Molecular Breeding for Rainfed Lowland Rice in the Mekong Region

Abstract

In the past 20 years, the rice-breeding program in Thailand had little success in developing new cultivars to replace Kao Dawk Mali 105 (KDML105) and Kao Khor 6 (RD6) for the rainfed lowland rice environments. The main reason for the poor adoption of new cultivars by farmers is the susceptibility to diseases and unacceptable grain qualities. The conventional breeding program also takes at least 15 years from initial crossing to the release of new cultivars. A new breeding strategy can be established to shorten the period for cultivar improvement by using marker-assisted selection (MAS), rapid generations advance (RGA), and early generation testing in multi-locations for grain yield and qualities. Four generation of MAS backcross breeding were conducted to transfer genes and QTL for bacterial blight resistance (BLB), submergence tolerance (SUB), brown plant hopper resistance (BPH) and blast resistance (BL) into KDML105. Selected backcross lines, introgressed with target gene/QTL, were tolerant to SUB and resistant to BLB, BPH and BL. The agronomic performance and grain quality of these lines were as good as or better than KDML105.     

Development of rice introgression lines with brown planthopper resistance and KDML105 grain quality characteristics through marker-assisted selection

‘Khao Dawk Mali 105’ (KDML105), a Thai aromatic rice cultivar, has been accepted in markets as a prime jasmine rice with premium prices. It has been extensively used as a parental line to develop new cultivars for rainfed lowland areas in Thailand because of its favorable quality and fragrance. However, this cultivar is highly susceptible to brown planthopper (BPH), a phloem sap-feeding insect pest of rice. The main objective of this study was to combine KDML105 essential grain quality traits with BPH resistance from the donor cultivar, ‘Rathu Heenati’. The linkage drag between Bph3 and Wx^a alleles was successfully broken by phenotypic and marker-assisted selections. All introgression lines (ILs) developed in this study showed a broad spectrum resistance against BPH populations in Thailand and had KDML105 grain quality standards. Finally this study was revealed that the ILs can be directly developed into BPH resistant varieties or can be used as genetic resources of BPH resistance to improve rice varieties with the Wx^b allele in rice breeding programs.     

Evaluation of Agronomic Traits in Chromosome Segment Substitution Lines of KDML105 Containing Drought Tolerance QTL under Drought Stress

Drought is a major abiotic constraint to rice production in rainfed lowland and insufficiently irrigated areas.The improvement of drought tolerant varieties is one of the strategies to reduce the negative effects of drought.Quantitative trait loci(QTLs) for primary and secondary traits related to drought tolerance(DT) on chromosomes 1,3,4,8 and 9 that determined from double haploid lines derived from a cross between CT9993 and IR62266 were introgressed and dissected into small pieces in the genetic background of Khao Dawk Mali 105(KDML105) to develop chromosome segment substitution line(CSSL) population.The CSSLs were evaluated at the reproductive stage for their agronomic performance and yield components under drought stress,and results were compared with irrigated condition.The flowering of CSSL lines was 6 to 7 d earlier than KDML105.The mean values of grain yields in the CSSLs were higher than KDML105 under drought and irrigated conditions.At irrigated condition,the grain yields of introgression lines carrying DT-QTLs from chromosomes 4 and 8 were higher than that of KDML105,whereas other traits showed little difference with KDML105.Analysis indicated that grain yield has positive correlation with plant height,tiller and panicle number per plant,and total grain weight per plant under drought stress while negatively correlated with days to flowering.As mentioned above,CSSLs showing good adaptation under drought stress can be used as genetic materials to improve drought tolerance in Thai rainfed lowland rice breeding program,and as materials to dissect genes underlying drought tolerance.     

