[11].

The expression form of leaf color in Renong 1A is different from other materials currently used in rice breeding.It has two main characteristics,①leaf color maker trait is intuitive at seedling stage,as the process of leaves turning from yellow to green leaf can be observed continuously;②the rice plant can grow and develop normally,based on the facts that every single leaf is yellow at early development stage,and then turns green from leaf tip to base,while the content of photosynthetic pigment increases gradually to normal level.So,Renong 1A is an ideal material with leaf color marker.Some of its combinations exhibited early maturity,short stem,good quality,outstanding yield and so on, presenting high application potential. Based on our previously studies,we also found that the leaf color mutation of Renong 1A was controlled by a recessive gene.According to the expression of leaf color and molecular mapping,this gene was a new leaf color mutation gene.It has better utilization value in new cultivar breeding with marker trait and further study of molecule mechanism of rice leaf color changing.

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Responsible editor:Qingqing YIN

Responsible proofreader:Xiaoyan WU

轉(zhuǎn)綠型新葉黃化秈稻不育系熱農(nóng)1A的選育

賀治洲1*，謝振宇1，沈建凱1，王悅2
（1.中國熱帶農(nóng)業(yè)科學院熱帶作物品種資源研究所，海南儋州571737；2.湖南農(nóng)業(yè)大學農(nóng)學院，湖南長沙410128）

熱農(nóng)1A是從T98B/金23B∥宜香1B雜交后代群體中獲得的轉(zhuǎn)綠型新葉黃化自然突變株系為父本，以豐源A為母本，經(jīng)測交和連續(xù)多代回交轉(zhuǎn)育而成的秈稻葉色標記不育系。熱農(nóng)1A全生育期每片葉都經(jīng)歷新葉黃化，隨后從葉尖向葉基部由黃轉(zhuǎn)綠的動態(tài)發(fā)育過程，是一種新的理想型水稻葉色標記材料;該不育系具有標記性狀明顯、農(nóng)藝性狀優(yōu)良、花粉敗育徹底、異交結(jié)實率高、米質(zhì)較優(yōu)、配合力好等特點，于2013年通過海南省農(nóng)作物品種審定委員會審定。

秈稻不育系；熱農(nóng)1A；葉色標記；選育leaves turning from yellow to green can be observed during leaf growth period.There is no doubt that Renong 1A is a new ideal leaf-color marker material.Furthermore,Renong 1A provides a new source for rapidly and accurately removing off-types,rapidly and accurately identifying seeds purity during seed production.

中央級公益性科研院所基本科研業(yè)務(wù)費專項（1630032014031）；海南省自然科學基金項目（313071）；2013年度留學人員科技活動擇優(yōu)資助項目；湖南省科技計劃項目（2013NK3037）；湖南省教育廳科學研究項目（13C392）。

賀治洲(1984-)，男，湖南邵陽人，助理研究員，博士,從事水稻遺傳育種方面研究，E-mail：hzzdata@163.com。*通訊作者。

2015-03-30

修回日期 2015-05-15

Supported by National Nonprofit Institute Research Grant of CATAS-TCGRI (1630032014031);Natural Science Foundation of Hainan Province(313071);Scientific Research Fund for Outstanding Overseas Chinese Scholars(2013);Hunan Provincial Science and Technology Project,China(2013NK3037);Research Foundation of Education Bureau of Hunan Province(13C392).

*Corresponding author.E-mail:hzzdata@163.com

Received:March 30,2015 Accepted:May 15,2015