Root plasticity under fluctuating soil moisture stress exhibited by backcross inbred line of a rice variety,Nipponbare carrying introgressed segments from KDML105 and detection of the associated QTLs

In rainfed lowland rice ecosystem, rice plants are often exposed to alternating recurrences of waterlogging and drought due to erratic rainfall. Such soil moisture fluctuation (SMF) which is completely different from simple or progressive drought could be stressful for plant growth, thereby causing reduction in yield. Root plasticity is one of the key traits that play important roles for plant adaptation under such conditions. This study aimed to evaluate root plasticity expression and its functional roles in dry matter production and yield under SMF using Nipponbare, KDML 105 and three backcross inbred lines (BILs) and to identify QTL(s) associated with root traits in response to SMF at two growth stages using Nipponbare/KDML105 F₂ plants. A BIL, G3-3 showed higher shoot dry matter production and yield than Nipponbare due to its greater ability to maintain stomatal conductance concomitant with greater root system development caused by promoted production of nodal and lateral roots under SMF. QTLs were identified for total nodal root length, total lateral root length, total root length, number of nodal roots, and branching index under SMF at vegetative and reproductive stages. The QTLs detected at vegetative and reproductive stages were different. We discuss here that relationship between root system of G3-3 and the detected QTLs. Therefore, G3-3 and the identified QTLs could be useful genetic materials in breeding program for improving the adaptation of rice plants in target rainfed lowland areas.     

Improvement of Upland Rice Variety by Pyramiding Drought Tolerance QTL with Two Major Blast Resistance Genes for Sustainable Rice Production

Varalu is an early maturing rice variety widely grown in the rainfed ecosystem preferred for its grain type and cooking quality. However, the yield of Varalu is substantially low since it is being affected by reproductive drought stress along with the blast disease. The genetic improvement of Varalu was done by introgressing a major yield QTL, qDTY_12.1, along with two major blast resistance genes i.e. Pi54 and Pi1 through marker-assisted backcross breeding. Both traits were transferred till BC₂ generation and intercrossing was followed to pyramid the two traits. Stringent foreground selection was carried out using linked markers as well as peak markers (RM28099, RM28130, RM511 and RM28163) for the targeted QTL (qDTY_12.1), RM206 for Pi54 and RM224 for Pi1. Extensive background selection was done using genome-wide SSR markers. Six best lines (MSM-36, MSM-49, MSM-53, MSM-57, MSM-60 and MSM-63) having qDTY_12.1 and two blast resistance genes in homozygous condition with recurrent parent genome of 95.0%-96.5% having minimal linkage drag of about 0.1 to 0.7 Mb were identified. These lines showed yield advantage under drought stress as well as irrigated conditions. MSM-36 showed better performance in the national coordinated trials conducted across India, which indicated that improved lines of Varalu expected to replace Varalu and may have an important role in sustaining rice production. The present study demonstrated the successful marker-assisted pyramiding strategy for introgression of genes/QTLs conferring biotic stress resistance and yield under abiotic stress in rice.     

Avoidance of Linkage Drag Between Blast Resistance Gene and the QTL Conditioning Spikelet Fertility Based on Genotype Selection Against Heading Date in Rice

Previous study showed that a linkage drag between a blast resistance gene Pi25(t) and QTLs conditioning spikelet fertility (qSF-6) and number of filled grains per panicle (qNFGP-6) was detected on the short arm of chromosome 6. A larger population was used for further verification, and the results confirmed the linkage drag between the blast resistance gene and QTL conditioning spikelet fertility, other than QTL conditioning number of filled grains per panicle. Breakdown or avoidance of the linkage drag could be achieved by selection against the genotype background of a heading-date gene (qHD-7) that resided in the region between RM2 and RM214 on chromosome 7. For further validation, two lines with almost identical genotypes on all chromosomal regions except the Pi25(t) region on chromosome 6 were chosen to develop a new population. The results showed that qSF-6 could be further subdivided into qSF-6-1 and qSF-6-2. When the genotype of the region between RM2 and RM214 was from rice variety Zhong 156, the linkage drag between Pi25(t) and qSF-6-2 was detected and the allele of qSF-6-2 from rice variety Gumei 2 reduced the spikelet fertility. When the genotype of the region between RM2 and RM214 was from Gumei 2, no linkage drag was detected. This indicates that the linkage drag between the blast resistance gene and the QTL conditioning spikelet fertility could be broken down or avoided under a certain background genotype selection against heading-date and provides a marker aided solution for high level of blast resistance and yield breeding in rice and other crops as well.     

分子标记辅助选择育成抗稻瘟病粳稻新品种扬农粳3091

通过杂交及不完全回交,结合分子标记辅助选择,育成携带广谱抗稻瘟病基因Pigm的粳稻新品种扬农粳3091。选育过程中的稻瘟病抗性鉴定结果显示,携带Pigm基因品系的抗性水平显著好于对照。该品种于2021年通过江苏省农作物品种审定委员会审定,具有综合抗性好、品质优、丰产稳产性好等特点。本文介绍了该品种的选育过程、特征特性及主要栽培技术要点。     

通过生育期基因型选择避免稻瘟病抗性与结实率的遗传累赘

利用重组自交系研究表明,在水稻第6染色体短臂上稻瘟病抗性基因Pi25(t)与控制结实率和每穗实粒数的QTL之间存在遗传累赘。为了验证这种关系,采用了更大的遗传群体进行分析,结果表明稻瘟病抗性与结实率存在遗传累赘,但未检测到稻瘟病抗性与每穗实粒数存在遗传累赘。通过对第7染色体长臂RM2-RM214区间抽穗期基因（qHD 7）型背景进行选择,可以打破或避免稻瘟病抗性与结实率的遗传累赘。为了进一步验证这种关系,选择Pi25(t)区间基因型不同、RM2-RM214区间基因型相同、其他染色体区间基本一致的两个株系发展新群体进行分析,除第6染色体的结实率QTL可以分解成2个效应较小的QTL（qSF 6 1和qSF 6 2）外,当第7染色体RM2-RM214区间基因型为中156背景时,Pi25(t)与结实率QTL（qSF 6 2）存在遗传累赘,且qSF 6 2来自父本谷梅2号的等位基因起减效作用;当第7染色体RM2-RM214区间基因型为谷梅2号背景时,第6染色体上没有检测到结实率QTL。上述结果说明在特定育种材料中对抽穗期基因进行选择可以成功打破或避免稻瘟病抗性与结实率的遗传累赘,为水稻以及其他作物的高产抗病育种提供了一种新途径。     

Breeding of R8012,a Rice Restorer Line Resistant to Blast and Bacterial Blight Through Marker-Assisted Selection

Genetic improvement is one of the most effective strategies to prevent rice from blast and bacterial blight(BB) diseases,the two most prevalent diseases jeopardizing rice production.Rice hybrids with dural resistance to blast and BB are needed for sustainable production of food.An incomplete diallele design resulted in 25 crosses between five blast and five BB resistant germplasm accessions.Only one pair of parents,DH146 × TM487,showed polymorphism for all the markers to identify one blast resistance gene Pi25 and three BB resistance genes,Xa21,xa13 and xa5,thus it was used in the marker-assisted selection(MAS).F2 individuals of DH146 × TM487 were genotyped using flanking markers of RM3330 and sequence tagged site(STS) marker SA7 for Pi25.The resistant F2 plants with Pi25 were used for pyramiding BB resistance genes Xa21,xa13 and xa5 identified by the markers pTA248,RM264 and RM153,respectively in subsequent generations.Finally,after selection for agronomic traits and restoration ability among 12 pyramided lines,we acquired an elite restorer line,R8012 including all four target genes(Pi25+Xa21+xa13+xa5).Hybrid Zhong 9A/R8012 derived from the selected line showed stronger resistance to blast and BB,and higher grain yield than the commercial checks uniformally in experimental plots,2007 state-wide yield trial and 2008 nation-wide yield trial.This study provides a paradigmatic example to show that MAS is a practically feasible tool in effectively pyramiding multiple resistance genes.The resultant restoring line and its hybrid would play an important role in securing rice production in China.     

Marker-Assisted Pyramiding of Genes Conferring Resistance Against Bacterial Blight and Blast Diseases into Indian Rice Variety MTU1010

Two major bacterial blight(BB) resistance genes(Xa21 and xa13) and a major gene for blast resistance(Pi54) were introgressed into an Indian rice variety MTU1010 through marker-assisted backcross breeding. Improved Samba Mahsuri(possessing Xa21 and xa13) and NLR145(possessing Pi54) were used as donor parents. Marker-assisted backcrossing was continued till BC2 generation wherein PCR based functional markers specific for the resistance genes were used for foreground selection and a set of parental polymorphic microsatellite markers were used for background selection at each stage of backcrossing. Selected BC2F1 plants from both crosses, having the highest recoveries of MTU1010 genome(90% and 92%, respectively), were intercrossed to obtain intercross F1(ICF1) plants, which were then selfed to generate 880 ICF2 plants possessing different combinations of the BB and blast resistance genes. Among the ICF2 plants, seven triple homozygous plants(xa13xa13Xa21Xa21Pi54Pi54) with recurrent parent genome recovery ranging from 82% to 92% were identified. All the seven ICF2 plants showed high resistance against the bacterial blight disease with a lesion lengths of only 0.53–2.28 cm, 1%–5% disease leaf areas and disease scoring values of ‘1' or ‘3'. The seven ICF2 plants were selfed to generate ICF3, which were then screened for blast resistance, and all were observed to be highly resistant to the diseases. Several ICF3 lines possessing high level of resistance against BB and blast, coupled with yield, grain quality and plant type on par with MTU1010 were identified and advanced for further selection and evaluation.     

Improving Rice Blast Resistance by Mining Broad-Spectrum Resistance Genes at Pik Locus

Magnaporthe oryzae is known for its genetic diversity and pathogenic variability, leading to rapid breakdown of resistance in rice. Incorporating multiple broad-spectrum blast resistance genes into rice cultivars would extend disease resistance longevity. Effective resistance breeding in rice therefore requires continual enrichment of the reservoir of resistance genes and alleles. We conducted a large-scale screen of rice blast resistance in about 2 000 rice accessions. Among them, 247 accessions showed at least medium resistance to the natural infection of rice blast and 7 novel Pik alleles were identified from them. Variations in gene sequences were then correlated with the phenotypic trait to enable the identification of favorable alleles. Among the seven novel Pik alleles, the resistant rate of Pik-R0/ME/7017 donors was greater than 80%, and the disease score was less than 3. Through molecular marker-assisted backcross breeding, we successfully transferred the three Pik alleles, Pik-R0/ME/7017, into an elite cultivated line Kongyu 131 to obtain BC₃F₂ lines, which showed enhanced resistance to rice blast compared with the recurrent parent. Assessment of these near-isogenic lines in the greenhouse using 31 isolates of M. oryzae from Heilongjiang Province of China revealed that the resistant levels of the BC₃F₂ lines with Pik-R0/ME/7017 were significantly higher than those of the established cloned resistance genes Pik-m and Pi1. Exploring such alleles will enrich our gene library for resistance to rice blast.     

回交法在杂交稻抗瘟育种中的应用

以ＩＲ２６、ＩＲ２４为轮回亲本，赤块矮，ＩＲ５０等为抗源供体品种，采用回交育种法，，成功地将多个抗源的不同抗性基因导入轮回亲本，育成了一批抗稻瘟病的恢复系；用这些新育成的恢复系配组成选育得到汕优６２１６等一批抗稻瘟病的新组合，目前已在生产大面积推广。回交育种中的有关技术也进行了讨论。     

水稻灌浆期耐热害的数量性状基因位点分析

 利用由98个家系组成的Nipponbare / Kasalath // Nipponbare回交重组自交系群体及其分子连锁图谱，以粒重感热指数\[（适温粒重-高温粒重）/适温粒重×100\]为评价指标，采用混合线性模型的QTL定位方法，对水稻灌浆期耐热性的主效、上位性数量性状基因位点及其与环境的互作进行分析。共检测到3个灌浆期耐热性主效QTL，分别位于第1、4和7染色体上，LOD值为8.16、11.08和12.86，贡献率8.94%、17.25%和13.50%。其中位于第4染色体标记C1100-R1783之间的QTL，没有显著的上位性和环境互作效应，表明在不同环境和遗传背景中的表达较为稳定，在水稻耐热性育种中可能具有较大的利用价值，其耐热性等位基因来自亲本Kasalath，高温热害时可减少粒重损失3.31%。位于第1染色体标记R1613-C970之间的QTL和第7染色体标记C1226-R1440之间的QTL，耐热性等位基因来自亲本Nipponbare，分别可减少粒重损失2.38%和2.92%。这两个QTL均具有与环境的互作效应，其中第7染色体上的QTL还和其他基因位点有互作。检测到8对加性×加性上位性互作QTL，分布于第1、2、3、5、7、8、10和12染色体上。没有检测到上位性QTL与环境的互作效应。     

稻瘟病抗性分子育种研究综述

对稻瘟病菌致病机理、抗性鉴定方法、抗源筛选、抗性基因定位和克隆及无毒基因研究、分子标记辅助育种和转基因育种等方面的研究进展进行了综述,同时对抗性基因的利用以及聚合育种进行了展望.     

Development of elite breeding lines conferring Bph18 gene-derived resistance to brown planthopper (BPH) by marker-assisted selection and genome-wide background analysis in japonica rice (Oryza sativa L.)

Brown planthopper (BPH) is a serious threat to rice production. In this study, we have used the novel resistance gene Bph18 derived from Oryza australiensis and incorporated it into an elite japonica cultivar, Junambyeo, which is highly susceptible to BPH. The Bph18 gene was introduced by marker-assisted backcross (MAB) breeding into Junambyeo. The backcrossed progenies were evaluated for desirable agronomic and grain quality traits and the selection of improved breeding lines while simultaneously evaluating BPH resistance by bioassays in the greenhouse and foreground selection. Of the 26 advanced backcross breeding lines (ABL), four lines showed agronomic traits similar to those of the recurrent parent, with strong resistance to BPH. Molecular genotyping of the four ABL revealed the conversion of genotypes closely resembling the genotype of Junambyeo. The percentage of donor chromosome segments in ABL decreased from 12.3% in the BC₂ to 9.4%, 8.4% and 5.3% in BC₃, BC₄ and BC₅ generations, respectively. ABL retained small sizes of the donor chromosome segments on chromosomes 1, 2, 10, 11 and 12 but the genomes of ABL2, ABL3 and ABL4 were homosequential to the recurrent parent on chromosomes 3, 4, 5, 6, 7 and 9 without donor chromosome segment introgression. The ABL1 and ABL2 retained only some small segments of the donor genome on chromosomes 9 and 8, respectively. Fine structure analysis of the Bph18 flanking region between RM511 and RM1584 markers on chromosome 12 showed a progressive elimination of donor-derived chromosome segments from BC₂ to BC₅ generations. The percentage of O. australiensis derived chromosome segment substitution in the recurrent parent background decreased from 28% of the donor parent to 6.7%, 3.9%, 3.4% and 3.4% in BC₂, BC₃, BC₄ and BC₅ generations, respectively. However, it was revealed that the O. australiensis-derived chromosome segment (1320 kb) in ABL containing the Bph18 gene was consistently maintained irrespective of advances in backcross generations. BPH resistant elite breeding lines with agronomic and grain quality traits similar to those of the recurrent parent were successfully developed by foreground and background analysis in japonica background without linkage drag.     

Genetic Diversity Analysis of Rice Germplasm in Tripura State of Northeast India Using Drought and Blast Linked Markers

We genotyped 74 rice germplasms including Tripura's local landraces, improved varieties, cultivars and breeding lines and other rice varieties using molecular markers for genetic diversity, drought QTLs, and blast resistance genes. The number of alleles per locus ranged from 2 to 5 with an average of 2.9. The polymorphic information content value per locus ranged from 0.059 (RM537) to 0.755 (RM252) with an average of 0.475. Cluster analysis based on 30 simple sequence repeat markers revealed 5 clusters and also indicated the presence of variability within the rice accessions. The drought QTL qDTY2.1 was found in 56.0% of germplasms and qDTY1.1 was detected in only 6.8% of the germplasms. Out of seven rice blast resistance genes screened, only two rice varieties, RCPL-1-82 and Buh Vubuk (Lubuk), were positive for four blast resistance genes while only Releng possessed two blast resistance genes. Among 74 rice germplasms, only three accessions, Releng, RCPL1-82 and Buh Vubuk (Lubuk), possessed both drought-related QTLs and blast resistance genes. Overall, the 74 indigenous rice genotypes showed low level of genetic diversity, which is in contrast to high level of genetic diversity among rice varieties in northeast India, where highlights the good farming practice, conservation of germplasms and the limitation of molecular markers employed in this study. The presence of both drought related QTLs and blast resistance genes in some of the germplasms can be useful in future breeding programmes.     

Race Specificity of Major Rice Blast Resistance Genes to Magnaporthe grisea Isolates Collected from indica Rice in Guangdong, China

Race-specific resistance and field resistance of 30 rice blast resistance monogenic lines dedved from different resources were evaluated. The spectra of resistance to 163 Magnaporthe grisea isolates collected from indica rice in Guangdong Province, China ranged from 0.6% to 89.6%. Most of the monogenic lines showed a narrow resistance spectrum and high susceptibility in rice blast area, whereas the lines with Pikh and Pi1(t) had the broad resistance spectra of 89.6% and 82.2% respectively, showing a high and stable blast resistance in fields. According to the cluster analysis of specific resistance to 163 blast isolates tested, the 30 monogenic lines were divided into 15 groups, and based on the principal factor analysis, nine kinds of race-specific resistance were identified. Pik, Piz5, Pi9 and Pish can be used as candidate resistance genes for rice breeding since their specific resistance differed from those of the backbone parents in Guangdong, China. Gene pyramiding of Pikh [or Pi1(t)], Pi9 (or Piz5) and Pish (or Pita2) will be effective to obtain broad-spectrum blast resistance in rice breeding program in Guangdong, China. The strategies for studying and application of rice blast resistance genes were discussed.     

具抗稻瘟病基因Pi25杂交稻恢复系的分子标记辅助选育

 从组合中156/谷梅2号所衍生的重组自交系群体中选择携带抗稻瘟病基因Pi25的3个株系,分别与高产恢复系9308和3 11配组,通过单粒传法获得的6个F6重组自交系群体用于研究。在Pi25先前定位的遗传图谱上,新增加与该基因紧密连锁的分子标记RM3330和A7,并结合RM3330和A7分子标记辅助选择的结果,共筛选到了109个Pi25基因纯合的株系。用现有的与恢复基因连锁的标记对这109个株系进行二次筛选,最终获得20个Pi25基因和恢复基因均纯合的株系。对育性恢复力鉴定试验表明,选育出的抗病恢复系具有较好的恢复能力,并已应用于育种实践;人工稻瘟病接种试验证实,所用标记不同,分子标记辅助选择的效率差异显著,单个标记辅助选择符合率均不高,而采用目标基因两侧连锁的标记同时进行辅助选择,则可以明显提高分子标记辅助选择符合率。     

应用分子标记辅助选择培育抗稻瘟病杂交稻亲本

多性状的分子标记辅助选择具有重要的育种应用价值。研究先应用与水稻抗稻瘟病基因Pi25紧密连锁的分子标记对394个重组自交系进行筛选,再应用育性恢复基因连锁标记检测中选株系,获得20份候选恢复系和27份候选保持系。经田间接种稻瘟病菌、考查测交F1育性,表明利用该研究策略可以同步对多个性状进行选择,提高杂交稻亲本选育的效率。     

稻瘟病抗性基因Pi1和Pi2的聚合及其育种价值分析

水稻稻瘟病的发病受外界条件影响较大,单纯依靠人工接种抗性鉴定比较困难。因此,人工接种抗性鉴定与分子标记辅助选择结合应用对提高抗性育种效率具有重要意义。本研究利用已建立的水稻抗稻瘟病基因Pi1和Pi2显性分子标记,对黑龙江省主栽品种空育131进行分子标记辅助选择,将Pi1和Pi2基因聚合到空育131中,对其原始亲本和不同基因组合聚合后代进行了稻瘟病田间抗性鉴定,对2个抗性基因的聚合效应进行了统计和评价,由此明确了Pi1和Pi2基因在寒地育种辅助选择中的应用价值。